Piperlongumine, a novel treatment option to overcome BRAF inhibitor resistance in melanoma


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Sharavan Ramachandran1,Neel Fofaria1,Sahdeo Prasad1,Sanjay Srivastava1
1Texas Tech University Health Sciences Center

Abstract

Background

Melanoma cells are highly proliferative and are shown to develop addiction towards the metabolic pathways like glycolysis and mitochondrial respiration to meet energy demands. Melanoma cells predominantly utilize cytosolic aerobic glycolysis or Warburg effect to develop resistance towards BRAF inhibitors (vemurafenib and dabrafenib).

Method

Sulforhodamine-B assay

Annexin-V assay

Western blotting

XF glycolysis stress test

Subcutaneous xenograft melanoma model

Results

Vemurafenib resistant (VR) cells possessed 1.5 fold higher extra-cellular acidification rate (ECAR), an indicator of glycolysis, when compared to sensitive cells. We also observed that STAT3, a known regulator of glycolysis was up-regulated in A375 and SKMEL28 BRAF inhibitor resistant cells when compared to the parental cell line. In addition, LDH-A, a key glycolytic enzyme was significantly overexpressed in A375VR cells when compared to its parental counterpart. Consequently, we observed that vemurafenib and dabrafenib treatment induces the expression of pSTAT3(Y705) in  melanoma cells. Our results showed that piperlongumine inhibits the survival of A375VR  cells. Moreover, the combination of piperlongumine with vemurafenib and dabrafenib shows synergistic effects by suppressing 75% - 85% of cell survival when compared to vemurafenib/dabrafenib and piperlongumine (25% - 45%) treatment alone in A375 and SKMEL-28 BRAF inhibitor resistant  cells.  The combination of piperlongumine with vemurafenib or dabrafenib induced 2-8 fold apoptosis when compared to vemurafenib/dabrafenib and piperlongumine treatment alone in A375 and SKMEL-28 BRAF inhibitor resistant cells. Piperlongumine treatment suppressed 50% ECAR in a concentration dependent manner after 12h of treatment in A375VR cells. Treatment of A375VR cells with piperlongumine for 48h inhibited the expression of LDH-A and its upstream regulators pSTAT3 (Y705), β-catenin, c-Myc and HIF-1α as evaluated by western blot. In addition, oral administration of piperlongumine and vemurafenib combination suppressed 86% of tumour growth when compared to vemurafenib treatment.

Conclusion

Our study thus indicate that piperlongumine potentiates the effects of BRAF inhibitors by inhibiting Warburg effect and its upstream regulators.