Caffeic acid phenethyl ester suppresses melanoma tumor growth by inhibiting PI3K/AKT/XIAP pathway

Abstract

Melanoma is highly metastatic and resistant to chemotherapeutic drugs. Our previous studies have demonstrated that caffeic acid phenethyl ester (CAPE) suppresses the growth of melanoma cells and induces reactive oxygen species generation. However, the exact mechanism of the growth suppressive effects of CAPE was not clear. Here, we determined the potential mechanism of CAPE against melanoma in vivo and in vitro. Administration of 10 mg/kg/day CAPE substantially suppressed the growth of B16F0 tumor xenografts in C57BL/6 mice. Tumors from CAPE-treated mice showed reduced phosphorylation of phosphoinositide 3-kinase, AKT, mammalian target of rapamycin and protein level of X-linked inhibitor of apoptosis protein (XIAP) and enhanced the cleavage of caspase-3 and poly (ADP ribose) polymerase. In order to confirm the in vivo observations, melanoma cells were treated with CAPE. CAPE treatment suppressed the activating phosphorylation of phosphoinositide 3-kinase at Tyr 458, phosphoinositide-dependent kinase-1 at Ser 241, mammalian target of rapamycin at Ser 2448 and AKT at Ser 473 in B16F0 and SK-MEL-28 cells in a concentration and time-dependent study. Furthermore, the expression of XIAP, survivin and BCL-2 was downregulated by CAPE treatment in both cell lines. Significant apoptosis was observed by CAPE treatment as indicated by cleavage of caspase-3 and poly (ADP ribose) polymerase. AKT kinase activity was inhibited by CAPE in a concentration-dependent manner. CAPE treatment increased the nuclear translocation of XIAP, indicating increased apoptosis in melanoma cells. To confirm the involvement of reactive oxygen species in the inhibition of AKT/XIAP pathway, cells were treated with antioxidant N-acetyl-cysteine (NAC) prior to CAPE treatment. Our results indicate that NAC blocked CAPE-mediated AKT/XIAP inhibition and protected the cells from apoptosis. Because AKT regulates XIAP, their interaction was examined by immunoprecipitation studies. Our results show that CAPE treatment decreased the interaction of AKT with XIAP. To establish the involvement of AKT in the apoptosis-inducing effects of CAPE, cells were transfected with AKT. Our results revealed that AKT overexpression attenuated the decrease in XIAP and significantly blocked CAPE-mediated apoptosis. Similarly, overexpression of XIAP further decreased CAPE-induced apoptosis. Taken together, our results suggest that CAPE suppresses phosphoinositide 3-kinase/AKT/XIAP pathway leading to apoptosis in melanoma tumor cells in vitro and in vivo.

Fig. 1.

CAPE suppresses tumor growth in C57BL/6 mice by inhibiting PI3K/AKT/XIAP pathway. C57BL/6 mice were kept on antioxidant-free diet for a week. Exponentially growing B16F0 (1 × 10⁶) cells were injected subcutaneously into the right flanks of 24 mice. When the tumor reached 70 mm³ in size, mice were randomly segregated into two groups with 12 mice in each group. Treated group of mice received 10 mg/kg/day CAPE in 150 µl of 50% dimethyl sulfoxide/saline by intraperitoneal injection for subsequent 7 days, whereas control mice received vehicle alone. The animals were weighed twice a week. Tumors were measured using vernier calipers. Effect of CAPE on (A) % tumor volume and (B) % tumor weight was evaluated. In order to determine the mechanism of tumor growth suppression, tumors were homogenized, lysed and subjected to western blot. (C) Representative immunnoblots showed the effect CAPE treatment on thephosphorylation of PI3K at Tyr 458, AKT at Ser 473, mTOR at Ser 2448 and protein levels of PI3K, AKT, mTOR, XIAP, cleaved caspase-3 and cleaved PARP. Each lane represents tumor data from eight control and eight treated mice, respectively. The blots were stripped and re-probed for actin to ensure equal protein loading. Values are mean ± SD of 12 samples. *P < 0.05, statistically significant compared with control.

Fig. 2.

CAPE inhibits the PI3K/AKT/XIAP pathways in melanoma cells. (A) B16F0, (B) and (C) SK-MEL-28 melanoma cells were treated with various concentrations or various time points with 20 µM of CAPE for 48 h. Cells were lysed and subjected to western blot. Immunoblots were probed for p-PI3K (Tyr 458), p-AKT (Ser 473), p-PDK1 (Ser 241) and p-m-TOR (Ser 2448), PI3K, AKT, PDK1, mTOR, XIAP, BCL-2, survivin, cleaved caspase-3 and cleaved PARP. Same blots were stripped and re-probed for actin to ensure equal protein loading. The experiments were repeated three times with similar results obtained. *P < 0.05, statistically significant compared with control.

Fig. 3.

Effect of CAPE on AKT kinase activity and the interaction of AKT/XIAP in melanoma cells. (A) B16F0 cells were pretreated for 1 h with LY294002 followed by 20 µM CAPE treatment for 48 h. Cell lysates were immunoblotted with p-PI3K (Tyr 458), p-AKT (Ser 473) and XIAP. Same blots were stripped and re-probed for actin to ensure equal protein loading. *P < 0.05, statistically significant compared with control. (B) SK-MEL-28 or B16F0 cells were treated with various concentrations of CAPE for 48 h. Cell lysates were prepared and AKT kinase activity was determined using a kit according to the manufacturer’s instruction. B16F0 and SK-MEL-28 cells were treated with 20 µM CAPE for 48 h and immunoprecipitated (C) with XIAP and (D) with AKT antibodies and immunoblotted with p-AKT (Ser 473) and XIAP antibodies, respectively. The experiments were repeated three times with similar results obtained.

Fig. 4.

Effect of CAPE in cells overexpressing with AKT and XIAP. B16F0 melanoma cells were transiently transfected with 2 µg AKT or 2 µg of XIAP plasmid by electroporation followed by 20 µM CAPE treatment for 48 h. (A and C) Cell lysates were subjected to western blots and immunoblotted with p-AKT (ser 473), XIAP, cleaved caspase-3 and cleaved PARP. Same blots were stripped and re-probed for actin to ensure equal protein loading. (B and D) Apoptosis wasdetermined using Annexin V/FITC and propidium iodide and analyzed by flow cytometry as described in Materials and methods section. Results are expressed as mean ± SD of three independent experiments. *P < 0.05, statistically significant compared with control. **P < 0.05, statistically significant compared with CAPE treatment.

Fig. 5.

CAPE increases nuclear translocation of XIAP. (A) SK-MEL-28 and (B) B16F0 melanoma cells were treated with 20 µM CAPE for 48 h and nuclear fraction were isolated using nuclear fractionation kit. Represented western blots were immunoblotted with XIAP antibody. Same blots were stripped and re-probed with actin and lamin B to ensure equal protein loading. (C and D) B16F0 cells were treated with dimethyl sulfoxide or 20 µM CAPE, immunostained with XIAP and p-AKT (Ser 473), and actin antibodies and visualized under fluorescence microscope (Olympus Inc). The experiments were repeated three times with similar results obtained.

Fig. 6.

CAPE-mediated ROS generation inhibits AKT/XIAP pathway. B16F0 and SK-Mel-28 melanoma cells were pretreated with 5 mM NAC followed by 20 µM CAPE treatment for 48 h. (A and B) whole-cell lysates were immunoblotted with p-AKT (Ser 473), XIAP and cleaved caspase-3. Same blots were stripped and re-probed for actin to ensure equal protein loading. (C and D) Apoptosis assay was determined using Annexin V/FITC and propidium iodide and analyzed by flow cytometry as described in Materials and methods section. Results are expressed as mean ± SD of three independent experiments. *P < 0.05, statistically significant compared with control. **P < 0.05, statistically significant compared with CAPE treatment.