The up-regulation of type I interferon receptor gene plays a key role in hepatocellular carcinoma cells in the synergistic antiproliferative effect by 5-fluorouracil and interferon-alpha

Combination therapy with interferon (IFN)-alpha and 5-fluorouracil (5-FU) has been reported to show an improved therapeutic efficacy in patients with advanced hepatocellular carcinoma (HCC) but the mechanism behind this has not been completely elucidated. We examined the molecular events underlying the antiproliferative effects of IFN-alpha and 5-FU in combination using six human HCC cell lines. When the antiproliferative effects of administering IFN-alpha and 5-FU together were analyzed using isobolograms, we found that the cell lines could be divided into two groups: the S-group containing three cell lines, which showed synergistic effects, and the A-group, containing the remaining three cell lines, which showed additive effects. Real-time RT-PCR and Western blot analyses revealed that the expression levels of type I IFN receptor subunits, IFNAR1 and IFNAR2, were specifically up-regulated by 5-FU in all three cell lines of the S-group with the exception of IFNAR2 in one cell line, but not in those of the A-group. IFN-alpha modulated the protein expression levels of six enzymes regulating sensitivity to 5-FU, but none of them were down- or up-regulated in the same way in all members of the S- or A-group. In conclusion, the 5-FU-induced modulation of IFN receptor expression could play a pivotal role in the therapeutic efficacy of IFN-alpha combined with 5-FU. Measuring the expression levels of IFN receptors, and their ability to be up-regulated, may be a promising method for selecting HCC patients for this type of combination therapy.     

Augmentation of antitumor activity of 5-fluorouracil by interferon alpha is associated with up-regulation of p27Kip1 in human hepatocellular carcinoma cells.

Several clinical trials have demonstrated the effectiveness of combination therapy with 5-fluorouracil (5-FU) and IFN-alpha in colon cancer, hepatocellular carcinoma (HCC), and other malignancies. In our preliminary clinical studies, we have observed outstanding effects with this combination therapy in patients with advanced HCC. However, the underlying mechanism by which IFN-alpha modulates the effects of 5-FU is unknown. We, therefore, conducted a mechanistic study using two HCC cell lines, PLC/PRF/5 and HuH7. IFN-alpha significantly enhanced the growth inhibitory effect of 5-FU in PLC/PRF/5 cells but not in HuH7 cells, and the isobolographic analysis indicated that this effect was synergistic. Flow cytometric analysis showed a delay in the progression of G0-G1 to S phase in PLC/PRF/5, and a sustained, induction of the cyclin-dependent kinase inhibitor p27-Kip1 and down-regulation of cyclin D1 was observed. Moreover, increased expression of p27Kip1 was associated with reduced CDK-2-associated kinase activity. Another difference in the two cell types was that PLC/PRF/5 expressed abundant IFN receptors, but HuH7 did not. Apoptosis assays were not helpful in explaining the mechanism. Our results suggest that the synergistic effects of 5-FU and IFN-alpha may in part be attributable to alterations in cell cycle progression via up-regulation of p27Kip1.     

Anti-tumor effects of various interferons-alpha +5-fluorouracil via downregulation of dihydropyrimidine dehydrogenase in hepatoma cells

BACKGROUND AND AIMS: Systemic chemotherapy has not seen widespread use in the treatment for hepatocellular carcinoma (HCC). Recently, it was reported that combination treatment of 5-fluorouracil (5-FU) and interferon (IFN)-alpha was effective for non-resectable HCC. The effect of 5-FU treatment is influenced by the activities of pyrimidine catabolic enzymes. The aim of this study was to investigate which IFN-alphais most effective in combination therapy via downregulation of dihydropyrimidine dehydrogenase (DPD). METHODS: Cell proliferation assay in human hepatoma cells (HepG 2) was performed using 5-FU and/or various IFNs-alpha (C-IFN, Intron A, OIF). At the same time,DPD mRNA levels were quantified by real-time polymerase chain reaction. RESULTS: 5-FU alone but not all the IFNs showed anti-cancer effects, and the combination of each IFN +5-FU had a greater anti-cancer effect than 5-FU alone. C-IFN +5-FU most effectively prevented cancer cell proliferation (p < 0.05). Also, the combination of C-IFN +5-FU most downregulated DPD mRNA expression. CONCLUSION: The combination of each IFN-alpha +5-FU showed anti-cancer effects for HCC via downregulation of DPD. C-IFN may be most effective in the combination therapy of IFN-alpha +5-FU for HCC.     

Interferon-alpha enhances antitumor activities of oncolytic adenovirus-mediated IL-24 expression in hepatocellular carcinoma

ABSTRACT: BACKGROUND: Hepatocellular carcinoma (HCC) has a dismal 5-year-survival rate of 10%, so novel strategies are warranted. IL-24 mediates anti-tumor activity reducing STAT3 expression, which suggests that interferon (IFN) alpha may augment tumor cell lysis and reduce angiogenesis. We investigated the antitumor activity of treatment with IFN-alpha, with the oncolytic adenovirus SG600-IL-24, or the combination of both in HCC in vitro and in vivo. RESULTS: RT-PCR, ELISA assay and Western-blot confirmed that the exogenous IL-24 gene was highly expressed in HCC cells infected with SG600-IL-24. Treatment with combined IFN-alpha and SG600-IL-24 suppressed growth and promoted apoptosis of the HepG2, MHCC97L, and HCCLM3 cell lines compared with the normal cell line L02. The combined therapy increased STAT1 and SOCS1 and apoptosis, but decreased the expression of the metastatic and angiogenic proteins MMP-2, XIAP, OPN, and VEGF, which are regulated by STAT3 in HCC cells in vitro. To assess the effects in vivo, the HCC cell line HCCLM3 was transplanted subcutaneously into the right flanks of nude mice. Mice in the IFN-alpha group, the SG600-IL-24 group, or the combined therapy group had significantly suppressed growth of the HCC xenografted tumors compared to the PBS control group of mice. Among the mice treated with the combination of IFN-alpha and SG600-IL-24, three of those eight mice had long-term survival and no evidence of a tumor. These mice also had decreased expression of the metastatic and angiogenic proteins MMP-2, XIAP, OPN, and VEGF. CONCLUSIONS: The present study demonstrated for the first time the potential antitumor activity of IFN-alpha combined with the oncolytic adenovirus SG600-IL-24 in HCC both in vitro and in vivo, and suggests its further development as a potential candidate for HCC cancer gene therapy.     

Treatment of hepatocellular carcinoma with major portal vein thrombosis by combined therapy with subcutaneous interferon-alpha and intra-arterial 5-fluorouracil; role of type 1 interferon receptor expression

We previously reported the beneficial effects of combination therapy of interferon (IFN)-alpha/5-fluorouracil (FU) for advanced hepatocellular carcinoma (HCC) with tumour thrombi in the major portal branches. This report describes the results of longer follow-up and includes more than double the number of patients relative to the original report, and evaluates the role of IFN-alpha/type 2 interferon receptor (IFNAR2) expression on the response to the combination therapy. The study subjects were 55 patients with advanced HCC and tumour thrombi in the major branches of the portal vein (Vp3 or 4). They were treated with at least two courses of IFN-alpha/5-FU without major complication. In the 55 patients, 24 (43.6%) showed objective response (eight (14.5%) showed complete response, 16 (29.1%) partial response), four (7.3%) showed no response, and 27 (49.1%) showed progressive disease. Immunohistochemically, IFNAR2 expression was detected in nine out of 13 (69.2%) patients. There was significant difference in the time-to-progression survival (P = 0.0002) and the overall survival (P < 0.0001) between IFNAR2-positive and -negative cases. There was a significant correlation between IFNAR2 expression and response to IFN-alpha/5-FU combination therapy in univariate analysis (P = 0.0070). IFN-alpha/5-FU combination therapy is a promising modality for advanced HCC with tumour thrombi in the major portal branches and could significantly depend on IFNAR2 expression.     

Genes associated with human hepatocellular carcinoma cell chemosensitivity to 5-fluorouracil plus interferon-alpha combination chemotherapy

Recently, combined chemotherapy with 5-fluorouracil (5-FU) and interferon (IFN)-alpha has been reported to show marked effects in patients with advanced hepatocellular carcinoma. We investigated the genes associated with susceptibility to this combination therapy. The gene expression profiles of eight human hepatocellular carcinoma cells (HepG2, Hep3B, Huh7, Huh6, PLC/PRF/5, HLE, HLF, and SK-Hep1) were evaluated using an oligonucleotide microarray that consisted of 3,800 genes. The 50% growth inhibitory concentration (GI50) values for 5-FU, IFN-alpha, and the combination of 5-FU plus IFN-alpha were determined by the MTT assay. We selected genes that were expressed differentially between the cells with increased susceptibility to the combination therapy and the remaining cells. Relevance networks of the gene expression patterns and GI50 values of the susceptible cells were constructed to find genes associated with susceptibility to the combination therapy. Of the eight cells tested, five showed increased susceptibility to 5-FU plus IFN-alpha compared with 5-FU treatment alone. Among the 3,800 genes, 25 were expressed differentially between susceptible cells and resistant cells. The relevance networks revealed that sensitivity to 5-FU plus IFN-alpha involved the expression of 27 independent genes, which included 10 genes that are commonly associated with sensitivity to 5-FU alone. We selected a set of genes to predict susceptibility to 5-FU plus IFN-alpha combination therapy. We also selected genes that play key roles in the synergistic effect of this combination therapy. These gene sets should prove useful in evaluations of the efficacy and underlying molecular mechanisms of this combination therapy.     

A case of recurrent hepatocellular carcinoma successfully treated with a combination therapy of interferon-alpha and intravenous continuous infusion of 5-fluorouracil

We report a case of recurrent hepatocellular carcinoma (HCC) successfully treated with a combination therapy of interferon-alpha (IFN-alpha) and 5-fluorouracil (5-FU), which was administered intravenously. A 49-year-old Japanese man who underwent a right hepatectomy for HCC developed tumor recurrence in the liver 19 months after surgery. Abdominal CT revealed multiple metastatic lesions in the liver. He received a combination therapy of 500 mg/day of 5-FU that was given intravenously by continuous infusion and 5 x 10(6) units of IFN-alpha, given three times weekly. The treatment resulted in a fall in serum PIVKA-II (protein induced by vitamin K antagonism) levels from 337 mAU/ml to 65 mAU/ml and disappearance of tumor stain in enhanced CT. 5-FU is usually administered by arterial infusion in a combination therapy of IFN-alpha and 5-FU. However, 5-FU infusion may be possible intravenously in the combination therapy of IFN-alpha and 5-FU for the treatment of advanced HCC.     

Growth inhibitory effects of pegylated IFN-alpha2b and 5-fluorouracil in combination on renal cell carcinoma cell lines in vitro and in vivo

We investigated the effects of pegylated IFN-alpha2b (PEG-IFN-alpha2b) alone and PEG-IFN-alpha2b plus 5-fluorouracil (5-FU) in vitro on the proliferation of renal cell carcinoma (RCC) cell lines. After the transplantation of RCC cells into nude mice, we administered IFN (PEG-IFN-alpha2b or IFN-alpha2b) alone, 5-FU alone, or IFN (PEG-IFN-alpha2b or IFN-alpha2b) plus 5-FU; and investigated tumor volume, tumor weight, the numbers of apoptotic cells and artery-like blood vessels, relative mRNA expression levels of enzymes which relate to 5-FU metabolism, angiogenesis factor, and type I interferon receptor. RCC cells in vitro were generally and relatively resistant to the anti-proliferative effects of PEG-IFN-alpha2b, but the addition of 5-FU augmented IFN-induced anti-proliferative effects with the induction of apoptosis. PEG-IFN-alpha2b in vivo presented stronger anti-tumor effects than IFN-alpha2b, and its combination with 5-FU augmented the effects. The significant anti-tumor effect of the combination treatment was the increase in apoptotic cell number, but there were no significant differences in the suppression of angiogenesis, expression of IFN receptor, and the actions of metabolic enzymes of 5-FU. In conclusion, PEG-IFN-alpha2b presents stronger anti-tumor effects than non-pegylated IFN, and the effects are augmented in the combination with 5-FU. Our findings suggest the clinical usefulness of PEG-IFN-alpha2b in the treatment of RCC.     

The progress of hepatic arterial infusion chemotherapy for hepatocellular carcinoma

Hepatic arterial infusion (HAI) chemotherapy has been selected as a therapeutic option for highly advanced hepatocellular carcinoma (HCC) with tumor thrombi in major portal branches or intrahepatic metastases. Conventional therapies have no clinical effect on highly advanced HCC. Recent advances of an implanted portcatheter system have facilitated repeated arterial infusion of chemotherapeutic agents, and HAI chemotherapy with several anticancer drugs provides a useful choice for advanced HCC. In various regimens of HAI chemotherapy, low-dose cisplatin and 5-fluorouracil (5-FU) therapy or combination therapy of interferon (IFN)-alpha/5-FU have been reported to improve the response rates for advanced HCC. However, the survival benefit of HAI chemotherapy may be affected by liver function. None of these regimens have been proved to be the standard for HAI chemotherapy. We previously reported that the beneficial effects of combination therapy of IFN-alpha/5-FU for advanced HCC. IFN-alpha/5-FU combination therapy may be a promising treatment modality for advanced HCC.     

Interferon-alpha and 5-fluorouracil combination therapy after palliative hepatic resection in patients with advanced hepatocellular carcinoma, portal venous tumor thrombus in the major trunk, and multiple nodules

BACKGROUND: The authors reported previously the beneficial effects of interferon (IFN)-alpha/5-fluorouracil (5-FU) combination therapy for patients with advanced hepatocellular carcinoma (HCC) who have tumor thrombi in the major portal branches. In this report, the authors describe the results from IFN/5-FU chemotherapy for patients who underwent palliative hepatic resection for advanced HCC with tumor thrombus in the main trunk of the portal vein and multiple nodules in the whole liver. In addition, they evaluated the correlation between the response to such therapy and expression of IFN-alpha type 2 receptor (IFNAR2). METHODS: From October 1999 to December 2004, 30 patients with advanced HCC, tumor thrombi in the main trunk of the portal vein, and multiple nodules in the whole liver (Vp4 and grade 3 intrahepatic metastases) were recruited for this study. They underwent palliative hepatic resection followed by at least 2 courses of IFN/5-FU. IFNAR2 expression levels were determined by immunohistochemistry. RESULTS: No major treatment-related complications were noted. An objective response was noted in 10 patients (33.3%) and included a complete response in 6 patients (20%), a partial response in 4 patients (13.3%), no response in 1 patient (3.3%), and progressive disease in 19 patients (63.4%). IFNAR2 expression was detected in 20 of 30 patients (66.7%). There was a significant difference in overall survival between patients with positive and negative IFNAR2 expression cases (P<.0025), and a significant correlation was observed between IFNAR2 expression and response to IFN/5-FU combination therapy (P=.0199). CONCLUSIONS: Adjunct IFN/5-FU therapy is a promising modality for patients with advanced HCC, tumor thrombi in the major trunk, and multiple nodules after palliative hepatic resection. The results from this study indicated that the response to such therapy seemed to be correlated with IFNAR2 expression.     

A report of two cases--two patients of the extremely advanced hepatocellular carcinoma have responded completely for a long time after a combination therapy consisting of arterial chemotherapy and injection of interferon-alpha

The prognosis of advanced hepatocellular carcinoma (HCC) is extremely poor. Patient 1 was a 43-year-old male with major portal tumor thrombi. He received combination therapy consisting of continuous arterial infusion (MTX 30 mg/m2, day 1, CDDP/5-FU 6 mg/m2: 250 mg/m2, day 1-14) and subcutaneous injection of IFN-alpha (500 x 10(4) U, 3 times a week, 4 weeks). Patient 2 was a 66-year-old male with major hepatic venous tumor thrombi. He received combination therapy consisting of continuous arterial infusion (5-FU 6 mg/m2: 250 mg/m2, day 1-14) and subcutaneous injection of IFN-alpha (500 x 10(4) U, 3 times a week, 4 weeks). Decrease in tumor was observed in both patients markers and marked regression of tumor was observed in both patients. They are still in complete response. This combination therapy is an effective strategy for advanced HCC.     

Combination of interferon-α and 5-fluorouracil induces apoptosis through mitochondrial pathway in hepatocellular carcinoma in vitro

Many clinical reports have proven that the combination therapy of interferon-alpha plus 5-fluorouracil is remarkably effective for advanced hepatocellular carcinoma (HCC). However, the mechanism of this therapy is not well understood. Here, we demonstrated that the combination therapy synergistically inhibited the growth of Fas-negative HCC cells, arrested cell-cycle progression and induced apoptosis. Moreover, the combination therapy significantly increased the protein expression of caspase-8, activated Bid and cytochrome c. Meanwhile, the expression of anti-apoptotic gene Bcl-xL was reduced and intracellular calcium elevated obviously during the early stage of treatment. Therefore, mitochondrial pathway was involved in the apoptosis of Fas-negative HCC cells induced by IFN-α/5-FU and Ca(2+) partially promoted the beneficial effect against HCC.     

Soluble VEGF receptor-2 may be a predictive marker of anti-angiogenic therapy with clinically available safe agents

The identification of biomarkers of anti-angiogenic therapy that predict clinical benefit is of vital importance. We previously reported that a combination treatment with clinically available safe agents, specifically angiotensin-converting enzyme inhibitor (ACE-I) and vitamin K (VK), inhibited the cumulative recurrence of hepatocellular carcinoma (HCC) via suppression of the vascular endothelial growth factor (VEGF). The present study aimed to identify non-invasive biological markers that predict the clinically beneficial effect of this combination regimen. A combination of ACE-I (perindopril; 4 mg/day) and VK (menatetrenone; 45 mg/day) was administered for 54 months following curative therapy for HCC. The cumulative recurrence and several indices, which are reportedly considered as biological markers of anti-angiogenic therapies, were analyzed. The combined treatment of ACE-I and VK markedly inhibited the cumulative recurrence of HCC during the 54-month follow-up. The serum VEGF and soluble VEGF receptor (sVEGFR)-2 were significantly suppressed with this combination regimen, whereas sVEGFR-1 was not. In HCC patients without recurrence, a significant suppression of VEGF and sVEGFR-2 was achieved within 6 and 3 months after treatment, respectively. In conclusion, the combination treatment of ACE-I and VK is a potentially novel anti-angiogenic strategy for secondary chemoprevention against HCC since the two agents are widely used in clinical practice without serious side effects. Furthermore, sVEGFR-2 may become a useful clinical predictive marker of this combination treatment.     

MicroRNA-21 induces resistance to the anti-tumour effect of interferon-α/5-fluorouracil in hepatocellular carcinoma cells

Background:

We reported recently the clinical efficiency of interferon (IFN)-α/5-fluorouracil (5-FU) combination therapy in advanced hepatocellular carcinoma (HCC). However, prediction of the response to the combination therapy remains unsatisfactory. The aim of this study was to investigate the anti-tumour effects of microRNA (miR)-21 on the sensitivity of HCC cells to IFN-α/5-FU and whether miR-21 can be used as a predictor of the response to such therapy in HCC.

Methods:

Changes in the sensitivity of HCC cells (PLC/PRF/5 and HepG2) to IFN-α/5-FU were examined after transfection with pre-miR-21 or anti-miR-21. The correlation between miR-21 expression level, evaluated by qRT–PCR, and response to the therapy was also investigated in clinical HCC specimens.

Results:

Hepatocellular carcinoma cells transfected with pre-miR-21 were significantly resistant to IFN-α/5-FU. Annexin V assay showed that the percentage of apoptotic cells was significantly lower in cells transfected with pre-miR-21 than control cells. Transfection of anti-miR-21 rendered HCC cells sensitive to IFN-α/5-FU, and such sensitivity was weakened by transfection of siRNAs of target molecules, PETN and PDCD4. miR-21 expression in clinical HCC specimens was significantly associated with the clinical response to the IFN-α/5-FU combination therapy and survival rate.

Conclusions:

The miR-21 in HCC cell lines and clinical HCC samples is a significant modulator of the anti-tumour effect of IFN-α and 5-FU. This suggests that miR-21 is a potentially suitable marker for the prediction of the clinical response to the IFN-α/5-FU combination therapy.     

Intra arterial infusion chemotherapy combined with interferon-alpha following palliative hepatic resection against advanced hepatoma with portal venous tumor thrombus in the major trunk and multiple nodules--a preliminary study

Recently, we reported the beneficial effects of intra arterial 5-FU infusion chemotherapy combined with interferon-alpha (IFN-alpha/5-FU combined chemotherapy) for advanced hepatocellular carcinoma (HCC). This report describes the preliminary results of treatment of IFN-alpha/5-FU combined chemotherapy following palliative hepatic resection for advanced hepatocellular carcinoma with tumor thrombus in the main trunk of the portal vein with multiple nodules in the whole liver. The 15 patients of HCC with portal venous tumour thrombi (PVTT) and multiple intra-hepatic multiple nodules (IM3) were treated with IFN-alpha/5-FU combined chemotherapy following palliative surgery in this study. No leukopenia, thrombocytopenia, or myelosuppression was observed in any of the 15 patients. Other adverse effects were, in general, clinically manageable. Concerning the anti-tumor effect, 6 showed an objective response and 9 showed a progressive disease; the response rate was 40.0% (6/15). The 1-year and 3-year survival rates were 48% and 21% in all 15 cases, respectively. In conclusion, IFN-alpha/5-FU combined therapy may be a promising modality for advanced HCC with tumor thrombi in the major trunk with multiple nodules after following palliative surgery.     

Activation of Wnt/��-catenin signalling pathway induces chemoresistance to interferon-��/5-fluorouracil combination therapy for hepatocellular carcinoma

Type I IFN receptor type 2 (IFNAR2) expression correlates significantly with clinical response to interferon (IFN)-α/5-fluorouracil (5-FU) combination therapy for hepatocellular carcinoma (HCC). However, some IFNAR2-positive patients show no response to the therapy. This result suggests the possibility of other factors, which would be responsible for resistance to IFN-α/5-FU therapy. The aim of this study was to examine the mechanism of anti-proliferative effects of IFN-α/5-FU therapy and search for a biological marker of chemoresistance to such therapy. Gene expression profiling and molecular network analysis were used in the analysis of non-responders and responders with IFNAR2-positive HCC. The Wnt/β-catenin signalling pathway contributed to resistance to IFN-α/5-FU therapy. Immunohistochemical analysis showed positive epithelial cell adhesion molecule (Ep-CAM) expression, the target molecule of Wnt/β-catenin signalling, only in non-responders. In vitro studies showed that activation of Wnt/β-catenin signalling by glycogen synthesis kinase-3 inhibitor (6-bromoindirubin-3′-oxime (BIO)) induced chemoresistance to IFN-α/5-FU. BrdU-based cell proliferation ELISA and cell cycle analysis showed that concurrent addition of BIO and IFN-α/5-FU significantly to hepatoma cell cultures reduced the inhibitory effects of the latter two on DNA synthesis and accumulation of cells in the S-phase. The results indicate that activation of Wnt/β-catenin signalling pathway induces chemoresistance to IFN-α/5-FU therapy and suggest that Ep-CAM is a potentially useful marker for resistance to such therapy, especially in IFNAR2-positive cases.     

A case of advanced hepatocellular carcinoma with lung metastases and cirrhosis that responded to combination chemotherapy with interferon-alpha and 5-fluorouracil

The prognosis for patients with hepatocellular carcinoma (HCC) with progressive liver cirrhosis or extrahepatic metastases remains dismal. We report a case of HCC with liver cirrhosis and lung metastases who had been treated successfully by combination chemotherapy of 5-fluorouracil (5-FU) and interferon-alpha (IFN-alpha). A 67-year-old male with a history of hepatitis C, liver cirrhosis and HCC was hospitalized because of cough and dyspnea. Computed tomography (CT) of chest revealed multiple lung metastases. Systemic combination chemotherapy with 5-FU and IFN-alpha was begun, and lung metastases disappeared after one course of treatment. He died of liver failure one year later, but no recurrence of lung metastases was seen. Although systemic combination chemotherapy of 5-FU and IFN-alpha induced the bone marrow suppression, it was effective for lung metastases and palliates symptoms and signs in our case of HCC.     

Andrographolide enhances 5-fluorouracil-induced apoptosis via caspase-8-dependent mitochondrial pathway involving p53 participation in hepatocellular carcinoma (SMMC-7721) cells

Despite recent significant advances in the treatment of human carcinoma (HCC), the results of chemotherapy to date remain unsatisfactory. 5-Fluorouracil (5-FU) still represents the cornerstone of treatment of carcinoma, and resistance to the actions of 5-FU is a major obstacle to successful chemotherapy. More effective treatment strategies may involve combinations of agents with activity against HCC. Andrographolide (ANDRO), a natural bicyclic diterpenoid lactone isolated from Andrographis paniculata, has been shown to suppress the growth of HCC cells and trigger apoptosis in vitro. To assess the suitability of ANDRO as a chemotherapeutic agent in HCC, its cytotoxic effects have been evaluated both as a single agent and in combination with 5-FU. ANDRO potentiates the cytotoxic effect of 5-FU in HCC cell line SMMC-7721 through apoptosis. ANDRO alone induces SMMC-7721 apoptosis with p53 expression, Bax conformation and caspase-3,8,9 activation. Surprisingly, the addition of ANDRO to 5-FU induces synergistic apoptosis, which could be corroborated to the increased caspase-8, p53 activity and the significant changes of Bax conformation in these cells, resulting in increased losses of mitochondrial membrane potential, increased release of cytochrome c, and activation of caspase-9 and caspase-3. Suppression of caspase-8 with the specific inhibitor z-IETD-fmk abrogates largely ANDRO/5-FU biological activity by preventing mitochondrial membrane potential disappearance, caspase-3,9 activation and subsequent apoptosis. The results suggest that ANDRO may be effective in combination with 5-FU for the treatment of HCC cells SMMC-7721.