Effect Of Melittin Sensitizes Human Hepatocellular Carcinoma Cells To TRAIL-induced Apoptosis

Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third leading cause of cancer-related mortality worldwide. In China, HCC is the second cause of death due to cancer. HCC usually develops in the presence of continuous inflammation and hepatocyte regeneration in the setting of chronic hepatitis and cirrhosis, although the molecular mechanisms linking chronic inflammation to malignant transformation remain to be further defined. Treatment of HCC is complex. Involvement of surgery,chemotherapy and radiotherapy, but up to now there has been no satisfying result. The majority of patients with HCC presented with an advanced stage beyond surgical treatment. In addition, chemotherapy and radiotherapy have limited efficacy in hepatocellular carcinoma of an advanced stage. So it is important to explore effective drugs and combination methods for therapy of this disease.Melittin is the principal toxic component in the venom of the European honey bee Apis mellifera and is a cationic, hemolytic peptide. It is a small linear peptide composed of 26 amino acid residues in which the amino-terminal region is predominantly hydrophobic whereas the carboxy-terminal region is hydrophilic. It has been reported that melittin has multiple effects, inculding antibacteria, antivirus and anti-inflammation, in various cell types. We and others have shown that melittin can induce cell cycle arrest, growth inhibition and apoptosis in various tumor cells. However, the mechanisms of the anti-cancer effects of melittin have not been fully elucidated.TNF-related apoptosis-inducing ligand (TRAIL) is a member of tumor necrosis factor (TNF) superfamily. In its soluble form,it is emerging as an attractive anticancer agent because of its cancer cell specificity and potent antitumor activity.TRAIL signals by interacting with its receptors. Thus far, five receptors (TRAIL-R) have been identified, namely, the two agonistic receptors, TRAIL-R1 and TRAIL-R2, and the three antagonistic receptors TRAIL-R3, TRAIL-R4, and osteoprotegerin. Binding of TRAIL to the extracellular domain of agonistic receptors results in the trimerization of the receptors and clustering of the intracellular death domains (DDs), which leads to the recruitment of the adaptor molecule Fas-associated protein with death domain (FADD). Subsequently, FADD recruits and activates initiator caspase-8 and caspase-10, leading to cellular disassembly.Meanwhile, TRAIL-initiatedapoptotic signaling requires an amplification loop by mitochondrial pathwayengagement 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.Similar to TNF-induced activation of the nuclear factorκB (NFκB) transcription factor and the mitogen-activated protein kinase (MAPK) pathway,TRAIL can also initiate the activation of signaling pathways that involve the adaptor molecules TNF receptor-associated factor 2 (TRAF2), receptor interacting protein(RIP) and transforming growth factor-β(TGF-β)-activated kinase 1 (TAK1), finally leading to the activation of the MAPK pathway (including extravellular signal-related kinase ERK1/2, c-Jun N-terminal kinase JNK1/2, and p38) and the IκBαkinase (IKK)-NFκB pathway.Unfortunately, human hepatoma-derived cell types exhibit a major resistance to TRAIL-induced cell death. In the current study, we tested the effects of melittin in the induction of apoptosis of HCC cells and explored the mechanisms involved in melittin-induced apoptosis of TRAIL-resistant HepG2 cells. We show that melittin can initiate an apoptotic machinery that depends on calcium influx and activation of Ca2+/calmodulin(CaM)-dependent protein kinase (CaMKⅡ)-TAK1-JNK/p38 signaling pathway. Moreover, we find that melittin can sensitize HCC cells to TRAIL-induced apoptosis by activating CaMKⅡ-TAK1-JNK/p38 but inhibiting IKK- NFκB pathways. The differential roles of melittin in activation of TAK1-JNK/p38 but inhibition of IKK-NFκB in response to TRAIL may hold true for other TNF superfamily members because we also find that melittin can potentiate the TNFα-induced activation of TAK1-JNK/p38 but inhibit TNFα-induced activation of IKK-NFκB. Additionally, we find that melittin can not potentiate the drug-induced cell death of BEL-7402 cells that have been selected under chemotherapeutic drugs adriamycin or 5-fluorouracil to establish multi-drug resistance. Therefore, the sensitization of HCC cells to apoptosis by melittin may only be applicable to a limited set of apoptotic stimuli that employ apoptotic machinery similarly to TRAIL. Whether melittin can potentiate apoptosis by the other cancer treatments, however, may need further investigations.In conclusion, we have demonstrated in the present study that melittin potentiated the apoptotic effects of TRAIL in human HCC cells by activating CaMKⅡ-TAK1-JNK/p38 pathway but inhibiting IKK-NFκB pathway. Our data suggest that melittin may exhibit anti-tumor activity by sensitizing HCC cells to TRAIL-mediated apoptosis, and that the combination of TRAIL with melittin may have therapeutic potential in the treatment of human HCC.