Ernest C.W. Chak

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.

Immunohistochemical analysis of pro-apoptotic Bid level in chronic hepatitis, hepatocellular carcinoma and liver metastases.

Bid, a member of the Bcl-2 family, mediates apoptosis by inducing the release of proapoptotic factors. The expression of Bid in liver diseases has not been investigated. This study evaluated Bid level in various liver diseases including hepatocellular carcinoma (HCC), liver metastases from colorectal cancer, chronic hepatitis and liver cirrhosis. The expression of Bid in tumorous tissues of HCC was lower than that in their corresponding non-tumorous tissues from the same patient. Heavy staining with Bid antibody was found in some localized tumorous liver tissues from patients with poorly differentiated tumors. In patients with chronic hepatitis and liver cirrhosis, there were gradient tumor-development centers, a gradient increase in reaction with Bid antibody from the middle of the center to its edge. The gradient tumor-development center was also found in non-tumorous tissues of HCC, suggesting that occurrence of this center in chronic hepatitis might be an early pathologic sign of HCC development. Bid was also expressed in the epithelial cells in tissues from liver metastases and their expression was often stronger than in the non-tumorous liver tissues. Heavy nuclear staining of Bid was not uncommon in these metastatic cells. The different patterns of staining between primary and secondary liver tumors may reflect a difference in tumor origin and in cell type. Nuclei of metastatic cells, though positive for Bid, still showed a considerable mitotic activity, indicating that they were in active proliferation rather than on a pathway deemed to be apoptotic. In conclusion, this study shows that the Bid level is decreased in HCC except in poorly differentiated HCC in which cells may undergo a process of apoptosis or necrosis. The existence of gradient tumor-development center in chronic hepatitis, liver cirrhosis and non-tumorous tissues from HCC may serve as a pathologic marker of a carcinogenic change of cell phenotypes.

Differential expression of inducible nitric oxide synthase and peroxisome proliferator-activated receptor gamma in non-small cell lung carcinoma

Both inducible nitric oxide synthase (iNOS) and peroxisome proliferator-activated receptor gamma (PPARgamma) are closely associated with the development of human cancer. Although the expression of iNOS has been studied in non-small cell lung carcinoma (NSCLC), the level of PPARgamma has not been examined in tumorous and non-tumorous tissues from NSCLC. The present study analysed the levels of both iNOS and PPARgamma in NSCLC tissues and in lung cell lines. The possible role of these two molecules in the carcinogenesis of lung cancer was investigated. The expression of iNOS was significantly higher in the tumorous tissues than in the non-tumorous ones. In contrast to this pattern of iNOS protein expression, the level of PPARgamma was much lower in the tumorous tissues than in the non-tumorous samples. A similar result was also obtained in vitro using human lung cancer cell lines and normal lung cells. Immunohistochemical examination revealed that PPARgamma expression in the non-tumorous tissues was more likely to be located in the nucleus whereas it was present in both the nucleus and cytoplasm of the tumorous tissues. The intensity of iNOS expression was stronger in the nucleus than in the cytoplasm of the tumorous tissues. More than 50% of the cases tested did not express iNOS protein in the non-tumorous tissues. Statistical analysis indicated a negative correlation between iNOS and PPARgamma levels in the NSCLC tissues. In conclusion, this study demonstrated differing expressions for iNOS and PPARgamma in NSCLC tissues. Since activated PPARgamma is able to inhibit the expression of iNOS and the generation of iNOS is particularly associated with the inflammatory and environmental factors of lung cancer risk, this discrepant expression pattern may be associated with the pathogenesis of NSCLC.

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.

Activation of kupffer cells inhibits tumor growth in a murine model system

Kupffer cells, a liver organ‐specific macrophage, play an important role in preventing the development of malignant tumors. The mechanism responsible for their tumoricidal activities is not completely known. In our study, we established in vivo models involving a rat malignant cell line, rat Kupffer cells and tumor implantation in nude mice. A series of relevant in vitro experiments were also carried out to determine possible pathways. LPS‐activated Kupffer cells produced significant amounts of NO, TNFα and IFNγ. Malignant cells treated with either Kupffer cells or culture supernatant of the activated Kupffer cells had an increase in caspase‐8 activity. Implanted tumors originated from malignant cells treated with either Kupffer cells or culture supernatant of the activated Kupffer cells grew much smaller than those from malignant cells without treatment or treated with control supernatants. The alteration of anti‐apoptotic Bcl‐2 was inversely associated with the change of pro‐apoptotic caspase‐8 and their levels in the tumor tissues matched the size of the tumors and treatments they received. It appeared that the above changes resulted in an increase in cellular DNA damage and apoptosis seen in malignant cells. Therefore, Kupffer cells execute their anti‐tumor effect via increasing the production of NO, TNFα and IFNγ and these cytotoxic molecules inhibit the growth of tumor by damaging cellular DNA and inducing apoptosis that was featured by downregulation of Bcl‐2 but upregulation of caspase‐8.

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.

Negative correlation between the ratio of Bax to Bcl-2 and the size of tumor treated by culture supernatants from Kupffer cells

Kupffer cells play an important role in keeping liver from occurrence of tumors. Apoptosis is thought to be a major mechanism responsible for the anti-tumor function of Kupffer cells. Previous studies have mainly concentrated on the direct contact and interaction between Kupffer cells and tumor cells. The present experiment is to investigate the apoptotic pathway in tumor induced by culture supernatant from activated Kupffer cells. The levels of Bax, Bcl-2 and iNOS were analyzed in an in vivo mouse tumor model, which was treated with culture supernatant from activated Kupffer cells. The results showed that the expression of Bax significantly increased while the expression of Bcl-2 decreased when tumor cells were treated with culture supernatants from activated Kupffer cells. The alteration of Bax and Bcl-2 levels resulted in an increase in the ratio of Bax to Bcl-2, which had negative correlation with the size of tumor and positive correlation with the expression of iNOS. The expression of TNFα and the occurrence of apoptosis were also increased in tumor treated with culture supernatants from activated Kupffer cells, compared with those which received no treatment. In conclusion, culture supernatants from activated Kupffer cells were able to change the balance between Bax and Bcl-2 in favor of the former. The ratio of Bax to Bcl-2 is a useful index to evaluate tumor apoptosis induced by Kupffer cells. Our experiment also suggests that alteration of the ratio of Bax to Bcl-2 may result from increased levels of iNOS and TNFα.

Ent-11α-hydroxy-15-oxo-kaur-16-en-19-oic-acid inhibits hepatocellular carcinoma in vitro and in vivo via stabilizing IkBα

SummaryEnt-11-hydroxy-15-oxo-kaur-16-en-19-oic-acid (5F) isolated from Pteris Semipinnata L is known to inhibit certain tumor cells in vitro. The information on the in vivo effect of 5F is limited and its effect on hepatocellular carcinoma (HCC) is unknown. In this study, the anti-tumor effect of 5F was investigated in a diethylnitrosamine (DEN)-induced mouse HCC model. In addition to therapeutic effect, the potential side effect was monitored. A panel of cultured HCC cells was used to confirm the in vivo data and explore the responsible molecular pathway. The result showed that 5F significantly inhibited the DEN-induced HCC tumors by reducing the number of tumor foci and the volume of tumors. Furthermore, 5F induced the death of cultured HCC cells in dose- and time-dependent manners. The cell death was confirmed to be apoptotic by in vivo and in vitro TUNEL assays. 5F inhibited NF-kB by stabilizing its inhibitor IkBα, reducing the nuclear p65 and inhibiting NF-kB activity. Subsequently it affected the NF-kB downstream molecules with a decrease in anti-apoptotic Bcl-2 and increase in pro-apoptotic Bax and Bak. During the whole period of the experiment, mice receiving 5F appeared to be healthy, though they suffered from a mild degree of hair loss. 5F did not damage liver and renal functions. In conclusion, 5F is effective against HCC with minimal side effects. It induces apoptosis in HCC cells via inhibiting NF-kB, leading to the decrease of Bcl-2 but the increase of Bax and Bak.

The expression of Bcl-2 family proteins and spontaneous apoptosis in laryngeal carcinomas.

Bcl-2 family proteins play an important role in the growth and biological behavior of tumors. This study aimed to determine Bcl-2 family proteins in laryngeal carcinoma and to examine their relationship with spontaneous apoptosis. The material studied was from 39 patients with laryngeal carcinoma. It was found that the expression of both Bak and Bax was lower in tumor tissues than in nontumor tissues. However, there was no difference in the expression of Bcl-2 between tumor and nontumor tissues. The frequency of spontaneous apoptosis was lower in tumor tissues than in nontumor tissues but was not significantly related to the expression of Bak, Bax, or Bcl-2. Bak was decreased in moderately differentiated tumors compared to well-differentiated tumors. In contrast to Bak, the expression of Bcl-2 was increased in moderately differentiated tumors compared to well-differentiated tumors. These results indicate that the reduction in Bak may be associated with an increase in tumor grade and dedifferentiation in laryngeal carcinomas. The lack of correlation between apoptosis and the expression of Bcl-2 family proteins suggests that spontaneous apoptosis in laryngeal carcinoma is a complex process and that molecules other than Bak, Bax, and Bcl-2 participate in it.