Melittin, a Major Component of Bee Venom, Sensitizes Human Hepatocellular Carcinoma Cells to Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL)-induced Apoptosis by Activating CaMKII-TAK1-JNK/p38 and Inhibiting IκBα Kinase-NFκB

Abstract

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 ...

Oxaliplatin Sensitizes Human Colon Cancer Cells to TRAIL Through JNK-Dependent Phosphorylation of Bcl-xL

“…8,26 On the one hand, Mucha et al 28 observed that JNK inhibition sensitizes hepatocellular carcinoma cells to TRAIL, but other groups have shown that JNK activation sensitizes hepatocellular carcinoma and breast cancer cells to TRAIL-induced apoptosis. 29,30 Our results would rather support the second conclusion because apoptosis induced by oxaliplatin/TRAIL combination was strongly suppressed by JNK inhibition or knockdown. Several studies have focused on the implication of JNK activation in apoptosis induced by the mitochondrial pathway.…”

High content analysis to determine cytotoxicity of the antimicrobial peptide, melittin and selected structural analogs

“…Melittin also inhibits Ca 2+ -ATPase in the sarcoplasmic reticulum, suggesting modulation of IC is not just related to cell membrane perturbation [36]. The peptide (1.8-3.5µM) did not alter membrane integrity of HepG2 cells when stained with annexin-FITC/PI dual staining, indicating an apoptotic process rather than necrosis [16]. Further assessment indicated that melittin increased IC followed by activation of a signaling cascade involving CAMKII-TAK1-MKK-JNK/p38.…”

“…It increased IC in osteoblast-like cells [32], fibroblasts [33], hepatocytes [16,34] and mast cells [35]. In human osteoblasts, melittin increased IC levels only when calcium was present extracellularly and not from intracellular stores [32].…”

“…National Clinical Laboratory standards), it is paramount that such an equally rapid cytotoxicity screen can be employed to detect selective toxicity for bacterial over human cells Cytotoxicity of melittin is well established in a range of mammalian cells, notably erythrocytes [12]. Its toxicity has also been detected in hepatocytes [16], enterocytes [13], intestinal goblet cells [13], and astrocytomas [29]. Incubation of Caco-2 cells for 24…”

“…Melittin is an amphipathic cationic linear 26-mer α-peptide that has cell penetrating activity as well as well-known cytotoxicity in erythrocytes [12] and enterocytes [13] and other mammalian cells [14]. Alongside phospholipase A2, it is a major constituent of the venom of Apis Mellifera, and has been found to cause apoptosis and necrosis in different cells [15,16], as well as agonist and antagonist actions on many cellular proteins including phospholipase A2 and ATPases (reviewed in [14]). Melittin itself cannot be used clinically as an antimicrobial because it is equally damaging to mammalian cells.…”