Study On Suppression Of Programmed Cell Death 4 (PDCD4) To Metastastic Potential Of Human Hepatocellular Carcinoma Cells

Background and Objective:Human hepatocellular carcinoma(HCC) is one of the most common human cancers worldwide and ranks the second leading cause of cancer death in China.The frequency of HCC in China is very high.More than 55%patients of the world are in China.The natural life spans of HCC patients are quite short(about 2-3 months).Since HCC is clinically silent at early stage,mostHCC patients(＞80%) are presented with advanced or unresectable disease.Without treatment,the 5-year survival rate of HCC is less than 5%.To those with resected disease, the recurrence rate can be as high as 50%at 2 years and the 5 year survival rate is only 25-39%.Despite of the advances in treatment, the prognosis of HCC remains very poor due to the frequent presence of recurrence and the high rate of metastasis.The programmed cell death 4(PDCD4) was found to be an inhibitor of neoplastic transformation.It was first found to be highly expressed during apoptosis,but the role of PDCD4 in programmed cell death was not clear.A comparative study on cells with different transformation response to tumor promoters revealed that PDCD4 was expressed more than ten folds higher in promotion-sensitive cells than in promotion-resistant cells.In less progressed mouse keratinocytes, higher level of PDCD4 was expressed.Later investigations demonstrated that loss of PDCD4 expression was associated with tumor progression in carcinomas of the lung,colon,prostate,and breast.The inhibition of PDCD4 on transformation is achieved through down-regulation of the JNK signal transduction pathway which is essential for cell migration. Decrease of JNK activity then leads to inhibition of cell migration.In the course of tumor metastasis of tumor is very complicated.There are many genes involved,including VEGF(vascular endothelial growth factor),MMPs(the matrix metalloproteinases) and MTA1(the metastasis tumor antigen 1).VEGF was initially found to be a prime regulator of angiogenesis and vasculogenesis.Since angiogenesis is essential for the growth of tumor,expression of VEGF is found to be associated with the progression of tumors.Previous studies have shown that'VEGF is able to induce permeabilization of blood vessels and contribute to metastasis of tumor cells.The extracellular matrix(ECM) consists of collagenⅣ,fibronectin, entactin,laminin,etc.Proteolytic degradation of ECM is a critical event during tumor invasion and metastases.The matrix metalloproteinases(MMPs),in particular the typeⅣcollagen specific collagenases,MMP-2(72 kDa collagenase) and MMP-9(92 kDa collagenase), participate in the degradation of ECM components including the basement membrane.Increased expression of MMP-2 and MMP-9 have been found in many kinds of human solid tumors.Previous in vitro and in vivo studies have demonstrated that production of MMP-2,MMP-9 and typeⅣcollagen content are correlated with metastatic potential.MTA1 was originally identified by differential expression in rat mammary adenocarcinoma metastatic cells.The expression of the MTA1 gene was found to be positively correlated with metastatic potential of some human cell lines and tissues,such as the breast,prostate,colon and pancreas.More and more researches on the effects of PDCD4 have been performed,among which little is about the relation of PDCD4 to metastatic potentials of HCC cells.Based on the previous studies,we hypothesized that PDCD4 might also play a role on the inhibition of HCC metastasis.Methods:1.The expression levels of PDCD4 were determined in three HCC cell lines with different metastatic potentials:MHCC-97H(high metastatic potential HCC cell line),MHCC-97L(low metastatic potential HCC cell line) and Hep3B(no metastatic potential HCC cell line).Immunocytochemical method,western blots analysis and real time PCR study were performed to analyze PDCD4 expression.2.Construction of a recombinant plasmid encoding the PDCD4 gene:RT-PCR method was used to construct the PDCD4 expression recombinant plasmid and then transfected into the HCC cell line with lower Pdcd4 expression level.Western blots analysis was used to intestify the effectiveness of transfection.3.Effects of PDCD4 on metastasis potentials of HCC:the palsmid encoding PDCD4 geng was transfected into HCC cells(MHCC-97H) with lowest PDCD4 expression level in study of Part 1.Series studies were performed after transfection:cell proliferation was detected with MTT method;cell cycle analysis was detected with flowcytometry method;cell apoptosis was analysed quantitatively with flowcytometry method and morphorlogically with Hoechst33258 staining;cell migration and invasion tests were performed with transwell chambers equipped with filter membranes with 8-μm pores,being coated with(for invasion test) or without(for migration test) Matrigel.Effect of PDCD4 on expression of metastasis associated genes,such as VEGF,MMP-2,MMP-9 and MTA1 were detected with real-time PCR assay. Results:1,Expression of PDCD4(1) Immunocytochemical analysis:the positive immunocytochemical staining for PDCD4 was brownish and localized in cytoplasm.HSCORE for MHCC-97H cells(Groupl),MHCC-97L cells(Group2) and Hep3B cells(Group3) was 0.85±0.17,1.46±0.36 and 1.97±0.29,respectively.Difference between Groupl and Group2(n=5,P＜0.05) orGroupl and Group3(n=5,P＜0.01) or Group2 and Group3(n=5,P＜0.05) was significant.(2) The quantitative assay by real time PCR:the results were reported in RQ units as compared with the noninvasive Hep3B cells.RQ for MHCC-97H cells and MHCC-97L cells was 0.126±0.023 and 0.385±0.084, respectively.The difference between the two groups was significant(n=3, p＜0.05).(3) Western blots assay:a band of 54 kD for PDCD4 expression was displayed.The relative densities(RD) of PDCD4 for Group1,Group2 and Group3 were 0.053±0.045,0.268±0.067 and 0.587±0.182,respectively (Fig.1E).Difference between Group1 and Group2 or Group1 and Group3 was significant(n=3,P＜0.05).There is no difference between Group2 and Group3(n=3,P＞0.05).Data of the above experiments showed that the highest metastatic potentialMHCC-97H cells expressed lowest level of PDCD4.The expression level of PDCD4 was inversely correlated with the metastasis potentials of HCC cells.2,Plasmid construction and efficiency of PDCD4 transfection A plasmid pcDNA3.1(-)-PDCD4 encoding the PDCD4 gene was constructed. The recombinant was identified by double digestion with restriction enzymes and sequencing analysis.DNA sequencing of the recombinant pcDNA3.1(-)-PDCD4 was also identified by Sangon.The efficiency of PDCD4 gene transfection was identified by western blot analysis. 3,Effects of PDCD4 on MHCC-97H cells proliferation(1) The MHCC-97H cell proliferation rate was assayed by MTT.The detected absorbance at 490 nm of the MHCC-97H-PDCD4 group was 0.543±0.150,which was lower than that of the MHCC-97H-vector group (1.343±0.268) or MHCC-97H group(1.278±0.258).The difference was significant(n=3,P＜0.05).No statistical difference was found between the two control groups(n=3,P＞0.05).(2) Cell cycle analysis with a flow cytometer:an increase of percentage both in G1 stage and in G2 stage was observed in MHCC-97H-PDCD4 cells, accompanied by a corresponding reduction in the percentage of cells in S phase.The proliferative indexes(PI) were calculated.PI was 27.83±0.95%42.47±2.90%and 44.47±2.37%for the MHCC-97H-PDCD4 cells,the MHCC-97H-vector and the MHCC-97H cells,respectively.The difference of G1,G2,or S percentage and PI between the MHCC-97H-PDCD4 cells and the MHCC-97H-vector or the MHCC-97H cells is significant(n=3, P＜0.05).No significant difference was found between the MHCC-97H-vector and the MHCC-97H cells.These data indicate that PDCD4 might promote both G 1 and G2 arrest in MHCC-97H cells and further block the proliferation of HCC cells.4,Effects of PDCD4 on MHCC-97H cell apoptosis(1) Cell apoptosis was analyzed by the flow cytometric assay:the apoptosisrate was 13.03±1.47%,2.99±0.33%and 2.47±0.15%in the MHCC-97H -PDCD4 cells(Groupl),the MHCC-97H-vector cells(Group2) and the MHCC-97H cells(Group3),respectively.The difference was significant between Groupl and Group2 or Group3(n=5;P＜0.01).There was no statistical difference between the two control groups.(2) Hoechst 33258 staining analysis.The nuclear alterations of apoptosis were showed to be condensed,coalesced,and segmented nuclei with a brighter blue fluorescence.The percentage of apoptosis cells was 29.84±3.80%in MHCC-97H-PDCD4 group(Groupl),5.666±0.44%in the MHCC-97H-vector group(Group2) and 4.62±0.43%in the MHCC-97H group(Group3),respectively.The difference was significant. between Groupl and Group2 or Group3(n=5,P＜0.01).There was no statistical difference between the two control groups.5,Effects of PDCD4 on MHCC-97H cell migration and invasion.(1) The migration assay:the average number of migrated cells per field of the MHCC-97H-PDCD4 group(Groupl) was 27.20±7.26,which was much lower than that of the MHCC-97H-vector group(Group2)(161.80±17.06) or the MHCC-97H group(Group3)(194.60±30.83).(2) The invasion assay:the average number of migrated cells per field was 19.0±3.18,64.40±9.61 and 69.80±12.32 for the Group1,Group2 and Group3,respectively.The difference was significant between Group1 and Group2 or Group3(n=5,P＜0.01).There is no difference betweenGroup2 and Group3.6,Real-time PCR study for effects of PDCD4 on expression of metastasis associated genes in MHCC-97H cells:The quantitative assay of real-time PCR was reported in RQ units as compared with the parental MHCC-97H cells.(1) RQ of VEGFmRNA expression in the recombinant group and the empty vector group 0.424±0.040 and 0.900±0.135,respectively.The difference was significant(n=3,P＜0.05)(2) RQ of MMP-2 mRNA expression in the recombinant group and the empty vector group was 0.437±0.020 and 0.717±0.120,respectively.The difference was significant(n=3,P＞0.05)(3) RQ of MMP-9 mRNA expression in the recombinant group and the empty vector group was 0.448±0.098 and 0.882±0.164,respectively.The difference was significant(n=3,P＜0.05).(4) RQ of MTA1 mRNA expression in the recombinant group and the empty vector group was 0.187±0.083 and 0.652±0.105,respectively.The difference was significant(n=3,P＜0.05).Conclusion:1 Expression of PDCD4 is inversely correlated with metastasis potential of hepatocellular carcinoma cells.2 PDCD4 can effectively suppress metastasis potential of HCC cells.Significance:So far to our knowledge,there is little study on relation of PDCD4 expression with or suppression of PDCD4 to metastasis potential of HCC cells.Our study shows that PDCD4 is an effective inhibitor of metastasis potential of HCC cells and maybe an effective target of gene therapy to HCC.