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*

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 Ca2+/calmodulin-dependent protein kinase, transforming growth factor-β-activated kinase 1 (TAK1), and JNK/p38 MAPK. We show that melittin-induced apoptosis can be inhibited by calcium chelator, by inhibitors for Ca2+/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 IκBα kinase-NFκB. 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 IκBα kinase-NFκB pathway. Therefore, the combination of melittin with TRAIL may be a promising therapeutic approach in the treatment of TRAIL-resistant human cancer.

Melittin is the principal toxic component in the venom of the European honey bee Apis mellifera and is a cationic, hemolytic peptide (13). It is a small linear peptide composed of 26 amino acid residues in which the amino-terminal region is predominantly hydrophobic, whereas the carboxyl-terminal region is hydrophilic. It has been reported that melittin has multiple effects, including antibacteria, antivirus, and anti-inflammation, in various cell types (13). We and others have shown that melittin can induce cell cycle arrest, growth inhibition, and apoptosis in various tumor cells (14 -18). However, the mechanisms of the anti-cancer effects of melittin have not been fully elucidated.
HCC is one of the most common cancers in the world. Unfortunately, human hepatoma-derived cell types exhibit a major resistance to TRAIL-induced cell death (19 -26). In this study, we tested the effects of melittin in the induction of apoptosis of HCC cells and explored the mechanisms involved in melittininduced apoptosis of TRAIL-resistant HepG2 cells. We show that melittin can initiate an apoptotic machinery that depends on calcium influx and activation of Ca 2ϩ /calmodulin-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-NFB pathways.
Apoptosis Assay-After treatments with melittin or TRAIL, cells were labeled with annexin V and propidium iodide (PI) provided by Molecular Probes (Eugene, OR). Mitochondrial membrane potential was measured by labeling cells with 1 M rhodamine 123 (Rho123, Molecular Probes) at 37°C for 15 min. The production of ROS was analyzed by labeling cells with 10 M dihydrorhodamine 123 (DHR123, Molecular Probes) for 15 min. Samples were examined by fluorescence-activated cell sorter (FACS) analysis, and the results were analyzed using CellQuest software (BD Biosciences) as described (29).
Mitochondrial Isolation-For the isolation of mitochondria after melittin treatments, the mitochondrial isolation kit for cultured cells (Pierce) was used as instructed.
RNA Quantification-Quantitative real time reverse transcription-PCR analysis was performed by LightCycler (Roche Applied Science) and SYBR reverse transcription-PCR kit (Takara, Dalian, China). Data were normalized by the level of ␤-actin. The experiments were performed as described previously (30).
Western Blot Assay-Western blot assay was performed as described by us previously (29). Bands were revealed using Supersignal West Femto maximum sensitivity substrate (Pierce).
Caspase-3 Activity Assay-For the examination of caspase-3 activation, whole cell lysates were subjected to ELISAs of cleaved caspase-3 by using sandwich ELISA kit (Cell Signaling Technology) as instructed.
Assay of Luciferase Reporter Gene Expression-Cells were transfected with the pGL3.5XB-luciferase reporter plasmid, RL-TK-Renilla-luciferase plasmid, and treated as indicated. Total amounts of plasmid DNA were equalized via empty control vector. Luciferase activities were measured with the dual luciferase reporter assay system (Promega) as described previously (30). Data are normalized for transfection efficiency by dividing firefly luciferase activity with that of Renilla luciferase.
In Vitro Kinase Assay-For the analysis of kinases activity, cells were lysed in lysis buffer containing 20 mM Tris, pH 7.5, 300 mM NaCl, 2 mM EDTA, 25 mM ␤-glycerophosphate, 2 mM p-nitrophenyl phosphate, 1 mM sodium orthovanadate, 10% glycerol, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride. Then the indicated kinases were immunoprecipitated with specific antibodies and protein A/G beads (Pierce). After washing extensively with the lysis buffer for three times, the beads were suspended in the kinase assay buffer containing 25 mM Tris, pH 7.5, 5 mM ␤-glycerol phosphate, 2 mM dithiothreitol, 0.1 mM sodium orthovanadate, and 10 mM MgCl 2 . Then 100 M ATP and 2 g of MKK4 or IB␣ was added and incubated for 30 min at 30°C. The reaction was terminated by adding SDS sample buffer. The phosphorylated substrates were detected by Western blot.
Tumor Establishment and in Vivo Treatments-HepG2 or SMMC-7721 cells (1 ϫ 10 7 cells/animal) were injected subcutaneously into BALB/c nude mice (SIPPR-BK Experimental Animal Co., Shanghai, China) to produce implanted tumors. For the treatments of the pre-established tumor, melittin (50 or 100 g/kg) or TRAIL (10 mg/kg) was intravenously injected via tail vein daily for 7 days (17,31). Then volumes of tumors were measured with a slide caliper and were evaluated by the following equation: a (the larger diameter) ϫ b (the smaller diameter) 2 /2.
Isolation of Tumor Cells from Tissue Samples-Tumor nodules were excised. The fresh tissues were then cut into small fragments and incubated in RPMI 1640 medium containing 10 mg/ml collagenase and DNase (4 ml/g of tumor tissue; Sigma) at 4°C for 1 h. Single cell suspension was prepared by pressing the digested tissues through a stainless 200-gauge mesh. After hypotonic lysis of red blood cells, cells were washed three times and resuspended in 10 ml of complete RPMI 1640 medium. Tumor cells were enriched by centrifugation on a Ficoll-Hypaque gradient. The collected tumor cells contained more than 95% purity of tumor cells, as confirmed by histology examination. Lysates were extracted and subjected to assays for caspase-3 activation.
Statistical Analysis-All the experiments were repeated at least three times. Results are given as mean Ϯ S.E. or mean Ϯ S.D. Comparisons between the two groups were done using Student's t test analysis. Multiple comparisons were done with a one-way analysis of variance followed by Fisher's least significant difference analysis. Statistical significance was determined as p Ͻ 0.05.

RESULTS
Melittin Induces Apoptosis of HCC Cells-To determine the effects of melittin on HCC cells, we treated HepG2 cells with melittin at the concentration of 5 or 10 g/ml and examined the apoptosis by annexin V/PI double staining. We found that melittin could induce apoptosis of HepG2 cells (Fig. 1, A and B). Similar effects were observed in SMMC-7721, BEL-7402, and Hep3B HCC cell lines (Fig. 1C).
Moreover, we found that the mitochondrial membrane potential of HepG2 cells was decreased by melittin, as evidenced by the increase in Rho123 low cells (Fig. 1D). The production of ROS (DHR123 high cells) was also significantly increased after melittin treatments (Fig. 1E).
Melittin Activates Caspase-3 and -9 and PARP and Promotes the Release of Cytochrome c and Smac/DIABLO from Mitochondria-To elucidate the mechanisms involved in melittin-induced apoptosis of HCC cells, we first examined the activation of caspases by melittin. We found that melittin could induce the cleavage of caspase-3 and -9 and PARP but not caspase-8, which could be blocked by the calcium chelator BAPTA and ROS inhibitor NAC, indicating that melittin-induced activation of caspase-3 and -9 and PARP was initiated by the calcium influx and the production of ROS ( Fig. 2A). Moreover, we found that the pretreatment of cells with caspase-3 inhibitor Z-DEVD-FMK, caspase-9 inhibitor Z-LEHD-FMK, BAPTA, or NAC could inhibit the melittin-induced apoptosis (Fig. 2B).
The data (Fig. 1D) suggested that melittin treatments may have disrupted the membrane permeability of mitochondria. To verify this mechanism, we isolated mitochondrial and cytosol fractions of HepG2 cells. We found that melittin treatments could induce the release of cytochrome c and Smac/ DIABLO from mitochondria to cytosol (Fig. 2C), indicating that melittin-induced apoptosis may also involve the mitochondrial pathway of apoptosis. Then we tested the roles of calcium influx and ROS production on mitochondrial release of cytochrome c and Smac/DIABLO. We found that both BAPTA and NAC could inhibit the release of cytochrome c and Smac/DIABLO into the cytosol (Fig. 2C), indicating that the mitochondrial disruption by melittin may be subsequent to calcium influx and ROS production.
Melittin Activates CaMKII-TAK1-JNK/p38 Pathway-Previously it has been demonstrated that melittin can activate the calcium channel and induce the influx of calcium from extracellular medium (13-15, 32, 33). However, the signaling pathways activated by melittin have not been fully understood.
CaMKII is a ubiquitous mediator of Ca 2ϩ -linked signaling that phosphorylates a wide range of substrates to coordinate and regulate Ca 2ϩ -mediated alterations in cellular function (34). We find that melittin treatments could activate CaMKII, which could be blocked by the calcium chelator BAPTA (Fig. 3A) in HepG2 cells, indicating that melittin could activate CaMKII by increasing cytosol Ca 2ϩ concentration.
TAK1 is a MAPK kinase kinase (MEKK) that has been shown to activate MKK3, -4, and -6 and subsequent JNK1/2 and p38 signaling pathway (35,36). Considering that TAK1 is a substrate for CaMKII (37), we hypothesized that melittin-induced activation of JNK/p38 may be due to activation of CaMKII. After melittin treatments of HepG2 cells, we found that the phosphorylated TAK1 (p-TAK1) was increased (Fig. 3B), which could be blocked by CaMKII inhibitor KN62, indicating that melittin could activate TAK1 via CaMKII. Correspondingly, we detected increased levels of phosphorylated MKK3/6, MKK4, JNK1/2, and p38 after melittin treatments, which could also be blocked by KN62 (Fig. 3B). To verify the roles of TAK1 in activation of JNK/p38, we transfected dominant negative TAK1K63W in HepG2 cells and treated the cells with melittin. We found that melittin-induced activation of MKK3/6, MKK4,  JNK1/2, and p38 was inhibited (Fig. 3C). The activation of JNK/ p38 by melittin and the involvement of TAK1 in melittin-induced JNK/p38 activation were also verified by ELISAs of the levels of phosphorylated JNK/p38 (Fig. 3, D and E). These data together suggest that melittin could activate a signaling pathway sequentially involving CaMKII-TAK1-MKK-JNK/p38.
Melittin-induced Apoptosis Is Dependent on the Activation of CaMKII-TAK1-JNK/p38 Signaling Pathway-The above results have demonstrated that melittin alone may induce apoptosis of HCC cells by activating caspase-3 and disruption of mitochondria. We then tested the relationship of the CaMKII-TAK1-JNK/p38 signaling pathway with melittin-induced apoptosis. When HepG2 cells were pretreated with kinase inhibitors KN62, SP600125, and SB203580, we found that melittin-induced apoptosis was significantly inhibited (Fig. 3F). Transient transfection of TAK1K63W in HepG2 could also significantly inhibit the apoptosis induced by melittin (Fig. 3G).
Melittin Inhibits TAK1-mediated Activation of IKK-NFB Signaling Pathway-To elucidate whether melittin-mediated activation of caspase-3 and CaMKII was dependent on the elevation of cytosol Ca 2ϩ concentration, we treated HepG2 cells with ionomycin (calcium ionophore) and examined its effects on melittin-induced apoptosis. We found that ionomycin alone was not sufficient to induce apoptosis of HepG2 cells but was sufficient to activate CaMKII-TAK1-JNK/p38 signaling pathway (data not shown). However, ionomycin alone could increase the kinase activity of IKK␤ (Fig. 4A) and the activation of NFB gene reporter (Fig. 4B), which could be inhibited by CaMKII inhibitor KN62 and the dominant negative TAK1K63W expression (Fig. 4, A-D), indicating that elevation of intracellular Ca 2ϩ could activate CaMKII-TAK1-IKK-NFB pathway.
Previously, it has been reported that melittin can bind IKK␣/␤ and inhibit the expression of inflammatory targets and intermediate mediators in RAW264.7 cells and synoviocytes (38). In HepG2 cells, we found that melittin treatments could not alter the kinase activity of IKK␤, phosphorylation of IB␣, and NFB gene reporter (Fig. 4, A and B), indicating that melittin alone may not exert inhibitory effects on IKK-NFB in HCC cells.
Considering that melittin alone could activate CaMKII-TAK1-MKK-JNK/p38 (Fig. 3, A and B) but did not affect IKK-NFB (Fig. 4, A and B), we tested whether melittin could inhibit TAK1-induced activation of IKK-NFB. We found that transient transfection of TAK1 and IKK␤ could activate the NFB gene reporter (Fig. 4, E and F). However, melittin could block TAK1-and IKK␤-induced NFB activation (Fig. 4, E and F), suggesting that melittin inhibited the NFB activation subsequent to CaMKII-TAK1 activation. However, we found that melittin could not inhibit the NFB activation induced by p65/ RelA expression (Fig. 4G). Therefore, it may be inferred that melittin could simultaneously activate CaMKII-TAK1-MKK-JNK/p38 and inhibit CaMKII-TAK1-IKK-NFB initiated by calcium influx, which was confirmed by the findings that melittin could inhibit ionomycin-induced activation of NFB (Fig. 4,  A and B).
Melittin Sensitizes HCC Cells to TRAIL-induced Apoptosis-TRAIL-associated strategy in cancer treatments is promis-ing and is being tested in clinical trials (2). However, most of the HCC cells were resistant to TRAIL treatments (19 -26). Because melittin could activate CaMKII-TAK1-JNK/p38 but inhibit IKK-NFB in HCC cells, it may be expected that melittin may increase the sensitivity of HCC cells to TRAIL. In HepG2 cells, TRAIL alone was inefficient in the induction of apoptosis (Fig. 5A). The combination of melittin with TRAIL could increase the TRAIL-induced apoptosis of HepG2 cells (Fig. 5A). To further investigate the possible synergistic effects of melittin and TRAIL in the induction of HCC apoptosis, we treated various HCC cells with melittin (5 g/ml) in combination with different doses of TRAIL (0 -200 ng/ml). We found that both in TRAIL-resistant HCC cells (HepG2, Hep3B, SMMC-7721, and BEL-7402, the sensitivity to TRAIL-induced apoptosis that was different) and in TRAIL-sensitive HeLa and Jurkat cells, melittin was capable of promoting TRAIL-induced apoptosis (Fig. 5, B and C). Moreover, we found that melittin could augment the effects of TRAIL in activation of caspase-3, cleavage of PARP, and mitochondrial release of cytochrome c and Smac/DIABLO, which confirmed that melittin could promote TRAIL-induced apoptosis (data not shown). Taken together, these results indicated that melittin could sensitize HCC cells (and possibly other tumor cells) to TRAIL-induced apoptosis.
Melittin Synergizes with TRAIL in Activation of TAK1-JNK/p38-TRAIL can induce apoptosis via FADD-caspase-8caspase-3 pathway and initiate the activation of MAPK and NFB (1). In the presence of melittin, TRAIL-induced activa-tion of JNK/p38 was augmented (Fig. 6A), as evidenced by ELISAs of the phosphorylated levels of JNK1/2 (p-JNK1/2) and p38 (p-p38). As further evidence, we also examined the activation of JNK/p38 30 min after melittin and TRAIL treatments by Western blot, and we found that melittin did potentiate the TRAIL-induced activation of JNK/p38 (data not shown). More importantly, we found that the melittin-mediated potentiation of TRAIL-induced apoptosis was attenuated by inhibitors specific for JNK1/2 (SP600125) and p38 (SB203580) (Fig. 6B), indicating that melittin-mediated sensitization of HepG2 cells to TRAIL may be exerted via activation of the JNK1/2 and p38 kinases. Similar results were obtained in BEL-7402 and SMMC-7721 HCC cells (data not shown).
TRAIL can activate the JNK/p38 signaling pathways via activation of TAK1-MKK (14 -18), whereas melittin may activate JNK1/2 and p38 via the CaMKII-TAK1 signaling pathways (Fig.  3, A-E). Therefore, we examined the effects of melittin on TRAIL-induced activation of TAK1. We found that melittin could increase the phosphorylated levels of TAK1 (p-TAK1)  and the kinase activity of TAK1 when compared with TRAIL or melittin treatments alone (Fig. 6C).
To investigate the potential roles of TAK1 in melittin-mediated potentiation of TRAIL-induced activation of JNK/p38, we examined the activation status of JNK/p38 after transient transfection of wild type TAK1 and TAK1K63W. We found that TAK1 overexpression could potentiate the activation of JNK/ p38 in melittin-and TRAIL-treated cells, but to a lesser extent than that observed in melittin ϩ TRAIL-treated cells (Fig. 6D). However, TAK1K63W overexpression could inhibit the activation of JNK/p38 in melittin-, TRAIL-, and melittin ϩ TRAILtreated cells. These data (Fig. 6D) together suggest that melittin potentiates TRAIL-induced activation of JNK/p38 via the synergistic activation of TAK1.
Next we examined the roles of TAK1 activation in the synergistic effects of melittin plus TRAIL on apoptosis of HCC cells by overexpressing TAK1 and TAK1K63W. We found that TAK1K63W overexpression inhibited the apoptosis induced by melittin alone and melittin ϩ TRAIL treatments (Fig. 6E), although TAK1 overexpression could partially reverse the melittin-induced promotion of TRAIL-induced apoptosis (Fig. 6E).
Melittin Inhibits TRAIL-induced Activation of IKK-NFB-NFB usually plays protective roles in apoptosis of various cells in response to TNF superfamily molecules (1). Previously it has been demonstrated that melittin can inhibit the IKK-IB␣-NFB signaling pathway in macrophages (38). Furthermore, we have detected the inhibition of TAK1-mediated activation of IKK-IB␣-NFB by melittin (Fig. 4, D and E). Therefore, we examined the effects of melittin on TRAIL-induced activation of IKK-IB␣-NFB. We found that TRAIL could increase the kinase activity of IKK␤ (Fig. 7A) and the phosphorylated levels of IB␣ (Fig. 7B). However, melittin pretreatments could inhibit TRAIL-induced activation of IKK␤ and IB␣ (Fig. 7, A  and B). As further evidence, we found that melittin could inhibit TRAIL-induced activation of the NFB gene reporter (Fig. 7C).
To investigate the roles of melittin-mediated inhibition of TRAIL-induced activation of NFB in the apoptosis of HCC cells, we examined the effects of melittin in TRAIL-induced apoptosis after overexpressing IKK␤ and p65/RelA. We found that melittin-mediated potentiation of TRAIL-induced apoptosis in HepG2 cells could be greatly reversed by overexpressing p65/RelA but not by overexpressing IKK␤ (Fig. 7E), indicating that melittin-mediated potentiation of TRAIL-induced apoptosis was partially through the inhibition of IKK␤-mediated activation of NFB.
NFB activation usually protects cells from apoptosis by upregulating the expression of anti-apoptotic molecules like Bclxl, cIAP, XIAP, and FLIP, etc. (24 -26, 39, 40). TRAIL alone could increase the expression of Bcl-xl and c-IAP1 in HepG2 cells, whereas melittin could inhibit the TRAIL-induced upregulation of these anti-apoptotic molecules (Fig. 7F), indicating that melittin had redirected the TRAIL signaling to inhibition of NFB pathway.
Melittin Synergizes with TRAIL in the Treatment of Human HCC-To examine the efficiency of combining melittin and TRAIL in treatment of HCC in vivo, we established HepG2 and SMMC-7721 tumor in nude mice and treated the pre-established tumor 7 days after tumor inoculation by intravenous injection of melittin and/or TRAIL daily for seven episodes. We found that the combination of melittin with TRAIL could significantly inhibit the growth of both tumors, which was more remarkable than melittin or TRAIL alone (Fig. 8A). To examine whether intravenous injection of melittin and TRAIL induced apoptosis in vivo, we isolated HepG2 tumor cells from excised tumor nodules on days 7 (without treatments), 10 (24 h after the third treatment), and 15 (24 h after the seventh treatment) of tumor inoculation and examined the activation of caspase-3 by ELISA. We found that melittin could synergize with TRAIL in caspase-3 activation (Fig. 8B), indicating that melittin may synergize with TRAIL in induction of apoptosis in vivo.

DISCUSSION
In this study, we have demonstrated that melittin can induce the apoptosis of HCC cells potentially by activating CaMKII-TAK1-JNK/p38 signaling pathway. More importantly, we have demonstrated that melittin can sensitize HCC cells to TRAILinduced apoptosis by activating CaMKII-TAK1-JNK/p38 pathway but inhibiting IKK-NFB pathway. Our study has suggested that the combination of melittin with TRAIL may be promising in the treatment of human HCC.
Melittin has been demonstrated to have multiple functions in various cell types (13). The intracellular targets for melittin have been reported by several groups, such as calcium channel, calmodulin, phospholipase A 2 and phospholipase D, mitochondrial F 1 -ATPase, sphingomyelinase, IKK␣/␤, and Rac1, etc. (13-15, 17, 32, 33, 38, 41, 42). However, the signaling mechanisms, especially the detailed events taking place from the cell membrane to the nuclear activation of transcription factors, responsible for melittin-mediated inhibitory effects in tumor cells have not been elucidated. Upon treatment of cells with melittin, the initial cascade may be the activation of calcium channels and phospholipase A 2 , leading to the elevation of intracellular Ca 2ϩ concentration and the activation of calciumsensitive CaMKII. In our study, we show that melittin can rapidly activate CaMKII, which was dependent on calcium elevation, mimicking the effects of the calcium ionophore ionomycin. TAK1 is one of the targets of CaMKII and has been shown previously to be widely involved in the activation of IKK␣/␤ by IL-1R, Toll-like receptors, and TNF receptors (35)(36)(37). TAK1 activation can lead to the subsequent activation of MKK-JNK/p38 and IKK␣/␤-IB␣-NFB pathways (35,36). We suggest that melittin can activate TAK1 via calcium-dependent activation of CaMKII, which may provide a linkage between calcium influx and activation of MAPK/NFB. The inhibition of melittin-induced apoptosis by kinase inhibitors KN62, SP600125, SB203580, and dominant negative TAK1 suggests that melittin-induced apoptosis may be dependent on the activation of the CaMKII-TAK1-MKK-JNK/p38 pathway.
One controversy in our study is that melittin alone does not affect the IKK-NFB pathways despite the activation of CaMKII-TAK1 by melittin. We show that ionomycin treatments or overexpression of TAK1 and IKK␤ can activate the IKK-NFB pathways, which can be inhibited by melittin, suggesting that melittin may exert inhibitory effects on IKK-NFB signaling pathways. The data that melittin cannot inhibit the p65/RelA-induced activation of NFB suggest that IKK may be the target of melittin in the IKK-NFB pathways in HCC cells. Previously melittin has been demonstrated to bind and inhibit the activity of IKK␣/␤ in RAW264.7 macrophages (38). However, a later report suggests that melittin does not affect the NFB p50-DNA interactions nor the activation of NFB in human synoviocytes and dermal fibroblasts (43), indicating that the effects of melittin on IKK-NFB pathways may varied between cell types. Our study suggests inhibitory effects of melittin on IKK-NFB pathways in human HCC cells, which may contribute to melittin-induced apoptosis of HCC cells.
Resistance of tumor cells to TRAIL treatments is a major barrier of TRAIL application in cancer (1,2). The combination of TRAIL reagents with other treatments may increase the efficiency of cancer treatments. It has been reported that TRAIL, in combination with chemotherapy, radiotherapy, and other bioactive reagents (such as IFN␣), can increase clinical response in in BALB/c nude mice (n ϭ 10 per group), TRAIL (10 mg/kg) and/or melittin (50 or 100 g/kg) was rapidly intravenously injected daily for 7 days. On the indicated days, the tumor volume was measured and evaluated by the following equation: (a ϫ b 2 /2). Results are presented as mean Ϯ S.E. (n ϭ 10). *, p Ͻ 0.05; **, p Ͻ 0.01; ***, p Ͻ 0.001. B, on day 7 (without treatments), 10 (24 h after the third treatment) or 15 (24 h after the seventh treatment) of tumor inoculation, HepG2 tumor cells were isolated and purified. Caspase-3 activity in the whole cell lysates (100 g of protein) was determined by ELISAs. Results were presented as mean Ϯ S.D. of triplicate samples. ***, p Ͻ 0.001. various human cancers (1, 2, 19 -26). HCC cells are resistant to TRAIL treatments both in vitro and in vivo (19 -26). Melittin is a bioactive component of bee venom, although its effects on HCC cells in combination with TRAIL have not been examined. In our study, we demonstrate that melittin can sensitize TRAIL-resistant HepG2 cells to TRAIL treatments by showing melittin can increase the apoptosis of HCC cells in the presence of lower concentrations of TRAIL (as low as 10 ng/ml). The observed synergistic induction of apoptosis by melittin plus TRAIL may not be due to a simple summary effect of individual agents because melittin can significantly increase the apoptosis of TRAIL-resistant HCC cells even when TRAIL alone (10 ng/ml) is unable to induce apoptosis. Additionally, we have detected the synergistic effects of melittin plus TRAIL in other TRAIL-sensitive cell lines. Therefore, melittin may be a novel agent capable of sensitizing HCC cells to TRAIL treatments, which thus awaits clinical trials.
TRAIL signals both apoptosis and survival, which may be differentially regulated by JNK/p38 and NFB (1). Inhibition of NFB may thus favor the TRAIL-induced apoptosis. In our study, we showed that melittin can synergize with TRAIL in activation of TAK1-JNK/p38 but inhibit TRAIL-induced activation of IKK-NFB. Moreover, we provide evidence that melittin can inhibit TRAIL-induced transcription of NFB-related Bcl-xl and c-IAP1. Therefore, melittin-induced sensitization of HCC cells to TRAIL may be contributed to the dual functions of melittin in the activation of TAK1-JNK/p38 and inhibition of IKK-NFB. The possible convergence of TRAIL signaling and melittin signaling may be TAK1. Proapoptotic and antiapoptotic roles have been suggested for TAK1 in various cells types under diverse conditions (12, 35, 44 -48). In melittin-induced apoptosis, melittin can activate CaMKII-TAK1-JNK/p38 and inhibit TRAIL-induced activation of IKK-NFB, which together synergize HCC cells to TRAIL-induced apoptosis.
The differential roles of melittin in activation of TAK1-JNK/ p38 but inhibition of IKK-NFB in response to TRAIL may hold true for other TNF superfamily members because we also find that melittin can potentiate the TNF␣-induced activation of TAK1-JNK/p38 but inhibit TNF␣-induced activation of IKK-NFB (data not shown). Additionally, we find that melittin cannot potentiate the drug-induced cell death of BEL-7402 cells that have been selected under the chemotherapeutic drugs adriamycin or 5-fluorouracil to establish multidrug resistance (data not shown). 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 investigation.
In conclusion, we have demonstrated in this study that melittin potentiated the apoptotic effects of TRAIL in human HCC cells by activating the CaMKII-TAK1-JNK/p38 pathway but inhibiting the IKK-NFB pathway. Our data suggest that melittin may exhibit anti-tumor activity by sensitizing HCC cells to TRAILmediated apoptosis, and that the combination of TRAIL with melittin may have therapeutic potential in the treatment of human HCC.