Unconjugated
Methyl methanesulfonate (MMS) is an alkylating agent that can induce cell death through apoptosis and necroptosis. The molecular mechanisms underlying MMS-induced apoptosis have been studied extensively; however, little is known about the mechanism for MMS-induced necroptosis. Therefore, we first established MMS-induced necroptosis model using human lung carcinoma A549 cells. It was found that, within a 24-h period, although MMS at concentrations of 50, 100, 200, 400, and 800 μM can induce DNA damage, only at higher concentrations (400 and 800 μM) MMS treatment lead to necroptosis in A549 cells, as it could be inhibited by the specific necroptotic inhibitor necrostatin-1, but not the specific apoptotic inhibitor carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-fmk). MMS-induced necroptosis was further confirmed by the induction of the necroptosis biomarkers including the depletion of cellular NADH and ATP and leakage of LDH. This necroptotic cell death was also concurrent with the increased expression of p53, p53-induced gene 3 (PIG-3), high mobility group box-1 protein (HMGB1), and receptor interaction protein kinase (RIP) but not the apoptosis-associated caspase-3 and caspase-9 proteins. Elevated reactive oxygen species (ROS) level was also involved in this process as the specific ROS inhibitor (4-amino-2,4-pyrrolidine-dicarboxylic acid (APDC)) can inhibit the necroptotic cell death. Interestingly, knockdown of PIG-3 expression by small interfering RNA (siRNA) treatment can inhibit the generation of ROS. Taken together, these results suggest that MMS can induce necroptosis in A549 cells, probably through the PIG-3-ROS pathway.
OBJECTIVE:
This study aimed to investigate the radiosensitivity of bortezomib to cervical cancer and the possible underlying mechanism.
METHODS:
HeLa and SiHa cell lines with or without hypoxia treatment were divided into control, radiation alone, bortezomib alone, and radiotherapy plus bortezomib groups. CCK8 assay, clone formation assay, flow cytometry, and immunofluorescence test were used to measure cell proliferation, colony formation, apoptosis, and DNA double-strand break (DSB). Western blot analysis was performed to detect the expression of HIF-1α, PARP-1, and caspase-3, -8, and -9.
RESULT:
Statistical analysis of data revealed that bortezomib at nanomolar level exerted a radiosensitization effect on both cervical cancer cell lines in normoxia or hypoxia. Western blot analysis showed that the drug could inhibit hypoxia-related HIF-1α expression to increase apoptosis-related caspase-3, -8, and -9 activation and DNA DSB-related PARP-1 cleavage.
CONCLUSIONS:
Radiotherapy sensitization of bortezomib on cervical cancer cell lines had a drug-dose relation, and sensitization in hypoxia was more remarkable than in normoxia. Bortezomib may be a potential radiotherapy sensitization drug for cervical cancer.
