a b s t r a c t a r t i c l e i n f oHepatocellular carcinoma (HCC) is one of the most lethal malignancies, and is also the fourth most common cancer worldwide with around 700,000 new cases each year. Currently, first line chemotherapeutic drugs used for HCC include fluorouracil, cisplatin, doxorubicin, paclitaxel and mitomycin, but most of these are non-selective cytotoxic molecules with significant side effects. Sorafenib is the only approved targeted therapy by the U.S. Food and Drug Administration for HCC treatment, but patients suffer from various kinds of adverse effects, including hypertension. The signal-transducer-and-activator-of-transcription 3 (STAT3) protein, one of the members of STATs transcription factor family, has been implicated in signal transduction by different cytokines, growth factors and oncogenes. In normal cells, STAT3 activation is tightly controlled to prevent dysregulated gene transcription, whereas constitutively activated STAT3 plays an important role in tumorigenesis through the upregulation of genes involved in anti-apoptosis, proliferation and angiogenesis. Thus, pharmacologically safe and effective agents that can block STAT3 activation have the potential both for the prevention and treatment of HCC. In the present review, we discuss the possible role of STAT3 signaling cascade and its interacting partners in the initiation of HCC and also analyze the role of various STAT3 regulated genes in HCC progression, inflammation, survival, invasion and angiogenesis.

Celastrol, a plant triterpene has attracted great interest recently, especially for its potential anti-inflammatory and anti-cancer activities. In the present report, we investigated the effect of celastrol on proliferation of various cancer cell lines. The mechanism, by which this triterpene exerts its apoptotic effects, was also examined in detail. We found that celastrol inhibited the proliferation of wide variety of human tumor cell types including multiple myeloma, hepatocellular carcinoma, gastric cancer, prostate cancer, renal cell carcinoma, head and neck carcinoma, non-small cell lung carcinoma, melanoma, glioma, and breast cancer with concentrations as low as 1 μM. Growth inhibitory effects of celastrol correlated with a decrease in the levels of cyclin D1 and cyclin E, but concomitant increase in the levels of p21 and p27. The apoptosis induced by celastrol was indicated by the activation of caspase-8, bid cleavage, caspase-9 activation, caspase-3 activation, PARP cleavage and through the down regulation of anti-apoptototic proteins. The apoptotic effects of celastrol were preceded by activation of JNK and down-regulation of Akt activation. JNK was needed for celastrol-induced apoptosis, and inhibition of JNK by pharmacological inhibitor abolished the apoptotic effects. Overall, our results indicate that celastrol can inhibit cell proliferation and induce apoptosis through the activation of JNK, suppression of Akt, and down-regulation of anti-apoptotic protein expression.

Background and Purpose Aberrant activation of STAT3 is frequently encountered and promotes proliferation, survival, metastasis and angiogenesis in hepatocellular carcinoma (HCC). Here, we have investigated whether emodin mediates its effect through interference with the STAT3 activation pathway in HCC. Experimental Approach The effect of emodin on STAT3 activation, associated protein kinases and apoptosis was investigated using various HCC cell lines. Additionally, we also used a predictive tumour technology to analyse the effects of emodin . The in vivo effects of emodin were assessed in an orthotopic mouse model of HCC. Key Results Emodin suppressed STAT3 activation in a dose‐ and time‐dependent manner in HCC cells, mediated by the modulation of activation of upstream kinases c‐Src, JAK1 and JAK2. Vanadate treatment reversed emodin‐induced down‐regulation of STAT3, suggesting the involvement of a tyrosine phosphatase and emodin induced the expression of the tyrosine phosphatase SHP‐1 that correlated with the down‐regulation of constitutive STAT3 activation. Interestingly, silencing of the SHP‐1 gene by siRNA abolished the ability of emodin to inhibit STAT3 activation. Finally, when administered i.p., emodin inhibited the growth of human HCC orthotopic tumours in male athymic nu/nu mice and STAT3 activation in tumour tissues. Conclusions and Implications Emodin mediated its effects predominantly through inhibition of the STAT3 signalling cascade and thus has a particular potential for the treatment of cancers expressing constitutively activated STAT3.

Accumulating evidence(s) indicate that CXCL12-CXCR4 signaling cascade plays an important role in the process of invasion and metastasis that accounts for more than 80% of deaths in hepatocellular carcinoma (HCC) patients. Thus, identification of novel agents that can downregulate CXCR4 expression and its associated functions have a great potential in the treatment of metastatic HCC. In the present report, we investigated an anthraquinone derivative, emodin for its ability to affect CXCR4 expression as well as function in HCC cells. We observed that emodin downregulated the expression of CXCR4 in a dose-and time-dependent manner in HCC cells. Treatment with pharmacological proteasome and lysosomal inhibitors did not have substantial effect on emodin-induced decrease in CXCR4 expression. When investigated for the molecular mechanism(s), it was observed that the suppression of CXCR4 expression was due to downregulation of mRNA expression, inhibition of NF-κB activation, and abrogation of chromatin immunoprecipitation activity. Inhibition of CXCR4 expression by emodin further correlated with the suppression of CXCL12-induced migration and invasion in HCC cell lines. In addition, emodin treatment significantly suppressed metastasis to the lungs in an orthotopic HCC mice model and CXCR4 expression in tumor tissues. Overall, our results show that emodin exerts its anti-metastatic effect through the downregulation of CXCR4 expression and thus has the potential for the treatment of HCC.

Recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is currently under clinical trials for cancer, however many tumor cells, including hepatocellular carcinoma (HCC) develop resistance to TRAIL-induced apoptosis. Hence, novel agents that can alleviate TRAIL-induced resistance are urgently needed. In the present report, we investigated the potential of emodin to enhance apoptosis induced by TRAIL in HCC cells. As observed by MTT cytotoxicity assay and the externalization of the membrane phospholipid phosphatidylserine, we found that emodin can significantly potentiate TRAIL-induced apoptosis in HCC cells. When investigated for the mechanism(s), we observed that emodin can downregulate the expression of various cell survival proteins, and induce the cell surface expression of both TRAIL receptors, death receptors (DR) 4 as well as 5. In addition, emodin increased the expression of C/EBP homologous protein (CHOP) in a time-dependent manner. Knockdown of CHOP by siRNA decreased the induction of emodin-induced DR5 expression and apoptosis. Emodin-induced induction of DR5 was mediated through the generation of reactive oxygen species (ROS), as N-acetylcysteine blocked the induction of DR5 and the induction of apoptosis. Also, the knockdown of X-linked inhibitor of apoptosis protein by siRNA significantly reduced the sensitization effect of emodin on TRAILinduced apoptosis. Overall, our experimental results clearly indicate that emodin can indeed potentiate TRAIL-induced Aruljothi Subramaniam and Ser Yue Loo contributed equally to this work.

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