Rocky L.K. Ho

Systemic Cytokine Response After Laparoscopic-Assisted Resection of Rectosigmoid Carcinoma: A Prospective Randomized Trial

OBJECTIVE To compare the systemic cytokine response in patients after laparoscopic-assisted resection with those after open resection of rectosigmoid carcinoma. SUMMARY BACKGROUND DATA Laparoscopic resection of colorectal carcinoma is technically feasible, but objective evidence of its benefit is scarce. Systemic cytokines are accepted as markers of postoperative tissue trauma and mediators of the host immune response. METHODS Thirty-four patients with rectosigmoid carcinoma, without evidence of metastatic disease and suitable for laparoscopic resection, were randomized to undergo either laparoscopic (n = 17) or conventional open (n = 17) resection of the tumor. Clinical parameters were recorded. Sera were collected before surgery and at appropriate time points afterward and assayed for interleukin-1beta, tumor necrosis factor-alpha, interleukin-6, and C-reactive protein. The primary end points were the cytokine and C-reactive protein levels. Data were analyzed by intention to treat. RESULTS The demographic data of the two groups were comparable. The clinical outcome of both groups was satisfactory, with no surgical deaths and a reasonable complication rate. Both interleukin-1beta and interleukin-6 levels peaked 2 hours after surgery, with the responses in the laparoscopic group significantly less than those in the open group. C-reactive protein levels peaked at 48 hours, and the difference was also statistically significant. Levels of tumor necrosis factor-alpha were not elevated after surgery, and there was no difference between the groups. CONCLUSIONS Tissue trauma, as reflected by systemic cytokine response, was less after laparoscopic resection than after open resection of rectosigmoid carcinoma. The difference in the systemic cytokine response may have implications on the long-term survival.

Sorafenib Inhibits Hypoxia-Inducible Factor-1α Synthesis: Implications for Antiangiogenic Activity in Hepatocellular Carcinoma

Purpose: The overexpression of hypoxia-inducible factor 1α (HIF-1α) is a common finding in hepatocellular carcinoma (HCC), and it leads to angiogenesis and poor prognosis. Sorafenib, a multikinase inhibitor, has shown significant improvement in survival in patients with advanced HCC in clinical trials. However, the mechanisms that account for the antiangiogenic efficiency of sorafenib have not been fully elucidated. The present study aims to explore the effect of sorafenib on HIF-1α expression and activation in HCC cells and xenografts. Experimental Design: HCC cells and xenografts were treated with sorafenib or vehicles. Western blotting and quantitative PCR array were used to determine protein and mRNA expression, respectively. HIF-1α activity, de novo protein synthesis, and VEGF secretions were determined using assay kits. Results: Sorafenib dose dependently decreased the hypoxia-induced accumulation and activation of HIF-1α protein. Further analysis revealed that such reduction of HIF-1α was associated with the inhibition of HIF-1α protein synthesis rather than the promotion of HIF-1α protein degradation or the reduction of HIF-1α mRNA. Moreover, the phosphorylation levels of mTOR, extracellular signal-regulated kinase (ERK), p70S6K, RP-S6, 4E-BP1, and eIF4E were significantly suppressed by sorafenib. In vivo studies further confirmed the inhibitory effect of sorafenib on the expression of HIF-1α and VEGF proteins, leading to a decrease in tumor vascularization and growth of the xenografts. Conclusions: Sorafenib-mediated inhibition of HIF-1α synthesis is associated with previously undefined pathways in which mTOR/p70S6K/4E-BP1 and ERK phosphorylation are downregulated. Our preclinical data expand our understanding of sorafenibs antiangiogenic mechanism of action by inhibiting HIF-1α and VEGF protein expression. Clin Cancer Res; 18(20); 5662–71. ©2012 AACR.

Decreased expression of Bid in human hepatocellular carcinoma is related to hepatitis B virus X protein

As a mitochondrial membrane death ligand, Bid oligomerises Bak to release cytochrome C and its deficiency renders hepatocytes resistant to apoptosis induced by Fas. The Bid level in hepatocellular carcinoma (HCC) is unknown. In this report, we examined the expression of Bid protein and mRNA in HCC cancerous tissues and their corresponding non-cancerous ones. The effect of the hepatitis B x protein (HBx) on the expression of Bid was also evaluated by transfecting hepatoma cells with the HBx gene. The results showed that the expression of Bid was significantly lower in cancerous tissues than that in their corresponding non-cancerous tissues. Immunohistochemical study revealed that Bid molecule was mainly localised in hepato-cytoplasm. Some nuclei were also positive for Bid antigen though to a lesser degree. In vitro experiments demonstrated that the expression of Bid in cells transfected with HBx was significantly lower than that in the cells without HBx transfection. This finding suggests that HBx may play a causative role in the reduction of Bid expression in HCC. This in vitro result is, to some degree, supported by clinical data that all the HCC examined are positive for hepatitis B virus (HBV). We conclude from this data that the expression of Bid in HCC is significantly decreased and the reduction of Bid may result from a mechanism associated with HBx, a major hepatocarcinogenic product from HBV. The imbalance of increased anti-apoptosis and decreased pro-apoptosis seen in HCC is a critical mechanism leading to the uncontrolled growth of tumour cells. Therefore, this study suggests that a deficiency in the expression of Bid may contribute to the development of such an imbalance in HCC.

Mutation of p53 in Recurrent Hepatocellular Carcinoma and Its Association with the Expression of ZBP-89

p53 has recently been identified as a downstream target of ZBP-89, a zinc finger transcription factor. ZBP-89 promotes growth arrest through stabilization of the p53 protein. The aim of this study is to determine the status of the p53 gene in recurrent human hepatocellular carcinoma (HCC) and test the link between the expression of ZBP-89 and the p53 gene. The results showed that mutations in the p53 gene were frequently detected in recurrent HCC. The interval between surgical resection and the recurrence of HCC was significantly longer in patients with the wild-type p53 gene than those with mutations, strongly suggesting a pathological role for the mutant p53 gene in HCC recurrence. Among those positive for the p53 protein, nearly 85% (18 of 21) showed nuclear localization of the p53 protein while only about 14% (3 of 21) were positive for the p53 protein in the cytoplasm. ZBP-89 co-localized with p53 in the nucleus in about 67% (12 of 18) of all cases positive for the nuclear p53 protein, suggesting that ZBP-89 may play a role in the nuclear accumulation of the p53 protein in a subset of recurrent HCC. With accumulation of p53 protein in the nucleus, tumor cells undergo apoptosis and thus are more susceptible to radiotherapy and chemotherapy. Therefore, co-localization of p53 protein with ZBP-89 may define a subgroup of recurrent HCC that is more sensitive to treatment.

Adenovirus-mediated tBid overexpression results in therapeutic effects on p53-resistant hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with a very high mortality. Because the success of the conventional therapies is limited, gene therapy may represent an alternative for HCC management. Our earlier study has shown that Bid plays a role in the development of HCC. The aim of our study is to evaluate the possibility of using truncated Bid (tBid) as a novel therapy for HCC treatment. Two HCC cell lines, Hep3B and PLC/PRF/5, were used in the experiment. Hep3B was a p53‐resistant while PLC/PRF/5 a p53‐sensitive. A recombinant adenovirus‐Ad/AFPtBid, which contained a tBid gene driven by an α‐fetoprotein (AFP) promoter, was constructed. Both Hep3B and PLC/PRF/5 cells infected with Ad/AFPtBid showed a significant decrease in cell viability. The decrease in cell viability by Ad/AFPtBid resulted from apoptosis of HCC cells, evident by enhanced activity of caspases and increased release of cytochrome c. In vivo experiment was performed by the intratumor injection of Ad/AFPtBid in nude mice inoculated with Hep3B. Ad/AFPtBid injection significantly inhibited tumor growth, and tumor tissues showed a marked increase in TUNEL‐positive cells. Our experiment also demonstrated that Ad/AFPtBid only targeted AFP‐producing cells but not those non‐AFP producing cells. In conclusion, these results indicate that the introduction of Ad/AFPtBid can not only significantly but specifically kill HCC cells that produce AFP. The cell death induced by Ad/AFPtBid in HCC cells is via an apoptotic pathway that can be independent of p53 status.

Identification and characterization of BH3 domain protein Bim and its isoforms in human hepatocellular carcinomas

BH3-only protein Bim is a critical regulator of apoptosis and plays an essential role in mammalian development, but the characterization of Bim and its isoforms in hepatocellular carcinoma (HCC) has not been studied before. Here we investigated the expression, distribution, regulation and role of Bim isoforms in HCC cells. Fifteen Bim isoforms were identified in HCC, with six newly identified isoforms and two newly identified exons. Among of them, Bim EL, L, S, a1, a2, a3, b2, b4 and b6 are abundant isoforms according to their mRNA levels. However only Bim EL, L and S proteins could be clearly detected. Bim mRNA and protein were strongly expressed in HCC tissues compared to relevant non-tumorous regions, but the ratio of variant isoforms showed no difference between tumorous and non-tumorous tissues. Bim isoforms were differentially regulated after chemotherapeutic drug 5-Fluorouracil (5-FU) treatment. Interestingly, Bim EL, L and S, the isoforms known to induce apoptosis strongly, are the least inducible isoforms at their mRNA levels when exposed to the stress, suggesting that post-transcriptional rather than transcriptional, modulations may play a role to enhance their functions. Finally, overexpression of Bim EL, L, S and all alpha isoforms induced apoptosis in HCC cells, while overexpression of Bim beta isoforms showed no effects on cell survival after 5-FU treatment. In conclusion, Bim alpha isoforms appears to have a role in the regulation of apoptosis in HCC cells, which may contribute to not only the growth of tumor cells but also the sensitivity of HCC cells to chemotherapy.

ZBP-89 enhances Bak expression and causes apoptosis in hepatocellular carcinoma cells

ZBP-89 can enhance tumor cells to death stimuli. However, the molecular mechanism leading to the inhibitory effect of ZBP-89 is unknown. In this study, 4 liver cell lines were used to screen for the target of ZBP-89 on cell death pathway. The identified Bak was further analyzed for its role in ZBP-89-mediated apoptosis. The result showed that ZBP-89 significantly and time-dependently induced apoptosis. It significantly upregulated the level of pro-apoptotic Bak. ZBP-89 targeted a region between -457 and -407 of human Bak promoter to stimulate Bak expression based on the findings of Bak promoter luciferase report gene assay and electrophoretic mobility shift assay. ZBP-89-induced Bak increase and ZBP-89-mediated apoptosis were markedly suppressed by Bak siRNA, confirming that Bak was specifically targeted by ZBP-89 to facilitate apoptosis. In conclusion, this study demonstrated that ZBP-89 significantly induced apoptosis of HCC cells via promoting Bak level.

Epigenetic upregulation of Bak by ZBP-89 inhibits the growth of hepatocellular carcinoma

Zinc-binding protein-89 regulates Bak to facilitate apoptosis in cancer cells. This study examined if zinc-binding protein-89 regulates Bak through an epigenetic mechanism in hepatocellular carcinoma. We first demonstrated that the expression of Bak was reduced but the levels of deoxyribonucleic acid methyltransferase 1 and histone deacetylase 3 were increased in hepatocellular carcinoma cancer tissues compared to the corresponding non-cancer tissues. Moreover, there was a negative correlation between Bak expression and deoxyribonucleic acid methyltransferase 1 levels in hepatocellular carcinoma. Administration of zinc-binding protein-89 downregulated histone deacetylase 3 expression and suppressed the activities of histone deacetylase and deoxyribonucleic acid methyltransferase, which led to maintenance of histone acetylation status, inhibited the binding of methyl-CpG-binding protein 2 to genomic deoxyribonucleic acid and demethylated CpG islands in the Bak promoter in hepatocellular carcinoma cells. Using the xenograft mouse tumor model, we demonstrated that zinc-binding protein-89 or inhibitors of either epigenetic enzymes could stimulate Bak expression, induce apoptosis, and arrest tumor growth and that the maximal effort was achieved when zinc-binding protein-89 and the enzyme inhibitors were used in combination. Conclusively, zinc-binding protein-89 upregulates the expression of Bak by targeting multiple components of the epigenetic pathway in hepatocellular carcinoma.

Therapeutic efficacy of improved α-fetoprotein promoter-mediated tBid delivered by folate-PEI600-cyclodextrin nanopolymer vector in hepatocellular carcinoma

SNPs in human AFP promoter are associated with serum AFP levels in hepatocellular carcinoma (HCC), suggesting that AFP promoter variants may generate better transcriptional activities while retaining high specificity to AFP-producing cells. We sequenced human AFP promoters, cloned 15 different genotype promoters and tested their reporter activities in AFP-producing and non-producing cells. Among various AFP variant fragments tested, EA4D exhibited the highest reporter activity and thus was selected for the further study. EA4D was fused with tBid and coupled with nano-particle vector (H1) to form pGL3-EA4D-tBid/H1. pGL3-EA4D-tBid/H1 could express a high level of tBid while retain the specificity to AFP-producing cells. In a HCC tumor model, application of pGL3-EA4D-tBid/H1 significantly inhibited the growth of AFP-producing-implanted tumors with minimal side-effects, but had no effect on non-AFP-producing tumors. Furthermore, pGL3-EA4D-tBid/H1 could significantly sensitize HCC cells to sorafenib, an approved anti-HCC agent. Collectively, pGL3-EA4D-tBid/H1, a construct with the AFP promoter EA4D and the novel H1 delivery system, can specifically target and effectively suppress the AFP-producing HCC. This new therapeutic tool shows little toxicity in vitro and in vivo and it should thus be safe for further clinical tests.

Cytochrome P450 1A2 Metabolizes 17β-Estradiol to Suppress Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) occurs more frequently in men than in women. It is commonly agreed that estrogen plays important roles in suppressing HCC development, however, the underlying mechanism remains largely unknown. Since estrogen is mainly metabolized in liver and its metabolites affect cell proliferation, we sought to investigate if the liver-specific cytochrome P450 1A2 (CYP1A2) mediated the inhibitory effect of estrogen on HCC. In this study, the expression of estrogen-metabolizing enzyme CYP1A2 was determined in HCC tissues and cell lines. Cell proliferation and apoptosis were assessed in cells with or without CYP1A2 overexpression. The levels of 17β-estradiol (E2) and its metabolite 2-methoxyestradiol (2-ME) were determined. A xenograft tumor model in mice was established to confirm the findings. It was found that CYP1A2 expression was greatly repressed in HCC. E2 suppressed HCC cell proliferation and xenograft tumor development by inducing apoptosis. The inhibitory effect was significantly enhanced in cells with CYP1A2 overexpression, which effectively conversed E2 to the cytotoxic 2-ME. E2 in combination with sorafenib showed an additive effect on HCC. The anti-HCC effect of E2 was not associated with estrogen receptors ERα and ERβ as well as tumor suppressor P53 but enhanced by the approved anti-HCC drug sorafenib. In addition, HDAC inhibitors greatly induced CYP1A2 promoter activities in cancer cells, especially liver cancer cells, but not in non-tumorigenic cells. Collectively, CYP1A2 metabolizes E2 to generate the potent anti-tumor agent 2-ME in HCC. The reduction of CYP1A2 significantly disrupts this metabolic pathway, contributing the progression and growth of HCC and the gender disparity of this malignancy.