| Cancer is still a "nightmare" for all mankind,and millions of patients are threatened by cancer every year.Gastric cancer(GC)ranks the fifth among all cancers and is the most commonly diagnosed cancer,while its mortality rate ranks the third.In recent years,despite the great progress has made in treatment of GC including surgery,radiotherapy and chemotherapy,the prognosis of patients with advanced GC is still very poor.Postoperative chemotherapy is an effective and widely used treatment approach.However,the high toxic side effects of chemotherapy drugs also bring additional pain to patients.Therefore,the development of low-toxicity,high-efficiency anti-cancer drugs and certain treatment strategies that reduce toxicity and increase efficacy appear to urgent.In 1950s,Canadian research team discovered a natural product-vinblastine(also translated as vinblastine)in the leaves of periwinkle(Madagascar periwinkle),which was subsequently found to have an ideal anti-cancer effect;since then,it has been used as an anti-cancer drug in treatment of clinical cancer patients.It is a potent cell division inhibitor and was used to treat lymphoma,testicular cancer,breast cancer,bladder cancer,and lung cancer.The vinca extracts or analogs currently approved for clinical use include Vinorelbine,Vindesine,Vincristine,Vinblasine and Vinflunine.These drugs exhibit ideal anti-cancer effects,while the strong toxic side effects often limit their further application.Cisplatin(Cis)is a metal-based drug that is used for testicular cancer,ovarian cancer,bladder cancer,lung cancer,cervical cancer,head and neck cancer,gastric cancer,and other solid tumors.Similarly,many literatures reported that it exhibits approximately 40 individual toxicities,of which nephrotoxicity is the most common one.There is no doubt that the anti-cancer effect of Cis on tumor inhibition,but its strong side effect faced by long-term use is undoubtedly take a chronic drug,which also greatly reduces the life quality of patients.In present study,we found that another extract from Catharanthus roseus,Catharanthine,was further modified into Catharanthine Sulfate(CAS)and easily soluble in organic solvents.Its anti-cancer effect and mechanism have not been reported.We found that CAS exhibited an ideal tumor inhibition;more interestingly,CAS combined with Cis greatly enhances the therapeutic effect on GC,and in turn reducing the toxic side effects on mice.We systematically evaluated the effect and feasibility of CAS combined with Cis in GC treatment,our results of the study are as follows:1.CAS exhibits an ideal anti-GC effect and without obvious organs toxicity.In vitro,we found that CAS inhibited GC cell(SGC-7901 and MKN-45)growth in a time-and dose-dependent manner.Unlike Vincristine(VCR),we found that CAS did not induced classic cell apoptosis and it just mediated obvious cell inhibition through flow cytometry and western blot analysis.To further explore the anti-GC mechanism of CAS,we detected the cell cycle using the flow cytometry,and the results showed that the cell cycle was blocked at G0/G1 phase;in addition,the positive regulatory protein of cycle CDK2,CyclinE1,and CyclinE2 were down-regulated in a time-and dose-depedent mananer,while the negative regulatory protein of p21 was up-regulated in a time-and dose-depedent manner,assessed by western blot.In vivo,to further verify the anti-GC effect of CAS,we conducted a mouse subcutaneous xenograft tumor model(including cell line-derived(CDX)subcutaneous xenograft models and patient-derived orthotopic xenograft(PDX)model).Our results showed that the tumor volume and mass were significantly inhibited by CAS in both models.What's more,the Ki67(a proliferation-related factor)postitive cells were significantly reduced in CAS-treated tumor tissues.In addition,compared with the control group,the tumor cells treated by CAS exhibited obvious nucleoplasmic heterogeneity,and with a larger and appeared empty blistered cytoplasm through eosin and hematoxylin staining(H&E staining).To explore whether the drug has toxic side effects on the organs of mice,we performed H&E staining to detect the pathological alterations on the mice organs(including heart,liver,spleen,lung,and kidney),and our results demonstrated that CAS had no obvious side effects on the organs.However,the other side effects are still unknown but need further exploration.2.CAS co-activated PERK/ATF4/eIF2?/CHOP and IRE1?/XBP1 signal axis to induce protective endoplasmic reticulum stress(ERS)To further explore the potential tumor inhibition mechanism of CAS,the mRNA levels of the two GC cell lines in the treated and untreated groups were investigated through transcriptome sequencing(RNA sequencing,RNA-seq).Gene Set Enrichment Analysis(GSEA)showed that the mRNA of GC cells treated with CAS was highly enriched in the unfolded protein response(UPR)related gene set.Further,the Go analysis showed that the CAS-treated cells were enriched in a variety of biological process(BP)changes,including the ER unfolded protein response,especially in SGC-7901 cells,indicating severe ERS.Using bioinformatics methods to retrieve and analyze the differential expression of genes related to ERS,we found that 43 associated with ERS genes were upregulated in SGC-7901 cells,while 33 genes were up-regulated in MKN-45 cells,and nearly 20 genes responded to CAS treatment,which resulted in ERS and caused these genes to be upregulated.Further cluster analysis indicated that the pathway changes mediated by PERK and IRE1? were the most obvious.Cluster analyses based on RNA-seq were consistent with the RT-qPCR results,indicating that the PERK,IRE1? and ATF6-mediated signalling pathways of ERS were activated by CAS,especially the PERK-and IRE1?-mediated pathways,which contained more gene alterations.The related protein markers,including PERK,ATF4,eIF2?,p-eIF2?,GRP78,CHOP and IRE1? were significantly increased in a dose-dependent manner,and these findings were further verified in tumour tissues.Moreover,it was more interesting that the drug enhance the cell viability to a certain extent when stress is relieved,suggesting that CAS-induced ERS plays a negative role in cell survival.3.CAS activates ATF4/AMPK?/ULKl and XBP1/Beclin-1(ATG)signaling pathways to induce autophagy.ERS is a stress protection mechanism in eukaryotic cells.Under stress condition,the cell initiates an UPR,which also activates autophagy.Therefore,to explore whether the UPR mediated by CAS could induce autophagy,we detected the expression level of LC3B(a marker for autophagy)through various methods,and our results showed that the expression level of LC3B in the cells and tumor tissues treated with CAS was significantly up-regulated.In addition,we found that a large number of autophagosomes appeared in the cells after CAS treatment through electron microscope(TEM)observation,indicating that autophagy was activated.However,it is more interesting that when the levels of PERK and IRE1? were down-regulated by using shRNA or gene-specific inhibitors,the LC3B level was reduced to a greater extent.The results indicate that CAS-mediated autophagy is closely related to ERS.Usually,the UPR activates autophagy through three pathways,among which the most classic pathways include mTOR,AMPK? and Beclin-1/ATG.To explore the downstream pathways of autophagy activation,we performed weatern blot to analyze the expression of ERS related pathways,and found that the both ATF4/AMPK?/ULK1 and XBP1/Beclin-1(ATG)signaling axes were activated in CAS-treated cancer cells,however it needed to point out was that autophagy activation was independent of the mTOR signaling pathway.In addityion,autophagy would be greatly eliminated when two pathways were blocked.Therefore,these findings indicate that CAS activates autophagy through two signal axes mediated by UPR.4.CAS damages the autophagic flux by targeting the cytoskeleton systemAutophagy activation is accompanied by two states,namely autophagic flux circulation and blocking,respectively.To explore the state of autophagic flux by CAS,we combined a variety of autophagy activators or inhibitors to explore the autophagic flux.Western blot analysis revealed that the autophagy level can be significantly reduced when the cells were co-treated with 3-MA;however,when cells were co-treated with the autophagy activator rapamycin(Rapamycin),the LC3B level was further increased or maintained a constant level,suggesting that CAS-mediated autophagic flux was blocking.In addition,western blot results showed that the LC3B level did not further increase when CAS combined with the autophagic flux blockers Bafilomycin A1(BafA1)or chloroquine(CQ),respectively.SQSTM1 protein,also known as P62,is an adaptor protein in the process of autophagy;it can not only participate in the maturation process of autophagy,but also serve as a substrate that is degraded during autophagy.Therefore,the expression level of SQSTM1 can be used as a manner to determine whether autophagosomes are fused with lysosomes.Obviously,the expression level of SQSTM1 was significantly up-regulated when CAS combined with autophagy inhibitors BafAl and CQ.What's more interesting was that the level of SQSTM1 was still up-regulated when CAS treatment alone.These results showed that CAS hinders the fusion of autophagosomes and lysosomes.To further verify above results,we transfected an LC3B fusion tag vector that simultaneously expressed green fluorescent protein(GFP)and red fluorescent protein(RFP)tags,and the results showed that unlike Rapa treatment,a large number of yellow puncta appeared in CAS treated cells.Rapa is a classic autophagy activator,has been reported to activate autophagic flux.Therefore,only the red fluorescently labeled LC3B was detected,and the relatively poorly stable green fluorescent protein was quickly degraded in acidic environment after being fused with the lysosome.To verify whether the autophagosome is fused with the lysosome,we also detected the colocalization of GFP-LC3B and LysoTracker Red and of GFP-LC3B and LAMP1(a marker for lysosomal and endosomal membranes)to assess whether the inhibitory effect of CAS on autophagy was due to blockade of autophagosome-lysosome fusion.Similar to that observed for CQ-treated cells,most GFP-LC3B puncta were not colocalized with LysoTracker Red puncta in the CAS-treated cells.Moreover,the CAS-treated group showed little colocalization between GFP-LC3B and LAMP1 puncta.In particular,the LC3B puncta were dispersed throughout the cytoplasm instead of being clustered near the nucleus in CAS-treated GC cells.Autophagosome delivery to the perinuclear region has been shown to be required for autophagosome maturation.Thus,these findings confirmed that CAS inhibited the fusion of autophagosomes with lysosomes to suppress autophagosome degradation.To explore the root cause of the blockage of autophagic flux,we analyzed big data set and found that CAS caused greater damage to the cytoskeletonal system,especially microtubules and microfilaments.The occurrence of autophagy is a very complex physiological process,which involved in many organelles.However,many reports have shown that the skeletal system plays an important role in the occurrence of autophagy.We found that the microfilaments of CAS treated cells labeled with phalloidin were significantly depolymerized in a time-dependent manner,and the depolymerization was different from the funcational depolymerization caused by autophagy activation,because we found that the down-regulation of ?-actin in CAS treated cells.What's more,unlike the depolymerization of the microtubule caused by VCR,CAS treatment resulted in the remodeling of the microtubule and the remodeling of the microtubule is the adaptive change of cells in response to external stimuli,and it would affect the functions microtubule performs.In addition,we found that the expression of ?-Tubulin protein was significantly reduced in CAS treated cells through western blot analysis.CAS also reduces the expression of many kinesins.These results indicate that CAS caused severe damage to the cytoskeleton system,thereby disrupting the fusion of autophagosomes and lysosomes,resulting in blockage of autophagy flux.Most vinblastine extracts exhibit microtubule targeting properties.To explore whether CAS is targeted to the cytoskeleton system,we performed the computer molecular simulation docking,and the result that the ligand molecule CAS and its receptor molecule ?-actin amino acid residues can be combined through hydrophobic force and aromatic ring stacking force;in addition,the docking analysis also showed that the ligand molecule CAS had a potential binding affinity with the ASP457,GLU502,LYS449,and LYS498 amino acid residues of the receptor molecule ?-tubulin through hydrogen bond forces,and the cellular thermal shift assay indicated that?-tubulin was physically engaged and stabilized against thermal changes under CAS treatment.Taken together,our results demonstrated that CAS physically binds to microtubules and that actin filaments impair the cytoskeleton system,thereby suppressing autophagosome maturation.5.CAS-mediated autophagy inhibition enhances the chemotherapy sensitivity of gastric cancer to cisplatin and significantly prolongs the survival of cancer-bearing miceCisplatin(Cisplatin,Cis)is a widely used metal-based chemotherapeutic drug.Because of its high efficiency and broad spectrum,it has always been an ideal drug for the treatment of many solid tumors.Unfortunately,the clinical use of Cis is limited by its severe,dose-dependent side effects.We explored whether the combination of CAS and Cis still mediates autophagy.We found that the combination treatment still caused strong autophagy through the detection of the expression of LC3B.More interestingly,the expression of SQSTM1 was also up-regulated,suggesting that the combination treatment still causes the autophagy flux to be blocked.Besides,the MTT and plate cloning experiments analysis showed that the combination treatment had a better inhibitory effect on gastric cancer cell lines.To further evaluate the feasibility and effectiveness of the combined strategy,we performed single-dose acute toxicity test and sub-chronic toxicity test,and our results showed the combined treatment of CAS and Cis can reduce the organ and tissue damage caused by Cis monotherapy,especially the damage to the kidneys.The Chou-Talalay method was used to evaluate whether CAS combined with Cis has the effect of synergistic treatment of gastric cancer.The results show that the combination of CAS and Cis has a synergistic inhibitory effect on gastric cancer in a wide range of drug concentrations(Combination Index,CI<1.0);further tests have found that combined chemotherapy could induce more servere DNA damage under lower concentrations of Cis,but in turn leads to the occurrence of apoptosis.Subcutaneous xenograft tumor experiments have shown that the combined strategy had a better inhibitory effect on tumors than any single-agent therapy.To explore whether the combined treatment strategy can prolong the survival of orthotopic tumor-bearing mice,we constructed orthotopic xenograft tumor models derived from two patients.We found that the combined treatment could further extend Survival time of mice with cancer.These results indicate that the blocking effect of CAS-mediated autophagy flux enhances the chemotherapy sensitivity of Cis to gastric cancer and prolongs the survival of mice with tumor in situ.In summary,these findings indicate that CAS activates ERS-dependent autophagy and physically binds to the cytoskeletal system to cause cytoskeletal dysfunction,thereby disrupting the autophagic flux.Our results also showed that CAS-mediated autophagy inhibition plays a negative role in cell survival.Importantly,CAS can play a synergistic anti-tumor effect with Cis,and antagonize the individual toxicity induced by Cis.Therefore,our results indicate CAS combined with Cis is a feasible approach for GC treatment,providing preclinical insights into its potential application in GC therapy. |
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