| BackgroundHepatocellular carcinoma(HCC)is the second leading cause of tumor death in China and the fifth most common malignant tumor worldwide.The incidence rate of HCC has been increasing in the past 10 years.In 2020,the number of new cases of HCC in China was approximately 0.41million,particularly among females,with an annual rate of 2.1%.Liver cancer mortality has increased in the past five years,making it the second leading cause of cancer-related death worldwide.Previous studies have reported that age,gender and race can affect the mortality of liver cancer.Hepatitis B or C virus infection and alcohol consumption are the main causes of>80%of liver cancer-related.The prognosis of liver cancer is poor,although the five-year survival rate is lower than that of other cancers,such as prostate,breast,and lung cancers;nevertheless,it seriously affects patient quality of life.The clinical treatment of liver cancer mainly includes surgically removed,liver transplantation,interventional therapy,radiotherapy and systematic treatment.Tanscatheter arterial chemoembolization(TACE)is an effective method for treating advanced liver cancer.Epirubicin(EPI)is one of the most commonly used drugs in TACE.Evidence-based studies have demonstrated that TACE can effectively prolong the survival of patients with advanced liver cancer,and that TACE combined with radioactive particle implantation and ablation can increase its therapeutic effect on HCC.In recent years,iodine-125 seed implantation technology has been widely used for the treatment of malignant tumors.In the treatment of HCC,iodine-125 seed implantation has demonstrated promising results,and many studies have confirmed the safety and effectiveness of this technology.Iodine-125 seed combined with lobaplatin,traditional Chinese medicine preparations,and other drugs can play a sensitizing role and significantly improve patient prognosis,and is commonly combined with TACE in the treatment of liver cancer.The present study demonstrated that the two-year overall survival rate of patients with liver cancer treated with iodine-125 seed combined with TACE was 4.72 times that of those treated with TACE alone.However,the exact mechanism of action of iodine-125 seed in the treatment of liver cancer,and whether chemotherapeutic drugs can enhance the radiosensitivity of cells to iodine-125 seed,requires further study.EPI is a traditional anthracycline drug with fewer side effects than those associated with doxorubicin.It has been widely used in the treatment of non-small cell lung cancer,and breast,liver,and gastric cancers.EPI can be embedded in cellular DNA,interfere with the transcription process,directly inhibit cell proliferation,or indirectly inhibit cell proliferation by interfering with messenger RNA synthesis and inhibiting topoisomerase Ⅱ activity.EPI can also inhibit tumor proliferation through polymeric micelles,hyaluronic acid,and other carriers.Studies have shown that EPI can inhibit the metastasis of tumor cells in breast cancer,thereby improving prognosis.EPI is less cardiotoxic than other anthracycline drugs.Combined treatment with trastuzumab,paclitaxel,and other chemotherapeutic drugs can significantly improve the efficacy of tumor treatment.Clinical studies have shown that the therapeutic effect of EPI combined with iodine-125 seed implantation in the treatment of liver cancer is significantly higher than that of iodine-125 seed alone.Therefore,we speculate that EPI may potentially exert a radiosensitization effect;however,the mechanism by which it acts on liver cancer cells remains to be further explored.In the early stages of this study,the mechanism of action of iodine-125 seed on liver cancer was explored using Isobaric tags for relative and absolute quantitation(iTRAQ),The results revealed a significant difference in Signal Transducers and Activators of Transcription 1(STAT1)expression level between the control and the experimental groups.Therefore,we speculated that iodine-125 seed play an anti-cancer role through Janus Kinase-Signal Transducers and Activators of Transcription 1(JAKSTAT1)pathway.Previous studies have shown that the JAK-STAT1 signaling pathway is closely associated with the occurrence and development of cancer.STAT1 is a member of the STAT family,which is involved in signal transduction inside and outside the cell,and in the regulation of gene transcription in the nucleus.Any abnormality or change in signal regulatory factors may lead to tumor formation.The role of STAT1 in tumorigenesis and development requires further investigation.Mounting evidence has shown that STAT1 is a tumor suppressor.Previous studies have found that STAT1 can participate in antiviral and immune defense,promote apoptosis,and inhibit tumor growth by regulating apoptosis-related genes,such as Bcl-XL,caspases,and Bax.STAT1 also mediates an important antitumor response in head and neck squamous cell carcinoma.Increased STAT1 expression inhibits the progression of ovarian cancer and improves prognosis of these two cancers.At the same time,STAT1 is expressed at low levels in liver,ovarian,and lung cancers,and in other solid tumors,which confirms the utility of STAT1 as a potential biomarker and prognostic indicator of liver cancer and other cancers.Based on previous research,combined with the role and function of the JAKSTAT1 pathway,this study speculated that iodine-125 seed may induce apoptosis of hepatoma cells and inhibit the proliferation of HCC cells by mediating the upregulation of the JAK-STAT1 pathway.Previous studies have shown that the effect of EPI combined with iodine-125 seed in the treatment of liver cancer may be more significant,and EPI may enhance radiosensitization.To verify our hypothesis,this study used a HCC cell line and nude mouse transplanted tumor model.Using lentivirus transfection,flow cytometry,western blotting,and external irradiation of iodine-125 seeds,we explored the anticancer mechanism of combining iodine-125 seed and EPI,and the involvement of the JAKSTAT1 pathway in the anticancer process at the in vivo and in vitro levels.Objective:1.To explore the mechanism of action of iodine-125 seed in inducing apoptosis and inhibiting the proliferation of HCC cells.2.To study the effect of EPI in enhancing the radiosensitivity of HCC cells to iodine-125 seed.3.To investigate the role of the JAK-STAT1 pathway in anticancer activity induced by the combination of iodine-125 seed and EPI.Methods1.To study the effect of EPI on the antitumor effect of iodine-125 seed1.1IC50 determination of EPI in HCC cellsCCK-8 method was used to detect the IC50 of EPI against liver cancer cells.After continuous culture for 72h,the absorbance value of each 96-well plate was detected using CCK-8 reagent.The IC50 of EPI against liver cancer cells was analyzed and calculated using GraphPad Prism 6.The sensitization concentration of EPI was determined to be 10%IC50.1.2To study the effect of EPI on inhibiting the proliferation of HCC cells by iodine-125 seedCCK-8 method was used to detect the effect of EPI combined with iodine-125 seed on the proliferation of HCC cells.According to the treatment,cells were divided into control group,EPI group,iodine-125 particle group and EPI combined iodine-125 seed group.Cells growing in 96-well plates were detected by cell proliferation experiment,and CCK-8 reagent was added to 96-well plates at 0h,24h,48h and 72h,respectively.After continuous culture for 2 hours,absorbance value of each well was detected.Flow cytometry was used to analyze the effect of EPI combined with iodine-125 seed on cell cycle of HCC.According to treatment methods,cells were divided into control group,EPI group,iodine-125 seed group and EPI combined iodine-125 seed group,respectively,and cell cycle arrest at G2/M phase in each group was detected.1.3To study the effect of EPI on inhibiting migration and invasion of HCC cells by iodine-125 seedTranswell assay was used to detect the effects of EPI combined with iodine-125 seed on the migration and invasion of HCC cells.According to the treatment,the cells were divided into control group,EPI group,iodine-125 seed group and EPI combined iodine-125 seed group.After 24 hours of culture,the cells were fixed and stained,and the number of cells penetrating was observed for further analysis.1.4To study the effect of EPI on apoptosis of liver cancer cells induced by iodine-125 seedFlow cytometry was used to detect the effect of EPI combined with iodine-125 on apoptosis of HepG2 and SMMC7721 cells.According to the treatment,cells were divided into control group,EPI group,iodine-125 seed group and iodine-125 seed combined with EPI group,and the percentage of cell apoptosis in each group was statistically analyzed to determine the effect of EPI on the apoptosis of HCC cells induced by EPI combined with EPI.2.Proteomic analysisHCC cells were irradiated with iodine-125 seed at the doses of 0Gy(control group),2Gy and 4Gy,respectively,and then protein extraction was carried out.Proteins in each group were divided into two equal parts on average for concentration determination,and then iTRAQ-labeled high-throughput proteomics analysis was performed to explore and select the differential proteins after iodine-125 seed irradiated HCC cells.The expression levels of differential proteins were determined,and the disease and functional status of HCC cells that might be affected were analyzed,and the signaling pathways of iodine-125 seed inducing apoptosis of HCC cells and inhibiting proliferation,migration and invasion were screened and determined.3.Positive and reverse verification of the role of JAK-STAT1 pathway in irradiated HCC cells with iodine-125 seed3.1To investigate the expression of JAK-STAT1 pathway in HCC cells irradiated with iodine-125 seedSMMC7721 was divided into three groups,and the cells were irradiated with different doses of 0Gy(control group),2Gy and 4Gy,respectively.After the iodine-125 seed irradiation treatment,protein extraction was conducted,and then Western Blot analysis was used to determine the expression of JAK-STAT1 pathway-related proteins in the iodine-125 seed treated SMMC7721.3.2Reverse verification of the role of JAK-STAT1 pathway in irradiated HCC cells with iodine-125 seedFirst,SMMC7721 was transfected with lentivirus,and STAT1 expression was exogenous reduced to obtain STAT1-RNAi cell line.After idling lentivirus,NC-RNAi group was obtained as the control group.According to the treatment methods,cells were divided into control group,iodine-125 seed group and iodine-125 seed combined lentivirus transfection group.Transwell experiment was used to verify the changes of invasion and migration ability of HCC cells before and after transfection.Flow cytometry was used to detect the changes of HCC cell cycle and apoptosis number before and after transfection.Cell proliferation assay was used to detect the changes of cell proliferation before and after transfection.4.To study the effect of JAK-STAT1 pathway on EPI and iodine-125 seed in anti-tumor process4.1 To study the changes of expression levels of JAK-STAT1 pathway related proteins after EPI combined with iodine-125 seedCells transfected with NC-RNAi or STAT1-RNAi were treated with iodine-125 seed or EPI alone or in combination.According to the treatment methods,cells were divided into control group,iodine-125 seed group,EPI group and EPI combined iodine125 seed group for 72 hours.Then protein extraction was performed for each group.Then Western Blot was used to detect the expression of JAK-STAT1 signaling pathway related proteins.4.2Reverse verification of JAK-STAT1 pathway changes in EPI combined with iodine-125 seed on the effect of HCC cellsCells transfected with NC-RNAi or STAT1-RNAi were treated with iodine-125 seed or EPI alone or in combination.According to the treatment methods,cells were divided into control group,iodine-125 seed group,EPI group and EPI combined iodine125 seed group.Cell proliferation assay was used to detect the changes in the proliferation ability of HCC cells after down-regulation of STAT1.Flow cytometry was used to detect the cell cycle arrest and the effect on the number of apoptosis,to further verify the effect of JAK-STAT1 signaling pathway on the anticancer process of iodine125 seed combined with EPI.5.1n vivo experiments demonstrated that down-regulation of STAT1 could attenuate the anticancer effects induced by iodine-125 seed and EPISMMC7721 was used to construct the xenograft tumor model in nude mice.Twenty-five male nude mice were randomly divided into 5 groups with 5 mice in each group.Subcutaneous tumor formation was performed with idle lentivirus cells NCRNAi or STAT1-RNAi transfected cells.When the tumor volume reached 400 mm3,intervention and treatment were performed with iodine-125 seed or EPI respectively.The body weight and tumor size of each nude mouse were measured every 3 days for 30 consecutive days in the control group(NC-RNAi),iodine-125 seed group,iodine125 seed+STAT1-RNAi group,EPI+iodine-125 seed group and EPI+iodine-125 seed+STAT1-RNAi group.Further in vivo verification of the effect of EPI on the antitumor effect of iodine-125 seed,the role of JAK-STAT1 pathway in the irradiation of iodine-125 seed on HCC cells,and the role of JAK-STAT1 pathway in the anti-tumor process of EPI and iodine-125 seed.Results:1.EPI can increase the irradiation sensitivity of iodine-125 seed1.1 Sensitization concentration of EPI to iodine-125 seedThrough cell proliferation experiment,CCK-8 method was used to detect the IC50 of EPI against HepG2 and SMMC7721 cells,and GraphPad Prism 6 was used to analyze and calculate the IC50 of EPI.The results showed that,The IC50 of HepG2 and SMMC7721 were 0.20μg/mL and 0.23 μg/mL,respectively.Therefore,the sensitization concentration of EPI to iodine-125 seed in HepG2 and SMMC7721 was determined to be 0.020μg/mL and 0.023μg/mL,respectively.1.2EPI can enhance the inhibitory effect of iodine-125 seed on the proliferation of HCC cellsThe results of cell proliferation experiment showed that compared with the treatment group alone,the number of cell survival in the EPI combined with iodine125 seed group was significantly reduced,indicating that EPI combined with iodine125 seed can significantly inhibit the proliferation ability of HCC cells.Flow cytometry results showed that the cell cycle G2/M arrest of EPI combined with iodide-125 seed was more significant than that of the iodide-125 seed alone or EPI treatment group,indicating that EPI combined with iodide-125 seed could significantly inhibit the proliferation of HCC cells,which was consistent with the results of cell proliferation experiment.1.3EPI can enhance the inhibitory effect of iodine-125 seed on migration and invasion of HCC cellsThe results of transwell invasion and migration experiment showed that the number of cells invaded and migrated by EPI combined with iodine-125 seed was significantly less than that by EPI alone and EPI alone,indicating that EPI combined with iodine-125 seed could significantly inhibit the invasion and migration of HCC cells.1.4EPI can enhance the effect of iodine-125 seed on HCC cell apoptosisFlow cytometry results show that the number of cell apoptosis of iodine-125 seed alone group was obviously greater than the control group,at the same time,EPI combined iodine-125 seed group is separate iodine-125 seed number of apoptosis cells also increased significantly,the difference had statistical significance(P<0.001),the said EPI can enhance the effect of iodine-125 seed induced HCC cell apoptosis.2.Proteomic analysis resultsHeat map analysis showed significant differences in protein expression levels between the iodine-125 seed treated group and the control group.Volcanic map data analysis and statistical results showed that there were 207 differentially expressed proteins in SMMC7721 cells treated with iodine-125 seed,including 119 up-regulated proteins and 88 down-regulated proteins.iTRAQ results showed that there was a significant difference in the expression level of STAT1 between the irradiated and nonirradiated iodine-125 seed groups.The expression level of STAT1 in HCC cells irradiated iodine-125 seed was significantly higher than that in the control group(P<0.05).At the same time,through the above analysis,this study found that iodine-125 seed can play a key role in the disease and functional state of HCC cells,which can not only affect the death and survival of HCC cells,but also affect the synthesis of proteins in HCC cells.Positive and reverse verification of the role of JAK-STAT1 pathway in irradiated HCC cells with iodine-125 seed.2.1Expression of JAK-STAT1 pathway-related proteins in HCC cells irradiated with iodine-125 seedWestern Blot results showed that p-JAK and p-STAT1 protein expression levels were significantly increased in SMMC7721 HCC cells treated with iodine-125 seed.Through data analysis,the results showed that the expression levels of p-JAK and pSTAT1 in the 4Gy group were significantly higher than those in the 2Gy group and the control group,P<0.05,the difference was statistically significant,indicating that the expression levels of p-JAK and p-STAT1 also showed a trend of gradual increase with the increase of iodine-125 seed irradiation dose.There is a dose-dependent increase.2.2Reverse verification of the role of JAK-STAT1 pathway in irradiated HCC cells with iodine-125 seedSMMC7721 were transfected with lentivirus,and STAT1 expression was exogenous reduced.Transwell assay results showed that the invasion and migration of SMMC7721 cells were significantly inhibited after transfection with STAT1-RNAi and irradiation with iodine-125 seed compared with the control group and iodine-125 seed alone group,suggesting that the effect of iodine-125 seed on the invasion and migration of SMMC7721 cells was weakened by down-regulation of STAT1 expression.The results of flow cytometry showed that STAT1 expression in SMMC7721 cells was decreased,and the cell cycle was blocked in G2/M phase after iodine-125 seed irradiation.Meanwhile,the number of apoptosis of SMMC7721 cells decreased significantly.In addition,the results of cell proliferation assay showed that reduced STAT1 expression in SMMC7721 HCC cells,and the proliferation ability of SMMC721 HCC cells after iodine-125 seed irradiation was enhanced compared with that of iodine-125 seed treatment group.In conclusion,iodine-125 seed can inhibit the proliferation of HCC cells and promote apoptosis of HCC cells through JAK-STAT1 pathway,and down-regulation of STAT1 expression can weaken the anticancer effect of iodine-125 seed.3.To study the role of JAK-STAT1 pathway in the anti-tumor process of EPI and iodine-125 seed3.1Changes in expression levels of JAK-STAT1 pathway related proteins after EPI combined with iodine-125 seedCells transfected with NC-RNAi or STAT1-RNAi were treated with iodine-125 seed or EPI alone or in combination.Western Blot results showed that the total amount of JAK and STAT1 proteins did not change significantly,but compared with other single treatment groups,p-JAK and p-STAT1 expression levels in EPI combined with iodine125 seed treatment group were significantly increased.Data analysis results also showed that,the expression of iodine-125 seed in combined group was significantly higher than that in iodine-125 seed group(P<0.05).3.2Reverse verification of the effect of JAK-STAT1 pathway in EPI combined with iodine-125 seed on HCC cellsThe results of cell proliferation assay showed that the proliferation ability of HCC cells after down-regulated STAT1 expression in SMMC7721 was higher than that in the iodine-125 seed treatment group alone,suggesting that down-regulated STAT1 expression can attenuate the anticancer effect of iodine-125 seed and EPI(P<0.05).In addition,flow cytometry results showed that down-regulation of STAT1 expression in SMMC7721 reduced the effect of iodine-125 seed combined with EPI on G2/M cell cycle arrest and apoptosis.4.In vivo experiments demonstrated that down-regulation of STAT1 attenuated the anticancer effects induced by iodine-125 seed and EPIThe nude mouse model of transplanted tumor was constructed with SMMC7721 and observed and measured for 30 days.The results showed that the combination of iodine-125 seed and EPI could effectively inhibit the growth of the transplanted tumor,and the volume and weight of the combined group were significantly reduced compared with the control group.Meanwhile,down-regulation of STAT1 weakened the inhibitory effect of iodine-125 seed combined with EPI on the growth of transplanted tumor.Conclusions:1.Iodine-125 seed inhibited proliferation and induced apoptosis of HCC cells by upregulating the JAK-STAT1 pathway.2.EPI enhanced radiosensitization of HCC cells to iodine-125 seed treatment.3.EPI can promote the upregulation of the JAK-STAT1 pathway induced by iodine-125 seed,thus enhancing the effect of iodine-125 seed on apoptosis and inhibiting the proliferation of HCC cells. |
