Overexpression of Mcl-1 confers resistance to BRAFV600E inhibitors alone and in combination with MEK1/2 inhibitors in melanoma

Melanoma harboring BRAF mutations frequently develop resistance to BRAF inhibitors, limiting the impact of treatment. Here, we establish a mechanism of resistance and subsequently identified a suitable drug combination to overcome the resistance. Single treatment of BRAF mutant melanoma cell lines with vemurafenib or dabrafenib (BRAF inhibitors) alone or in combination with trametinib (MEK1/2 inhibitor) resulted in overexpression of Mcl-1. Overexpression of Mcl-1 in A375 and SK-MEL-28 by transfection completely blocked BRAF and MEK1/2 inhibitor-mediated inhibition of cell survival and apoptosis. Melanoma cells resistant to BRAF inhibitors showed massive expression of Mcl-1 as compared to respective sensitive cell lines. Silencing of Mcl-1 using siRNA completely sensitized resistant melanoma cells to growth suppression and induction of apoptosis by BRAF inhibitors. In vivo, vemurafenib resistant A375 xenografts implanted in athymic nude mice showed substantial tumor growth inhibition when treated with a combination of vemurafenib and Mcl-1 inhibitor or siRNA. Immunohistochemistry and western blot analyses demonstrated enhanced expression of Mcl-1 and activation of ERK1/2 in vemurafenib-resistant tumors whereas level of Mcl-1 or p-ERK1/2 was diminished in the tumors of mice treated with either of the combination. Biopsied tumors from the patients treated with or resistant to BRAF inhibitors revealed overexpression of Mcl-1. These results suggest that the combination of BRAF inhibitors with Mcl-1 inhibitor may have therapeutic advantage to melanoma patients with acquired resistance to BRAF inhibitors alone or in combination with MEK1/2 inhibitors

Therapeutic Potential of Black Pepper Compound for BRaf Resistant Melanoma

Malignant melanoma is significant problem for Caucasian population in the western countries. Mutations in BRAF gene in 60% of patients is responsible for developing resistance to BRAF inhibitors. Our results delineated the mechanism of resistance and identified a suitable drug combination to overcome the resistance. Treatment of BRAF mutant melanoma cells with vemurafenib or dabrafenib (BRAF inhibitors) alone or in combination with trametinib (MEK1/2 inhibitor) resulted in induced expression of Mcl-1. Melanoma cells resistant to BRAF inhibitors exhibited substantial expression of Mcl-1 as compared to sensitive cell lines. Silencing of Mcl-1 using siRNA completely sensitized resistant cells to growth suppression and induction of apoptosis by BRAF inhibitors. Piperlongumine, an active component of black pepper substantially suppressed the growth of vemurafinib resistant melanoma cells by inducing apoptosis and inhibiting Mcl-1 expression. Vemurafenib resistant A375 xenografts showed substantial tumor growth inhibition in mice when treated with a combination of vemurafenib and Mcl-1 inhibitor or siRNA. Oral administration of piperlongumine also suppressed the growth vemurafinib resistant tumor xenografts in athymic nude mice. Immunohistochemistry and western blot analyses confirmed enhanced expression of Mcl-1 and activation of ERK1/2 in vemurafenib-resistant tumors whereas level of Mcl-1 or p-ERK1/2 was reduced in the tumors of mice treated with either of the combination or piperlongumine. Biopsied tumors from the patients treated with or resistant to BRAF inhibitors revealed overexpression of Mcl-1. These results suggest that the combination of BRAF inhibitors with Mcl-1 inhibitor such as piperlongumine may have therapeutic advantage to melanoma patients with acquired resistance to BRAF inhibitors alone or in combination with MEK1/2 inhibitors

Piperine causes G1 phase cell cycle arrest and apoptosis in melanoma cells through checkpoint kinase-1 activation.

In this study, we determined the cytotoxic effects of piperine, a major constituent of black and long pepper in melanoma cells. Piperine treatment inhibited the growth of SK MEL 28 and B16 F0 cells in a dose and time-dependent manner. The growth inhibitory effects of piperine were mediated by cell cycle arrest of both the cell lines in G1 phase. The G1 arrest by piperine correlated with the down-regulation of cyclin D1 and induction of p21. Furthermore, this growth arrest by piperine treatment was associated with DNA damage as indicated by phosphorylation of H2AX at Ser139, activation of ataxia telangiectasia and rad3-related protein (ATR) and checkpoint kinase 1 (Chk1). Pretreatment with AZD 7762, a Chk1 inhibitor not only abrogated the activation of Chk1 but also piperine mediated G1 arrest. Similarly, transfection of cells with Chk1 siRNA completely protected the cells from G1 arrest induced by piperine. Piperine treatment caused down-regulation of E2F1 and phosphorylation of retinoblastoma protein (Rb). Apoptosis induced by piperine was associated with down-regulation of XIAP, Bid (full length) and cleavage of Caspase-3 and PARP. Furthermore, our results showed that piperine treatment generated ROS in melanoma cells. Blocking ROS by tiron protected the cells from piperine mediated cell cycle arrest and apoptosis. These results suggest that piperine mediated ROS played a critical role in inducing DNA damage and activation of Chk1 leading to G1 cell cycle arrest and apoptosis

Piperine suppresses the survival of melanoma cells.

<p>Effect of various concentrations of piperine at different time periods in (A) SK MEL 28, (B) B16 F0, (C) A375 and (D) Aspc-1 cells was determined by Sulforhodamine B cell survival assay. Values are the means ± S.D. of three independent experiments with eight replicates; *<i>p</i><0.05 when compared with control.</p

Piperine induces G1 phase cell cycle arrest in melanoma cells.

<p>(A) and (B) are representative cell cycle profiles of control and 150 µM piperine treated SK MEL 28 and B16 F0 cells for 48 h. FL2-A represents the intensity of propidium iodide, and the <i>y</i>-axis represents the cell counts. (C) And (D) represents concentration-dependent effects of piperine on number of cells in G1 phase in both SK MEL 28 and B16 F0 respectively. Values are means ± S.D. of three independent experiments, each conducted in triplicate. *p<0.05 when compared with control.</p

Piperine generates ROS in melanoma cells.

<p>(A) Represents time dependent generation of ROS in SK MEL 28 cells and (B) represents ROS in B16 F0 cells in response to 150 µM piperine treatment and subsequently analysed using flow cytometer. (C) SK MEL 28 and (D) B16 F0 cells were treated piperine following which the cells were analyzed for ROS using flow cytometer. (E) SK MEL 28 cells were pre-treated with either 10 mM tiron or NAC for 1 h and then treated with 150 µM piperine for 48 h. The cells were processed for ROS analysis by flow cytometry. (F) SK MEL 28 cells were pre-treated with either 10 mM tiron or NAC for 1 h followed by 150 µM piperine for 48 h after which the cell survival was evaluated by sulphorhodamine B assay. (G) SK MEL 28 and (H) B16 F0 cells were pre-treated with 10 mM tiron for 1 h followed by 150 µM piperine for 48 h. The cells were then processed for cell cycle analysis by flow cytometry. In another experiment, SK MEL 28 cells were pre-treated with (I) tiron or (J) NAC as described above and analyzed by western blotting for p.H2A.X, p.Chk1 and cleavage of PARP. Each experiment was performed at least three times independently, and the results were comparable. The values are means ± S.D. *<i>p</i><0.05 when compared with control. **p<0.05 when compared to piperine treatment.</p

Piperine causes DNA damage and modulates G1 cell cycle regulatory proteins.

<p>SK MEL 28 (A) and B16 F0 (B) cells were treated with different concentrations of piperine for 48 h. Cells were lysed and total lysate was prepared as described under <i>Materials and Methods</i> and analyzed by western blotting. Representative immunoblots show the effect of piperine on the phosphorylation of H2A.X (Ser139), ATR (Ser428), Chk1 (Ser296) and p-Rb (Ser795), and the protein levels of DNA Polymerase β, p53, p21, Cyclin D1 and E2F1. Each blot was stripped and reprobed with anti-actin antibody to ensure equal protein loading. (C)Representative immunofluorescence images of p. Chk1 (Ser 296) in control and 150 µM piperine treated SK MEL 28 cells. Alexafluor 594 (Red) represents p.Chk1, Alexafluor 488(green) represents β-actin and DAPI (blue) represents nucleus. Each experiment was performed at least three times independently and the results were comparable.</p

Piperine induces apoptosis in melanoma cells. SK MEL-28 and B16 F0 cells were treated with different concentrations of piperine for 48 h.

<p>Cells were stained with Annexin V and PI and analysed using flow cytometer. (A) and (B) shows representative apoptosis profile of SK MEL 28 and B16 F0 respectively. It also shows the concentration-dependent increase in the percent of apoptotic cells in both the cell lines. Figure (C) and (D) shows western blot analysis of SK MEL 28 and B16 F0 cell lysates upon piperine treatment respectively. Representative immunoblots show the effect of piperine on the protein levels of XIAP, Bid (full length), Cleaved Caspase 3 and Cleaved PARP. Each blot was stripped and reprobed with anti-actin antibody to ensure equal protein loading. Each experiment was performed at least three times independently and the results were comparable.</p