Decrease In Necroptosis Sensitivity Contributes To Cisplatin Resistance In Lung Cancer

Background and Objective:Cancer is a major public health problem worldwide.Due to its high incidence rate and mortality rate,lung cancer is one of the most threatening cancers.Chemotherapy is frequently used in lung cancer treatment and cisplatin remains a first-line agent in lung cancer chemotherapy.It has been years since the beginning of the studies on the mechanisms of its cytotoxicity and drug resistance,while the emergence of a new type of programmed cell death known as necroptosis brings new directions to the studies on cisplatin.It has been shown that cisplatin trigger necroptosis in some cell lines,but whether it can kill lung cancer cells by inducing necroptosis has not been reported.It is widely known that apoptosis resistance is an important mechanism of cisplatin resistance.However,whether necroptosis resistance contributes to the cisplatin-induced chemoresistance in lung cancer is not fully understood.Our study aims to answer the two questions above.This study is composed of the following three parts.Part 1.Mechanism research on cisplatin-induced necroptosis in lung cancer cellsMethods:1)Cell death was observed by light microscopy,and the morphology of the dead cells was observed by electron microscope.2)MTT assay,LDH release assay and flow cytometry were used to measure cytotoxicity after drug treatment.3)Western blotting was used to detect the changes in protein expression levels.4)Quantitative-PCR?q-PCR?was used to determine the changes in expression at mRNA levels.5)Immunocytochemistry was used to determine the changes in the distribution of molecules and in protein expression levels after drug treatment.6)TNF-?in cell culture supernatant was measured by ELISA.7)Immunohistochemistry was used to detect p-MLKL expression in lung cancer tissues.8)PI and Hoechst staining were used to detect cell death.9)Immunoprecipitation was performed to detect intracellular protein interactions.Results:We examined the expression level of RIPK3 in five lung cancer cell lines,and the results showed that RIPK3 expression level was higher in A549 cells.zVAD was introduced to inhibit cisplatin-triggered apoptosis.However,we still noticed dead cells in A549 cells with zVAD pretreatment and the morphology of the dead cells suggested that they were necroptotic cells.Necroptosis inhibitors Nec-1 and NSA were able to decrease cisplatin-induced A549 cell death.By immunoprecipitation,we confirmed that RIPK1,RIPK3 and MLKL can form a complex in cisplatin-treated A549 cells,and the expression level of p-MLKL was up-regulated in A549 cells after cisplatin treatment.The results of immunohistochemistry showed that the expression level of p-MLKL in samples from patients treated with neoadjuvant chemotherapy?cisplatin was included in the chemotherapeutic regimens?was higher than that in samples from patients without neoadjuvant chemotherapy.We found that TNF-?expression level was elevated in A549cells after cisplatin treatment and cisplatin promoted autocrine TNF-?in A549 cells.Pretreatment with a neutralizing anti-TNF-?antibody decreased cisplatin-triggered necroptosis.Conclusion:Cisplatin induces necroptosis in lung cancer A549 cells.Part 2.PITP?interacts with MLKL to regulate necroptosis Methods:1)Immunoprecipitation was performed to detect intracellular protein interactions.2)Fluorescence resonance energy transfer experiment was conducted to detect the direct interaction of two molecules in living cells.3)Non-reducing SDS-PAGE was performed to detect MLKL oligomerization.4)Cell lysate was divided into cytosolic fraction and membrane fraction to measure MLKL plasma membrane translocation.5)RNA interference was used to determine if PITP?participates in the function of MLKL.6)Immunocytochemistry was used to determine MLKL distribution.7)MTT assay was used to measure cytotoxicity after treatment.8)Western blotting was used to detect the changes in protein expression levels.Results:We found that MLKL directly interacts with PITP?in A549 cells.We constructed two MLKL truncations and it was confirmed by fluorescence resonance energy transfer experiments that the N-terminal domain of MLKL interacts with PITP?.Based on the MLKL-PITP?complex model obtained by RossetaDock,we designed MLKL double-point mutants and found that the MLKL^Q135P/Q138P mutant showed significantly decreased affinity towards PITP?.Knocking down the expression of PITP?not only affected the plasma membrane translocation and oligomerization of MLKL overexpressed in HEK-293T cells,but also affected that of MLKL in cisplatin-treated A549 cells.Interference with PITP?expression could attenuate both MLKL overexpression-induced HEK-293T cell death and cisplatin-triggered A549 cell death.Conclusion:PITP?interacts with MLKL to regulate necroptosis.Part 3.Regulatory mechanism research on the involvement of necroptosis resistance in cisplatin resistance of lung cancer cellsMethods:1)CCK-8 assay,LDH release assay and flow cytometry were used to measure cytotoxicity after drug treatment.2)Western blotting was used to detect the changes in protein expression levels.3)q-PCR was used to determine the changes in expression at mRNA levels.4)RNA interference was used to determine the function of a molecule in cell death signaling pathway.5)Immunoprecipitation was performed to detect intracellular protein interactions.Results:We found that the expression level of RIPK3 was lower in A549/DDP cells than that in the parent cells,and A549/DDP cells were not sensitive to cisplatin-induced necroptosis.RIPK3 overexpression could sensitize A549 cells and A549/DDP cells to cisplatin-induced cell death and necroptosis could be triggered in A549/DDP cells.We confirmed that the expression of RIPK3 in lung cancer cells is negatively regulated by CNOT3.Thus,the lower expression of RIPK3 in A549/DDP cells was a result of CNOT3 up-regulation.Furthermore,we demonstrated that the expression level of CNOT3 was negatively correlated with the sensitivity of lung cancer cells to cisplatin.Depletion of CNOT3sensitized A549/DDP cells to cisplatin-triggered apoptosis through a mechanism of RIPK3up-regulation and the increased RIPK3 drove more assembly of FADD and Caspase 8 to transduce extrinsic apoptotic signal.By analyzing the data in the database,we found that CNOT3 mRNA expression in lung cancer tissues was higher than that in normal lung tissues,and its high expression was associated with poor prognosis of lung cancer patients.In addition,CNOT3 was able to regulate the proliferation of lung cancer cells.Conclusions:1.Necroptosis resistance caused by RIPK3 down-regulation is involved in the mechanisms of cisplatin resistance.2.CNOT3 regulates RIPK3 expression and its depletion sensitizes cisplatin-resistant lung cancer cells to cisplatin-triggered apoptosis.