Sensitization of TRAIL-Resistant Malignant Melanomas by Ellagic Acid
The incidence of malignant melanoma has been on the rise for the past 30 years with a lifetime risk of 1 in 75. In its early stage, melanoma is easily cured, but the prognosis associated with metastatic malignant melanoma remains very poor and is one of the most treatment-refractory malignancies. FDA-approved melanoma therapeutics, Dacarbazine (DTIC) and Interleukin-2 (IL-2) are associated with response rates of only 10-20% and do not yield long-lasting remissions. A direct and selective p53-independent cancer therapy is the application of Tumor necrosis factor-Related Apoptosis-Inducing Ligand (TRAIL). TRAIL can induce apoptosis in a broad range of transformed human cells while showing minimal toxicity towards non-malignant cells. However, some cancers are resistant to rhTRAIL-induced apoptosis as a direct result of overexpressed antiapoptotic proteins. One key driver mediating the expression of such antiapoptotic proteins is Casein Kinase 2 (CK2). Upregulated in the nucleus of all cancer cells, CK2 serves as an oncoprotein, promoting the expression of inhibitors of apoptosis resulting in resistance to cell death. By inhibiting CK2 and, therefore, downregulating its antiapoptotic products, TRAIL-resistant cells may become more sensitive to TRAIL-induced apoptosis. To date, Ellagic Acid (EA) is the most potent, naturally-occurring CK2 inhibitor, possessing a Ki of 20 nM. To test whether the addition of EA could sensitize cancer cells to the effects of TRAIL, we utilized cotreatment with TRAIL and EA against the V600E BRAF-mutated melanoma cell lines A375 and WM164, which have previously been reported as TRAIL-resistant. Our in vitro analyses showed that the cotreatment of TRAIL plus EA does not augment the ability of TRAIL to induce apoptosis in A375 as determined by Annexin-V Apoptosis assays and Western Blot analysis. TRAIL-alone was able to induce apoptosis in A375 characterized by the formation of Annexin-V positive cells, PARP fragmentation and the activation of caspase 3. TRAIL initiated both the extrinsic and intrinsic pathways of apoptosis marked by caspase 8-activation and the mitochondrial release of cytochrome c, respectively. However, EA treatment alone did not induce apoptosis marked by the absence of Annexin-V positive cells and PARP fragmentation. Additionally, both TRAIL and EA, as single-agents, inhibited the growth of A375 in a dose-dependent manner, and the combination treatment of TRAIL plus EA enhanced the cytostatic effects of the single-agent treatments as determined by Sulforhodamine-B (SRB) colorimetric assays. Cell cycle analysis through Propidium Iodide (PI) staining and FACS analysis revealed that EA impeded cell growth through an EA-mediated S-phase arrest in A375. Microarray analysis showed that EA downregulates antiapoptotic proteins survivin, XIAP, cIAP-2 and cIAP-1 and upregulates the death receptor DR5. In vivo, we showed that the combination treatment of TRAIL plus EA did not potentiate the ability of TRAIL to inhibit the growth of established A375 tumors. TRAIL alone inhibited tumor growth by ∼80% and H&E staining of TRAIL-treated tumors presented evidence of apoptotic cells. EA was not able to stop the growth of established A375 tumors and did not augment the in vivo antiproliferative effects of TRAIL. Lastly, we showed that TRAIL is cytotoxic towards WM164 cells and can reduce the viability of A375 as determined by Trypan Blue exclusion assays. Whereas EA, although it can inhibit the growth of WM164, does not reduce the viability of the cells. Additional analysis of the potential of EA to sensitize WM164 to the effects of TRAIL is ongoing. These data demonstrate that EA can enhance the cytostatic but not the cytotoxic or proapoptotic effects of TRAIL in vitro. In vivo, TRAIL can inhibit the growth of established tumors as a single-agent but is not augmented by cotreatment with EA.
Turner, K. A.; Lindner, D.; Kalafatis, M. Abstract B34: Sensitization of TRAIL-resistant malignant melanomas by ellagic acid. Cancer Res. 2015, 75, B34-B34.