Expressions of B7-H4 and B7-H3 Proteins and Effect of Sorafenib on their Expressions in Different Human Hepatoma Cell Lines

Purpose: To study whether B7-H3 and B7-H4 proteins can be candidate drug targets for preventing and treating hepatoma. Methods: Western blot assay was used to study the expressions of B7-H3 and B7-H4 proteins and the effect of sorafenib on their expressions in different human hepatoma cell lines (HepG2, Hep3B, BEL-7402, BEL-7404, BEL-7405, QGY-7701, QGY-7703, SMMC-7721, MHCC97H, MHCC97L, HCCLM3 and HCCLM6). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to study the cytotoxicity of sorafenib against different human hepatoma cell lines. Results: The expressions of B7-H3 (0.26 - 0.84 μM) and B7 -H4 (0.18 - 0.78 μM) proteins in different human hepatoma cell lines were significantly (p < 0.01) up-regulated, compared with that of the normal human liver cell line (HL- 7702) (0.09 and 0.08 μM). Sorafenib was cytotoxic on Hep3B, BEL -7404, MHCC97H, HCCLM3 and HCCLM6 cells with half-maximal inhibitory concentration (IC 50 ) of 14.56, 9.14, 9.46, 17.21 and 9.29 μM, respectively. After treatment with sorafen ib at concentrations of 5, 10 and 20 μM, the expressions of B7 -H3 protein in MHCC97H, HCCLM3 and HCCLM6 cells and the expressions of B7-H4 protein in Hep3B, BEL-7404 and MHCC97H cells were significantly (p < 0.01) down-regulated, compared with that of the control. Conclusion: Over-expression of B7-H3 and B7-H4 proteins is common in different human hepatoma cell lines and thus, B7-H3 and B7-H4 proteins may be regarded as candidate drug targets for preventing and treating hepatoma.


INTRODUCTION
Primary hepatoma is the 15th most common cancer and the 2nd leading cause of cancer mortality worldwide [1]. Primary hepatoma remains the 8th most common malignancy in women and the 5th in men according to the estimates of incidence of cancer in 2000 [2].
There were 749 thousand new cases and 695 thousand deaths from hepatoma in 2008 [3]. Therefore, finding novel therapeutic methods are urgent for preventing and treating hepatoma.

MTT assay
MTT assay was used to evaluate the antiproliferative activities of sorafenib against Hep3B, BEL-7404, MHCC97H, HCCLM3 and HCCLM6 cells. Different human hepatoma cells were separately seeded on 96 well plates for 24 h, and then cells were treated with sorafenib at the concentrations of 0 (for control), 1.25, 2.5, 5, 10, 15, 20 and 40 μM. After 48 h, the MTT was added into each well and the cells were cultured for another 3 h. Then the DMSO was added into each well and the optical density (OD) of the DMSO solution was measured at 570 nm using a microplate reader (Perkin-Elmer, USA) [13]. The inhibition rate of sorafenib against different human hepatoma cells were calculated as in Eq 1 [14].

Statistical analysis
All data are presented as mean ± standard deviation. A one-way ANOVA was used to analyze the differences among different groups using Dunnett test on SPSS 19.0. When p-value was less than 0.05, the differences were recognized as statistically significant.

Cytotoxicity of sorafenib against different human hepatoma cell lines
The cell lines (Hep3B, BEL-7404, MHCC97H, HCCLM3 and HCCLM6), which had the higher expressions of B7-H3 and B7-H4 proteins than other cell lines, were used to study the effect of sorafenib on the expressions of B7-H3 and B7-H4 proteins. However, before beginning the study, MTT assay was first used to determine the cytotoxicity of sorafenib against these cell lines to ensure the effectiveness of the drug. As depicted in Figure 2, sorafenib was cytotoxic to Hep3B, BEL-7404, MHCC97H, HCCLM3 and HCCLM6 with half maximal inhibitory concentration (IC 50 ) values of 14.56, 9.14, 9.46, 17.21 and 9.29 μM, respectively.

Inhibitory effects of sorafenib on the expressions of B7-H3 and B7-H4 proteins
The effects of sorafenib on the expressions of B7-H3 protein in MHCC97H, HCCLM3 and HCCLM6 cells and the expressions of B7-H4 protein in Hep3B, BEL-7404 and MHCC97H cells were studied using western blot. As shown in Figure 3, after treatment with sorafenib at the concentrations of 5, 10 and 20 μM for 48 h, the expressions of B7-H3 protein in MHCC97H, HCCLM3 and HCCLM6 cells were significantly (p < 0.01) down-regulated, compared with that of the control. As shown in Figure 4, after treatment with sorafenib at the concentrations of 5, 10 and 20 μM for 48 h, the expressions of B7-H4 protein in Hep3B, BEL-7404 and MHCC97H cells were significantly (p < 0.01) down-regulated, compared with that of the control.
Defect in the immune response may lead to the growth of tumor. T cell plays an important role in immune response. Co-stimulatory ligands such as CD80 and CD86 play an important role in the startup and maintenance of immune response, and their non-expressions in tumor cells has been regarded as a reason for immune evasion [17,18].
The expressions of inhibitory cofactors such as CD152 and PD-1 in immune cells, and the expression of their ligands in tumor cells are related to tumor pathogenesis [19]. Nowadays, inhibiting the interaction of inhibitory cofactors with their ligands has been successfully used to treat tumor [20]. Blocking the expressions of inhibitory cofactors in immune cells and the expressions of their ligands in tumor cells may be important in suppressing the immune evasion of tumor cells [21].
B7-H4 and B7-H3 are two major co-stimulators or ligands of inhibitory cofactors in B7 protein family, which is related to the activation of T cells and immunologic functions [22]. B7-H4 is expressed at the transcriptional level in the thymus, lung and spleen [23] while B7-H3 is widely expressed at the transcriptional level in lymphoid organs, liver, testis, uterus, lung, skeletal muscle, etc [24]. Therefore, B7-H4 and B7-H3 seem to be limited at the translational level [25]. It was reported that the B7-H4 and B7-H3 proteins were expressed in non-small-cell lung cancer [26].
Some studies indicate that B7-H4 is overexpressed at the transcriptional level in human hepatoma cell line BEL-7404 [27]. However, the expressions of B7-H3 and B7-H4 proteins in different human hepatoma and normal human liver cell lines and the effect of sorafenib on their expressions were still unknown. Because sorafenib can prevent both tumor cell proliferation and angiogenesis, and has been successfully used to treat hepatoma [28,29], it was considered as positive drug in the present study.
The results indicate that the expressions of B7-H3 and B7-H4 proteins in all the human hepatoma cell lines were significantly upregulated, compared with that of the normal human liver cell line (HL-7702). Sorafenib was cytotoxic to Hep3B, BEL-7404, MHCC97H, HCCLM3 and HCCLM6 with IC 50 values of 14.56, 9.14, 9.46, 17.21 and 9.29 μM, respectively.
After treatment with sorafenib, the expression of B7-H3 protein in MHCC97H, HCCLM3 and HCCLM6 cells and the expression of B7-H4 proteins in Hep3B, BEL-7404 and MHCC97H cells were significantly down-regulated, compared with that of the control. Although overexpression of B7-H3 and B7-H4 proteins was observed in the human hepatoma cell lines, expressions of B7-H3 and B7-H4 proteins in liver cancer tissues, the relationship between them, and the classification of liver cancer requires further study.

CONCLUSION
Over-expression of B7-H3 and B7-H4 proteins in human hepatoma cell lines are common. Sorafenib is cytotoxic to human hepatoma cell lines and inhibit t over-expression of B7-H3 and B7-H4 proteins in human hepatoma cell lines. These findings indicate that B7-H3 and B7-H4 proteins may be regarded as candidate drug targets for preventing and treating hepatoma.