Effects And Mechanism Of Homoharringtonine On Hepatocellular Carcinoma

Background:Liver cancer seriously threatens human health.The morbidity and mortality of liver cancer rank 5th and 2nd among malignant tumors,respectively.Hepatocellular carcinoma?HCC?accounts for 75-85%of primary liver cancer.The incidence of liver cancer is high in China with more male patients than female patients.70%of liver cancer patients were first diagnosed as terminal liver cancer and have lost the opportunity of radical treatment.Moreover,they are prone to recurrence and metastasis after surgery.Currently,the chemotherapy drugs for liver cancer show low sensitivity and efficacy,and more adverse reactions.Therefore,it is urgent to develop new effective and safe drugs for the treatment of liver cancer.Homoharringtonine?HHT?is a kind of plant alkaloid with anti-tumor effects.It has been widely used in the treatment of chronic myeloid leukemia?CML?,acute myeloid leukemia?AML?and myelodysplastic syndrome?MDS?.Its adverse effects are relatively small and the incidence of drug resistance is low.However,the effects on solid tumors and the mechanism are still unclear,and the effects and mechanism on liver cancer have not been reported.Moreover,hippo pathway has been shown to play an important regulatory role in the occurrence and development of a variety of cancers.Objectives:The present study aimed to investigate the effects of HHT on hepatocellular carcinoma and the mechanism related to Hippo signaling pathway,so as to provide a basis for clinical treatment.Methods:1.The effect of HHT on the viability of human hepatocellular carcinoma cells:Human hepatocellular carcinoma cell lines,Huh7 and HepG2,were cultured.HHT at different concentrations?0.01,0.02,0.05,0.1,0.2,0.5,1,2,5?M?were added.After culture for 24h,48h and 72h,CCK8 assay was used to detect cell viability.The wavelength of 450nm was selected,and the light absorption values were measured.The cell viability and the half-inhibitory concentration(IC₅₀)were calculated.2.The influence of HHT on the colony formation of human hepatocellular carcinoma cells:Control group and 0.05?M HHT groups were divided.Cells were inoculated in a 6-well plate at a density of 2000 cells/well and cultured for 1-2 weeks.When visible clones appeared in the six-well plates,the culture was stopped and the clones were stained with crystal violet dye.Image J software was used to count the number of clone formation and the clone formation percentage was calculated.3.The effect of HHT on human hepatocellular carcinoma cell cycle:Control group,0.05?M,0.1?M and 0.2?M HHT groups were divided.After 48h,PI staining were performed and flow cytometry were used for analysis.4.The effect of HHT on apoptosis of HCC cells by flow cytometry:Control group,0.05?M,0.1?M and 0.2?M HHT groups were divided.After 48h of treatment,FITC Annexin V and PI staining were performed and flow cytometry was used for analysis within 1 hour.5.The effect of HHT on apoptosis of HCC cells by TUNEL&DAPI combined staining assay:Control group,0.05?M,0.1?M and 0.2?M HHT groups were divided.Cell apoptosis was observed by TUNEL&DAPI combined staining after 48h treatment of HHT.6.The mechanism of HHT on human hepatocellular carcinoma cells:Control group,0.05?M,0.1?M and 0.2?M HHT groups were divided.Western Blot?WB?method was used to detect the relative expression of apoptotic proteins,Pro-caspase-9,cleaved-caspase-9,Pro-caspase-3,and cleaved-caspase-3 and the proteins related to Hippo signaling pathways(Mst1,p-Mst1,Sav1,Lats1,p-Lats1,YAP,p-YAP?Ser127?after 48h of HHT treatment at different concentrations.7.The effect of HHT on migration ability of human hepatocellular carcinoma cells:Control group,0.05?M,0.1?M and 0.2?M groups were divided.Cells were inoculated in six-well plates,and the migration ability of cells was detected with cell scratch assay after treating Huh7 with different concentrations of HHT.8.The effect of HHT on the invasion ability of human hepatocellular carcinoma cells:Control group,0.05?M,0.1?M and 0.2?M HHT group were divided.Transwell Boyden cells were used to observe the effect of different concentrations of HHT on the invasion ability of Huh7 cells.After being incubated with HHT at different concentrations for 48 hours,0.1%crystal violet was used for staining and the area of the invaded cells were calculated.9.The effects of HHT on BALB/C male nude mice with Huh7 transplanted tumor:Control group,0.05mg/kg and 1mg/kg HHT groups were divided.Huh7 cells?5×10⁶/each?were injected into the armpit of nude mice.From the 11th day,the experimental group were injected with HHT?0.05mg/kg?and HHT?1mg/kg?respectively until day 25,while the control group received only the same amount of PBS.Meanwhile,body weight and tumor volume of nude mice were measured every other day.At the end of the experiment,the transplanted tumor was removed,photographed and measured.Hematoxylin and eosin staining were performed,and the expression of cleaved-caspase-3 and p-YAP?ser127?protein was analyzed by immunohistology.Results:1.The effect of HHT on the viability of human hepatocellular carcinoma cells:HHT has an inhibitory effect on hepatocellular carcinoma cells.HHT significantly inhibited viability of HCC cell lines Huh7 and HepG2 in a time-dependent and dose-dependent manner?P<0.05?.Effective concentration range from 0.02?M?P<0.001?.The IC₅₀ of HHT on Huh7 and HepG2 cells at 24h,48h and 72h were 0.4685?M,0.08124?M,0.01697?M,0.0804?M,0.02909?M and 0.00134?M,respectively.2.The effects of HHT on the colony formation of human hepatocellular carcinoma cells:0.05?M HHT significantly inhibited the proliferation and the colony formation percentage of Huh7 and HepG2 cell lines?P<0.001?.3.The effect of HHT on human hepatocellular carcinoma cell cycle:Cell cycle detection by flow cytometry showed that HHT could block Huh7 in S phase.4.The effects and mechanism of HHT on apoptosis of human hepatocellular carcinoma cells:With the flow cytometry and TUNEL&DAPI staining,the degree of apoptosis in Huh7 cell line gradually increased at different concentrations of HHT.HHT down-regulated the expression of total Pro-caspase-9 and Pro-caspase-3 proteins,and up-regulated the expression of total cleaved caspase-9 and cleaved caspase-3 proteins.Moreover,HHT increased the expression of Mst1,p-Mst1,Sav,Lats1,p-Lats1 and p-YAP?Ser127?total protein,and down-regulated the expression of YAP total protein.5.The effect of HHT on migration ability of human hepatocellular carcinoma cells:0.1?M and 0.2?M HHT significantly inhibited the migration of Huh7 cells in cell scratch assay.6.The effect of HHT on invasion ability of human hepatocellular carcinoma cells:HHT at 0.05?M,0.1?M and 0.2?M significantly inhibited the invasion of Huh7 cells.7.The effects of HHT on BALB/C nude mice with Huh7 transplanted tumor:HHT inhibited the size of the tumor and increased the expression of cleaved-caspase-3 and p-YAP?ser127?proteins.Conclusions:Both in vitro and in vivo experiments showed that HHT had an inhibitory effect on hepatocellular carcinoma.The in vitro experiments showed that HHT may regulate Hippo signaling pathway by upregulation of Mst1,p-Mst1,Sav,Lats1,p-Lats1,p-YAP?Ser127?proteins,down-regulation of YAP protein expression to inhibit proliferation and promote apoptosis of hepatocellular carcinoma.It also has a certain inhibitory effect on the metastasis of hepatocellular carcinoma.The in vivo experiments further support the inhibitory effects of HHT on hepatocellular carcinoma as demonstrated by inhibiting the size of the tumor and increasing the expression of cleaved-caspase-3 and p-YAP?ser127?proteins.Moreover,the detailed mechanism need further investigation.