The Role Of Nutrient-Free Induced Autophagy And Telomerase In Insensitivity Of Hepatocellular Carcinoma To Chemotherapy

The role of nutrient-free induced autophagy and telomerase in insensitivity of hepatocellular carcinoma to chemotherapyHepatocellular carcinoma (HCC) is one of the most common malignancies; the incident rate of it is growing every year in our country. The most common way to cure it is undergoing combined therapy including chemotherapy. But the curative chemotherapy is far from satisfaction, because hepatocellular carcinoma is more resistant to chemotherapeutics. How to increase hepatocellular carcinoma sensitive to chemotherapy is more significant to hepatocellular carcinoma (HCC) therapy and improving patients'survival. Althoμgh there are a lot of drμg-resistant mechanisms about HCC, it is still out of fully understanding. Recently, growing evidence show that autophagy and telomerase were involved in tumor drμg resistance.Autophagy is a regulated lysosomal degradation pathway。Autophagy is the cell's major regulated mechanism for degrading long-lived proteins and the only known pathway for degrading organelles. Autophagy widely exists in the normal physiological process, and maintains the intracellular homeostatisis .autophagy is the rapid adaptive response of cells to exogenous stress. Autophagy is rapidly upregulated when cells need to generate intracellular nutrients and energy (e.g., during starvation or trophic factor withdrawal), or rid themselves of damaging cytoplasmic components (e.g., during oxidative stress, infection, and accumulation of protein aggregates). There are many gene products essential for autophagy and related pathways referred to as the ATG genes .The ATG genes encode proteins needed for the induction of autophagy, and the generation, maturation, and recycling of autophagosomes. Several pathways are involved in regulation of autophagy, for example PI-3K-AKT-mTOR or p38-MAPK.The deregulation of autophagy is related to many human diseases, including infections, cancer, neurodegeneration, aging, and heart disease.Telomerase is a rib nucleoprotein enzymatic complex that adds repeated telomere sequences (TTAGGG) onto the chromosome ends, compensates for the telomeric loss that occurs with cell division and stabilizes. telomerase consists of three components: telomerase reverse transcriptase (hTERT), an RNA template—telomerase RNA component (hTR), an other subunit is telomerase associate protein (TEP1) .the hTR and TEP1 is ubiquitously exist in both normal and malignant tissues and cells, but the hTERT component is undetectable in most normal human tissues and somatic cell but almost expressed in human cancer cell/immortally cell line and malignant tissues. So the expression of hTERT is thoμght to be paralleled with telomerase activity and considered as a rate-limiting determinant of enzymatic activity. Data of our lab revealed more than 85% HCC with much stronger telomerase activity than cirrhosis. Based on the other spurring observation that normal hepatocellular tissue without telomerase activity, many researcher propose that telomerase plays an important role in the initiation and development of HCC. In the preliminary experiment, we find that low concentrate cisplatin not only can't kill HCC cell line but also up-regulate telomerase and induce drμg insensitivity to cisplatin. So our object is to study the role of autophagy and telomerase in insensitivity of hepatocellular carcinoma to chemotherapy.PartI: The role of nutrient-free induced autophagy in insensitivity of hepatocellular carcinoma to chemotherapyHCC cell lines (HepG2,Hep3B,SMMC7721) were cultured in the full nutrient and nutrient free media ,and then were treated with chemotherapeutic agents (5FU and cisplatin) for12h. Cell viability was measured by cck8 assay, cell morphological change was detected under phase contrast microscope and apoptosis cell was stained with Annixin V/PI double staining. The results showed that cells grown in nutrient free media demonstrated more resistant to chemotherapeutic agents than that in full nutrient media.It has been demonstrated that nutrient starvation is the natural inducer of autophagy. Autophagy was also reported to be contributing to chemoresistance in tumor. Thus we want to investigate whether autophagy was invovled in hepatocarcinoma insensitivity to chemotheraputic agents.HCC cell lines (HepG2,Hep3B,SMMC7721) were cultured in the full nutrient and nutrient free media. Electron microscope was performed to detect autophagysome and realtime PCR assays were used to detect the expression of autophagy related gene (LC3,ATG5,ATG 7,DRAM,beclin1). LC3-GFP plasmid was transfected into HCC cell lines, and the green dot was detected and measured. The results demonstrated that the expression of autophagy related gene was significant upregulated and the green dots were also increased during nutrient starvation which suggested that HCC cells showed more activated autophagy. Futher reseach suggested that inhibition of autophagy increased cell death and chemosensitivity during nutrient starvation.HCC cell lines (HepG2,Hep3B,SMMC7721) were cultured in the full nutrient and nutrient free media. cells were pretreated with autophagy inhibitor(3MA,LY294002) for 2h and then were incubated with chemotherapeutic agents (5FU and cisplatin) for another 12h. Cell viability was measured by cck8 assay, cell morphological change was detected under phase contrast microscope and apoptosis cell was stained with Annixin V/PI double staining and DAPI staining. The results showed that cells treated with autophagy inhibitor demonstrated more insensitivity to chemotherapeutic during nutrient starvation.Taken together our data suggested that autophagy was one of the suvival mechnism of hepatocarcinoma during nutrient starvation. Meanwhile, autophagy was also contributed to chemotherapy resistance. But the mechnism of how autophagy contributed to chemotherapy resistance is largely unknown.it has been reported that mitochondria and p53 were connected with autophagy, thus we investigated the possible role of mitochondria and p53 in heaptocarcinoma autophagy. HCC cell lines (HepG2,SMMC7721) were pretreated with p53 inhibitor(PFT-a) for 1h, and then were cultured in the full nutrient and nutrient free media for another 24h. Electron microscope was performed to detect autophagysome and realtime PCR assays were used to detect the expression of mitocondria gene (Nd2,Nme1). LC3-GFP plasmid was transfected into HCC cell lines, and the green dot was detected and measured. The results demonstrated that the expression of mitocondria gene Nd2 was significant downregulated and the autophagysome and green dots were also decreased during nutrient starvation when p53 was inhibited. To further investigate the role of p53 in autophagy activation,p53 widetype HCC cell lines (HepG2,SMMC7721) were cultured in the full nutrient and nutrient free media. Cells were pretreated with p53 inhibitor (PFT-a) for 1h or transfected with si-p53 plasmid, and then were incubated with chemotherapeutic agents (5FU and cisplatin) for another 24h. Cell viability was measured by cck8 assay, cell morphological change was detected under phase contrast microscope and apoptosis cell was stained with Annixin V/PI double staining and DAPI staining. The results showed that cells treated with p53 inhibitor demonstrated more insensitivity to chemotherapeutic during nutrient starvation. Thus,our results suggested that mitocondria and p53 were involved in chemotherpy resistance in hepatocarcinoma. PartII: The role of telomerase in insensitivity of hepatocellular carcinoma to chemotherapyTelomerase reactivation is considered to be a landmarker of cancer. Telomerase was also reported to have antiapotosis function otherwise the role of maintainance of telomere. In this section we found that Low-dose cisplatin induces up-regulation of telomerase activity and hTERT expressionHuman HCC cells SMMC7721 were treated with various concentrations of cisplatin for 24 h. TRAP assay was used to test telomerase activity. Western blotting analysis and semi-quantitative RT–PCR assays were performed to examine whether activation of telomerase by cisplatin was due to up-regulation of the hTERT expression. Telomerase activity was found to reach a plateau at 1μg/ml, and declined thereafter.Treatment of SMMC7721cells with indicated concentrations of cispaltin for 24 h led to up-regulations of hTERT protein and hTERT mRNA. To examine the effect of cisplatin on the transcriptional activity of the hTERT promoter, luciferase assay in which hTERT luciferase reporter plasmid (pGL3B-TRTP) was transfected into SMMC7721cells was performed. Cisplatin increased the transcriptional activity of the hTERT promoter for up to 3.5-fold. These results sμggest that cisplatin can induce the hTERT expression at transcriptional level. hTERT expression was regulated by lots of transcriptional factors. Cispaltin was reported to activate the NF-кB. Thus, we investigated whether NF-кB is involved in the induction of hTERT expression by cisplatin. Our results showed cisplatin can activate NF-кB and inhibition of NF-кB can reduce the hTERT expression induced by cisplatin.Data of our lab revealed more than 85% HCC with much stronger telomerase activity than cirrhosis. Based on the other spurring observation that normal hepatocellular tissue without telomerase activity, many researcher propose that telomerase plays an important role in the initiation and development of HCC. Based on the antiapoptosis role of telomerase and hepatocarcinoma insensetivity to chemotherapy, we hypothsis that telomerase is invovled in chemotherapy insensetivity of hepatocarcinoma. SMMC7721cells were preincubated in 1μg/ml cisplatin for 24 h, and then treated with 5-FU (65μg/ml) and epirubicin (1μg/ml) for another 24 h respectively. The morphology of cells was observed, and pictures were taken under the microscope. Then, the cells were harvested and used to prepare cell lysates. The lysates were subjected to SDS–PAGE and blotted with anti-PARP antibody.the result revealed that control cells, but not preincubated cells, show significantly cell death. Control cells revealed morphologic features typical of apoptosis, including cytoplasmic condensation, nuclear fragmentation, and blebbing. These results were confirmed by immunoblotting, where control cells resulted in increased level of cleaved PARP. Thus these data sμggested Low concentrate cisplatin induce insensitivity of HCC to chemotherapeutic agents. Furthermore, we also demonstrated Inhibition of hTERT can enhance the chemosensitivity of SMMC-7721 cellsSMMC7721cells were infected with Ad-si/hTERT virus for 24 h, and then preincubated in 1μg/ml cisplatin for another 24 h. After preincubation, cells were treated with 5-FU (65μg/ml) and epirubicin (1μg/ml) for 24 h respectively.WST-8 assay and DAPI staining was performed to detected cell death. The results suggested Ad-si/hTERT can significantly inhibit hTERT expression and telomerase activity. Meanwhile, SMMC7721cells infected with Ad-si/hTERT showed dramatically sensitivity to chemotherapeutic agents.To determine the effect of Ad-si/hTERT plus cisplatin on tumor growth in vivo, SMMC-7721 and HepG2 cells were injected into the right back of BALB/c nude mice. After 4 days, the xenograft tumor started to grow and after about 15 days the model was successfully established to initiate treatment. Ad-si/hTERT and empty vector Ad-null (1x 109/30 ul/time, separately) was directly injected into the tumor, and cisplatin (3 g/Kg/100 ul/time) was intraperitoneally injected. All types of treatment were administered at the same time every 5 days for 4 administrations. Tumor sizes were subsequently measured daily. The results indicate that combination Ad-si/hTERT with cisplatin can significantly inhibit tumor growth in vivo.To be conclued, our results sμggested1,Nutrient starvation can activate autophagy in hepatocarcinoma cells, and autophagy contributes to chemoresistance; inhibtion of autophagy increased the senstivity to chemotherpeutic agents of hepatocarcinoma during nutrient starvation.2,P53 was invovled in autophagy activation of hepatocarcinoma during nutrient starvation. Inhibition of p53 increased cell death and chemosensitivity during nutrient starvation.3,NF-кB is involved in low-dose cisplatin inducd up-regulation of telomerase activity and hTERT expression Inhibition of hTERT enhances the chemosensitivity of hepatocarcinoma cells to chemotherpeutic agents...