Project Title

Delta-tocotrienol and simvastatin induces differential cytotoxicity and synergy in BRAF wild-type SK-MEL-2 and mutant BRAF SK-MEL-28 melanoma cancer cells

Authors' Affiliations

Nagaishwarya Moka, Department of internal medicine1, Quillen college of medicine, ETSU2 Kelley cross, Quillen college of medicine, ETSU1 Marianne Brannon, ETSU1 Janet Lightner, ETSU1 Megan Dycus, ETSU1 William Stone, ETSU1 Victoria Palau, Bill Gatton College of Pharmacy1, ETSU2 Koyamangalath Krishnan, Department of oncology1, ETSU2

Location

Clinch Mtn. Room 215

Start Date

4-5-2018 8:00 AM

End Date

4-5-2018 12:00 PM

Poster Number

161

Name of Project's Faculty Sponsor

Dr.Krishnan

Faculty Sponsor's Department

Department of oncology

Type

Poster: Competitive

Classification of First Author

Medical Resident or Clinical Fellow

Project's Category

Biomedical Case Study

Abstract Text

Targeting the mutant BRAF and immunotherapy are new approaches to the treatment of metastatic malignant melanoma that has significantly improved survival but is associated with significant toxicity and cost. Potent and specific BRAF inhibitors like vemurafenib and dabrafenib are superior to chemotherapy in treatment of BRAF mutant melanomas which represent nearly 50% of all melanomas. A less toxic approach to treatment of malignant melanoma is hence appealing. Delta-tocotrienol (DT3), an unsaturated vitamin E isoform, and simvastatin, an HMG-CoA reductase inhibitor have been shown to have anti-neoplastic properties. We studied the effects of these chemicals in both BRAF-mutated SK-MEL-28 and BRAF-wild type SK-MEL-2 melanoma cells. MTS assays were used to analyze cytotoxicity. SK-MEL-28 and SK-MEL-2 cells were cultured in MEM media containing 10% serum and plated in 96-well culture plates for 48 hours then treated with DT3 (0-80 µM), simvastatin (0-10 µM), or a combination and dosed again at 72 hours. SK-MEL-28 and SK-MEL-2 cells were grown in 60 mm plates and treated with DT3 at concentrations of 30 µM, simvastatin at concentrations of 10 µM and combination of DT3 and simvastatin at concentrations of 10 µM and 2 µM. Cell were lysed with RIPPA buffer with protease and phosphatase inhibitor after 6 hours of treatment. Protein concentration of cell lysates was measured spectrophotometrically (GLO Max Multi+, Promega), using a BCA protein assay kit. The samples were run in SDS PAGE and blotted onto nitrocellulose membranes. Membranes were incubated with antibodies against Hsp 70 (Enzo Life Sciences, Farmingdale, NY), Hsp 90 (Santa Cruz, Dallas, TX), pS6 and pERK (Cell Signaling, Danvers, MA) and pAKT. Using MTS assay, we found that DT3 (IC50 75.2 μM) and simvastatin (IC50 8.3μM) have cytotoxic effects on melanoma cell line SK-MEL-2, but not on the SK-MEL-28 cells DT3 and simvastatin at the concentrations studied (10-80 μM DT3) and (0.625- 10 μM simvastatin). Further studies determined that simvastatin decreased expression of pS6, pERK on SK-MEL-2 and not DT3. However, these effects are different in SK-MEL-28 cells where there is only decrease in expression of pS6; treated SK-MEL-2 cells also show over-expression of Hsp70 suggestive of a rescue effect leading to lesser cytotoxic activity. The selective cytotoxicity observed in wild type BRAF melanoma cell lines by DT3 and simvastatin warrants further research into the potential therapeutic use of these drugs. A differential cytotoxicity is shown by DT3 and simvastatin in malignant melanoma cells with selective more potency in wild type BRAF melanoma compared to mutant BRAF melanoma cells. Further studies will be undertaken to dissect the mechanistic basis of this differential response.

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Apr 5th, 8:00 AM Apr 5th, 12:00 PM

Delta-tocotrienol and simvastatin induces differential cytotoxicity and synergy in BRAF wild-type SK-MEL-2 and mutant BRAF SK-MEL-28 melanoma cancer cells

Clinch Mtn. Room 215

Targeting the mutant BRAF and immunotherapy are new approaches to the treatment of metastatic malignant melanoma that has significantly improved survival but is associated with significant toxicity and cost. Potent and specific BRAF inhibitors like vemurafenib and dabrafenib are superior to chemotherapy in treatment of BRAF mutant melanomas which represent nearly 50% of all melanomas. A less toxic approach to treatment of malignant melanoma is hence appealing. Delta-tocotrienol (DT3), an unsaturated vitamin E isoform, and simvastatin, an HMG-CoA reductase inhibitor have been shown to have anti-neoplastic properties. We studied the effects of these chemicals in both BRAF-mutated SK-MEL-28 and BRAF-wild type SK-MEL-2 melanoma cells. MTS assays were used to analyze cytotoxicity. SK-MEL-28 and SK-MEL-2 cells were cultured in MEM media containing 10% serum and plated in 96-well culture plates for 48 hours then treated with DT3 (0-80 µM), simvastatin (0-10 µM), or a combination and dosed again at 72 hours. SK-MEL-28 and SK-MEL-2 cells were grown in 60 mm plates and treated with DT3 at concentrations of 30 µM, simvastatin at concentrations of 10 µM and combination of DT3 and simvastatin at concentrations of 10 µM and 2 µM. Cell were lysed with RIPPA buffer with protease and phosphatase inhibitor after 6 hours of treatment. Protein concentration of cell lysates was measured spectrophotometrically (GLO Max Multi+, Promega), using a BCA protein assay kit. The samples were run in SDS PAGE and blotted onto nitrocellulose membranes. Membranes were incubated with antibodies against Hsp 70 (Enzo Life Sciences, Farmingdale, NY), Hsp 90 (Santa Cruz, Dallas, TX), pS6 and pERK (Cell Signaling, Danvers, MA) and pAKT. Using MTS assay, we found that DT3 (IC50 75.2 μM) and simvastatin (IC50 8.3μM) have cytotoxic effects on melanoma cell line SK-MEL-2, but not on the SK-MEL-28 cells DT3 and simvastatin at the concentrations studied (10-80 μM DT3) and (0.625- 10 μM simvastatin). Further studies determined that simvastatin decreased expression of pS6, pERK on SK-MEL-2 and not DT3. However, these effects are different in SK-MEL-28 cells where there is only decrease in expression of pS6; treated SK-MEL-2 cells also show over-expression of Hsp70 suggestive of a rescue effect leading to lesser cytotoxic activity. The selective cytotoxicity observed in wild type BRAF melanoma cell lines by DT3 and simvastatin warrants further research into the potential therapeutic use of these drugs. A differential cytotoxicity is shown by DT3 and simvastatin in malignant melanoma cells with selective more potency in wild type BRAF melanoma compared to mutant BRAF melanoma cells. Further studies will be undertaken to dissect the mechanistic basis of this differential response.