| Part Ⅰ The effects of platelet and its releasate on HCC proliferation and metastasisAims:Platelets in the primary tumor microenvironment play crucial roles in the regulation of tumor proliferation,metastasis and angiogenesis.Clinical and experimental evidences support that platelets influence progression of various tumors,including prostate cancer,breast cancer and melanoma.However,it is not clear whether platelet mediated the growth and metastasis of hepatocellular carcinoma(HCC)cells.Here,we studied the effect of platelet and its releasate on proliferation and migration of HCC cells.Method:Human blood was collected from health volunteers,washed platelets were prepared and resuspended by fresh medium.Then platelets were stimulated with its specific agonist collagen-related peptide(CRP,0.8μ/ml).After fully activated,platelet suspensions were centrifuged to obtain platelet releasates from the supernatant,and the sediments,which were consisted of activated platelets pellets,was resuspended in fresh DMEM.SMMC.7721 and HepG2 cells were incubated with resting platelets,CRP-activated platelets,platelet releasates or resuspended activated platelets for 12 or 24 hours.After co-cultured,proliferation of HCC cells was determined with the EdU assay.Cell cycle and apoptosis analysis were also conducted.Cell scratch experiment was performed to detect migration of HCC cells in vitro.Additionally,for in vivo tumorigenicity assay,SMMC.7721 cells were treated with platelet releasates or resuspended platelets for 24 hours and subcutaneously tumors were established.Expression of Ki67,Bax and Bcl-2 in tumor tissues was determined by immunohistochemistry assay.Results:After incubation with either platelets or platelet granule contents,SMMC.7721 and HepG2 cells exhibited significant increases in proliferation and decreases in apoptosis.However,no effect was observed when incubating cancer cells with resuspended activated platelet pellets which exhausted of releasates.Platelet releasates also increased the population of HCC cells in the S and G2/M phases of the cell cycle and reduced the cell population in the G0/G1 phase.In in-vivo experiment,it was platelet releasates not resuspended activated platelet pellets promoted SMMC.7721 tumor growth and inhibited tumor cell apoptosis.In contrast,not only platelet releasates but also resuspended activated platelet pellets increased migration of SMMC.7721 cells in vitro.Conclusions:In this study,we found that washed human platelets actively signal to HCC cells to promote their proliferation both in vitro and in vivo.This effect is most dependent on the secretions of activated platelets because resuspended activated platelets in fresh medium cannot alter tumor growth,cell cycle or apoptosis.However,it dose differ from the role of platelet in HCC migration,which depended not only on platelet releasates,but also on platelet adhesion receptors.Part Ⅱ The role of platelet derived TGF-β and KLF6 in platelets’ promoting HCC growthAims:Previous studies showed platelet releasates significantly increased proliferation of HCC cells,but the mechanisms underlying are poorly understood.Platelet contains rich TGF-β in its a-granules,and is the main source of bioavailable TGF-(3.Researches suggested that TGF-(3 cooperates with Smad3-Spl(specificity protein 1)-KLF6 to regulate tumor cell growth.KLF6,a tumor suppressing gene,was one of the factors to regulate tumor proliferation,cell cycle and apoptosis.Therefore,we explored the role of platelet derived TGF-β and KLF6 in platelet’s promoting HCC growth by using TGF-β receptor inhibitor SB431542 and KLF6 siRNA.Method:After co-incubated with platelet and its releasates for 12 or 24 hours,expression of KLF6 in HCC cells was examined by western blot.SMMC.7721 and HepG2 cells were transfected with KLF6 shRNA to knockdown KLF6 expression.Cell proliferation assay and cell cycle analysis were conducted in Scrambled shRNA-transfected cells and KLF6 shRNA-transfected cells after treatment with platelet and its releasates.Also,subcutaneously tumors were established by Scrambled shRNA-transfected cells and KLF6 shRNA-transfected cells with or without treatment of platelet releasate or resuspended activated platelet pellets.Tumor growth was monitored and expression of Ki67,Bax,and Bcl-2 was determined by immunohistochemistry assay.In addition,TGF-β receptor inhibitor SB431542 was preincubated with HCC cells to inspect the role of TGF-β in platelet mediated HCC proliferation and KLF6 expression.Results:KLF6,a zinc finger transcription factor,was expressed in both SMMC.7721 and HepG2 cells,but deficient in platelets.Hepatic tumor cells showed a significant reduction in KLF6 expression after treated with platelet and its releasates.Compared with scrambled shRNA-transfected HCC cells,KLF6 shRNA-transfected cells exhibited an improved growth rate.Moreover,after 12 or 24 hours of treatment with platelets and their releasates,a significant increase in the proliferation and cell populations in the S and G2/M phases of control shRNA-transfected HCC cells was observed.However,after treated with platelets and their releasates,no additional pro-proliferative effect was observed in KLF6 silenced HCC cells.In vivo tumorigenicity assay also exhibited that platelet releasates markedly promoted scrambled shRNA-transfected SMMC.7721 cell growth and inhibited tumor cell apoptosis.But there were no effects of platelets on KLF6 shRNA-transfected SMMC.7721 cells.In addition,blocking TGF-β signaling with the TGF-β receptor inhibitor SB431542 counteracted the effect of platelets on KLF6 expression and proliferation of HCC cells.Conclusions:Based on these findings,we conclude that platelet releasates,especially TGF-β in a-granules,promote the proliferation of SMMC.7721 and HepG2 cells by decreasing expression of KLF6.Part Ⅲ The effects and underlying mechanism in HCC cells induced platelet aggregationAims:Clinical and experimental evidences suggest that HCC patients always come with hyper platelet activity.Our previous study also indicated that hepatic tumor cells may induce platelet activation,but the mechanism is still unknown.In this part,we aim to investigate the role and molecular mechanism of HCC cells induced platelet aggregation.Method:The effect of HCC cells on inducing platelet aggregation was examined with an aggregometer.In order to reveal the mechanism,inhibitors or antibodies were pretreated with platelet in platelet aggregation assay.Phosphorylation levels of proteins of platelet GP Ⅵ signaling pathway in the presence or absence of HCC cells were also inspected.Results:Both SMMC.7721 and HepG2 cells could induced platelet aggregation.This promoting aggregation effect couldn’t be inhibited by ADP scavenger Apyrase,but could be abolished by GP Ⅵ antibody JAQ-1 and αⅡbβ3 inhibitor Tirofiban.Furthermore,PP2 and Ly294002,blockers for Src-family kinases and PI3 kinase respectively,could partially surpress platelet aggregation induced by HCC cells.Additionally,PLCγ2 and integrin β3 were phosphorylated in HCC cells induced platelet aggregation.Conclusions:Hepatocellular carcinoma cells induced platelet aggregation through GP VI-SFKs-PI3K-PLCγ2 pathway in platelet and integrin αⅡbβ3. |
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