The cyclin-dependent kinase inhibitor flavopiridol sensitizes human hepatocellular carcinoma cells to TRAIL-induced apoptosis.

Flavopiridol was one of the first cyclin-dependent kinase inhibitors demonstrated to have an antitumor effect in several cancer types. Here, we investigated the effects of flavopiridol on TNF-related apoptosis-inducing ligand (TRAIL) in the human hepatocellular carcinoma (HCC) cell lines HLE and HepG2, and evaluated the role of flavopiridol in apoptosis. To better understand the mechanism of increased TRAIL sensitivity in HCC cells, we determined the effect of flavopiridol on cell surface expression of TRAIL and TRAIL receptors using flow cytometry analysis. The levels of survivin, FLIP, Bcl-xL and X-chromosome-linked IAP (XIAP) in treated and untreated cells was also determined. Flavopiridol decreased cell viability in a dose-dependent manner in the two HCC cell lines tested. The pan-caspase inhibitor z-VAD-FMK did not inhibit the effect. However, subtoxic levels of flavopiridol dramatically enhanced TRAIL-induced apoptosis in both cells. Flavopiridol up-regulated TRAIL, TRAIL-R1 and TRAIL-R2 in both cell lines. In addition, flavopiridol down-regulated expression of survivin in both cell lines, and expression of FLIP and Bcl-xL were down-regulated in HLE cells. In summary, flavopiridol augmented TRAIL sensitivity by up-regulation of TRAIL receptors and down-regulation of survivin, FLIP and Bcl-xL. Thus, combining flavopiridol with a TRAIL agonist may prove to be an effective new strategy for treatment of HCC.

Adenovirus-mediated transfection of caspase-8 sensitizes hepatocellular carcinoma to TRAIL- and chemotherapeutic agent-induced cell death.

Caspase-8 belongs to the cysteine protease family and is known to be activated at the initial step in the cascade of TRAIL-induced apoptosis. The activation of procaspase-8 can be blocked by a relatively large amount of c-FLIP, which renders resistance to death receptor-mediated apoptosis in many types of cancer cells. To ask if extrinsic over-expression of caspase-8 contributes to the induction of apoptosis, we introduced the caspase-8 gene into HCC cells using an adenoviral (Adv) vector (Adv-Casp8). We demonstrated that Adv-Casp8 increased expression of active forms of caspase-8 in MOI-dependent manner. A large amount of Adv-Casp8 (MOI of 50) induced apoptosis significantly in HCC cells and resulted in downregulation of c-FLIP (in SK-Hep1, HLE, and HepG2 cells), XIAP, survivin, and Bcl-xL (in HLE cells) and dynamic release of cytochrome c and Smac from the mitochondria into the cytosol. On the other hand, a small amount of Adv-Casp8 (MOI of 10) causes a slight but detectable increase in the level of apoptosis with only a small effect on anti-apoptotic proteins and mitochondrial activation. However, small amounts of Adv-Casp8 augmented TRAIL- or chemotherapeutic agent-induced cell death (with an MOI of 10 or 20, respectively). These results suggest both that exogenous over-expression of caspase-8 by Adv-Casp8 may be essential for induction of HCC cell death and that the combination of Adv-Casp8 and TRAIL or chemotherapeutic agents could provide a useful strategy for treatment of HCC.

COX-2 inhibitors sensitize human hepatocellular carcinoma cells to TRAIL-induced apoptosis.

Cyclooxygenase (COX)-2 is upregulated in a variety of human cancers, including in hepatocellular carcinoma (HCC), whereas it is undetectable in most normal tissue. Evidence suggests that COX-2 is likely to be involved in hepatocarcinogenesis and, thus, COX-2 may be involved in an early process in carcinogenesis, dedifferentiation. To address this possibility, we investigated the effect of COX-2 inhibitors on TNF-related apoptosis, inducing ligand (TRAIL) sensitivity and its molecular mechanisms, with special attention to anti-apoptotic proteins. We used the highly selective COX-2 inhibitors, NS398 and CAY10404. We also used the MTT assay and cytological analysis of DAPI-stained DNA to assess viability and apoptosis in two HCC cells (SK-Hep1 and HLE). In order to ask what led to increased sensitivity to TRAIL in HCC cells, cell surface expression of TRAIL and TRAIL-receptors was investigated using flow cytometry analysis. Expression of survivin, X-chromosome-linked IAP (XIAP), Bcl-xL, AKT and phospho-AKT was also investigated using immunoblotting. COX-2 inhibitors resulted in a concentration-dependent decrease in cell viability in the two HCC cell lines tested. Subtoxic levels of COX-2 inhibitors did not significantly augment TNFalpha-induced apoptosis but did dramatically enhance TRAIL-induced apoptosis in both cell lines. TRAIL receptor 2/death receptor 5 (TRAIL-R2/DR5) expression was significantly up-regulated in SH-Hep1 and HLE cells. TRAIL receptor 1/death receptor 4 (TRAIL-R1/DR4) expression was up-regulated only in SK-Hep1. Expression of survivin and Bcl-xL was down-regulated in SK-Hep1 and HLE cells in the presence of CAY10404 but XIAP was not affected. Expression of survivin, Bcl-xL and XIAP was down-regulated in SK-Hep1 cells in the presence of NS398. Survivin expression was also down-regulated in the presence of NS398 in HLE cells. Finally, NS398 also decreased phospho-AKT in SK-Hep1 cells. These results demonstrate that COX-2 inhibitors can induce apoptosis and augment TRAIL sensitivity by up-regulation of TRAIL receptors and down-regulation of both survivin and AKT signaling.

Proteasome inhibition sensitizes hepatocellular carcinoma cells to TRAIL by suppressing caspase inhibitors and AKT pathway.

The ubiquitin-proteasome pathway is responsible for regulating cell cycle proteins, tumor-suppressor molecules, oncogenes, transcription factors, and pro- and anti-apoptotic proteins. The aim of this study is to evaluate the effects of proteasome inhibitors on human hepatocellular carcinoma (HCC) cells. HCC cells SK-Hep1, HLE and HepG2 were treated with the proteasome inhibitors MG132 and MG115. Our data showed that both inhibitors induce apoptosis in the three cell types tested in a dose-dependent manner. Moreover, subtoxic levels of MG132 and MG115 sensitized HCC cells to TRAIL-induced apoptosis. To investigate the mechanism of increased TRAIL sensitivity in HCC cells, we first examined surface expression of TRAIL and its receptors. MG132 upregulated TRAIL and its receptors (TRAIL-R1 and -R2) in SK-Hep1 and HLE, whereas MG115 upregulated them in SK-Hep1. MG132 downregulated expression of X-linked inhibitor of apoptosis protein (XIAP) in SK-Hep1 and HLE, and of survivin in all three cell-types. MG115 downregulated expression of XIAP in SK-Hep1, and survivin in SK-Hep1 and HepG2. Furthermore, MG132 downregulated phospho-AKT and its downstream target phospho-BAD, indicating that MG132 activated the mitochondrial apoptosis pathway by inhibiting phosphorylation of AKT and BAD. In conclusion, proteasome inhibitors induced apoptosis and augmented TRAIL sensitivity via both the IAP family and AKT pathways. Thus, combining proteasome inhibitors with a TRAIL agonist may provide a new therapeutic strategy for HCC.

Primary biliary cirrhosis after aortoiliac reconstruction surgery using a Y-graft.

Primary biliary cirrhosis (PBC) is an autoimmune disease characterized by anti-mitochondrial antibodies and destruction of intra-hepatic bile ducts. Though little is known about the etiology of PBC, some reports suggest that xenobiotics and viral infections may induce PBC. We describe a case of PBC after the aortoiliac reconstruction surgery using a Y-graft.

Involvement of tumor necrosis factor-related apoptosis-inducing ligand and tumor necrosis factor-related apoptosis-inducing ligand receptors in viral hepatic diseases.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in tumor cells, but not in most normal cells. The role of TRAIL in hepatic cell death and hepatic diseases is not well understood. The present study investigated the expression of TRAIL and TRAIL receptors (TRAIL-Rs) in patients with hepatitis C virus infection using immunohistochemistry and examined physiological roles under viral infection in the HepG2 cell line. Staining of TRAIL or TRAIL-Rs was prominent in the cytoplasm and membrane of hepatocytes in the periportal area. Some liver-infiltrating lymphocytes also displayed positive staining for TRAIL. Staining intensity was significantly increased with disease progression, particularly in the periportal area. AdCMVLacZ (Q-BIOgene, Carisbad, Calif) infection was also found to induce apoptosis in HepG2 cells and significantly augment TRAIL-induced apoptosis. Anti-TRAIL antibody significantly inhibited apoptosis induced by AdCMVLacZ infection. Flow cytometry analysis revealed that both TRAIL-R2 and TRAIL were up-regulated on the cell surface of HepG2 cells with AdCMVLacZ infection. Transforming growth factor-beta1 also enhanced TRAIL expression in HepG2 cells. These results indicate that TRAIL/TRAIL-R apoptotic pathways play important roles in the hepatic cell death during viral infection.

Targeting of X-linked inhibitor of apoptosis protein or survivin by short interfering RNAs sensitize hepatoma cells to TNF-related apoptosis-inducing ligand- and chemotherapeutic agent-induced cell death.

The inhibitors of apoptosis (IAPs) family regulate apoptosis by preventing the action of the central execution phase, and function as mediators and regulators of the anti-apoptotic activity of the v-Rel and NF-kappaB transcription factor families. The targeting of IAPs may be a promising strategy, but it is not well elucidated in human hepatocellular carcinomas (HCCs). We have therefore investigated the effects of the down-regulation of IAPs (XIAP or survivin) on the TNF-related apoptosis-inducing ligand (TRAIL) and chemotherapeutic agents that induced apoptosis in human HCC cells. To inhibit the IAPs gene expression, we designed small interfering RNA (siRNA) against the X-chromosome-linked IAP (XIAP) or survivin and investigated their efficacy in the suppression of the XIAP or survivin expression in two HCC cells (SK-Hep1 and HLE), and their consequent antitumor potential. We found that the designed siRNAs against the XIAP and survivin downregulated the protein expression of respective genes by almost 50%. The suppression of IAPs resulted in a significant decrease in procaspase-3 levels, especially by suppression of the XIAP. The apoptosis cell count was small in cells transfected with control siRNA and siRNA against the XIAP or survivin, but after treatment with 10 ng/ml of TRAIL, the apoptosis cells increased 2-3 times by the suppression of IAPs as control. The cytotoxicity of doxorubicin and camptothecin was augmented by the suppression of the XIAP in SK-Hep1 cells, whereas the suppression of survivin did not affect cytotoxicity. In conclusion, downregulation of the XIAP or survivin enhances cell death by TRAIL and increases sensitivity against some chemotherapeutic agents in HCC cells. In particular, the XIAP may be a potential target to increase therapeutic sensitivity.

Different effects of bile acids, ursodeoxycholic acid and deoxycholic acid, on cell growth and cell death in human colonic adenocarcinoma cells.

Secondary bile acids have been implicated as an important etiological factor in colorectal cancer. We investigated the effects of ursodeoxycholic acid (UDCA) and deoxycholic acid (DCA) on the growth and cytotoxicity in HT29 human colonic adenocarcinoma cells. Proliferation assay, cell cycle analysis and cell death characterization by bile acids were performed. Both UDCA and DCA reduced their proliferation rate of HT29 over 48 h in a concentration- and time-dependent manner compared with control cultures. In terms of cell cycle effects, however, UDCA induced G2/M arrest, while DCA induced G1 arrest in a concentration- and time-dependent manner. As for the effects of each bile acid on cell toxicity, UDCA induced early apoptosis and DCA induced both early apoptosis and necrosis. Bile acids play an important role in regulating cell survival and cell death in colon adenocarcinoma cells.

The right hepatic artery syndrome.

Various benign and malignant conditions could cause biliary obstruction. Compression of extrahepatic bile duct (EBD) by right hepatic artery was reported as a right hepatic artery syndrome but all cases were compressed EBD from stomach side. Our case compressed from dorsum was not yet reported, so it was thought to be a very rare case. We present here the first case of bile duct obstruction due to the compression of EBD from dorsum by right hepatic artery.

Risk factors for the recurrence of hepatocellular carcinoma after radiofrequency ablation of hepatocellular carcinoma in patients with hepatitis C.

AIM: To analyze the risk factors of hepatocellular carcinoma (HCC) recurrence after radiofrequency ablation (RFA) treatment with HCV-associated hepatitis. METHODS: Twenty-six patients with HCV-associated HCC who were followed-up for more than 12 mo were selected for this study. Risk factors for distant intrahepatic recurrences of HCC were evaluated for patients in whom complete coagulation was achieved without recurrence in the same subsegment as the primary nodule. Twelve clinical and tumoral factors were examined: Age, gender, nodule diameter, number of primary HCC nodule, Child-Pugh classification, serum platelet, serum albumin, serum AST, post RFA AST, serum ALT, post RFA ALT, post RFA treatment. RESULTS: Distant recurrences of HCC in remnant liver after RFA were observed in 14 cases and in the number of primary HCC nodules (P = 0.047), and the serum platelets (P = 0.030), the clear difference came out by the recurrence group and the non-recurrence group. The cumulative recurrence rates after 1 and 2 years were 30.8% and 86.8%, respectively for primary multinodular HCC, and 15.4% and 29.5% respectively, for primary uninodular HCC. In addition the 1-year recurrence rates for patients with serum albumin more than 3.4 g/dL and less than 3.4 g/dL were 23.1% for both, but the 2-years recurrence rates were 89.0% and 23.1%, respectively. The number of primary HCC nodules (relative risk, 6.970; P = 0.016) were found to be a statistically significant predictor for poor distant intrahepatic recurrence by univariate analysis. CONCLUSION: Patients who have multiple HCC nodules, low serum platelets and low serum albumin accompanied by HCV infection, should be carefully followed because of the high incidence of new HCC lesions in the remnant liver, even if coagulation RFA is complete.

Cardiac metastases of gallbladder carcinoma.

This report describes the case of a 68-year-old woman diagnosed with advanced gallbladder cancer, whose autopsy revealed multiple metastases, including cardiac metastases.

Augmentation of tumor necrosis factor family-induced apoptosis by E3330 in human hepatocellular carcinoma cell lines via inhibition of NF kappa B.

AIM: To investigate the reduction of cell viability in human hepatocellular carcinoma (HCC) cell lines induced by inhibition of nuclear factor kappa B (NF kappa B). METHODS: HLE, SKHep1, and HepG2 were incubated and E3330 was used to compare the stimulation of some chemotherapeutic drugs with that of TNF family, Fas ligand, TNF alpha and TNF-related apoptosis-inducing ligand (TRAIL) at the point of the reduction of cell viability by inhibiting NF kappa B. RESULTS: E3330 decreased NF kappa B levels in HLE cells stimulated by TNF and TRAIL. The cytotoxicity of the combination of TRAIL, TNF alpha, Fas ligand, and E3330 increased synergistically in a dose-dependent manner compared to either E3330 alone in all HCC cell lines by MTT assay. However, the combination of some chemotherapeutic drugs and E3330 did not decrease the cell viability. CONCLUSION: Inhibition of NF kappa B sensitizes human HCC cell lines to TNF-mediated apoptosis including TRAIL, and TRAIL-based tumor therapy might be a powerful potential therapeutic tool in the treatment of human HCC.

[Adjuvant chemotherapy for unresectable locally advanced pancreatic cancer in light of its characteristics].

The use of neoadjuvant chemotherapy for pancreatic cancer has been advocated for its potential ability to optimize patient selection for surgical resection and to downstage locally advanced tumors, especially for patients with Stage IV b (Japan criteria). We report our experience with a six-drug chemotherapeutic regimen that resulted in sufficient downstaging of the tumor in some patients to justify surgical resection. From Jan. 2001 through December 2003, 6 patients received 5-FU as a continuous infusion (200 mg/m2/day), calcium leucovorin weekly by intravenous bolus injection (30 mg/m2), mitomycin-C every 6 weeks (10 mg/m2 intravenously), and dipyridamole daily orally (75 mg), according to the UCLA regimen and gemcitabine weekly (600 mg/m2) and heparin as a continuous infusion (0-3,000 U/day) for locally advanced unresected pancreatic cancer. All of these patients were evaluable for response and survival. There were 5 partial responses (83% response rate) and 1 no response. Four of 5 responding patients had sufficient tumor regression to meet clinical criteria for resectability, three of whom underwent a curative resection. All patients who underwent downstage operation were still alive for the follow-up period (4-23 months).