Inhibition Of PI3K/mTOR Increase The Sensitivity Of Hepatocellular Carcinoma Cells To Cisplatin Via Interference With Mitochondrial-lysosomal Cross-talk

BackgroundThe hepatocellular carcinoma cells used in this experiment are Huh7 and Hep G2.Hepatocellular carcinoma(HCC)is a common malignant tumor of the digestive system with a poor prognosis.Surgical resection is the most effective treatment for improving the prognosis of patients with early-stage HCC;however,HCC is susceptible to recurrence and metastasis even after surgery.Effective systemic therapy during the perioperative period can decrease the recurrence rate and improve prognosis.Nonetheless,HCC cells have relatively low sensitivity to systemic chemotherapy,thereby making it essential to understand the mechanisms underlying drug resistance in hepatocellular carcinoma and to find new targets for improving the sensitivity of HCC cells to chemotherapeutic drugs.Cisplatin is reported to produce cytotoxicity by damaging the mitochondrial function of tumor cells.However,cancer cells will induce a series of mitochondrial quality control to response to cisplatin.The mitophagy-lysosomal pathway scavenges the damaged mitochondria caused by chemotherapeutic drugs,thereby maintaining cell homeostasis and participating in chemotherapy resistance of tumor cells..Whether this mitochondrial-lysosomal cross-talk plays a key role in HCC cells against cisplatin remains to be determined.The stability of lysosomal membrane is a prerequisite for the mitophagy-lysosomal pathway.Therefore,lysosomal homeostasis plays an important role in mitochondrial-lysosomal cross-talk.When the lysosomal membrane stability is damaged,it can lead to lysosomal membrane permeabilization(LMP).In this scenario,the lysosomal hydrolase and cathepsin can get released into the cytoplasm,damaging the mitochondria and other organelles.This can induce lysosomal cell death.There are many factors that can affect the stability of lysosomal membranes,including the abnormal active PI3K/m TOR pathway in HCC cells.PI3K/m TOR signaling has been reported to be involved in the control of the activity and stability of lysosomes.Inhibition of PI3 K may induce LMP.m TOR is also involved in the regulation of autophagy and lysosomal biogenesis.Moreover,m TOR has a negative regulation effect in lysosomal function and inhibition of m TOR can enhance the lysosomal biogenesis.Therefore,the role of PI3K/m TOR pathway in the regulation of lysosome homeostasis is very important.Based on the abovementioned points,we hypothesized that HCC cells might activate the mitophagy-lysosomal pathway,resulting in a mitochondrial-lysosomal cross-talk in response to cisplatin treatment,which can contribute cisplatin resistance of HCC,when combined with cisplatin PI3K/m TOR inhibitor,on one hand it can increase the number of lysosomes in HCC cells,on the other hand it may cause LMP in HCC cells,thereby inducing the release of lysosomal enzymes into the cytoplasm;this may affect the mitochondrial-lysosomal cross-talk of HCC cells induced by cisplatin,leading to an improved sensitivity in HCC cells to cisplatin.MethodsThe hepatocellular carcinoma cells used in this experiment are Huh7 and Hep G2.(1)MTT assay was used to evaluate cell viability.MitoSOX Red is a specific dye for mitochondrial reactive oxygen species(mt ROS)and its fluorescence level was analyzed using flow cytometry.After mt ROS inhibition,MTT assay was also used to evaluate the changes in cell viability.(2)We utilized Human Mitochondrial Energy Metabolism PCR Array to examine the transcriptional level of energy metabolism related genes.Mitochondrial oxygen consumption rate was used to assess the mitochondrial respiratory function.Mitotracker Green is a specific dye used to label mitochondria and its fluorescence level was analyzed using flow cytometry.(3)We stained mitochondria with Mitotracker Red and observed mitochondrial morphology using laser confocal microscope.RT-q PCR was used to examine the transcriptional level of mitochondrial fusion and fission related genes including MFN1,MFN2,OPA1,DRP1,and FIS1.Western blotting was used to determine the protein levels.(4)We utilized Western blotting to measure the expression of mitophagy-relatedproteins including PINK1,Parkin,LC3,p62,LAMP1,cathepsin B,and cathepsinD.(5)Lysotracker Green and DP BSA RED were used as specific markers for lysosomes and its fluorescence level was analyzed using flow cytometry to assess the quality and function of lysosomes.(6)We immunostained lysosome biogenesis transcription factor TFEB and observed its activation and transkocation to nucleus using laser confocal microscope.RT-q PCR was used to examine the transcriptional level of its downstream genes.(7)After the activation or inhibition of mitophagy,we used flow cytometry to examine mt ROS,mitochondrial quality,mitochondrial membrane potential,and apoptosis by staining using Mito SOX Red,Mito SOX Red,Mito SOX Red,Annexin V/PI,respectively.(8)After inhibition of PI3K/m TOR,fluorescence levels were analyzed using flow cytometry,which describes the changes in apoptosis(marker by Annexin V/PI),lysosome quality,and function(marked by Lysotracker Green and DQ BSA).(9)After inhibition of PI3K/mTOR pathway,we used flow cytometry to examine red fluorescence(stained by acridine orange)and Western blotting to measure the expression of Cytochrome C and lysosome cathepsin,including cathepsin B and cathepsin D.(10)After inhibition of PI3K/m TOR,we used flow cytometry to examine mt ROS (stained using Mito SOX Red)and mitochondrial membrane potential(stained using JC-1).Results(1)Cisplatin can target mitochondrial function in HCC cells and lead to mt ROS accumulation,a decrease in mitochondrial membrane potential,and accumulation of damaged mitochondria.Inhibition of formation of mitochondrial ROS can relief the cytotoxicity caused by cisplatin in HCC cells.(2)Confocal laser scanning microscopy showed that cisplatin can induce mitochondrial fission in HCC cells.The results of RT-q PCR and Western blot showed that the mitochondrial fission related molecules Fis1 and Drp1 were upregulated by cisplatin in HCC cells;however,the mitochondrial fusion related molecules Mfn1 and Mfn2 were down-regulated.Western blot results also showed that cisplatin upregulated the expression of the mitophagic-lysosomal pathway related proteins PINK1,Parkin,LC3-II/I,cathepsin B,cathepsin D,and LAMP1.However,the expression of p62,one kind of mitochondria autophagy substrate,was down-regulated.(3)Cisplatin induced the translocation of the transcription factor TFEB into the nucleus.RT-q PCR results indicated that the lysosome biogenesis related genes were also activated and the quality and function of lysosome were improved.(4)Chloroquine(CQ)caused the accumulation of mitophagy-related proteins(PINK1,Parkin,LC3,and p62),and considerably inhibited the mitophagic-lysosomal pathway.Combination CQ aggravated cisplatin-induced changes,such as mt ROS accumulation,impaired mitochondrial accumulation,mitochondrial respiratory function damage,and further reduction of the mitochondrial membrane potential,to increase apoptosis rate.A combination of cisplatin with the autophagy activator drug rapamycin could improve the above indices.(5)The combination of cisplatin and PI3K/m TOR inhibitor PKI-402 induced apoptosis,whereas cathepsin inhibitor E-64 inhibited this apoptosis.(6)The combination of cisplatin and the PI3K/mTOR inhibitor PKI-402 increased lysosome quantity;however,it destroyed the lysosome phagocytosis and degradation function.(7)AO staining showed that the red fluorescence level decreased when cisplatin was combined with PKI-402,and the level of cathepsin B,cathepsin D,and cytochrome C protein in cytoplasm increased considerably,indicating the level of LMP.(8)The combination of cisplatin and the PI3K/m TOR inhibitor PKI-402 induced increased accumulation of mt ROS and a decrease in the mitochondrial membrane potential;however,cathepsin inhibitor E-64 could reverse the situation.Conclusions(1)Cisplatin can target mitochondria and then cause cytotoxicity in HCC cells.(2)HCC cells can induce mechanisms for mitochondrial quality control,such as mitochondrial fission and mitophagy,to resist cisplatin action.(3)The mechanisms of resistance to cisplatin in HCC involve lysosomal biogenesis activation and increased lysosomal number and function.These can maintain mitophagy and develop the mitochondrial-lysosomal cross-talk.(4)Inhibition of mitophagy-lysosomal pathway can interfere with the mitochondrial-lysosomal cross-talk in HCC,thereby enhancing the sensitivity to cisplatin in HCC.(5)PKI-402 combined with cisplatin can induce LMP in HCC cells,which interferes with the mitochondrial-lysosomal cross-talk in HCC,resulting in an increased sensitivity to cisplatin in HCC.In summary,the mitochondrial-lysosomal cross-talk induced by cisplatin is one of the mechanisms of resistance to cisplatin employed by HCC cells.Inhibition of mitophagy-lysosomal pathway can interfere with this cross-talk and enhance the sensitivity to cisplatin in HCC.In addition,damaging the lysosomal membrane stability by inhibition of PI3K/m TOR can increase the sensitivity to cisplatin and change protective role of lysosomes into cell death promoter.The alteration in the mitochondrial-lysosomal cross-talk can be used as a strategy to increase cisplatin sensitivity in HCC.