| Part I Intratumoral intervention of MyD88 inhibits hepatocellular carcinoma development[Objective]To explore the expression of MyD88 in hepatocellular carcinoma(HCC),and text the effects of MyD88 inhibitor in hepatocellular carcinoma treatment.[Methods](1)We used western blotting and RT-PCR to investigate protein and mRNA expression levels of Myd88 in murine H22 cells,liver cells of normal BALB/c mice,hep G2 cells,hep 3B cells,human HCC tissues and human liver tissues.The immunofluorescence was used to test the expression of MyD88 in normal murine liver and H22 tumor.We used co-immunoprecipitation assay was to detect the effect of MyD88 inhibitor on MyD88 homodimerization.H22,hep G2 and hep 3B treated with or without MyD88 inhibitor were analyzed by flow cytometry for cell proliferation,apoptosis,necrosis and cell cycle.Cell cyclin and Erk/MAPK signals were detected by western blot.(2)H22 and hep G2 tumor-bearing BALB/c murine models were established.The mice were randomly divided into the control and MyD88 inhibitor treated groups.The treatment group mice received intratumoral injections of MyD88 inhibitor(50 mg/kg)once per day,until the end of the observation(day 21 in H22 model and day 28 in hep G2 model),while the control groups received the same doses of vehicle in the same way at the same time.The tumor growth and body weight were monitored.At the end of the observation,Ki-67 and Tunel were detected by immunofluorescence;CD31 expression was detected by immunohistochemistry;the proportion of Treg and CD3+CD69+ T cells in spleen and local lymph-nodes was detected by flow cytometry.Tumor infiltrating lymphocytes were separated from tissue samples,and macrophages were labeled and analyzed using flow cytometry.The effect of MyD88 on macrophages was detected by flow cytometry.RT-PCR was used to detect the mRNA level of IL-10,Arg and IL-12.[Results]Myd88 was significantly overexpressed in H22 cells,hep G2 cells,HCC tissues and H22 tumor tissues(P<0.05).MyD88 inhibitor decreased the percentage of MyD88 homodimerization to 45.69± 1.52%and 37.23±2.54%of the untreated cells,respectively(P<0.05).MyD88 inhibitor significantly inhibit the proliferation of H22 cells and hep G2 cells(P<0.05).But MyD88 inhibitor did not affect the apoptosis and necrosis of H22 cells(P>0.05).MyD88 inhibitor could cause G0/G1 phase cell cycle arrest.Decreasing expressions of cyclin Dl,CDK6,cyclin E,CDK2,p-MEK1/2 and p-Erkl/2 and increasing expressions of p18 and p27,were observed in MyD88 inhibitor treated H22 cells.In vivo,MyD88 inhibitor inhibited the development of H22 tumor and hep G2 tumor(P<0.05).Ki-67 and CD31 was downregulated and Tunel was upregulated in MyD88 inhibitor treated tumor tissues.No significant difference was observed in proportion of Treg and CD3+CD69+ T cells in spleen and local lymph-nodes of H22 tumor-bearing mice.But the difference of F4/80+CD206-CD11c+ macrophages was observed in H22 tumor tissues(MyD88 inhibitor treated group,1.14±0.051%vs control group,0.52±0.034%;P<0.05),MyD88 inhibitor had no directly effects in macrophages(MyD88 inhibitor treated group,32.13±5.61%vs control group,31.20±3.41%;P>0.05).However,the percentage of F4/80+CD206-CD11c+ macrophages was increased after cultured with the supernatant of MyD88 inhibitor treated H22 cells(MyD88 inhibitor treated group,54.67±4.93%vs control group,42.63±3.40%;P<0.05).[Conclusion]Intratumoral intervention of MyD88 suppresses hepatocellular carcinoma development.And MyD88 inhibitor induces G0/G1 phase cell cycle arrest through the disruption of Erk/MAPK signaling,and that this MyD88 inhibitor indirectly increases the proportion of M1 macrophages in the tumor microenvironment.Part ? Systemic inhibition of MyD88 promotes hepatocellular carcinoma development[Objective]To investigate the role of MyD88 inhibitor in the promotion of hepatocellular carcinoma development.[Methods]H22 tumor-bearing BALB/c murine and Myd88-BALB/c murine models were established.The mice were randomly divided into the control and MyD88 inhibitor treated groups.And the development of H22 tumor in each group was observed.At the end of the observation,the proportion of Treg and CD3+CD69+ T cells in spleen and local lymph-nodes of the treated mice was detected by flow cytometry.In vitro,bone marrow-derived DCs were incubated with different concentrations(10 ?M and 40 ?M)of MyD88 inhibitor for 2 h before stimulation with LPS(1 ?g/ml).Forty-eight hours later,the suspended and half-suspended cells were harvested and stained using APC-conjugated CD11c antibody,FITC-conjugated CD80 antibody,and PE-conjugated CD86 antibody,and then analyzed using flow cytometry.T cells(1 × 106/ml from the spleens of B6 mice)were stained with CFSE and co-cultured with BMDCs pretreated with the above conditions.And the cells were then harvested and stained using antibodies of PE-conjugated anti-CD4 and APC-conjugated anti-CD8.Cell proliferation was analyzed using flow cytometry,and the numbers of CD4 and CD8 T cell were analyzed.Lymph nodes were obtained from female NOD mice and separated for lymphocytes.The cells form lymph nodes werestimulated with 2 ?g/ml anti-CD3e and 1 ?g/ml anti-CD28e,and co-cultured with different concentrations of MyD88 inhibitor(10 ?M and 40 ?M).Forty-eight hours later,T cell proliferation was determined by Cell Proliferation ELISA,BrdU according to the manufacturer's instructions.[Results]Systemic administration of MyD88 inhibitor promotes the tumor growth(P<0.05).At the same time,the administration of MyD88 inhibitor does not influence body weight of the mice(P>0.05).In agreement with MyD88 inhibitor intraperitoneal injection promotes tumor growth,H22 cell-derived tumor in My88-/-female BALB/c mice also develops faster than the control(P<0.05).Besides,systemic administration of MyD88 inhibitor does not reduce the tumor in MyD88-/-female BALB/c mice compared with its control group(P>0.05).These are significant lower T cell activation(7.54×0.36%vs 5.98×0.513%,P>0.05)and higher proportion of CD4+CD25+Foxp3+ Tregs(12.00×0.64%vs 15.60× 1.06%,P<0.05)in the spleen of TJ-M2010-5 systemic administration group compared with its control group.T cell activation in MyD88 inhibitor treated group,presented by CD69 in local tumor draining lymph nodes has significant lover than control group(13.9±1.99%vs 6.91±0.70%,P<0.05).In vitro,significantly suppression of the bone-marrow DCs maturation is observed in MyD88 inhibitor treated group(P<0.05).MyD88 inhibitor could also reduce the frequency of reactive T cells during the mix lymphocyte culture with bone-marrow DCs.[Conclusion]MyD88 inhibitor inhibits DCs maturation and subsequently suppresses the proliferation of reactive T cells,and then promotes HCC development. |
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