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

Abstract

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.

Figure 1. Piperine suppresses the survival of melanoma cells.

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; *p<0.05 when compared with control.

Figure 2. Piperine induces G1 phase cell cycle arrest in melanoma cells.

(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 y-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.

Figure 3. Piperine causes DNA damage and modulates G1 cell cycle regulatory proteins.

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 Materials and Methods 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.

Figure 4. Piperine induces apoptosis in melanoma cells. SK MEL-28 and B16 F0 cells were treated with different concentrations of piperine for 48 h.

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.

Figure 5. Blocking Chk1 activation suppress the effects of piperine.

SK MEL 28 cells were (A) and (B) treated with AZD 7762 or (C) and (D) transfected with Chk1 siRNA prior to treatment with 150 µM piperine for 48 hours. Subsequently, cells were processed for flow cytometric analysis. Each experiment was performed at least three times independently and the results were comparable. Values are mean ± S.D of three independent experiments. *p<0.05 when compared with control, **p<0.05 when compared with piperine treatment.

Figure 6. Piperine generates ROS in melanoma cells.

(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. *p<0.05 when compared with control. **p<0.05 when compared to piperine treatment.