Unconjugated
A series of 12 novel acylhydrazone, chalcone and amide-bridged analogues of combretastatin A-4 were designed and synthesized toward tubulin. All these compounds were determined by elemental analysis, (1)H NMR, and MS. Among them, compound 7 with acylhydrazone-bridge, bearing a benzyl at the indole-N position, was identified as a potent antiproliferative agent against a panel of cancer cell lines with IC50 values ranging from 0.08 to 35.6 μM. In contrast, its cytotoxic effects on three normal human cells were minimal. Cellular studies have revealed that the induction of apoptosis by compound 7 was associated with a collapse of mitochondrial membrane potential, accumulation of reactive oxygen species, alterations in the expression of some cell cycle-related proteins (Cyclin B1, Cdc25c, Cdc2, P21) and some apoptosis-related proteins (Bax, PARP, Bcl-2, Caspase3). The docking mode showed the binding posture of CA-4 and compound 7 are similar in the colchicine-binding pocket of tubulin, as confirmed by colchicine-tubulin competitive binding assay, tubulin polymerization inhibitory activity, extracellular protein expression determination assay and confocal immunofluorescence microscopy. In vivo study, compound 7 effectively inhibited A549 xenograft tumor growth without causing significant loss of body weight suggesting that compound 7 is a promising new antimitotic agent with clinical potential.
Cyclin D2 is involved in the pathology of vascular complications of type 2 diabetes mellitus (T2DM). This study investigated the role of cyclin-D2-regulated miRNAs in endothelial cell proliferation of T2DM. Results showed that higher glucose concentration (4.5 g/l) significantly promoted the proliferation of rat aortic endothelial cells (RAOECs), and significantly increased the expression of cyclin D2 and phosphorylation of retinoblastoma 1 (p-RB1) in RAOECs compared with those under low glucose concentration. The cyclin D2-3' untranslated region is targeted by miR-98, as demonstrated by miRNA analysis software. Western blot also confirmed that cyclin D2 and p-RB1 expression was regulated by miR-98. The results indicated that miR-98 treatment can induce RAOEC apoptosis. The suppression of RAOEC growth by miR-98 might be related to regulation of Bcl-2, Bax and Caspase 9 expression. Furthermore, the expression levels of miR-98 decreased in 4.5 g/l glucose-treated cells compared with those treated by low glucose concentration. Similarly, the expression of miR-98 significantly decreased in aortas of established streptozotocin (STZ)-induced diabetic rat model compared with that in control rats; but cyclin D2 and p-RB1 levels remarkably increased in aortas of STZ-induced diabetic rats compared with those in healthy control rats. In conclusion, this study demonstrated that high glucose concentration induces cyclin D2 up-regulation and miR-98 down-regulation in the RAOECs. By regulating cyclin D2, miR-98 can inhibit human endothelial cell growth, thereby providing novel therapeutic targets for vascular complication of T2DM.
Probiotics actively participate in neuropsychiatric disorders. However, the role of gut microbiota in brain disorders and vascular dementia (VaD) remains unclear. We used a mouse model of VaD induced by a permanent right unilateral common carotid arteries occlusion (rUCCAO) to investigate the neuroprotective effects and possible underlying mechanisms of Clostridium butyricum. Following rUCCAO, C. butyricum was intragastrically administered for 6 successive weeks. Cognitive function was estimated. Morphological examination was performed by electron microscopy and hematoxylin-eosin (H&E) staining. The BDNF-PI3K/Akt pathway-related proteins were assessed by western blot and immunohistochemistry. The diversity of gut microbiota and the levels of butyrate in the feces and the brains were determined. The results showed that C. butyricum significantly attenuated the cognitive dysfunction and histopathological changes in VaD mice. C. butyricum not only increased the levels of BDNF and Bcl-2 and decreased level of Bax but also induced Akt phosphorylation (p-Akt) and ultimately reduced neuronal apoptosis. Moreover, C. butyricum could regulate the gut microbiota and restore the butyrate content in the feces and the brains. These results suggest that C. butyricum might be effective in the treatment of VaD by regulating the gut-brain axis and that it can be considered a new therapeutic strategy against VaD.
Phloretin (Ph) existing in apples, pears and various vegetables is known to have antitumor activities in several cancer cell lines. However, little is known about its effect on human lung cancer cells. The aim of the present study was to see whether Ph could induce apoptosis of non-small cell lung cancer (NSCLC) cells, and explore the possible underlying mechanism of action. We found that Ph markedly induced cell apoptosis of NSCLC cell line A549, and inhibited the migration of A549 cells in a dose-dependent manner. The expression level of BAX, cleaved caspase-3 and -9, and degraded form of PARP was increased and Bcl-2 was decreased after Ph treatment. In addition, the phosphorylation of P38 MAPK, ERK1/2 and JNK1/2 was increased in a dose‑dependent manner in parallel with Ph treatment. Inhibition of P38 MAPK and JNK1/2 by specific inhibitors significantly abolished the Ph-induced activation of the caspase-3 and -9. In vivo tumor-suppression assay further indicated that Ph (20 mg/kg) displayed a more significant inhibitory effect on A549 xenografts in tumor growth. All these findings indicate that Ph is able to inhibit NSCLC A549 cell growth by inducing apoptosis through P38 MAPK and JNK1/2 pathways, and therefore may prove to be an adjuvant to the treatment of NSCLC.
Low-density lipoprotein receptor-related protein 1 (LRP1) is known to regulate cell survival and inflammation. The present study investigated the involvement of LRP1 in the regulation of tumor necrosis factor (TNF)-α-induced expression of matrix metalloproteinase (MMP)-13. Furthermore, the study aimed to elucidate the mechanisms underlying the effects of LRP1 on TNF-α-induced inflammation and apoptosis of chondrocytes. Lentivirus-mediated RNA interference techniques were used to knockdown the LRP1 gene. Subsequently, the effects of LRP1 on TNF-α-induced MMP-13 expression were determined using quantitative polymerase chain reaction, western blot analysis and ELISA. Furthermore, the TNF-α-induced intracellular pathway was investigated using a nuclear factor (NF)-κB inhibitor (Bay 11-7082). In addition, the effect of LRP1 regulation on growth and apoptosis in chondrocytes was investigated using western blot analysis and a TUNEL assay. LRP1 knockdown was shown to increase TNF-α-induced MMP-13 expression via the activation of the NF-κB (p65) pathway, which reduced the expression of collagen type II and cell viability. In addition, LRP1 inhibited cell apoptosis by increasing the expression of phospho-Akt and B-cell lymphoma 2 (Bcl-2), while suppressing the expression of caspase-3 and Bcl-2-associated X protein. The results of the present study indicated that LRP1 was able to inhibit TNF-α-induced apoptosis and inflammation in chondrocytes. Therefore, LRP1 may be an effective osteoarthritis inhibitor, potentially providing a novel approach for antiarthritic therapeutics.
Forkhead box C2 (Foxc2) protein is a transcription factor in regulation of development, metabolism, and immunology. However, the regulatory mechanisms of Foxc2 on proliferation and apoptosis of preadipocytes are unclear. In this study, we found that high-fat-diet-induced obesity elevated the expression of Foxc2 and cyclin E after 6 weeks. Additionally, Foxc2 suppressed preadipocyte differentiation, increased cell counts and augmented G1-S transition of preadipocytes, along with the elevation of cyclin E expression and the reduction levels of p27 and p53. Furthermore, Foxc2 knockdown reduced early apoptotic cells with accompanying reduction of mitochondrial membrane potential and increased fragmentation of genomic DNA. We show that Foxc2 reduces the expression of Bax, caspase-9, and caspase-3 in both serum-starved and palmitic acid-induced cell apoptotic models, which confirms the anti-apoptotic role of Foxc2. Moreover, the protein kinase B (Akt)/mammalian target of rapamycin (mTOR)C1 signaling pathway and the ERK/mTORC1 signaling pathway were activated along with preadipocyte proliferation in response to Foxc2 overexpression, whereas apoptosis marker genes were downregulated during this process. Those effects were blocked by the interference of Foxc2 or signal pathways specific inhibitors. These data collectively reveal that Foxc2 enhances proliferation of preadipocytes and inhibits apoptosis of preadipocytes by activating the Akt/mTORC1 and ERK/mTORC1 signaling pathways.
Sodium butyrate (NaB) is a dietary microbial fermentation product of fiber and serves as an important neuromodulator in the central nervous system. In this study, we further investigated that NaB attenuated cerebral ischemia/reperfusion (I/R) injury in vivo and its possible mechanisms. NaB (5, 10 mg/kg) was administered intragastrically 3 h after the onset of reperfusion in bilateral common carotid artery occlusion (BCCAO) mice. After 24 h of reperfusion, neurological deficits scores were estimated. Morphological examination was performed by electron microscopy and hematoxylin-eosin (H&E) staining. The levels of oxidative stress and inflammatory cytokines were assessed. Apoptotic neurons were measured by TUNEL; apoptosis-related protein caspase-3, Bcl-2, Bax, the phosphorylation Akt (p-Akt), and BDNF were assayed by western blot and immunohistochemistry. The results showed that 10 mg/kg NaB treatment significantly ameliorated neurological deficit and histopathology changes in cerebral I/R injury. Moreover, 10 mg/kg NaB treatment markedly restored the levels of MDA, SOD, IL-1β, TNF-α, and IL-8. 10 mg/kg NaB treatment also remarkably inhibited the apoptosis, decreasing the levels of caspase-3 and Bax and increasing the levels of Bcl-2, p-Akt, and BDNF. This study suggested that NaB exerts neuroprotective effects on cerebral I/R injury by antioxidant, anti-inflammatory, and antiapoptotic properties and BDNF-PI3K/Akt pathway is involved in antiapoptotic effect.
Steroid alkaloids have been suggested as potential anticancer compounds. However, the underlying mechanisms of how steroid alkaloids inhibit the tumor growth are largely unknown. Here, we reported that solanine, a substance of steroid alkaloids, has a positive effect on the inhibition of pancreatic cancer cell growth in vitro and in vivo. In pancreatic cancer cells and nu/nu nude mice model, we found that solanine inhibited cancer cells growth through caspase-3 dependent mitochondrial apoptosis. Mechanically, solanine promotes the opening of mitochondrial membrane permeability transition pore (MPTP) by downregulating the Bcl-2/Bax ratio; thereafter, Cytochrome c and Smac are released from mitochondria into cytosol to process the caspase-3 zymogen into an activated form. Moreover, we found that the expression of tumor metastasis related proteins, MMP-2 and MMP-9, was also decreased in the cells treated with solanine. Therefore, our results suggested that solanine was an effective compound for the treatment of pancreatic cancer.
Musk has been traditionally used in East Asia to alleviate the symptoms of angina pectoris. However, it remains unclear as to whether muscone, the main active ingredient of musk, has any beneficial effects on persistent myocardial ischemia in vivo. The aim of the present study was to investigate whether muscone can improve cardiac function and attenuate myocardial remodeling following myocardial infarction (MI) in mice. Mice were subjected to permanent ligation of the left anterior descending coronary artery to induce MI, and then randomly treated with muscone (2 mg/kg/day) or the vehicle (normal saline) for 3 weeks. Sham-operated mice were used as controls and were also administered the vehicle (normal saline). Treatment with muscone significantly improved cardiac function and exercise tolerance, as evidenced by the decrease in the left ventricular end-systolic diameter, left ventricular end-diastolic diameter, as well as an increase in the left ventricular ejection fraction, left ventricular fractional shortening and time to exhaustion during swimming. Pathological and morphological assessments indicated that treatment with muscone alleviated myocardial fibrosis, collagen deposition and improved the heart weight/body weight ratio. Muscone inhibited the inflammatory response by reducing the expression of transforming growth factor (TGF)‑β1, tumor necrosis factor (TNF)-α, interleukin (IL)-1β and nuclear factor (NF)-κB. Treatment with muscone also reduced myocardial apoptosis by enhancing Bcl-2 and suppressing Bax expression. Muscone also induced the phosphorylation of protein kinase B (Akt) and endothelial nitric oxide synthase (eNOS). Our results demonstrate that muscone ameliorates cardiac remodeling and dysfunction induced by MI by exerting anti-fibrotic, anti-inflammatory and anti-apoptotic effects in the ischemic myocardium.
It has been documented in in vitro studies that zinc oxide nanoparticles (ZnO NPs) are capable of inducing oxidative stress, which plays a crucial role in ZnO NP-mediated apoptosis. However, the underlying molecular mechanism of apoptosis in neurocytes induced by ZnO NP exposure was not fully elucidated. In this study, we investigated the potential mechanisms of apoptosis provoked by ZnO NPs in cultured primary astrocytes by exploring the molecular signaling pathways triggered after ZnO NP exposure. ZnO NP exposure was found to reduce cell viability in MTT assays, increase lactate dehydrogenase (LDH) release, stimulate intracellular reactive oxygen species (ROS) generation, and elicit caspase-3 activation in a dose- and time-dependent manner. Apoptosis occurred after ZnO NP exposure as evidenced by nuclear condensation and poly(ADP-ribose) polymerase-1 (PARP) cleavage. A decrease in mitochondrial membrane potential (MMP) with a concomitant increase in the expression of Bax/Bcl-2 ratio suggested that the mitochondria also mediated the pathway involved in ZnO NP-induced apoptosis. In addition, exposure of the cultured cells to ZnO NPs led to phosphorylation of c-Jun N-terminal kinase (JNK), extracellular signal-related kinase (ERK), and p38 mitogen-activated protein kinase (p38 MAPK). Moreover, JNK inhibitor (SP600125) significantly reduced ZnO NP-induced cleaved PARP and cleaved caspase-3 expression, but not ERK inhibitor (U0126) or p38 MAPK inhibitor (SB203580), indicating that JNK signaling pathway is involved in ZnO NP-induced apoptosis in primary astrocytes.
The present study aims to investigate the pharmacological effect of the exopolysaccharides from Aphanothece halophytica GR02 (EPSAH) on the HeLa human cervical cancer cell line. HeLa cells were cultured in RPMI-1640-10% FBS medium containing with or without different concentrations of EPSAH. Cell viability was assessed by methylthiazol tetrazolium (MTT) assay. Cell apoptosis was elevated with Wright-Giemsa staining, AO/EB double staining, and DNA fragmentation assay. Apoptosis-associated molecules from cultured HeLa cells were quantified using Western blot analysis. Our results suggest that EPASH induces apoptosis in HeLa cells by targeting a master unfolded protein response (UPR) regulator Grp78. Grp78 further promotes the expression of CHOP and downregulates expression of survivin, which leads to activate mitochondria-mediated downstream molecules and p53-survivin pathway, resulting in caspase-3 activation and causing apoptosis. These findings provide important clues for further evaluating the potential potency of EPSAH for use in cancer therapy.
Our previous study revealed that Type II cGMP-dependent protein kinase (PKG II) inhibits epidermal growth factor (EGF)-induced MAPK/ERK and MAPK/JNK-mediated signal transduction through the inhibition of the phosphorylation/activation of the EGF receptor (EGFR). As EGFR also mediates several other signal transduction pathways besides MAPK-mediated pathways, the present study was designed to investigate whether PKG II was able to inhibit EGF/EGFR-induced phosphatidylinositol-3-kinase (PI3K)/Akt-mediated signal transduction. The AGS human gastric cancer cell line was infected with adenoviral constructs encoding a cDNA of PKG II (Ad-PKG II) to increase the expression of PKG II, and treated with 8-pCPT-cGMP to activate the enzyme. Western blotting was used to detect the phosphorylation/activation of the key components of the signal transduction pathway, including EGFR, PI3K, Akt, mTOR and NF-κB. The levels of apoptosis-related proteins, including Bax, Bcl-2, caspase 9 and DNA fragment factor (DFF), were also determined by western blotting. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining was used to detect the apoptosis of the AGS cells. The results revealed that EGF treatment increased the phosphorylation (activation) of EGFR, PI3K, Akt and mTOR, and increased the nuclear localization (activation) of NF-κB. EGF treatment also reduced the apoptosis of the AGS cells and increased the expression of the anti-apoptotic protein, Bcl-2, but had no effect on the expression of the pro-apoptotic protein, Bax, and did not alter the levels of caspase 9 and DFF. Increasing the PKG II activity of AGS cells by infecting them with Ad-PKG II and stimulating them with 8-pCPT-cGMP inhibited the EGF-induced activation of EGFR, PI3K, Akt, mTOR and NF-κB; caused an increase in caspase 9 breakdown (activation) and DFF levels; and reversed the anti-apoptotic effect of EGF. The results suggest that PKG II may also inhibit EGF-induced signal transduction of PI3K/Akt-mediated pathways, and further confirm that PKG II is able to block the activation of EGFR.
The fruit of Schisandra chinensis has been used in the traditional Chinese medicine for thousands of years. Accumulating evidence suggests that Schisandrin B (Sch B) has cardioprotection effect on myocardial ischemia in vitro. However, it is unclear whether Sch B has beneficial effects on continuous myocardial ischemia in vivo. The aim of the present study was to investigate whether Sch B could improve cardiac function and attenuate myocardial remodeling after myocardial infarction (MI) in mice. Mice model of MI was established by permanent ligation of the left anterior descending (LAD) coronary artery. Then the MI mice were randomly treated with Sch B or vehicle alone. After treatment for 3 weeks, Sch B could increase survival rate, improve heart function and decrease infarct size compared with vehicle. Moreover, Sch B could down-regulate some inflammatory cytokines, activate eNOS pathway, inhibit cell apoptosis, and enhance cell proliferation. Further in vitro study on H9c2 cells showed similar effects of Sch B on prevention of hypoxia-induced inflammation and cell apoptosis. Taken together, our results demonstrate that Sch B can reduce inflammation, inhibit apoptosis, and improve cardiac function after ischemic injury. It represents a potential novel therapeutic approach for treatment of ischemic heart disease.
OBJECTIVE:
Alpinetin is a novel flavonoid that has demonstrated potent antitumor activity in previous studies. However, the efficacy and mechanism of alpinetin in treating lung cancer have not been determined.
METHODS:
We evaluated the impact of different doses and durations of alpinetin treatment on the cell proliferation, the apoptosis of lung cancer cells, as well as the drug-resistant lung cancer cells.
RESULTS:
This study showed that the alpinetin inhibited the cell proliferation, enhanced the apoptosis, and inhibited the PI3K/Akt signaling in lung cancer cells. Moreover, alpinetin significantly increased the sensitivity of drug-resistant lung cancer cells to the chemotherapeutic effect of cis-diammined dichloridoplatium. Taken together, this study demonstrated that alpinetin significantly suppressed the development of human lung cancer possibly by influencing mitochondria and the PI3K/Akt signaling pathway and sensitized drug-resistant lung cancer cells.
CONCLUSION:
Alpinetin may be used as a potential compound for combinatorial therapy or as a complement to other chemotherapeutic agents when multiple lines of treatments have failed to reduce lung cancer.
BACKGROUND AND PURPOSE:
Non-small cell lung cancer (NSCLC) is one of the most commonly diagnosed malignancies in the world. Small-molecule inhibitors of the EGF receptor's tyrosine kinase domain (TKIs), including gefitinib and erlotinib, have been widely used for treating NSCLC. Unfortunately, nearly all patients after initially experiencing a marked improvement while on these drugs, eventually progress to acquire resistance to TKIs. Because there is no effective therapeutic strategy to treat TKI-resistant NSCLC, we evaluated the effects of luteolin, a naturally occurring flavanoid, on T790M mutant NSCLC cells.
EXPERIMENTAL APPROACH:
The effect of luteolin on the viability of NSCLC and normal cell lines was investigated using the Cell Counting Kit-8 (CCK-8) assay. Luteolin-induced apoptosis was assessed by bivariate FITC-annexin V/PI assay, and Western blots were used to measured apoptotic proteins. Co-immunoprecipitation was used to determine the effect of luteolin on the interaction between Hsp90 and mutant EGF receptors. The effect of luteolin on the Akt/mTOR pathway was studied using Western blotting analysis. Its anti-tumour efficacy in vivo was examined in a mouse xenograft model.
KEY RESULTS:
Luteolin exerted significant anti-tumourigenic effects on the EGF receptor L858R/T790M mutation and erlotinib-resistant NSCLC both at the cellular and animal levels. Mechanistically, luteolin induced degradation of the EGF receptor by inhibiting the association of Hsp90 with the mutant EGF receptor, and, therefore, prevented PI3K/Akt/mTOR signalling, which resulted in NSCLC cell apoptosis.
CONCLUSION AND IMPLICATIONS:
Luteolin may be a potential candidate for NSCLC therapy, especially for treatment of patients with acquired erlotinib-resistant NSCLC.
© 2014 The British Pharmacological Society.
BACKGROUND:
Human herpesvirus 6 (HHV-6) is a T-lymphtropic and neurotropic virus that can infect various types of cells. Sequential studies reported that apoptosis of glia and neurons induced by HHV-6 might act a potential trigger for some central nervous system (CNS) diseases. HHV-6 is involved in the pathogenesis of encephalitis, multiple sclerosis (MS) and fatigue syndrome. However, the mechanisms responsible for the apoptosis of infected CNS cells induced by HHV-6 are poorly understood. In this study, we investigated the cell death processes of primary human fetal astrocytes (PHFAs) during productive HHV-6A infection and the underlying mechanisms.
RESULTS:
HHV-6A can cause productive infection in primary human fetal astrocytes. Annexin V-PI staining and electron microscopic analysis indicated that HHV-6A was an inducer of apoptosis. The cell death was associated with activation of caspase-3 and cleavage of poly (ADP-ribose) polymerase (PARP), which is known to be an important substrate for activated caspase-3. Caspase-8 and -9 were also significantly activated in HHV-6A-infected cells. Moreover, HHV-6A infection led to Bax up-regulation and Bcl-2 down-regulation. HHV-6A infection increased the release of Smac/Diablo, AIF and cytochrome c from mitochondria to cytosol, which induced apoptosis via the caspase-dependent and -independent pathways. In addition, we also found that anti-apoptotic factors such as IAPs and NF-κB decreased in HHV-6A infected PHFAs.
CONCLUSION:
This is the first demonstration of caspase-dependent and -independent apoptosis in HHV-6A-infected glial cells. These findings would be helpful in understanding the mechanisms of CNS diseases caused by HHV-6.
The YiQiFuMai powder injection (YQFM), a traditional Chinese medicine (TCM) prescription re-developed based on the well-known TCM formula Sheng-maisan, showed a wide range of pharmacological activities in cardiovascular diseases in clinics. However, its role in protection against myocardial ischemia/reperfusion (MI/R) injury has not been elucidated. The present study not only evaluated the cardioprotective effect of YQFM from MI/R injury but also investigated the potential molecular mechanisms both in vivo and in vitro. The myocardium infarct size, production of lactate dehydrogenase (LDH), creatine kinase (CK), cardiac function, TUNEL staining, and caspase-3 activity were measured. Cell viability was determined, and cell apoptosis was measured by Hoechst 33342 staining and flow cytometry. Mitochondrial membrane potential (ΔΨm) was measured, and ATP content was quantified by bioluminescent assay. Expression of apoptosis-related proteins, including Caspase-3, Bcl-2, Bax, AMPKα, and phospho-AMPKα, was analyzed by western blotting. AMPKα siRNA transfection was also applied to the mechanism elucidation. YQFM at a concentration of 1.06 g/kg significantly reduced myocardium infarct size and the production of LDH, CK in serum, improved the cardiac function, and also produced a significant decrease of apoptotic index. Further, combined treatment with compound C partly attenuated the anti-apoptotic effect of YQFM. In addition, pretreatment with YQFM ranging from 25 to 400 μg/mL markedly improved cell viability and decreased LDH release. Moreover, YQFM inhibited H9c2 apoptosis, blocked the expression of caspase-3, and modulated Bcl-2 and Bax proteins, leading to an increased mitochondrial membrane potential and cellular ATP content. Mechanistically, YQFM activated AMP-activated protein kinase (AMPK) signaling pathways whereas pretreatment with AMPK inhibitor Compound C and application of transfection with AMPKα siRNA attenuated the anti-apoptotic effect of YQFM. Our results indicated that YQFM could provide significant cardioprotection against MI/R injury, and potential mechanisms might suppress cardiomyocytes apoptosis, at least in part, through activating the AMPK signaling pathways.
Lung cancer, especially non-small-cell lung cancer (NSCLC), plays the leading role in cancer which is closely related to a myriad of fatal results. Unfortunately, current molecular mechanisms and clinical treatment of NSCLC still remain to be explored despite the fact that intensive investigations have been carried out in the last two decades. Recently, growing attention to finding exploitable sources of anticancer agents is refocused on quinolone compounds, an antibiotic with a long period of clinic application, for their remarkable cell-killing activity against not only bacteria, but eukaryotes as well. In this study, we found LZ-106, an analog of enoxacin, exhibiting potent inhibitory effects on NSCLC in both cultured cells and xenograft mouse model. We identified apoptosis-inducing action of LZ-106 in NSCLC cells through the mitochondrial and endoplasmic reticulum (ER)-stress apoptotic pathways via Annexin-V/PI double-staining assay, membrane potential detection, calcium level detection and the expression analysis of the key apoptotic proteins. Through comet assay, reactive oxygen species (ROS) detection, the expression analysis of DNA damage response (DDR) marker γ-H2AX and other DDR-related proteins, we also demonstrated that LZ-106 notably induced ROS overproduction and DDR. Interestingly, additional evidence in our findings revealed that DDR and apoptosis could be alleviated in the presence of ROS scavenger N-acetyl-cysteine (NAC), indicating ROS-dependent DDR involvement in LZ-106-induced apoptosis. Thus our data not only offered a new therapeutic candidate for NSCLC, but also put new insights into the pharmacological research of quinolones.
Oleuropein (OLE) was found to have anti-inflammatory and anti-oxidant effects. The latest study has shown that it can resist myocardial injury that follows an acute myocardial infarction and can rescue impaired spinal nerve cells. In this study, we investigated the neuroprotective effects of OLE on cerebral ischemia and reperfusion injury in a middle cerebral artery occlusion model in mice.OLE (100 mg/kg) was injected intraperitoneally 1h before ischemia. We found that the volume of cerebral infarction was significantly reduced after 75 min of ischemia and 24 h of reperfusion compared with the I/R (ischemia/reperfusion) group. This protective function occurred in a dose-dependent manner. We also found that treatment with OLE could reduce the cerebral infarct volume. The neuroprotective effect was prolonged from 2 h to 4 h when we injected OLE intracerebroventricularly after reperfusion. We then found that OLE can decrease the level of cleavedcaspase-3, an important marker of apoptosis, in the ischemic mouse brain. Finally, we explored the role of OLE in providing anti-apoptotic effects through the increased expression of Bcl-2 and the decreased expression of Bax, which are important markers in apoptosis. As shown above, the function and safety of OLE in cardiovascular disease may indicate that it is a potential therapeutic for stroke.
Inflammation is emerging as a new hallmark of cancer. Arachidonic acid (AA) metabolism, the family of cyclooxygenases (COXs) and lipoxygenase (LOX) play important roles in AA-related inflammatory cascades. In 94 colorectal cancer samples collected from the Han population, the immunohistochemical results indicated that 68% of the patients with colorectal cancer had a co-expression of both COX-2 and 5-LOX, while both displayed low expression in the matched normal tissues. In cell lines, three colorectal cancer cell lines exhibited high expression of COX-2 and 5-LOX. During stable silencing of the expression of COX-2 or 5-LOX in LoVo cancer cells, we found that downregulation of either COX-2 or 5-LOX significantly diminished the growth, migration and invasion of the colon cancer cells and specifically, downregulation of COX-2 could elicit upregulation of 5-LOX protein and vice versa. The above results suggested that the simultaneous blocking of COX-2 and 5-LOX activity may bring more potential benefits in managing the progression of colon cancer. Therefore, we sought to explore the effectiveness of a dual COX-2/5-LOX inhibitor darbufelone on the proliferation, migration, invasion and apoptosis of colon cancer cells, as well as the underlying mechanism of action. The results indicated that darbufelone significantly decreased the proliferative and invasive abilities of the colon cancer cells, in a dose-dependent manner. During the study of the related mechanisms, we found an upregulation of p27 and downregulation of cyclin D1 as well as CDK4 after darbufelone treatment, which indicated that darbufelone could arrest the cell cycle of LoVo cells at the G0/G1 phase. Furthermore, the activation of caspase-3 and -9, upregulation of Bax and downregulation of Bcl-2 demonstrated the occurrence of apoptosis by darbufelone. Finally, darbufelone also prevented the migration and invasion of LoVo cells, which may be ascribed to the upregulation of E-cadherin and ZO-1. In summary, our data suggest that the inhibition of both COX-2/5-LOX may be an effective therapeutic approach for colon cancer management, particularly for those patients with high expression of COX-2/5-LOX.
Apelin has been proved to be protective against apoptosis induced by ischemic reperfusion. However, mechanisms whereby apelin produces neuroprotection remain to be elucidated. AMP-activated protein kinase (AMPK) is a master energy sensor that monitors levels of key energy metabolites. It is activated via AMPKαThr172 phosphorylation during cerebral ischemia and appears to be neuroprotective. In this study, we investigated the effect of apelin on AMPKα and tested whether apelin protecting against apoptosis was associated with AMPK signals. Focal transient cerebral ischemia/reperfusion (I/R) model in male ICR mice was induced by 60 min of ischemia followed by reperfusion. Apelin-13 was injected intracerebroventricularly 15 min before reperfusion. AMPK inhibitor, compound C, was injected to mice intraperitoneally at the onset of ischemia. In experiment 1, the effect of apelin-13 on AMPKα was measured. In experiment 2, the relevance of AMPKα and apelin-13' effect on apoptosis was measured. Data showed that apelin-13 significantly increased AMPKα phosphorylation level after cerebral I/R. Apelin-13, with the co-administration of saline, reduced apoptosis cells, down-regulated Bax and cleaved-caspase3 and up-regulated Bcl2. However, with the co-administration of compound C, apelin-13 was inefficient in affecting apoptosis and Bax, Bcl2 and cleaved-caspase3. The study provided the evidence that apelin-13 up-regulated AMPKα phosphorylation level in cerebral ischemia insults and AMPK signals participated in the mechanism of apelin-mediated neuroprotection.
Histone deacetylases (HDAC) are involved in diverse biological processes and therefore emerge as potential targets for pancreatic cancer. Silibinin, an active component of silymarin, is known to inhibit growth of pancreatic cancer in vivo and in vitro. Herein, we examined the cytotoxic effects of TSA in combination with silibinin and investigated the possible mechanism in two pancreatic cancer cell lines (Panc1 and Capan2). Our study found that combination treatment of HDAC inhibitor and silibinin exerted additive growth inhibitory effect on pancreatic cancer cell. Annexin V-FITC/PI staining and flow cytometry analysis demonstrated that combination therapy induced G2/M cell cycle arrest and apoptosis in Panc1and Capan2 cells. The induction of apoptosis was further confirmed by evaluating the activation of caspases. Moreover, treatment with TSA and silibinin resulted in a profound reduction in the expression of cyclinA2, cyclinB1/Cdk1 and survivin. Taken together, our study might indicate that the novel combination of HDAC inhibitor and silibinin could offer therapeutic potential against pancreatic cancer.
Evidence has shown that the activation of the autophagy pathway after experimental subarachnoid hemorrhage (SAH) protects against neuronal damage. Tert-butylhydroquinone (tBHQ), a commonly used nuclear factor erythroid 2-related factor 2 (Nrf2) activator, was found to significantly enhance autophagy activation. The aim of this study was to explore the effect of tBHQ treatment on early stage brain injury at 24 h after SAH. The results showed that tBHQ treatment failed to stimulate an effective anti-oxidative effect at 24 h after the SAH operation, but succeeded in ameliorating early brain injury, including alleviated brain edema, BBB disruption, neuronal degeneration and neurological deficits. Further exploration found that tBHQ treatment significantly increased the expression of Beclin-1 and the ratio of microtubule-associated protein 1 light chain 3 (LC3)-II to LC3-I, suggesting that autophagy was enhanced after tBHQ treatment. Moreover, tBHQ treatment restored Bcl-2 and Bax expression and reduced caspase-3 cleavage, suggesting the protective effect of tBHQ treatment in ameliorating brain injury after SAH. Furthermore, tBHQ enhanced autophagy activation, decreased neuronal degeneration and improved the neurological score after SAH in Nrf2-deficient mice. Taken together, these findings suggest that tBHQ treatment exerts neuro-protective effects against EBI following SAH by enhancing Nrf2-independent autophagy. Therefore, tBHQ is a promising therapeutic agent against EBI following SAH.
In this study, the anticancer effect of a newly synthesized flavonoid FV-429, against human breast cancer MDA-MB-231 cells, and the underlying mechanisms were investigated. FV-429 triggered the apoptosis and simultaneously inhibited the glycolysis of MDA-MB-231 cells. Both the HK II activity and its level in mitochondria were significantly down regulated by FV-429. Moreover, FV-429 weakened the interaction between HKII and VDAC, stimulated the detachment of HK II from the mitochondria, and resulted in the opening of the mitochondrial permeability transition pores. Thus FV-429 induced the mitochondrial-mediated apoptosis, showing increased Bax/Bcl-2 ratio, loss of mitochondrial membrane potential (MMP) and activation of caspase-3 and -9, cytochrome c (Cyt c) release, and apoptosis inducing factor (AIF) transposition. Further research revealed that the phosphorylation of mitochondrial HKII via Akt was responsible for the dissociation of HKII and the decreased HKII activity induced by FV-429. Taken together, FV-429 inhibited the phosphorylation of HKII, down-regulated its activity, and stimulated the release of HKII from the mitochondria, resulting the inhibited glycolysis and mitochondrial-mediated apoptosis. The studies provide a molecular basis for the development of flavonoid compounds as novel anticancer agents for breast cancer.
© 2015 Wiley Periodicals, Inc.
Nasopharyngeal carcinoma (NPC) is primarily treated by chemoradiation. However, how to promote radiation sensitivity in NPC remains a challenge. Salinomycin is potentially useful for the treatment of cancer. This study aimed to explore the radiosensitivity of salinomycin on human nasopharyngeal carcinoma cell line CNE-2. CNE-2 were treated with salinomycin or irradiation, alone or in combination. The cytotoxicity effects of salinomycin were measured using CCK-8 assay. Clonogenic survival assay was used to evaluate the effects of salinomycin on the radiosensitivity of CNE-2. The changes of cell cycle distribution and apoptosis were assayed using flow cytometry. The expression of Caspase3/Bax/Bal-2 was detected by Western blotting. DNA damage was detected via γ-H2AX foci counting. The results showed that salinomycin induced apoptosis and G2/M arrest, increased Bax and cleaved Caspase3, decreased Bcl-2 expression, and increased the formation of γ-H2AX nuclear foci. These data suggest that salinomycin may be a radiosensitizer for NPC radiotherapy.
O(2)-(2,4-dinitro-5-{[2-(12-en-28-b-d-galactopyranosyl-oleanolate-3-yl)-oxy-2-oxoethyl]amino}phenyl)1-(N-hydroxyethylmethylamino)diazen-1-ium-1,2-diolate (NOAD), a novel NO-releasing derivative of oleanolic acid (OA), is an active cytotoxic component. In this study, NOAD induced a rise in intracellular NO levels and showed cytotoxic effects which were prevented by hemoglobin (NO scavenger). Meanwhile, NOAD induced G2/M phase cell cycle arrest in a concentration-dependent manner. Analysis of the cell cycle regulatory proteins demonstrated that NOAD did not change the steady-state levels of cyclin A, cyclin B, cyclin E, Cdk2 and Cdk4, but decreased the protein levels of Cdk1 and Cdc25C. Meanwhile, the levels of phosphorylation of Cdc25C and Cdk1 were significantly increased by NOAD in a concentration-dependent manner. Moreover, NOAD modulated the phosphorylation of protein kinases Chk2. During the G2/M arrest, cyclin-dependent kinase inhibitors (CDKIs), p21(WAF1/CIP1) and p27(kip1) were increased in a concentration-dependent manner. In addition, NOAD also caused a marked increase in the apoptotic cells, as characterized by fragmented nuclei, sub G1 formation, the level of 8-OHDG increase and poly (ADP-ribose) polymerase (PARP) cleavage, which was associated with activation of caspase-3, caspase-8 and caspase-9. Up-regulation of Bax and down-regulation of Bcl-2 were also observed in Bel-7402 cells treated with NOAD. These data suggest that NOAD produces anti-tumor effect via induction of G2/M cell cycle arrest and apoptosis.
Although tumor-associated fetal protein AFP has demonstrated utility as a clinical tumor marker, the significance of intracellular AFP is still unclear. The aim of this study was to explore the role of cytoplasmic AFP during HBx induced carcinogenesis, which had not previously been recognized; 614 HCC patients were analyzed for correlation of HBV infection with AFP level, and much higher AFP levels were found in HBsAg positive patients. Tumor tissue specimens from 20 HCC patients were used for analysis of AFP and GADD45α. Analysis of HCC specimens showed that upregulation of cytoplasmic AFP is associated with down-regulation of GADD45α in neoplastic tissue. Transfected HBx promotes transcription of AFP by acting on the elements in the AFP gene regulatory region. HBx itself did not directly impact transcription of GADD45α. However, the obstruction of RAR signaling by HBx induced elevation of AFP, which led to down-regulation of GADD45α. Cytoplasmic AFP was able to interact with RAR, disrupting its entrance into the nucleus and binding to the elements in the regulatory region of the GADD45α gene. Knockdown of AFP in siRNA-transfected AFP positive cell lines was synchronously associated with an incremental increase of RAR binding to DNA, as well as upregulation of GADD45α and it was contrary in AFP gene-transfected AFP negative cell lines. These results indicate cytoplasmic AFP is not only a histochemical tumor biomarker for human hepatoma but is also an intracellular signal molecule and potential participant in HBx induced hepatocarcinogenesis.
Multiple sclerosis (MS) has been associated with a history of sub-optimal exposure to ultraviolet light, implicating vitamin D3 as a possible protective agent. We evaluated whether 1,25(OH)2D3 attenuates the progression of experimental autoimmune encephalomyelitis (EAE), and explored its potential mechanisms. EAE was induced in C57BL/6 mice via immunization with MOG35-55, and some mice received 1,25(OH)2D3. 1,25(OH)2D3 inhibited EAE progression. Additionally, 1,25(OH)2D3 reduced inflammation, demyelination, and neuron loss in the spinal cord. The protective effect of 1,25(OH)2D3 was associated with significantly elevated expression of Beclin1, increased Bcl-2/Bax ratio, and decreased LC3-II accumulation. Thus, 1,25(OH)2D3 may represent a promising new MS treatment.
Human non-small cell lung carcinoma (NSCLC) is one of the most common cancer worldwide. In previous studies, lovastatin, acting as an inhibitor of 3-hydroxy-3-methylglutaryl Co A (HMG-CoA) reductase, exhibited significant antitumor activity during tumorigenesis. However, whether or not this effect is mediated through changes in minichromosome maintenance (MCM) 2 expression remains unclear. The present study investigated whether lovastatin inhibits proliferation due to MCM2 in NSCLCs. We first assessed the effects of lovastatin on cell anti-proliferation, cell cycle progression and apoptosis in NSCLC cells. We found, by quantitative RT-PCR and western blot analysis, that lovastatin treatment markedly and consistently inhibited the expression of MCM2. Then, to further explore the anticancer mechanism of lovastatin involving MCM2, we silenced MCM2 by siRNA in two cell lines (A549 and GLC-82). Silencing of MCM2 triggered G1/S arrest. Following further examination of cell cycle-related factors, MCM2 knockdown inhibited protein retinoblastoma (Rb), cyclin D1 and CDK4 expression, but increased p21 and p53 expression, suggesting that siMCM2 indeed triggered cell cycle arrest. In addition, siMCM2 induced apoptosis. Finally, lovastatin treatment increased p-JNK, which is involved in the downregulation of MCM2. In conclusion, our data suggest that MCM2 may be a novel therapeutic target of lovastatin treatment in NSCLCs.
Ischemic stroke is the third leading cause of death and the main reason for severe disabilities in the world today. N2, 4 - (2 - (1H - imidazol - 1 - yl) ethoxy) - 3 - methoxybenzoic acid is considered as a novel potent agent for cerebral ischemia due to its effect in preventing neuronal cell death after ischemic stroke. In the present study, we investigated the post-ischemic neuroprotective effect of N2 and its underlying mechanisms. Using a MCAO rat model, we found that N2 reversed brain infarct size, reduced cerebral edema and decreased the neurological deficit score significantly. Moreover, N2 diminished TUNEL positive cells, down-regulated bax expression and up-regulated bcl-2 expression notably. In addition, we evaluated the oxygen glucose deprivation/reoxygenation (OGD/R) injury induced neuron cell death in rat primary cortical neuron and assessed the neuroprotective effect of our drug. N2 increased cell viability, ameliorated neuron cell injury by decreasing LDH activity, and inhibited cell apoptotic rate while suppressed apoptotic signaling via inhibiting the bax expression, and elevating the bcl-2 expression. Furthermore, the neuroprotective effect of N2 was associated with the PI3K/Akt pathway which was proved by the use of PI3K inhibitor LY294002. The combination of our findings disclosed that N2 can be used as an effective neuroprotective agent for ischemic stroke due to its significant effect on preventing neuronal cell death after cerebral ischemia both in vivo and in vitro and the effectiveness was dose dependent.
Injury potential, which refers to a direct current voltage between intact and injured nerve ends, is mainly caused by injury-induced Ca2+ influx. Our previous studies revealed that injury potential increased with the onset and severity of spinal cord injury (SCI), and an application of applied electric field stimulation (EFS) with the cathode distal to the lesion could delay and attenuate injury potential formation. As Ca2+ influx is also considered as a major trigger for secondary injury after SCI, we hypothesize that EFS would protect an injured spinal cord from secondary injury and consequently improve functional and pathological outcomes. In this study, rats were divided into three groups: (1) sham group, laminectomy only; (2) control group, subjected to SCI only; and (3) EFS group, received EFS immediately post-injury with the injury potential modulated to 0±0.5 mV by EFS. Functional recovery of the hind limbs was assessed using the Basso, Beattie, and Bresnahan (BBB) locomotor scale. Results revealed that EFS-treated rats exhibited significantly better locomotor function recovery. Luxol fast blue staining was performed to assess the spared myelin area. Immunofluorescence was used to observe the number of myelinated nerve fibers. Ultrastructural analysis was performed to evaluate the size of myelinated nerve fibers. Findings showed that the EFS group rats exhibited significantly less myelin loss and had larger and more myelinated nerve fibers than the control group rats in dorsal corticospinal tract (dCST) 8 weeks after SCI. Furthermore, we found that EFS inhibited the activation of calpain and caspase-3, as well as the expression of Bax, as detected by Western blot analysis. Moreover, EFS decreased cellular apoptosis, as measured by TUNEL, within 4 weeks post-injury. Results suggest that early EFS could significantly reduce spinal cord degeneration and improve functional and historical recovery. Furthermore, these neuroprotective effects may be related to the inhibition of secondary apoptotic responses after SCI. These findings support further investigation of the future clinical application of EFS after SCI.
Microenvironmental hypoxia gives many tumor cells the capacity for drug resistance. Thioredoxin family members play critical roles in the regulation of cellular redox homeostasis in a stressed environment. In this study, we established a hypoxia-drug resistance (hypoxia-DR) model using HepG2 cells and discovered that the overexpression and nuclear translocation of thioredoxin-1 (Trx-1) are closely associated with this resistance through the regulation of the metabolism by the oxidative stress response to glycolysis. Intranuclear Trx-1 enhances the DNA-binding activity of HIF-1α via its interaction with and reducing action on Ref-1, resulting in increased expression of glycolysis-related proteins (PDHK1, HKII, and LDHA), glucose uptake, and lactate generation under hypoxia. Meanwhile, we found that GL-V9, a newly synthesized flavonoid derivative, shows an ability to reverse the hypoxia-DR and has low toxicity both in vivo and in vitro. GL-V9 could inhibit the expression and nuclear translocation of Trx-1 and then suppress HIF-1α DNA-binding activity by inhibiting the Trx-1/Ref-1 axis. As a result, glycolysis is weakened and oxidative phosphorylation is enhanced. Thus, GL-V9 leads to an increment in intracellular ROS generation and consequently intensified apoptosis induced by DDP.
Angiogenin (ANG) is a multifunctional secreted protein that belongs to the pancreatic ribonuclease A super family, which has been conceived to play a more important role in cell survival, growth and proliferation than the mediation of angiogenesis. Accumulating evidences suggest that the expression and activity of ANG increased significantly in a variety of human cancers. Recent studies showed that ANG activates cell signaling pathway through the putative receptor on endothelial cells. However, the underlying mechanisms remain largely unknown. AKT/mTOR signaling pathway participates in cell growth, cell-cycle progression and cell apoptosis. The purpose of our study was to determine whether ANG implicated in growth and metastasis of bladder cancer cells through regulating AKT/mTOR signaling pathway. In this study, we constructed ANG siRNA plasmids that transfected into human bladder cancer T24 cells. We demonstrated that knockdown of ANG could inhibit cell proliferation, regulate cell cycle and induce apoptosis. We also found that down-regulation of ANG remarkably reduced the phosphorylation of signaling targets AKT, GSK-3β and mTOR. Furthermore, down-regulation of ANG increased expression of ribonuclease inhibitor, which is a cytoplasmic acidic protein with many functions. Finally, ANG siRNA led to the suppression for tumorigenesis and metastasis in vivo. Taken together, these findings highlight for the first time that ANG could play a pivotal role in the development of bladder cancer through regulating AKT/mTOR signaling pathway. The targeting of ANG and associated factors could provide a novel strategy to inhibit human bladder cancer.
The aim of this study was to explore the effects of Astragalus polysaccharides (APS) on the mRNA expression of epidermal growth factor-like domain 7 (EGFL7) in lung tissue in newborn rats with bronchopulmonary dysplasia (BPD). For this purpose, a total of 96 newborn SD rats were randomly divided into 4 groups (n=24): the control group, air room plus APS group, BPD group and the APS group (20 mg/kg/day). Lung tissues were obtained on days 4, 10 and 14 after birth. Morphological changes were observed and the protein and mRNA expression levels of EGFL7, Bax and Bcl-2 were determined. The rats in the BPD group (BPD induced by hyperoxia) presented with an arrest in alveolar and vascular development and low mRNA and protein expression levels of of EGFL7, Bcl-2 and high levels of Bax compared with the rats in the control group. However, lung damage in the APS intervention group was attenuated compared with the BPD group. The protein and mRNA expression levels of EGFL7 and Bcl-2 were also increased and the level of Bax was decreased in the APS intervention group (P<0.01) compared with the BPD model group after birth on days 4, 10 and 14. Our data demonstrate that APS reduce airway remodeling and alveolar damage by upregulating the expression of EGFL7 and exert protective effects against BPD in neonatal rats. Thus, APS may have potential for use as a therapeutic strategy for BPD.
NG, O(2)-(2,4-dinitro-5-{[2-(12-en-28-β-D-galactopyranosyl-oleanolate-3-yl)-oxy-2-oxoethyl] amino} phenyl) 1-(N-hydroxyethylmethylamino) diazen-1-ium-1,2-diolate, was identified in our laboratory as a novel nitric oxide-releasing prodrug with antitumor effects. A previous study showed that NG inhibited cell growth, and induced apoptosis in HepG2 cells. In this study, the inhibitory effects of NG on the viability of MGC803 cells were examined using methylthiazolyl tetrazolium biomide (MTT) assay, neutral red assay and trypan blue exclusion test. The results showed that NG had strong cytotoxicity to induce apoptosis, which was characterized by a significant externalization of phosphatidylserine, nuclear morphological changes and enhanced Bax-to-Bcl-2 ratio. Moreover, the release of cytochrome c (Cyt c) from mitochondria and the activation of caspase-9/3 were also detected, indicating that NG may induce apoptosis through a mitochondrial-mediated pathway. NG induced mitochondrial dysfunction in MGC803 cells by altering membrane potential (△Ψm), the inhibition of complexes I, II and IV consequently decreasing ATP level. Furthermore, the treatment of MGC803 cells with NG caused a marked rise in oxidative stress as characterized by accumulation of reactive oxygen species (ROS), excessive malondialdehyde (MDA) production and a reduction in glutathione hormone (GSH) level and superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activity. In addition, pretreatment with N-acetylcysteine (NAC), a GSH synthesis precursor, was partially protective against the NG-induced ROS generation and cell apoptosis. In contrast, pretreatment of MGC803 cells with L-buthionine-S, R-sulfoximine (BSO), a GSH synthesis inhibitor, increased the ROS levels, and aggravated cell apoptosis by NG. These results suggest that NG-induced apoptosis in MGC803 cells is mediated, at least in part, by the increase in ROS production, oxidative stress and mitochondrial dysfunction.
Stellate ganglion block (SGB) is a blockade of sympathetic ganglia innervating the head and neck, and is known to function through vasodilation of the target region. However, the effectiveness of SGB in relieving cerebral vasospasm (CVS) through dilation of intracerebral vessels has not been evaluated. The aim of the present study is to investigate the therapeutic effects of SGB in a rat model of subarachnoid hemorrhage (SAH) complicated by delayed CVS, and explore the underlying mechanisms. The SAH model was established by double injection of autologous arterial blood into the cisterna magna. We simulated SGB by transection of the cervical sympathetic trunk (TCST), and measured changes in the diameter, perimeter and cross-sectional area of the basilar artery (BA) and middle cerebral artery (MCA) to evaluate its vasodilatory effect. To investigate the underlying mechanisms, we determined the expression level of vasoactive molecules endothelin-1 (ET-1) and calcitonin gene-related peptide (CGRP) in the plasma, and apoptotic modulators Bcl-2 and Bax in the hippocampus. We found a significant increase in the diameter, perimeter and cross-sectional area of the BA and right MCA in SAH rats subjected to TCST. Application of SGB significantly reduced the expression of ET-1 while increasing that of CGRP in SAH rats. We also found a significant increase in the expression of Bcl-2 and decrease in the expression of Bax in the hippocampus of SAH rats subjected to TCST, when compared to untreated SAH rats. The mechanism of action of SGB is likely mediated through alterations in the ratio of ET-1 and CGRP, and Bax and Bcl-2. These results suggest that SGB can alleviate the severity of delayed CVS by inducing dilation of intracerebral blood vessels, and promoting anti-apoptotic signaling. Our findings provide evidence supporting the use of SGB as an effective and well-tolerated approach to the treatment of CVS in various clinical settings.
Bone is the primary site of skeletal metastasis in prostate cancer (PCa). Atelocollagen (ATE)-mediated siRNA delivery system can be used to silence endogenous genes involved in PCa metastatic tumor cell growth. However, we hope that the delivery system can target PCa cells to reduce damage to the bone tissue and improve the therapeutic effect. RNA aptamer (APT) A10-3.2 has been used as a ligand to target PCa cells that express prostate-specific membrane antigen (PSMA). APT was investigated as a PSMA-targeting ligand in the design of an ATE-based microRNA (miRNA; miR-15a and miR-16-1) vector to PCa bone metastasis. To observe the targeted delivery and transfection efficiency of ATE-APT in PSMA-overexpressing cells, luciferase activity and biodistribution of nanoparticles in Balb/c mice was analyzed. The anticancer effect of nanoparticles in vivo was investigated using the survival times of human PCa bone metastasis mice model. Luciferase assays of pGL-3 expression against PC3 (PSMA(-)) and LNCaP (PSMA(+)) cells showed that the transfection efficiency of the synthesized DNA/ATE-APT complex was higher than that of the DNA/ATE complex. The anticancer efficacy of miRNA/ATE-APT was superior to those of other treatments in vivo. This PSMA-targeted system may prove useful in widening the therapeutic window and allow for selective killing of PCa cells in bone metastatic foci.
Green tea polyphenol (GTP) is one of the most promising chemopreventive agent for cancer; it can inhibit cancer cell proliferation and induce apoptosis through p53-dependent cell signaling pathways. Unfortunately, many tumor cells lack the functional p53, and little is known about the effect of GTP on the p53-deficient/mutant cancer cells. To understand the p53-independent mechanisms in GTP-treated p53-dificient/mutant cancer cells, we have now examined GTP-induced cytotoxicity in human hepatoma Hep3B cells (p53-deficient). The results showed that GTP could induce Bax and Bak activation, cytochrome c release, caspase activation, and necroptosis of Hep3B cells. Bax and Bak, two key molecules of mitochondrial permeability transition pore (MPTP), were interdependently activated by GTP, with translocation and homo-oligomerization on the mitochondria. Bax and Bak induce cytochrome c release. Importantly, cytochrome c release and necroptosis were diminished in Hep3B cells (Bax(-/-)) and Hep3B cells (Bak(-/-)). Furthermore, overexpression of Bcl-2 could ameliorate GTP-induced cytochrome c release and necroptosis. Together, the findings suggested that GTP-induced necroptosis was modulated by the p53-independent pathway, which was related to the translocation of Bax and Bak to mitochondria, release of cytochrome c, and activation of caspases.
Benzo(a)pyrene-7,8-diol-9,10-epoxide (BPDE) is a highly reactive DNA damage agent and can induce cell death through both p53-independent and -dependent pathways. However, little is known about the molecular mechanisms of p53-independent pathways in BPDE-induced cell death. To understand the p53-independent mechanisms, we have now examined BPDE-induced cytotoxicity in p53-deficient baby mouse kidney (BMK) cells. The results showed that BPDE could induce Bax and Bak activation, cytochrome c release, caspases activation, and necrotic cell death in the BMK cells. Bax and Bak, two key molecules of mitochondrial permeability transition pore, were interdependently activated by BPDE, with Bax and Bak translocation to and Bax/Bak homo-oligomerization in mitochondria, release of cytochrome c was induced. Importantly, cytochrome c release and necrotic cell death were diminished in BMK cells (Bax(-/-)), BMK cells (Bak(-/-)), and BMK cells (Bax(-/-)/Bak(-/-)). Furthermore, overexpression of Bcl-2 could ameliorate BPDE-induced cytochrome c release and necrosis. Together the findings suggested that BPDE-induced necrosis was modulated by the p53-independent pathway, which was related to the translocation of Bax and Bak to mitochondria, release of cytochrome c, and activation of caspases.
Human ribonuclease inhibitor (RI), a cytoplasmic protein, is constructed almost entirely of leucine rich repeats. RI could suppress activities of ribonuclease and angiogenin (ANG) through closely combining with them. ANG is a potent inducer of blood vessel growth and has been implicated in the establishment, growth, and metastasis of tumors. ILK/PI3K/AKT signaling pathway also plays important roles in cell growth, cell-cycle progression, tumor angiogenesis, and cell apoptosis. Our previous experiments demonstrated that RI might effectively inhibit some tumor growth and metastasis. Our recent study showed that ILK siRNA inhibited the growth and induced apoptosis in bladder cancer cells as well as increased RI expression, which suggest a correlation between RI and ILK. However, the exact molecular mechanism of RI in anti-tumor and in the cross-talk of ANG and ILK signaling pathway remains largely unknown. Here we investigated the effects of up-regulating RI on the growth and apoptosis in murine melanoma cells through angiogenin and ILK/PI3K/AKT signaling pathway. We demonstrated that up-regulating RI obviously decreased ANG expression and activity. We also discovered that RI overexpression could remarkably inhibit cell proliferation, regulate cell cycle and induce apoptosis. Furthermore, up-regulation of RI inhibited phosphorylation of ILK downstream signaling targets protein kinase B/Akt, glycogen synthase kinase 3-beta (GSK-3β), and reduced β-catenin expression in vivo and vitro. More importantly, RI significant inhibited the tumor growth and angiogenesis of tumor bearing C57BL/6 mice. In conclusion, our findings, for the first time, suggest that angiogenin and ILK signaling pathway plays a pivotal role in mediating the inhibitory effects of RI on melanoma cells growth. This study identifies that RI may be a useful molecular target for melanoma therapy.
Apelin has been proved to protect the heart against ischemia/reperfusion (I/R) injury via the activation of phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK) signaling pathways. Whether this protective effect applies to brain I/R injury needed to be explored. We therefore investigated the potential neuroprotective role of Apelin-13 and the underlying mechanisms. Focal transient cerebral I/R model in male ICR mice was induced by 60min of ischemia followed by reperfusion. Apelin-13 intracerebroventricular injection was performed 15 min before reperfusion. Neurological function, infarct volume, brain edema and apoptosis were measured at 24h after stroke. To further test the mechanism of Apelin-13, PI3K inhibitor LY294002 and ERK1/2 inhibitor PD98059 were injected into the lateral cerebral ventricle 15min before ischemia. Compared with the Vehicle group, Apelin-13 significantly ameliorated neurological deficit, infarct volume, brain edema and reduced TUNEL-positive cells. Bax, caspase-3 and cleaved caspase-3 were down-regulated and Bcl-2 up-regulated. While, the effect of Apelin-13 on Bax, Bcl-2, caspase-3 and cleaved caspase-3 was attenuated by LY294002 and PD98059. Apelin protected the brain against I/R insult injury, and this effect may be through activation of PI3K/Akt and ERK1/2 signaling pathways.
Millet is an important cereal food and exhibits multiple biological activities, including immunodulatory, antioxidant, antifungal and anti-hyperglycemia effects. Herein, we describe a novel 35kDa protein with anti-cancer properties, named FMBP, which was extracted and purified from foxtail millet bran by cell-based screening. FMBP is highly homologous to peroxidase as revealed by mass spectrometry and gene sequencing analysis. Furthermore, in vivo anti-tumor results implicated that the novel protein FMBP had the ability to suppress xenografted tumor growth in nude mice. Mechanistically, FMBP is able to suppress colon cancer cell growth through induction of G1 phase arrest and also causes a loss of mitochondrial transmembrane potential which results in caspase-dependent apoptosis in colon cancer cells. Notably, FMBP has much lower toxicity in normal colon epithelial cells. Taken together, FMBP has targeted anti-colon cancer effects and may serve as a therapeutic agent against colon cancer.
Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by pronounced synovial inflammation and hyperplasia, in which there may be an imbalance between the growth and death of fibroblast-like synoviocytes (FLS). Norisoboldine (NOR), the main active constituent in the alkaloid fraction isolated from Radix Linderae, was previously demonstrated to alleviate arthritis severity in experimental RA. This study aimed to evaluate the effects of NOR on proliferation and apoptosis of FLS from adjuvant-induced arthritis (AIA) rats to elucidate the mechanism of its inhibitory effect on inflammatory synovial hyperplasia in RA. Our results indicated that NOR exhibited a pro-apoptotic effect on AIA FLS but only slightly affected cell proliferation and the cell cycle. Following treatment with NOR for 24h, the activation of caspase 3 and caspase 9 and the cleavage of poly (ADP-ribose) polymerase (PARP) in AIA FLS were observed; however, caspase 8 remained unaffected. Meanwhile, a flow cytometric assay revealed that NOR significantly increased the percentage of apoptotic cells, causing the loss of the depolarized mitochondrial membrane potential and the release of cytochrome C. The expression of Bax and Bcl-2 was also regulated by NOR treatment. Additionally, the expression of p53 protein was up-regulated by NOR, and pretreatment with PFT-α, a p53 specific inhibitor, reversed the increase in FLS apoptosis caused by NOR. These findings indicated that NOR-induced apoptosis in AIA FLS is achieved via a mitochondrial-dependent pathway, which may be mediated by promoting the release of cytochrome C and by regulating the expression of Bax and Bcl-2 proteins, and p53 might also be required for NOR-induced apoptosis in AIA FLS.
In Asia, the incidence of colorectal cancer has been increasing gradually due to a more Westernized lifestyle. The aim of study is to determine the interaction between melatonin-induced cell death and cellular senescence. We treated HCT116 human colorectal adenocarcinoma cells with 10 μm melatonin and determined the levels of cell death-related proteins and evaluated cell cycle kinetics. The plasma membrane melatonin receptor, MT1, was significantly decreased by melatonin in a time-dependent manner, whereas the nuclear receptor, RORα, was increased only after 12 hr treatment. HCT116 cells, which upregulated both pro-apoptotic Bax and anti-apoptotic Bcl-xL in the early response to melatonin treatment, activated autophagic as well as apoptotic machinery within 18 hr. Melatonin decreased the S-phase population of the cells to 57% of the control at 48 hr, which was concomitant with a reduction in BrdU-positive cells in the melatonin-treated cell population. We found not only marked attenuation of E- and A-type cyclins, but also increased expression of p16 and p-p21. Compared to the cardiotoxicity of Trichostatin A in vitro, single or cumulative melatonin treatment induced insignificant detrimental effects on neonatal cardiomyocytes. We found that 10 μm melatonin activated cell death programs early and induced G1-phase arrest at the advanced phase. Therefore, we suggest that melatonin is a potential chemotherapeutic agent for treatment of colon cancer, the effects of which are mediated by regulation of both cell death and senescence in cancerous cells with minimized cardiotoxicity.
(S)-ZJM-289, a novel nitric oxide (NO)-releasing derivative of 3-n-butylphthalide, induces the neuroprotection in a rat model of focal cerebral ischemia/reperfusion (I/R). However, much is unknown about the late phase effect in the neuroprotection of (S)-ZJM-289 preconditioning. The purpose of this study is to explore the late phase neuroprotection of (S)-ZJM-289 preconditioning, as well as underlying mechanisms involved. Preconditioning with 40-160 mg/kg, (S)-ZJM-289 significantly reduces brain damage after I/R. (S)-ZJM-289 preconditioning is effective when applied 1-3 days before I/R. Moreover, the degrees of neuroprotection offered by (S)-ZJM-289 preconditioning and ischemic preconditioning are virtually identical. (S)-ZJM-289 preconditioning also protects primary cultured cortical neurons against oxygen-glucose deprivation and recovery-induced cytotoxicity in vitro. (S)-ZJM-289 preconditioning significantly increases the generation of NO, but has no effect on the nitric oxide synthase activities. Additionally, (S)-ZJM-289 preconditioning promotes the dissociation between nuclear-factor-E2-related factor (Nrf2) and kelch-like ECH-associated protein 1, and induces Nrf2 nuclear localization. The neuroprotection of (S)-ZJM-289 preconditioning is blocked by Nrf2-siRNA in vitro. (S)-ZJM-289 preconditioning up-regulates antioxidant enzymes against nervous injury. (S)-ZJM-289 preconditioning significantly activates extracellular regulated protein kinases (ERK) and inhibits c-Jun N-terminal kinases signaling cascade. The neuroprotection is abolished by the ERK inhibitor PD98059 in vitro. Subsequently, (S)-ZJM-289 preconditioning increases the levels of anti-apoptotic protein B cell lymphoma 2 (Bcl-2) and inhibited the translocation of Bcl-2 associated X to the mitochondria, thus attenuating the release of cytochrome c from the mitochondria and the activation of downstream caspase. These results suggest that (S)-ZJM-289 preconditioning exerts the late phase protection against nervous injury induced by transient cerebral ischemia and oxygen-glucose deprivation.
SALL4, a zinc-finger transcriptional factor for embryonic stem cell self-renewal and pluripotency, has been suggested to be involved in tumorigenesis. The role of SALL4 in human gastric cancer, however, remains largely unknown. In this study, we demonstrated that SALL4 was aberrantly expressed at both mRNA and protein levels in human gastric cancer tissues, and SALL4 level was highly correlated with lymph node metastasis. Enforced expression of SALL4 enhanced the proliferation and migration of human gastric cancer cells, whereas knockdown of SALL4 by siRNA led to the opposite effects. In addition, SALL4 overexpression promoted the growth and metastasis of gastric xenograft tumor in vivo. SALL4 overexpression induced epithelial-mesenchymal transition (EMT) in gastric cancer cells, with increased expression of Twist1, N-cadherin and decreased expression of E-cadherin. Moreover, SALL4 promoted the acquirement of stemness in gastric cancer cells through the induction of Bmi-1 and Lin28B. Taken together, our findings indicate that SALL4 has oncogenic roles in gastric cancer through the modulation of EMT and cell stemness, suggesting SALL4 as a novel target for human gastric cancer diagnosis and therapy.
2,2',4,4'-tetrabromodiphenyl ether (PBDE-47)-elicited neurotoxicity is associated with neural apoptosis; however the underlying mechanisms remain unclear. To investigate whether the mitochondrial p53 pathway is involved in neuronal apoptosis induced by PBDE-47 and to correlate DNA hypomethylation with p53 activation, human neuroblastoma (SH-SY5Y) cells were treated with different concentrations of PBDE-47 (1, 5, 10 μmol/L) for 24h in vitro. The apoptosis and ultrastructural alterations in cells, levels of p53, Bcl-2, Bax, cytochrome c (Cyt c), caspase-3 and methylation status of p53 promoter were determined. Hoechst 33258 staining and transmission electron microscopy analysis showed that PBDE-47 induced SH-SY5Y cells apoptosis characterized by the typical apoptotic morphological changes. In addition, PBDE-47 activated the p53-dependent mitochondrial apoptotic pathway as evidenced by up-regulation of p53 and Bax, down-regulation of Bcl-2 and Bcl-2/Bax ration, enhancement of Cyt c release from mitochondria into the cytosol, activation of caspase-3 as well as ultrastructural abnormalities of mitochondria. However, no obvious decrease in p53 promoter methylation levels was observed in any of the treatment groups by bisulfite genomic sequencing. Collectively, these results suggest that the mitochondrial p53 pathway is involved in PBDE-47-induced SH-SY5Y cells apoptosis, nevertheless p53 promoter hypomethylation may not be implicated in this process.
Reactive oxygen species (ROS)-induced oxidative stress increases in skeletal muscle with aging and decreases the viability of implanted cells. Type 1 insulin-like growth factor (IGF-1) promotes the survival of skeletal muscle cells under oxidative stress. It is unknown whether IGF-1 protects muscle-derived stem cells (MDSCs) from oxidative stress. In this study, we genetically engineered rat MDSCs to overexpress IGF-1 and determined cell viability, apoptosis, and VEGF secretion under oxidative stress. Overexpression of IGF-1 prevented MDSCs from H2O2-induced caspase-dependent apoptotic cell death by upregulating the PI3K/AKT pathway, accompanied with an increase of NF-κB, p-NF-κB, Bcl-2, and VEGF, as well as a decrease of Bax. In contrast, pre-administration of picropodophyllinb, wortmannin, 1L-6-hydroxymethyl-chiro-inositol-2-((R)-2-O-methyl-3-O-octadecylcarbonate), or pyrrolidine-dithiocarbamate, specific inhibitors of IGF-1R, PI3K, AKT, and NF-κB, respectively, followed by treatment with H2O2, resulted in cell death of MDSCs. Our data indicated that IGF-1 suppresses apoptosis and enhances the paracrine function of MDSCs under oxidative stress via enhancing IGF-1R/PI3K/AKT signaling. Thus, IGF-1 gene-modified MDSCs present a potential application in the treatment of muscle wasting, such as urethra intrinsic sphincter deficiency.
3-Oxo-29-noroleana-1,9(11),12-trien-2,20-dicarbonitrile (ONTD) is a novel synthetic derivative of glycyrrhetinic acid (GA), which has the ability to inhibit the proliferation of human hepatocellular carcinoma (HCC) cells. However, the mechanisms by which ONTD exerts its inhibitory effects remain elusive. The present study was conducted to investigate the cytotoxicity of ONTD in Bel-7402 cells and its molecular mechanisms. We found that ONTD depleted intracellular GSH, increased the level of ROS, and consequently induced mitochondrial permeability transition (MPT) leading to the release of apoptosis-inducing factor (AIF) and cytochrome c (Cyt c) to the cytosol. Mitochondrial alteration and subsequent apoptotic cell death in ONTD-treated Bel-7402 cells could be blocked by addition of exogenous antioxidants N-acetylcystein (NAC), GSH and the MTP inhibitor cyclosporin A (CsA). In addition, ONTD activated the phosphorylation of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinases (MAPK) but not extracellular signal-regulated protein kinases (ERK 1/2). When the cells were exposed to SP600125 (a JNK inhibitor) and SB203580 (a p38 inhibitor), the deregulation of the expression of apoptotic proteins was attenuated. Furthermore, 40 mg/kg ONTD significantly reduced tumor weight (-70.62%, p<0.01) in the H22 tumor-bearing mouse model in vivo. Taken together, these findings provide the first experimental evidence supporting that ONTD could induce apoptosis of Bel-7402 cells via MAPK-mediated mitochondrial pathway and ONTD has the potential to be developed as a therapeutic agent for the treatment of HCC.
Nicorandil, an ATP-sensitive potassium (KATP) channel opener, is known to have protective effects on ischemic injury in heart and brain. One of the most important protective mechanisms is the anti-apoptotic effect on cardiomyocytes and neurons. This study explored the anti-apoptotic effect of nicorandil against neurotoxicity in SH-SY5Y cells overexpressing the Swedish mutant APP (APPsw) and the possible mechanisms involved. We used SH-SY5Y cells transiently transfected with APPsw as a cellular model of Alzheimer's disease. Cells were treated with nicorandil (0.1, 0.5, 1 mM) for 24 h with and without glibenclamide (10 μM), a KATP channel inhibitor. The cells were then collected for MTT, apoptosis assay, and Western blot. In addition, we also investigated the potential involvement of the PI3K/Akt pathway in nicorandil-mediated neuroprotection of APPsw cells. Our results showed that nicorandil dose-dependently increased cell viability and reduced the rate of apoptosis as measured by MTT assay and annexin V/PI staining. Western blot showed that nicorandil could upregulate Bcl-2 levels and downregulate Bax and caspase-3 expression. Further studies showed that nicorandil increased the levels of phospho-Akt and upregulated element-binding protein activity by PI3K activation. Applying a PI3K inhibitor, LY294002 blocked the protection. All these findings suggest that nicorandil might be a potential treatment option for Alzheimer's disease.
Fenofibrate, a peroxisome proliferator-androgen receptor-alpha agonist, is widely used in treating different forms of hyperlipidemia and hypercholesterolemia. Recent reports have indicated that fenofibrate exerts anti-proliferative and pro-apoptotic properties. This study aims to investigate the effects of fenofibrate on the prostate cancer (PCa) cell line LNCaP. The effects of fenofibrate on LNCaP cells were evaluated by flow cytometry, reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assays, Western blot analysis, and dual-luciferase reporter assay. Fenofibrate induces cell cycle arrest in G1 phase and apoptosis in LNCaP cells, reduces the expressions of androgen receptor (AR) and AR target genes (prostate-specific antigen and TMPRSS2), and inhibits Akt phosphorylation. Fenofibrate can induce the accumulation of intracellular reactive oxygen species and malondialdehyde, and decrease the activities of total anti-oxidant and superoxide dismutase in LNCaP cells. Fenofibrate exerts an anti-proliferative property by inhibiting the expression of AR and induces apoptosis by causing oxidative stress. Therefore, our data suggest fenofibrate may have beneficial effects in fenofibrate users by preventing prostate cancer growth through inhibition of androgen activation and expression.
It is well established that both hematoporphyrin monomethyl ether-sonodynamic therapy (HMME-SDT) and doxorubicin (DOX) can induce cell apoptosis but each alone has its own limitations. To date, the combined effects of HMME-SDT and DOX on inducing cell apoptosis are little known and the mechanism for the combined effects remains poorly understood. In the present study, we reported the synergistic effects of HMME-SDT and DOX on inhibiting the proliferation of human cholangiocarcinoma QBC939 cells and investigated the mechanism of this synergy. The data from MTT assay, flow cytometer, Hoechst staining and cell arrest analysis showed that the combination of HMME-SDT and DOX exhibited higher inhibiting effects on proliferation of QBC939 cells than the sole application of HMME-SDT or DOX. In addition, the synergistic effects were shown to result from the DNA damage as demonstrated by single cell gel electrophoresis and DNA fragmentation. Furthermore, the expression of p53, Fas, Bax and activated caspase-3 protein was significantly upregulated in cells treated with HMME-SDT and DOX, whereas Bcl-2 protein was downregulated. Taken together, our data suggested that the application of HMME-SDT combined with DOX had better inhibiting effects on QBC939 cells and the effects were caused mainly by DNA damage.
Mesenchymal stem cells (MSCs) have been considered as an attractive tool for the therapy of diseases. Exosomes excreted from MSCs can reduce myocardial ischemia/reperfusion damage and protect against acute tubular injury. However, whether MSC-derived exosomes can relieve liver fibrosis and its mechanism remain unknown. Previous work showed that human umbilical cord-MSCs (hucMSCs) transplanted into acutely injured and fibrotic livers could restore liver function and improve liver fibrosis. In this study, it was found that transplantation of exosomes derived from hucMSC (hucMSC-Ex) reduced the surface fibrous capsules and got their textures soft, alleviated hepatic inflammation and collagen deposition in carbon tetrachloride (CCl4)-induced fibrotic liver. hucMSC-Ex also significantly recovered serum aspartate aminotransferase (AST) activity, decreased collagen type I and III, transforming growth factor (TGF)-β1 and phosphorylation Smad2 expression in vivo. In further experiments, we found that epithelial-to-mesenchymal transition (EMT)-associated markers E-cadherin-positive cells increased and N-cadherin- and vimentin-positive cells decreased after hucMSC-Ex transplantation. Furthermore, the human liver cell line HL7702 underwent typical EMT after induction with recombinant human TGF-β1, and then hucMSC-Ex treatment reversed spindle-shaped and EMT-associated markers expression in vitro. Taken together, these results suggest that hucMSC-Ex could ameliorate CCl4-induced liver fibrosis by inhibiting EMT and protecting hepatocytes. This provides a novel approach for the treatment of fibrotic liver disease.
Ghrelin has a protective role in a rat model of myocardial infarction (MI), but the underlying mechanism is not clear. Here, we investigated the effects of ghrelin treatment on angiogenesis in an experimental rat MI model. Adult male Sprague-Dawley rats were subjected to MI by ligating the anterior descending coronary artery. The rats were then treated with a subcutaneous injection of ghrelin (100 μg/kg) or saline (control group) for 4 weeks. Sham animals underwent thoracotomy and pericardiotomy, but not LAD ligation. At 28 days after ligation, the ghrelin treatment group showed a higher density of α-SMA positive vessels than the saline treatment MI group in myocardial infarct (6±2.1/mm(2) vs 4±1.8/mm(2), P<0.05) and peri-infarct zones (25±9.5/mm(2) vs 15±5.7/mm(2), P<0.05). RT-PCR and western-blot analyses showed that ghrelin significantly increased vascular endothelial growth factor (VEGF) expression in the peri-infarct zone compared with the control group. Moreover, there was a two-fold increase of Bcl-2 and a 3.5-fold reduction of the Bax protein in the ghrelin-treated MI group compared to the saline treatment MI group. Taken together, ghrelin could induce angiogenesis in rats after MI, the process that may be associated with the enhancement of VEGF and an anti-apoptosis effect.
ZL11n is a novel furoxan-based nitric oxide (NO)-releasing derivative of farnesylthiosalicylic acid. In this study, we examined the anticancer effects and the potential mechanism of action of ZL11n in vitro and in vivo. It was found that ZL11n exhibited a favorable, selective cytotoxic effect in the HepG2 cell line. The yield of NO in the ZL11n treated HepG2 cells was much higher than in the control group and the normal human liver L-02 cells. Furthermore, the NO concentration was correlated to the degree of cytotoxicity observed. The ZL11n-induced apoptosis was assessed by Annexin V-FITC/propidium iodide flow cytometry assay. ZL11n triggered the mitochondrial/caspase apoptotic pathway by decreasing mitochondrial membrane potential, cytochrome c release from mitochondrial, and reducing the Bcl-2-to-Bax ratio, in addition to activating the caspase cascade. Simultaneously, we found that ZL11n treatment led to an increase in JNK and ERK1/2 phosphorylation. Furthermore, treatment with SP600125 (a JNK inhibitor) and PD98059 (an ERK1/2 inhibitor) prior to ZL11n treatment was found to significantly reverse ZL11n-induced apoptosis. The in vivo findings also revealed that ZL11n significantly reduced tumor volume and weight in the H(22) solid tumor mouse model examined. In short, our findings suggest that ZL11n induced apoptosis through the coordination of the mitochondrial apoptotic pathway (activated by NO) and MAPKs signaling pathway (triggered by JNK or ERK).
In pancreatic islets, free radical formation produced upon exposure to proinflammatory cytokines mediates β cell destruction, which ultimately leads to type 1 diabetes (T1D). In this study, we examined whether laccase, a family of the blue copper protein, can be successfully used to prevent β cells from cytokine-mediated apoptosis. Non-obese diabetic (NOD) mice were used for these experiments. In parallel, the RINm5f β cell line was employed as a model system for in vitro experiments. The results demonstrated that laccase effectively scavenged peroxinitrite, which can be formed by nitric oxide, and upregulated the expression of antioxidant enzymes, such as manganese superoxide dismutase (MnSOD) and catalase. Interestingly, laccase balanced pro- (Bax) and anti-apoptotic (Bcl-2) proteins in terms of both the mRNA and protein levels with a downregulation of cytochrome c protein in RINm5f cells. In addition, laccase maintained blood glucose concentrations at a normal level with a simultaneous increase in plasma insulin levels during the spontaneous induction of diabetes in NOD mice. In conclusion, the antioxidant potentials of laccase in scavenging free radicals and upregulation of antioxidant enzymes may exert its pro-survival effect by counteracting the increased intracellular oxidative stress, and, consequently, by inhibiting apoptosis induced by cytokine-mediated activation during the course of T1D.
OBJECTIVES:
The aim of this study was to investigate the cardioprotective effect of salvianolic acid B (Sal B) on acute myocardial infarction (AMI) in rats and its potential mechanisms.
METHODS:
The AMI model was established in rats to study the effect of Sal B on AMI. Haematoxylin-eosin (HE) staining was used to evaluate the pathological change in AMI rats. Immunofluorescence and TUNEL staining were used to detect autophagy and apoptosis of myocardial cells in hearts of AMI rats, respectively. Protein expression of apoptosis-related, autophagy-related and angiogenesis-related proteins were examined by Western blot.
KEY FINDINGS:
Sal B attenuated myocardial infarction significantly compared with that of the model group. Rats administered with Sal B showed higher inhibition rate of infarction and lower infarct size than those of the model group. Moreover, Sal B decreased the serum levels of creatine kinase, lactate dehydrogenase and malondialdehyde, while increased such level of superoxide dismutase significantly compared with those of the model group. Sal B inhibited the expression of Bax, cleaved caspase-9 and cleaved PARP, while promoted the expression of Bcl-2, LC3-II, Beclin1 and VEGF.
CONCLUSIONS:
Sal B has cardioprotective effect on AMI and Sal B may be a promising candidate for AMI treatment.
© 2016 Royal Pharmaceutical Society.
BACKGROUND:
Paclitaxel (PTX) is a first line chemotherapy drug for breast cancer. There have been few studies reported concerning the therapeutic efficacy of paclitaxel-conjugated polymeric micelles in breast cancer in vivo.
METHODS:
Two kinds of PTX conjugate micelles, one of which (M(PTX)) contained 25 wt.% of PTX and the other (M(FA/PTX)) contained 22.5 wt.% of PTX and 1.4 wt.% of folate (FA), were prepared for cell apoptosis and anti-tumor activity evaluation on EMT-6 mice breast cancer models in comparison with 0.9 wt.% saline (control) and equivalent PTX. Cell apoptosis was analyzed by flow cytometry. Breast tumors were examined histologically with H&E staining and immunohistochemically by examining Bax and Bcl-2 expression. The survival status of tumor-bearing mice with different treatments was also examined.
RESULTS:
On day 5 of the drug administration, the average tumor masses were 0.49, 0.33, 0.22, and 0.18 g for the control, PTX, M(PTX) and M(FA/PTX) groups, respectively. The inhibition rates of tumor growth calculated for the three drug groups were 32.6%, 51.6% and 62.3%, respectively. The percentage of cell apoptosis based on flow cytometry was 1.0%, 36.6%, 55.9% and 66.1%, respectively, which showed statistically significant differences (p<0.05) between three drug groups and the control group. Bcl-2 expression of PTX and M(FA/PTX) groups was lower than control group (p<0.05). Bax expression of drug groups was higher than control group (p<0.05). At an equivalent paclitaxel dose of 26.7 mg/kg, the average survival time was 33 days, 31 days, 34 days and 42 days, respectively (p<0.05).
CONCLUSION:
The M(FA/PTX) have better anti-tumor activity and are promising in treatment of human breast cancers.
Copyright © 2015 Elsevier B.V. All rights reserved.
BACKGROUND INFORMATION:
Microtubule affinity-regulating kinase 4 (MARK4) deficiency has been reported to negatively regulate diet-induced obesity and to mitigate insulin resistance in knockout mice, and thus may play a role in metabolic syndrome. However, the details of the molecular mechanism have yet to be revealed and the impacts of MARK4 on apoptosis remain unexplored. This study investigated the role of Mark4 in the regulation of lipid accumulation and apoptosis in adipocytes and analysed signalling pathways involved.
RESULTS:
We found that Mark4 significantly up-regulated the expression of gene sterol regulatory element binding protein-1c (SREBP-1c), fatty acid synthase (FAS), acetyl-CoA carboxylase-α (ACCα) and peroxisome proliferator activated receptor-γ (PPARγ); and reduced the protein contents of adipose triglyceride lipase (ATGL), as evidenced by the dramatic increasing lipid droplet accumulation in 3T3-L1 cells. Furthermore, a terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) apoptosis assay showed that Mark4 triggered apoptosis of adipocytes; and apoptosis was confirmed by the decreased protein contents of B-cell lymphoma-2 (Bcl-2), full-length caspase-3 and full-length caspase-9, as well as the increased expression of Bax, cleaved caspase-3 and cleaved caspase-9. Analysis of special inhibitors allowed us to offer the following explanation for these impacts of Mark4: activation of Jun N-terminal kinase1 (JNK1) promoted both apoptosis and adipogenesis, whereas inhibition of the p38 mitogen-activated protein kinase (p38MAPK) pathway contributed to lipid accumulation alone.
CONCLUSIONS:
Mark4 promotes adipogenesis in 3T3-L1 adipocytes by activating the JNK1 and inhibiting the p38MAPK pathway, and triggers apoptosis by activating the JNK1 pathway. We conclude that anti-Mark4 therapy targetted to inhibit lipid accumulation and apoptosis of adipocytes shows potential as a novel therapeutic strategy for treatment of obesity-associated metabolic complications.
© 2014 Société Française des Microscopies and Société de Biologie Cellulaire de France. Published by John Wiley & Sons Ltd.
PURPOSE:
This study was designed to investigate the role of PDGF-DD secreted by gastric cancer-derived mesenchymal stem cells (GC-MSCs) in human gastric cancer progression.
METHODS:
Gastric cancer cells were indirectly co-cultured with GC-MSCs in a transwell system. The growth and migration of gastric cancer cells were evaluated by cell colony formation assay and transwell migration assay, respectively. The production of PDGF-DD in GC-MSCs was determined by using Luminex and ELISA. Neutralization of PDGFR-β by su16f and siRNA interference of PDGF-DD in GC-MSCs was used to demonstrate the role of PDGF-DD produced by GC-MSCs in gastric cancer progression.
RESULTS:
GC-MSC conditioned medium promoted gastric cancer cell proliferation and migration in vitro and in vivo. Co-culture with GC-MSCs increased the phosphorylation of PDGFR-β in SGC-7901 cells. Neutralization of PDGFR-β by su16f blocked the promoting role of GC-MSC conditioned medium in gastric cancer cell proliferation and migration. Recombinant PDGF-DD duplicated the effects of GC-MSC conditioned medium on gastric cancer cells. Knockdown of PDGF-DD in GC-MSCs abolished its effects on gastric cancer cells in vitro and in vivo.
CONCLUSIONS:
PDGF-DD secreted by GC-MSCs is capable of promoting gastric cancer cell progression in vitro and in vivo. Targeting the PDGF-DD/PDGFR-β interaction between MSCs and gastric cancer cells may represent a novel strategy for gastric cancer therapy.
ETHNOPHARMACOLOGICAL RELEVANCE:
Nephrotic syndrome (NS) is a clinical syndrome with a variety of causes, mainly characterized by heavy proteinuria. Podocyte injury plays a key role in proteinuria, one of the principal means for the control of NS is to prevent podocyte injury. Qi-Dan Fang consists of two of the most extensively applied herbal remedies among Traditional Chinese Medicine (TCM) (Radix Astragali Mongolici and Radix Salviae Miltiorrhizae, with a weight ratio of 5:1) which are specifically used for the treatment of various kidney diseases. In previous studies, we found that Qi-Dan Fang provides improvement to patients with adriamycin-induced nephrotic syndrome by alleviating proteinuria and serum lipid. The aim of this study is to study the efficiency of Qi-Dan Fang on NS model rat with renal dysfunction and podocyte injury, something which has not been carried out yet.
MATERIALS AND METHODS:
The rats were divided into Normal, Model, Jin Gui Shen Qi Pill (4.12 g/kg), Qi-Dan Fang (3.09, 6.17 and 12.34 g/kg/d) groups, they were each given a single tail intravenous injection of Adriamycin (6.0 mg/kg) except for the Normal group and were orally administered dosages of Qi-Dian Fang and Jin Gui Shen Qi pills once daily for 7 weeks. Following the treatment, the content of cystation C (CysC), blood urea nitrogen (BUN), serum creatinine (Scr) were measured with an autobiochemical analyser. The pathomorphological changes to the glomeruli, the mRNA expressions of nephrin, podocin, CD2AP genes and p53, bax, bcl-2 proteins expressions were also carried out to probe the effects of Qi-Dan Fang.
RESULTS:
(1) Qi-Dan Fang treatment raised the level of CysC in blood serum while lowering the content of BUN and Scr in the adriamycin-induced nephrotic syndrome rat model; (2) Long-term administration of Qi-Dan Fang was able to ameliorate pathomorphological change of glomeruli and repair the organization structure of Glomerulus; (3) Qi-Dan Fang could increase the mRNA expression of nephrin, podocin and CD2AP genes, down-regulate the expression of p53, bax proteins, while increased bcl-2 protein to protect the podocyte and restore Glomerular selective filtration function.
CONCLUSIONS:
Results of our present studies reveal that Qi-Dan Fang is able to enhance renal function, inhibit podocyte injury to provide improvements to the Adriamycin-induced nephrotic syndrome.
Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.
BACKGROUND/AIMS:
Colorectal carcinoma is one of the most common cancers world-wide, with high morbidity and mortality rates. Arginine ADP-ribosyltransferase 1(ART1) is an important ecto-ADP-ribose transferase and has been proven to be intimately involved in a number of biological processes. However, the influence of ART1 on survival and apoptosis of colorectal carcinoma cells and the potential mechanism of action of ART1 remain uncharacterized.
METHODS:
ART1 was silenced via lentiviral vector-mediated short hairpin RNA (shRNA) in CT26 colon carcinoma cells, and cisplatin (CDDP) was applied to induce apoptosis. Survival and apoptosis rate of CT26 cells was assessed by CCK8 assay, flow cytometry and Hoechst 33342 staining. Expression and activity of signaling proteins were detected by Western blot.
RESULTS:
ART1 knockdown enhanced the inhibition of cell survival and increased the apoptosis induced by CDDP. Furthermore, the reduced survival rate correlated with reduced levels of phos-Akt(Thr308) and phos-IκBα and reduced NF-κB p65 nuclear translocation. A decline in Bcl-2 and Bcl-xl expression and an increase in Bax expression may explain the enhanced apoptosis.
CONCLUSION:
This study provides a molecular mechanism for the function of ART1 in colorectal carcinoma and defines a potential therapeutic target for the enhanced treatment of this prominent world-wide disease.
© 2013 S. Karger AG, Basel.
BACKGROUND:
Renal ischemia-reperfusion (I/R) injury is a major cause of acute kidney injury. The pathogenetic mechanisms underlying I/R injury involve oxidative stress and apoptosis. Osthole, a natural coumarin derivative, has been reported to possess antioxidant and antiapoptotic activities. This study aimed to investigate the potential effects of osthole on renal I/R injury in an in vivo rat model.
MATERIALS AND METHODS:
We induced renal I/R injury by clamping the left renal artery for 45 min followed by reperfusion, along with a contralateral nephrectomy. We randomly assigned 54 rats to three groups (18 rats/group): sham-operated, vehicle-treated I/R, and osthole-treated I/R. We treated rats intraperitoneally with osthole (40 mg/kg) or vehicle (40 mg/kg) 30 min before renal ischemia. We harvested serum and kidneys at 1, 6, and 24 h after reperfusion. Renal function and histological changes were assessed. We also determined markers of oxidative stress and cell apoptosis in kidneys.
RESULTS:
Osthole treatment significantly attenuated renal dysfunction and histologic damage induced by I/R injury. The I/R-induced elevation in kidney malondialdehyde level decreased, whereas reduced kidney superoxide dismutase and catalase activities were markedly increased. Moreover, osthole-treated rats had a dramatic decrease in apoptotic tubular cells, along with a decrease in caspase-3 and an increase in the Bcl-2/Bax ratio.
CONCLUSIONS:
Osthole treatment protects murine kidney from renal I/R injury by suppressing oxidative stress and cell apoptosis. Thus, osthole may represent a novel practical strategy to prevent renal I/R injury.
Copyright © 2013 Elsevier Inc. All rights reserved.
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