The Anti-pancreatic Cancer Role And Mechanism Studies Of IMB-6G And Resibufogenin

Autophagy is an evolutionary conserved,dynamic cellular homeostatic process that exerts cytoprotective effects by removing damaged organelles and protein aggregates.However,autophagy acts as both a tumor inducer by providing nutrients to tumor cells for survival under hypoxic,low-energy conditions and as a tumor suppressor by preventing ROS-induced tumorigenesis.The king of tumor-pancreatic cancer is an aggressive and malignant tumor with a 5-year survival rate between 3%and 11%.The most important factors affecting its poor prognosis are the difficulty of early diagnosis and high resistance to most existing chemotherapy drugs.Current researches have shown that the level of autophagy in pancreatic cancer is relatively higher than that of other epithelial tumors,which promotes tumor growth.Therefore,it is a new strategy to search for anti-tumor drugs by targeting autophagy.Our previous studies have found that the new compound of sophoridine,IMB-6G,can exert a good anti-tumor effect by inducing endoplasmic reticulum stress and cell cycle arrest in liver cancer and lung cancer.In addition,IMB-6G also has many advantages likes novel structure,good pharmacokinetic and high safety.At present,the impact of IMB-6G on autophagy and pancreatic cancer is not yet clear,so we have conducted studies on its anti-pancreatic cancer activity and its relationship with autophagy in pancreatic cancer cells.First,we studied the activities of IMB-6G in vitro.The pancreatic cancer cells MiaPaCa-2 and HupT-3 were selected as the cell lines.We confirmed that IMB-6G inhibits the growth of pancreatic cancer cells and induces caspsase-dependent cell death by MTT,Western blot,flow cytometry and other methods.Later,through GFP-LC3 aggregation experiments,it was found that IMB-6G can cause a significant increase in spot aggregation of LC3.Simultaneously,by immunoblotting,IMB-6G was detected to increase the expression of LC3-Ⅱ and p62 in a time-and concentration-dependent manner.These experiments indicate initially that IMB-6G inhibits autophagy activity in pancreatic cancer cells.Then,the LC3 turnover experiment was carried out by using autophagy inhibitor CQ and inducer Rapamycin.After joint use of IMB-6G and CQ,there was no further elevation of intracellular LC3-Ⅱ,indicating that IMB-6G might have similar effect to CQ that inhibits the degradation of LC3-Ⅱ by inhibiting the lysosomal pathway of autophagy.However,the level of LC3-Ⅱ increased significantly after the combination of IMB-6G and Rapamycin,confirming that IMB-6G inhibits autophagosome degradation in pancreatic cancer cells.Subsequently,we expressed the dual-fluorescent plasmid mCherry-GFP-LC3 to determine the change of autophagic activity in MiaPaCa-2 cells.Since the red fluorescence of mCherry was relatively stable in the acidic environment,it was not easy to quench,while the green fluorescence of GFP was unstable and quenched,the difference between mCherry fluorescence and GFP fluorescence was taken into account.Confocal microscopy results showed that the green fluorescence and red fluorescence were both increased significantly by IMB-6G treatment,indicating that the number of autophagosomes in the cells increased,but the degradation activity of autolysosomes was inhibited.These results suggested that IMB-6G inhibited autophagic flow in pancreatic cancer cells.After that,the activity of lysosomes was detected using the lysosomal fluorescent probe LysoSensor Green and LysoTracker Red.It was found that the green fluorescence of LysoSensor and the fluorescence of LysoTracker did not change significantly after IMB-6G treatment,indicating that the acidity of lysosomes was not influenced by IMB-6G.Since the degrading activity is mainly affected by the acidic environment of the lysosome and the acid hydrolase,we detected the effects of IMB-6G on lysozyme hydrolyzing enzymes.By DQ-BSA staining,the red fluorescence in the lysosome was significantly reduced after IMB-6G treatment,indicating that the activity of lysozyme hydrolase was inhibited.Cathepsins,which are the main hydrolases in lysosomes,were detected by cathepsin activity assay and immunoblotting.The results indicated that IMB-6G blocks autophagic flux by inhibiting the activity of cathepsin in lysosomes in pancreatic cancer cells.In addition,by AO staining,we found that IMB-6G induces lysosomal membrane permeabilization(LMP),which releases CTSB/CTSD from the lysosome to the cytoplasm.siAtg5 was used to inhibit autophagosome formation and it was found to attenuate IMB-6G-induced LMP and apoptosis.At the same time,cathepsin inhibitors also reduce IMB-6G-induced apoptosis.The above results indicate that IMB-6G can induce autophagy-dependent apoptosis through inhibiting the autophagy-cathepsin pathway in pancreatic cancer cells.It is a novel autophagy inhibitor and provides scientific basis for IMB-6G as a new anti-tumor drug candidate.As a key enzyme in the glycogen synthesis pathway,GSK-3 is known to play an important role in many biological functions,such as glycogen metabolism,protein synthesis,Wnt,mitosis and apoptosis.Studies have reported that GSK-3 activity is relatively high in pancreatic cancer,which can promote the survival of pancreatic cancer cells by activating the NF-κB signaling pathway.Resibufogenin(RB)is one of the major active compounds of traditional Chinese medicine Chansu and has great potential in the treatment of cancer.Studies have shown that the cytotoxic activity of RB in human liver cancer,gastric cancer,and ovarian cancer is comparable to or stronger than that of taxol.However,the anti-tumor effect and underlying mechanism of RB on pancreatic cancer is still unclear.So in this study,we investigated the anticancer effects and related molecular mechanisms of RB on pancreatic cancer in vitro and in vivo.Our experiment selected non-transformed pancreatic epithelial cell HPDE and pancreatic cancer cells Panc-1 and Aspc as the cell lines.Firstly,inhibition of RB on pancreatic cancer cell proliferation was detected by MTT,colony formation and other methods,while the inhibition of HPDE is weak,indicating that RB effect is selective.Subsequently,RB induced caspase-dependent apoptosis through Hochest fluorescence observation,flow cytometry,and WB experiments.Next we tested the activity of NF-κB.NF-κB Luciferase,RT-PCR and WB results showed that RB inhibited the expression of downstream substrates by inhibiting the transcriptional activity of NF-κB.Both classical and non-canonical NF-κB signaling pathways were inhibited by RB.Microarray analysis showed that RB induced the down-regulation of 49 NF-κB target genes,most of which had been shown to have tumor promoting effects in pancreatic cancer,such as anti-apoptotic genes PARP1,c-FLIPL,and TRAF5.Since the activity of TAK1 plays an important role in the activation of NF-κB,we detected the effect of RB on TAK1 level by WB.The results showed that RB can inhibit the expression of TAK1 and its binding protein TAB1/TAB2,thereby inhibiting the downstream phosphorylation of IKK and IκBα.Simultaneously,overexpression of TAK1 can reduce the apoptosis induced by RB,indicating that RB induces apoptosis of pancreatic cancer cells by inhibiting the activity of TAK1,thereby inhibiting the downstream IKK-NF-κB signaling pathway.Furthermore,we detected that RB induced phosphorylation inactivation of GSK-3 by WB and dual luciferase reporter gene assays,following inhibition of downstream substrates.After GSK-3 WT and GSK-3 S9A were over-expressed in Panc-1 cells,the apoptosis induced by RB was reduced,while over-expression of GSK-3 K85A had no effect on apoptosis,indicating that RB-induced apoptosis was associated with GSK-3 inactivation.Studies have shown that both AKT and PKC can cause phosphorylation of GSK-3,so we used inhibitors and siRNA.The results proved that RB-induced GSK-3 phosphorylation is dependent on PKC rather than AKT,and it is likely that PKCβ isoform plays a major role in it.In addition,we found that RB inhibited the growth of pancreatic tumor model by constructing a xenograft model of pancreatic cancer in nude mice,indicating that it has better anti-tumor effect in vivo.In summary,our findings reveal the possible mechanism that suppresses TAK1-mediated NF-κB activity via PKC-dependent inhibition of GSK-3,subsequently inducing apoptosis in pancreatic cancer cells.It provides a theoretical basis for the potential application of RB in the treatment of pancreatic cancer.