Effects Of Glutamine Metabolism Inhibition On Cisplatin-or Etoposide-induced Tumor Cell Apoptosis

Most tumor cells displayed increased glutamine metabolism. Glutamine metabolism can provide tumor cells energy and macromolecular substances, and maintain the redox homeostasis for their growth and proliferation. Increased glutamine metabolism also provides a new strategy for cancer therapy. Interfering glutamine metabolism has been proved to be effective in inhibiting tumor cell growth both in vivo and in vitro. In tumor research, at least two kinds of drugs are usually used. Thus, it is very important to understand the effects of glutamine metabolism inhibition on chemotherapy. However, there are few studies about this. And apoptosis is the main mechanism for cancer cell chemotherapy. To investigate the molecular mechanism of inhibiting glutamine metabolism in tumor therapy, our aim was to study the influences of glutamine deprivation and glutaminase inhibitors on cisplatin-and etoposide-induced apoptosis in neuroblastoma cells (NB) and breast cancer cells (BC) by using flow cytometry, western blotting and fluorescence confocal, etc.EXP.1 Effects of glutamine metabolism inhibition on cisplatin-or etoposide-induced apoptosis in neuroblastoma cells and breast cancer cells1. Effects of glutamine metabolism inhibition on cisplatin-or etoposide-induced apoptosis in breast cancer cellsBT-549, HCC1937 and HCC38 cells were treated with glutamine free medium for 24 hours or BPTES for 6 hours, and followed by cisplatin or etoposide treatment for 48 hours. Results showed that cisplatin and etoposide induced cleaved-PARP and cleaved- caspase3 expressions. Glutamine deprivation or BPTES pretreatment had no effects on cisplatin- or etoposide- induced HCC38 cells apoptosis, but can promote cisplatin- or etoposide- induced HCC1937 cells apoptosis and etoposide (5?M)-induced BT-549 cells apoptosis. However, glutamine deprivation or BPTES pretreatment inhibited etoposide (1?M)-induced BT-549 and ciaplatin (10?M)-induced HCC1937 cells apoptosis, which indicated does-specific.2. Effects of glutamine metabolism inhibition on cisplatin- or etoposide- induced apoptosis in Neuroblastoma cellsTET21N, SK-N-BE(2) and SK-N-AS cells were treated with glutamine free medium or glutaminase inhibitors for 24 hours or 6 hours respectively, and followed by cisplatin or etoposide treatment for 24 hours. Results of cleaved-PARP expression and caspase-3 activity showed that treatments of glutaminase inhibitors combined with drugs induced antagonism effects. Glutamine deprivation had no effects on cisplatin- or etoposide-induced SK-N-BE(2) and SK-N-AS cells caspase-3 activities. However, glutamine deprivation significantly increased cisplatin-induced but decreased etoposide-induced cleaved-PARP expression and caspase-3 activity in TET21N cells, and did not show does-specific.EXP.2 Effects of glutamine deprivation on cisplatin- or etoposide- induced apoptosis in TET21N cells1. Effects of glutamine deprivation combined with cisplatin or etoposide in TET21N cells apoptosisTET21N cells were treated with glutamine-free medium for 24 hours, followed by cisplatin or etoposide treatment for 24 hours. Caspase-3 activity, expressions of cleaved-caspase9, cleaved-caspase3, cleaved-PARP, p-yH2Ax, caspase-8, Bid, FLIP-S and p53 and release of cytochrome c showed that glutamine deprivation significantly increased cisplatin-induced apoptosis but decreased etoposide-induced apoptosis in TET21N cells, which was independent of NMYC expression. These results indicated that the effects of glutamine deprivation on cisplatin and etoposide were involved in intrinsic and caspase-8-mediated apoptosis pathways.2. The effects of glutamine deprivation on etoposide and cisplatin showed time-dependentTET21NMYC- cells were cultured in glutamine free medium for 24 hours, and then they were treated with etoposide or cisplatin for 6 hours,10 hours,14 hours and 18 hours, respectively. Cleaved-PARP expression and caspase-3 activity results showed that the effects of glutamine deprivation on etoposide were time-dependent, which was increased and then decreased. However, the effects of glutamine deprivation on cisplatin were gradually increased over time.EXP.3 Mechanism of TET21N cells apoptosis induced by glutamine deprivation combined with cisplatin or etoposide1. The effects of glutamine deprivation combined with cisplatin or etoposide on cell and mitochondrial respiratory function in TET21N cellsMitochondrial oxygen consumption and cell calcium intake results showed that glutamine deprivation or glutamine deprivation combined with cisplatin significantly decreased oxygen consumption and cell calcium intake. Besides, glutamine deprivation decreased BAX expression induced by etoposide or cisplatin, and increased cisplatin-induced but decreased etoposide-induced Bid(p15) expression, but had no effects on cyclophilin D expression. Cyclosporine A and bongkrekic acid were used to inhibit cyclophilin D and amide acids transferase. Results showed that both inhibitors can inhibit glutamine deprivation combined with cisplatin- or etoposide- induced apoptosis slightly. These results indicated that glutamine deprivation influenced mitochondrial respiration and cell membrane calcium channel, and mitochondrial permeability transition pore and BAX, Bid(p15) were involved in increasing mitochondrial permeability and thus induced apoptosis.2. The functions of caspase, AIF and ROS in glutamine deprivation combined with cisplatin- or etoposide- induced TET21N cells apoptosisCaspase inhibitor ZVAD was used to treat cells, and we found that ZVAD significantly inhibited apoptosis induced by cisplatin combined with glutamine deprivation. AIF release results said that glutamine deprivation could promote AIF release from mitochondria, which indicated that both AIF and caspase were involved in apoptosis in our case. ROS production detected by MitoSOX suggested that glutamine deprivation increased ROS level. Then NAC was used to inhibit ROS production, which showed that inhibition of ROS production deleted the glutamine deprivation combined with cisplatin-induced apoptosis and DNA damage. But NAC didn't eliminate effects of glutamine deprivation on etoposide, which was only decreased. These results indicated that ROS production was the key factor in cisplatin-induced apoptosis under glutamine deprivation condition. ROS was the reason but not the result of DNA damage.In conclusion, inhibition of glutamine metabolism promotes cisplatin-and etoposide-induced HCC1937 cells apoptosis, which showed does-specific. However, glutamine metabolism inhibition had no significant effects on BT-549 and HCC38 cells apoptosis. Glutaminase inhibitors combined with cisplatin or etoposide displayed antagonism effects in neuroblastoma cells. Glutamine deprivation can promote cisplatin-but inhibit etoposide-induced TET21N cells apoptosis, which was independent of NMYC expression. The effects of glutamine deprivation combined with cisplatin or etoposide were involved with intrinsic, caspase-8- and AIF-mediated apoptosis pathways. Glutamine deprivation stimulated ROS production, which induced DNA damage and then increased cytotoxicity of cisplatin. In this study, we used glutamine metabolism inhibition combined with cisplatin or etoposide to treat neuroblastoma cells and breast cancer cells to study apoptosis for the first time. This study provides theoretical basis for usage of drugs combination in cancer therapy and utilization of glutamine metabolism inhibition in cancer chemotherapy.