The tumor volume of shASPP1 or shASPP2 modified HCC-LM3 xenografts was 52% or 72% larger than that of shNon-treated xenografts 30 days after implantation (Fig. 5E). To investigate the effects of ASPP1 and ASPP2 on apoptosis, ASPP1 and ASPP2 genes were transfected into HCC cells with different p53 status. Serum-starvation caused a 3-fold increase of apoptotic cells in HCC-LM3 cells that had endogenous wildtype p53. Overexpression of p53 did not further enhance apoptosis
in HCC-LM3 cells. In contrast, overexpression of ASPP1 and ASPP2 caused 100% and 70% increases of apoptotic cells, respectively (Fig. 6A). This indicates that ASPP1 and ASPP2 could enhance apoptosis in HCC cells harboring the wildtype p53 gene. Interestingly, introduction of ASPP2 but not ASPP1 into Hep3B cells with p53 Barasertib manufacturer gene null induced apoptosis to a similar level as p53 did under serum-starvation (Fig. 6B). Introduction of ASPP1 and ASPP2 genes into Huh-7 with p53220Cys induced apoptosis to an extent similar to that of p53 (Fig. 6C). Knock-down of ASPP1 or Trichostatin A mw ASPP2 significantly reduced the apoptotic cells induced by serum starvation in HepG2 or HCC-LM3 cells that had wildtype p53 (Fig. 6D) and attenuated cisplatin-induced apoptosis in HepG2 cells (Fig. 6E). Consistent with
the in vitro experimental results, fewer apoptotic cells were found in HCC-LM3 xenografts with shASPP1 or shASPP2 treatment (Fig. 6F). These data indicate that down-regulation of ASPP1 and ASPP2 in HCC may promote tumor progression through inhibition of cell apoptosis. Dysregulation of apoptosis is closely related to the expansion of tumor cells, metastasis, and resistance to chemotherapy.26–28p53 is a key regulator for apoptosis and frequently mutates in various human cancers.29 The proapoptotic function of p53 is closely linked to its antitumor effects. All of the tumor-derived p53 mutants have lost their ability to induce apoptosis. However, only 30% of HCC contains p53 gene mutations. It remains unclear why wildtype p53 fails to suppress tumor growth in the remaining 70% of HCC. ASPP1 and ASPP2 proteins interact
with p53 and its ifenprodil family members, p63 and p73, to promote apoptosis.1, 3 In this study we describe for the first time that ASPP1 and ASPP2 genes are frequently inactivated by hypermethylation in HCC that is HBV-positive. In HCC tumor tissues, ASPP1 and ASPP2 were frequently found methylated, which contributed to the down-regulation of ASPP1 and ASPP2 in HCCs. Importantly, methylation of ASPP1 and ASPP2 in the surrounding nontumor tissues was closely related to the size and the stage of HCCs. A previous study showed that ASPP1 and ASPP2 were frequently down-regulated in breast cancer expressing wildtype p53.1 In this study we found that HCC tumors with the p53 gene wildtype more frequently had ASPP1 and/or ASPP2 gene methylation.