Mechanisms Of Oxaliplatin-induced Necrosis In Gastric Cancer Cells

Backgroud Gastric cancer is a common malignancy with high morbidity and mortality in China. Surgical resection and chemotherapy are the main therapeutics. Platinum anticancer drugs, such as cisplatin and oxaliplatin, are widely used in the treatment of gastric cancer. As alkylating agents, platinum-based drugs work by binding to DNA, leading to platinum-DNA adducts. Intrastrand and interstrand crosslinks cause DNA damage. The mechanism whereby DNA adducts kill cells is not fully understood. Intrinsic apoptotic pathway is considered to be responsible for platinum cytotoxicity. However, increasing evidences have demonstrated that DNA-damaging agents may kill cells via another way, that is, necrosis. Necrosis has been considered as a purely passive, accidental and unregulated event as a result of overwhelming insults for a long time. In 2005, Degterev et al. introduced the term of “necroptosis” in murine ischemia and reperfusion model, and identified a specific and potent small-molecule inhibitor of necroptosis, necrostatin-1(Nec-1). In the past decade, it was found that necroptosis is implicated in various pathophysical processes, either as a pathological consequence or as an etiological determinant. Nowadays the most extensively studied models are tumor necrosis factor-induced receptor interacting protein kinase 3(RIP3)-dependent pathway and DNA alkylating agents-induced poly(ADP-ribose) polymerase-1(PARP-1) pathway. In our previous work, we found that Nec-1 could protect SGC-7901 cells from oxaliplatin-induced cell death, confirming the participation of necrosis. The aim of thisstudy is to explore the mode of cell death after oxaliplatin treatment, especially the necrotic cell death. RIP3-dependent pathway and PARP-1-mediated pathway are equally examined. Apoptosis and necrosis are not mutually exclusive processes, they may function as reciprocal backup mechanisms of cellular demise. In contrast to the extensively studied apoptotic pathway, necrosis is a newly uncharted territory. Figuring out the mechanism of necrosis and its relationship with apoptosis will provide clues for overcoming resistance to chemotherapeutic drugs.Methods Apoptosis and necrosis were discriminated using Annexin V and PI staining by flow cytometry. Changes of plasma membrane and nucleus were observed under fluorescence microscope after Hoechst 33342 and PI staining. Mitochondrial membrane potential was determined by JC-1 staining on a flow cytometer. Activation of caspase-9, caspase-3, cleavage of PARP-1 and the expression level of anti-apoptotic Bcl-2 family members and IAP members, were measured by Western blot analysis. DNA fragmentation was monitored in agarose gel electrophoresis. Activity of lactate dehydrogenase(LDH) released from damaged cells was measured using a colorimetric method. The release of cyclophilin A(Cyp A) and high mobility group box 1(HMGB1) was determined by Western blot. Ultrastructural alterations were observed under transmission electron microscope. The production of reactive oxygen species(ROS) was monitored using DCF-DA staining on a flow cytometer. Transition of LC3-Ⅰto LC3-Ⅱ was also measured by Western blot. The expression level of Bmf at m RNA and protein level were determined by real-time PCR and Western blot. The effects of RIP1 inhibitor(Nec-1), MLKL inhibitor(NSA), Drp1 inhibitor(Mdivi-1), pan-caspase inhibitor(z-VAD-fmk) and PARP-1 inhibitor(olaparib) on oxaliplatin-induced cell death were examined by MTS assay. After transfection with RIP1 si RNA, RIP3 si RNA and PARP-1 si RNA, transfection efficiency was measured by real-time PCR and Western blot, and the effect of transfection on cell death was determined by MTS assay.The formation of PAR and phosphorylation of H2 AX at Ser139 were observed using fluorescence microscope and Western blot. Intracellular NAD+ was determined by a colorimetric method, and ATP level was measured by the luciferin/luciferase method. Translocation of t AIF was monitored by confocal laser scanning microscope and Western blot analysis as well. Phosphorylation(activation) of p38、JNK and ERK1/2 at the early stage of oxaliplatin exposure was also determined by Western blot. Pretreatment with p38 inhibitor SB203580, JNK inhibitor SP600125 or MEK1/2 inhibitor U0126 on oxaliplatin-induced cell death was measured by MTS assay.Results The majority of dead cells became positive for both Annexin V and PI, only a small proportion of cells are single positive for Annexin V. There were only smear bands in agarose gel electrophoresis after exposure to oxaliplatin for 48 h, no DNA ladder was observed. Mitochondrial membrane potential collapsed after oxaliplatin treatment. Oxaliplatin induced the activation of caspase-9 and caspase-3, as well as PARP-1 cleavage after 24 h. The expression levels of Bcl-2, Bcl-XL, Mcl-1, c IAP1 and c IAP2 were not altered after oxaliplatin treatment, except slight decrease of XIAP. Furthermore, z-VAD-fmk could partially suppress oxaliplatin-induced cell death. The activity of LDH in the supernatant increased dramatically after 36 h, and the release of Cyp A was earlier than that of HMGB1. Ultrastructural changes under transmission electron microscope demonstrated the rupture of the plasma membrane, loss of cellular contents and chromatin condensation. Oxaliplatin administration did not stimulate ROS production and autophagy, but elevated the level of Bmf. Nec-1 blocked oxaliplatin-induced cell death nearly completely, about 90 % of cells were rescued in the presence of Nec-1. But NSA or Mdivi-1 could barely inhibit oxaliplatin-induced necrosis. Moreover, Nec-1 could inhibit the release of LDH, Cyp A and HMGB1 into the supernatant. Transfection with RIP1 si RNA, but not RIP3 si RNA, could attenuate oxaliplatin-induced cell death. Exposure to oxaliplatin resulted in PARP-1 overaction,as indicated by the formation of PAR, which led to NAD+ and ATP depletion. Phosphorylation of H2 AX at Ser139 and translocation of t AIF from mitochondria to cytoplasm, eventually to the nucleus, were also observed. Employment of PARP-1 inhibitor, olaparib, or transfection with PARP-1 si RNA could suppress oxaliplatin-induced necrosis. Furthermore, olaparib could inhibit PARP-1 activation, LDH release, cellular energy depletion and t AIF relocalization. Stimulation of cells with oxaliplatin resulted in activation of the p38 pathway as determined by the detection of activating phosphorylation of the kinase. In untreated cells, ERK1/2 and JNK were constitutively phosphorylated, after treatment with oxaliplatin for 6 h, the expression level of p ERK1/2 was downregulated, while p JNK was not altered. Pretreatment with U0126 or SB203580, respectively, caused significant resistance to oxaliplatin-induced cell death. By contrast, pretreatment with SP600125 did not alter the sensitivity of cells to oxaliplatin-induced cell death.Conclusions Oxaliplatin triggers necrosis as well as apoptosis in gastric cancer SGC-7901 cells. Although caspase cascade is activated, it appears dispensable for killing. ROS production and autophagy are not the execution mechanisms of necrosis, but Bmf seems to be crucial to this process. RIP1 is a key molecule in oxaliplatin-induced cell death, and Nec-1 could block this death process nearly completely. RIP3 and its downstream components MLKL, PGAM5 and Drp1 do not participate in the signaling pathway. Oxaliplatin-induced DNA damage causes the disproportionate activation of PARP-1, which results in energy depletion and cell-viability loss. Phosphorylation of H2 AX at Ser139 and translocation of t AIF are critical for this death process. JNK is not required for oxaliplatin-induced cell death, while p38 and ERK1/2 play important roles in oxaliplatin-induced necrosis.