Melittin, a water-soluble toxic peptide derived from bee venom of Apis mellifera was reported to have inhibitory effects on hepatocellular carcinoma (HCC). However, its role in antimetastasis and the underlying mechanism remains elusive. By utilizing both HCC cell lines and an animal model based assay system, we found that Rac1, which has been shown to be involved in cancer cell metastasis, is highly expressed in aggressive HCC cell lines and its activity correlated with cell motility and cytoskeleton polymerization. In addition, Rac1-dependent activity and metastatic potential of aggressive HCC cells are remarkably high in both cellular and nude mouse models. We provide evidence here that melittin inhibits the viability and motility of HCC cells in vitro, which correlates with its suppression of Rac1-dependent activity, cell motility, and microfilament depolymerization. Furthermore, melittin suppresses both HCC metastasis and Rac1-dependent activity in nude mouse models. The specificity of the effect of melittin on Rac1 was confirmed in HCC cells both in vitro and in vivo. Conclusion: Melittin inhibits tumor cell metastasis by reducing cell motility and migration via the suppression of Rac1-dependent pathway, suggesting that melittin is a potential therapeutic agent for HCC. (HEPATOLOGY 2008;47:1964-1973 H epatocellular carcinoma (HCC) is one of the most aggressive malignant tumors highly prevalent in Asia and Africa. 1 Recent studies show that the incidence of HCC in the United States and the United Kingdom has increased substantially in the past 2 decades. 2,3 Despite intense efforts to improve its prognosis, the overall survival rate of patients with HCC is very low. [4][5][6] The major obstacles to survival are metastasis and recurrence after HCC resection. 7 However, the molecular mechanism for HCC metastasis remains unclear, and the means to inhibit HCC metastasis are limited.It has been shown that HCC cell invasiveness and metastasis are regulated by multiple cues, including extracellular molecules such as cell adhesion molecules, proteases, angiogenesis factors, cytokines, and growth factors, and the underlying signaling transduction components such as Rac1. Rac1, a member of the Ras superfamily of small GTP (guanosine triphosphate)-binding protein is known to play important roles in the regulation of distinct microfilament-based structures, which is required for cell adhesion, migration, and invasion. It has been demonstrated that Rac1 can promote tumor cell migration and invasion for multiple types of cancer such as renal, breast, and liver carcinomas. Rac1 is involved in the activation of c-Jun N-terminal kinase (JNK) and JNK-dependent cell motility. 8,9 A dominant-negative form of Rac1 (Rac12 DN or Rac1 N17) blocks the changes in cell shape and the formation of adhesion complexes induced by growth factors. Conversely, microinjection of a constitutively active form of Rac1 (Rac1 DA or Rac1 V12) into fibroblasts induces changes in cytoskeleton and cell morphology in the absence of growth factors. 10 T...

Promoting apoptosis is a strategy for cancer drug discovery. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in a wide range of malignant cells. However, several cancers, including human hepatocellular carcinoma (HCC), exhibit a major resistance to TRAIL-induced cell death. Melittin, a water-soluble 26-amino acid peptide derived from bee venom of Apis mellifera, can exert toxic or inhibitory effects on many types of tumor cells. Here we report that melittin can induce apoptosis of HCC cells by activating Ca 2؉ / calmodulin-dependent protein kinase, transforming growth factor-␤-activated kinase 1 (TAK1), and JNK/p38 MAPK. We show that melittin-induced apoptosis can be inhibited by calcium chelator, by inhibitors for Ca 2؉ /calmodulin-dependent protein kinase, JNK and p38, and by dominant negative TAK1. In the presence of melittin, TRAIL-induced apoptosis is significantly increased in TRAIL-resistant HCC cells, which may be attributed to melittin-induced TAK1-JNK/p38 activation and melittin-mediated inhibition of IB␣ kinase-NFB. Our data suggest that melittin can synergize with TRAIL in the induction of HCC cell apoptosis by activating the TAK1-JNK/p38 pathway but inhibiting the IB␣ kinase-NFB pathway. Therefore, the combination of melittin with TRAIL may be a promising therapeutic approach in the treatment of TRAIL-resistant human cancer.At present about 20 different ligands that belong to the tumor necrosis factor (TNF) 4 superfamily have been identified, among which TNF␣, lymphotoxin ␣, Fas ligand, apo3L, and TNF-related apoptosis-inducing ligand (TRAIL) have been characterized as major mediators of apoptosis (1, 2). TRAIL, in its soluble form, is emerging as an attractive anticancer agent because of its cancer cell specificity and potent antitumor activity (1, 2).TRAIL signals by interacting with its receptors (1). Thus far, five receptors (Fas ligand) have been identified, namely the two agonistic receptors, TRAIL-R1 and TRAIL-R2, and the three antagonistic receptors TRAIL-R3, TRAIL-R4, and osteoprotegerin (3-6). Binding of TRAIL to the extracellular domain of agonistic receptors results in the trimerization of the receptors and clustering of the intracellular death domains, which lead to the recruitment of the adaptor molecule Fas-associated protein with death domain (FADD) (1). Subsequently, FADD recruits and activates initiator caspase-8 and caspase-10, leading to cellular disassembly (1). Meanwhile, TRAIL-initiated apoptotic signaling requires an amplification loop by mitochondrial pathway engagement through impairment of the mitochondrial membrane permeability regulated by Bcl-2 family members, which sequentially leads to cytochrome c or Smac/DIABLO (second mitochondrial activator of caspases/direct IAP-binding protein with low isoelectric point (pI)) release, apoptosome formation, and the final DNA fragmentation (7).Similar to TNF-induced activation of the nuclear factor B (NFB) transcription factor and the MAPK pathway (1, 2), TRAIL can also initiate the activation of ...

Glucocorticoids (GCs) are widely used to treat inflammatory diseases. However, they cause debilitating side effects, which limit the use of these compounds. In the past decade, many researchers have attempted to find so-called dissociated GCs that have separate distinct transactivation and transrepression activities. Anti-inflammation of GCs is a result of glucocorticoid receptor (GR)-mediated transactivation and transrepression in some tissues, similar to their side effects; therefore, the goal to discover a compound that has anti-inflammatory properties, but lacks the negative side effects seen with GCs, has yet to be achieved. In the present study, we introduce a plant-derived compound, ginsenoside Rg1, which possesses GC and estrogen-like activities. In this study, we show that Rg1 downmodulates LPS-induced proinflammatory cytokine release and inhibits NF-κB nuclear translocation and DNA binding activity. The negative effects on NF-κB activation are due to a decrease in IκB phosphorylation and protein stabilization. Furthermore, the inhibitory effect of Rg1 on NF-κB is GR-dependent, as small interfering RNA knockdown of GR abrogated this function. Rg1 also displayed profound inhibitory effects on LPS-induced MAPK activation. Importantly, Rg1 did not impair proliferation or differentiation of mouse osteoblasts. Finally, we show that Rg1 can effectively inhibit acute and chronic inflammation in vivo, but it does not cause hyperglycemia or osteoporosis as seen with dexamethasone. These results suggest that ginsenoside Rg1 may serve as a novel anti-inflammatory agent and may exhibit a potential profile for therapeutic intervention in inflammatory diseases.

BackgroundHepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. Frequent tumor recurrence after surgery is related to its poor prognosis. Although gene expression signatures have been associated with outcome, the molecular basis of HCC recurrence is not fully understood, and there is no method to predict recurrence using peripheral blood mononuclear cells (PBMCs), which can be easily obtained for recurrence prediction in the clinical setting.MethodsAccording to the microarray analysis results, we constructed a co-expression network using the k-core algorithm to determine which genes play pivotal roles in the recurrence of HCC associated with the hepatitis B virus (HBV) infection. Furthermore, we evaluated the mRNA and protein expressions in the PBMCs from 80 patients with or without recurrence and 30 healthy subjects. The stability of the signatures was determined in HCC tissues from the same 80 patients. Data analysis included ROC analysis, correlation analysis, log-lank tests, and Cox modeling to identify independent predictors of tumor recurrence.ResultsThe tumor-associated proteins cyclin B1, Sec62, and Birc3 were highly expressed in a subset of samples of recurrent HCC; cyclin B1, Sec62, and Birc3 positivity was observed in 80%, 65.7%, and 54.2% of the samples, respectively. The Kaplan-Meier analysis revealed that high expression levels of these proteins was associated with significantly reduced recurrence-free survival. Cox proportional hazards model analysis revealed that cyclin B1 (hazard ratio [HR], 4.762; p = 0.002) and Sec62 (HR, 2.674; p = 0.018) were independent predictors of HCC recurrence.ConclusionThese results revealed that cyclin B1 and Sec62 may be candidate biomarkers and potential therapeutic targets for HBV-related HCC recurrence after surgery.

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Bufalin has significant anti-tumor activities in the orthotopic transplantation tumor model of human hepatocellular carcinoma in nude mice with no marked toxicity and was able to induce apoptosis of transplanted tumor cells. This apoptosis may be mediated mainly via up-regulating the expression of apoptosis-regulated gene bax, which may be involved in its anti-tumor mechanism of bufalin.

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