Alteration Of Invasive/Metastatic Potential Of The Residual Hepatocellular Carcinoma Following Palliative Liver Cancer Resection And Its Intervention

Primary liver cancer,mostly hepatocellular carcinoma(HCC),is the third cancer killer in the world.More and more HCC patients with long-term survival are benefited from various types of therapy aimed at tumor elimination including hepatic resection,one of the most effective therapies for HCC.Unfortunately,due to the high rate of metastatic recurrence,further improve of 5-year survival after small HCC resection was not found in the recent 20 years.Analysis of 2333 cases of HCC patients following hepatic resection in Liver Cancer Institute of Fudan University indicated that palliative resection amounted to 37.2%,and the 1,3,5,8 and 10-year survival in the palliative resection group were lower than that in the radical resection group.Therefore,it is important to seek for new target and new approach for intervention.Some reports revealed that surgical operation could promote tumor growth and metastasis.Unfortunately,little information is available about the alteration of invasive/metastatic potential of the residual HCC following palliative liver cancer resection.This study was to identify the metastasis-related genes in the residual HCC and provide the intervention regimen for those patients.Recently, Chinese herbs have become a focus of interest in cancer treatment.Experimental studies have shown that extracts from herbal medicines or mixtures have anticancer potential,some herbal extracts have been applied in clinical trials aimed at preventing DNA damage or proliferation;and researchers reported that they have achieved some success with the biotherapy and the therapy of Traditional Chinese Medicine(TCM) combination.The Chinese herbal compound extract "Songyou Yin" contains Salvia miltiorrhiza Bge.(danshen) and other four herbs,which was granted by Chinese Food and Drug Administration(FDA,Grant No.G20070160) with the fingerprints by highperformance liquid chromatography(HPLC-FP) determined by Shanghai Institute of Materia Medica(SIMM),Chinese Academy of Sciences(CAS).This study evaluates the effect of "Songyou Yin" on the tumor growth,invasion,and metastasis of HCC in vitro and in vivo;the further study was applied to investigate the effect of the combined therapy with "Songyou Yin(with the same batch number,#060601)" and interferon alfa(IFNα) on the tumor growth,metastasis and gene expression of the residual HCC following palliative liver cancer resection.Ⅰ.The effect of palliative liver cancer resection on the invasive/metastatic potential of the residual hepatocellular carcinomaLittle is known about the alteration of invasive/metastatic potential of the residual HCC following palliative liver cancer resection.Human HCC tumor models produced by MHCC97H(a human HCC cell line with a lung metastatic rate up to 100%) were established in the BALB/C-nu/nu mice by orthotopic inoculation.Then,the mice were randomized into different groups.The mice were performed a palliative resection with preservation of a small part of tumor(2 mm in diameter),or received a sham operation(control group).The methods of cell primary culture,invasion assay, zymography,immunohistochemistry assay(IHA) and Enzyme-linked immunosorbent assay(ELISA),were used to investigate the alteration of invasive/metastatic potential of the residual HCC.As a result,the invasive/metastatic potential of the residual HCC was increasing slowly.The obvious difference in invasiveness could be found between the palliative group and the control group 14 days after operation(p =0.015),the number of the lung metastatic nodule was 14 in the palliative group, whereas it was 9 in the control 35 days after operation(p=0.041),which indicated that palliative resection enhanced the invasive/metastatic potential of the residual tumor. Ⅱ.The effect of palliative liver cancer resection on the invasive/metastatic -related genes expression in the residual hepatocellular carcinomaWe applied a metastatic human HCC model of palliative resection in this study. Briefly,under anesthesia,the left lobe of the liver was exposed,and a part of the liversurface was mechanically injured with scissors,a piece of MHCC97H tumor tissue(size 2 mm×2 mm×2 mm) was fixed within the left liver tissue.Then,the mice were randomized into palliative resection group(with preservation of 2 mm tumor),control groupl(sham operation),and control group2(without any intervention) 14 days later.All mice were sacrificed by cervical dislocation 14 days following palliative liver cancer resection.Oligo Tumor Metastasis Microarray and GEArray Expression Analysis Suite soft ware were adopted for gene analysis.By the method of support vector machine(SVM),we found the markers composed of 12 genes(BAI1,MTA2,MTA1,SMAD2,GNRH1,CDH8,ITGB3,CHD4,GZMA, ITGA7,CXCR4,and TSHR,) could differentiate the invasive/metastatic potential of HCC with positive predictive rate of 88.9%and 93.5%in the groups with high and low invasive/metastatic potential.In addition,we found MTSS1,TGFβ1,SMAD2, IL1βand MMP7 were situated in the central position of the gene function net with high invasive/metastatic potential,which may be served as therapeutic targets.The analysis of gene expression that was up-or down-regulated more than 2 folds revealed that two differentially expressed genes,in which MTA2 was positively correlated,while BAI1 was negatively correlated with the invasive/metastatic potential,which was confirmed by Real-time PCR.Ⅲ.Inhibition effeets and possible mechanism of Chinese herbal compound extraet "Songyou Yin" on hepatocellular carcinoma in vitro and in vivoIn vitro,Chinese herbal compound extract "Songyou Yin"(containg Salvia miltiorrhiza Bge.-danshen and other four herbs) could cause dramatic attenuation of tumor proliferation by induction of apoptosis that was associated with caspase-3 activation,and inhibit invasiveness of MHCC97H via reducing matrix metalloproteinase-2(MMP2) activity and vascular endothelial growth factor(VEGF) expression.In vivo,"Songyou Yin" minimized cancer-related body weight loss of mice with tumors.Inhibition of tumor growth was observed with stepwise increased dosage of "Songyou Yin" and accorded with the expression of proliferating cell nuclear antigen(PCNA),VEGF and MMP2.The life span of nude mice bearing xenografts was(75.0±3.9) days in "Songyou Yin" group,whereas it was(52.0±2.3) days in the control(p＜0.001).The lung metastatic extent was decreased(p＜0.01, compared with control);and "Songyou Yin" inhibited tumor growth was associated with an increased TUNEL-positive apoptosis as well as a decreased microvessel density(MVD) and VEGF abundance,and inhibited tumor invasion via down-regulation of MMP2.Moreover,there was no animal experienced "Songyou Yin"-related weight loss＞10%,or serum transaminases abnormal after the treatment regimen in this study.Ⅳ.The reinforced antitumor effect of "Songyou Yin" and Interferon alfa combination on the growth and metastasis of residual hepatocellular carcinomaHuman HCC model of palliative resection was established for the study of the intervention strategies.The mice were randomized into 4 groups according to the different therapies:control,"Songyou Yin," interferon alfa(IFNα),and the combination of the "Songyou Yin" and IFNα.The residual tumor volume was 1205.2, 724.9,507.6,and 245.3 mm~3;MVD(n) was 44,35,23,and 18;pulmonary metastatic nodule(n) was 7,6,4,and 3;and life span(d) was 57,68,77,and 89,respectively. The residual tumor growth and invasion was inhibited,accorded with a decreased MVD and VEGF abundance.The combined treatment of "Songyou Yin" and IFNαwas more effective than "Songyou Yin" or IFNαalone with tumor inhibition rate of 79.6%(tumor volume,mm~3,245.3 Vs 1205.2;p=0.000).The combined therapy produced more effective reductions of the number of MVD(n) by 59.1%(MVD,n, 18 Vs 44;p=0.000) and lung metastatic nodules by 57.1%(nodule,n,3 Vs 7;p =0.001),prolonged the life span(d) of nude mice by 56.1%,being 89 days Vs 57 days (p=0.000).Moreover,BAI1 mRNA expression was up-regulated by "Songyou Yin" (%,5.7 Vs 5.2;p=0.948) without significance,but significantly up-regulated by IFNα (%,30.0 Vs 5.2;p=0.006),and more significantly up-regulated by the combined therapy(%,39.5 Vs 5.2;p=0.000) when compared with control group.Conclusions1.Palliative liver cancer resection promotes the invasive/metastatic potential of the residual HCC.2.Palliative liver cancer resection causes differential invasive/metastatic-related gene expression in the residual HCC when compared with the control group.3.Chinese herbal compound extract "Songyou Yin" inhibited HCC growth by inducing apoptosis that was associated with caspase-3 activation,and inhibited tumor invasion via reducing MMP2 activity and VEGF expression.4.Combination of "Songyou Yin" and IFNαreinforced the antitumor effect each other,and significantly improved the survival time of mice with tumor following palliative liver cancer resection.The underlying mechanism may be related to the changes of gene expression in mRNA level in the MHCC97H cells.The potential application of this work1.The differential expressed genes identified by Oligio microarray analysis provide a valuable resource for the next basic and clinical studies of invasive/metastatic potential of HCC,and may be served as potential biomarkers and individualized therapeutic targets for some HCC patients.2.Combination of "Songyou Yin" and IFNαreinforced the antitumor effect each other,which laid a foundation for clinical application. The novelty of this work1.Confirmed the invasive/metastatic potential of the residual tumor was promoted by palliative resection,and analyzed its gene expression profile.The identified gene, BAI1 and MTA2 may be served as potential predictive markers and therapeutic targets for some certain type of HCC patients.2.Discovered Chinese herbal compound extract "Songyou Yin" could inhibit HCC growth and invasive/metastatic potential,and the combined therapy with "Songyou Yin" and IFNαsignificantly reinforced the antitumor effect each other on the residual hepatocellular carcinoma following palliative liver cancer resection.