Effect of ghost pepper on cell proliferation, apoptosis, senescence and global proteomic profile in human renal adenocarcinoma cells

Abstract

Chili peppers are an important constituent of many foods and contain medicinally valuable compounds, such as capsaicin and dihydrocapsaicin. As various dietary botanicals have anticancer properties, this study was aimed to examine the effect of Ghost pepper (Bhut Jolokia), one of the hottest chili peppers in the world, on cell proliferation, apoptosis, senescence and the global proteomic profile in human renal cell adenocarcinoma in vitro. 769-P human renal adenocarcinoma cells were cultured on RPMI-1640 media supplemented with fetal bovine serum (10%) and antibiotic-antimycotic solution (1%). Treatment stock solutions were prepared in ethanol. Cell proliferation was tested with phenol red-free media with capsaicin (0-400 μM), dihydrocapsaicin (0-400 μM), capsaicin + dihydrocapsaicin (5:1), and dry Ghost peppers (0-3 g L-1) for 24, 48 and 72 h. Polycaspase and senescence associated-beta-galactosidase (SA-beta-gal) activities were tested with capsaicin (400 μM), dihydrocapsaicin (400 μM), capsaicin (400 μM) + dihydrocapsaicin (80 μM), and ghost pepper (3 g L-1) treatments. Global proteomic profile of cells in control and ghost pepper treatment (3 g L-1) was analyzed after 6 h by a shotgun proteomic approach using tandem mass spectrometry. At 24 h after treatment (24 HAT), relative to control, cell proportion with capsaicin (400 μM), dihydrocapsaicin (400 μM), capsaicin (400 μM) + dihydrocapsaicin (80 μM), and ghost pepper (3 g L-1) treatments was reduced to 36%, 18%, 33% and 20%, respectively, and further reduced at 48 and 72 HAT. All treatments triggered an early polycaspase response. SA-beta-gal activity was normal or suppressed with all treatments. About 68,220 protein isoforms were identified by shotgun proteomic approach. Among these, about 8.2% were significantly affected by ghost pepper. Ghost pepper regulated various proteins involved in intrinsic and extrinsic apoptotic pathways, Ras, Rb/E2F, p53, TGF-beta, WNT-beta catenin, and calcium induced cell death pathways. Ghost pepper also induced changes in proteins related to methylation, acetylation, genome stability, cell cycle check points, carbohydrate, protein and other metabolism and cellular mechanisms. Ghost pepper exhibited antiproliferation activity by inducing apoptosis through a complex network of proteins in human renal cell adenocarcinoma in vitro.

Fig 1. Ghost pepper and its major capsaicinoids.

(A) Commercially available dried ripened Ghost pepper fruits. Bar = 1 cm. (B) Capsaicin and dihydrocapsaicin levels in the commercial dried ripened Ghost pepper powder. Values are means ± SD; n = 3; **p ≤ 0.01 (as compared to capsaicin).

Fig 2. Effect of ghost pepper on human renal adenocarcinoma cell proliferation.

Percentage of cells at 24, 48 and 72 H with different concentrations of capsaicin (Cap), dihydrocapsaicin (DCap), Cap + DCap (5:1), and ghost pepper. Control (media without any treatment compound) is adjusted to 100%. Values are means ± SD; n = 4; *p ≤ 0.05 and **p ≤ 0.01 (as compared to control); ^p ≤ 0.05 and ^^p ≤ 0.01 (as compared to lowest concentration with in each compound group).

Fig 3. Effect of solvent on human renal adenocarcinoma cell proliferation.

Different controls with different levels of ethanol, the solvent used for dissolving capsaicinoids in the study, were tested at 24 and 72 H. Control-1 contains culture media without ethanol. All other controls contain culture media with different levels of ethanol. Control-2, -3, -4 and -5 contained 0.2, 0.4, 0.6 and 0.8% ethanol, respectively. These were the corresponding controls for 100, 200, 300 and 400 μM capsaicin as well as dihydrocapsaicin treatments; and 0.75, 1.50, 2.25 and 3.00 g L^-1 ghost pepper treatments, respectively. Control-6, -7, -8 and -9 contained 0.24, 0.48, 0.72 and 0.96% ethanol, respectively. These were the corresponding controls for 100 + 20; 200 + 40; 300 + 60; and 400 + 80 μM capsaicin + dihydrocapsaicin treatments, respectively (refer Fig 2 for treatments). Values are mean ± SD; n = 4; NS = Not significantly different from control-1 (p ≤ 0.05).

Fig 4. Cellular polycaspase activity in human renal adenocarcinoma cells at 0.5, 1, 2 and 4 H.

Control-1 contained culture media without ethanol. Control-2 and control-3 contained culture media with different levels of ethanol (0.80 and 0.96%, respectively). Control (+ve) contained staurosporine (6 μM). Control-2 was a corresponding control for capsaicin (Cap 400 μM), dihydrocapsaicin (DCap 400 μM), and Ghost pepper (3 g L^-1) treatments. Control-3 was a corresponding control for capsaicin + dihydrocapsaicin treatment (Cap 400 μM + DCap 80 μM) (5:1). Polycaspase activities were normalized with respective total number of cells. Values are means ± SD; n = 4; p ≤ 0.05 (*, ^, + or ! ); p ≤ 0.01 (**, ^^, ++ or !!); * or ** as compared to control-1; ^ or ^^ as compared to cap 400 μM; + or ++ as compared to DCap 400 μM; ! or !! as compared to Cap 400 μM + DCap 80 μM.

Fig 5. SA-beta-gal activity in human renal adenocarcinoma cells at 0.5, 1 and 2 H.

Control-1 contained culture media without ethanol. Control-2 and control-3 contained culture media with different levels of ethanol (0.80 and 0.96%, respectively). Control-2 was a corresponding control for capsaicin (Cap 400 μM), dihydrocapsaicin (DCap 400 μM), and ghost pepper (3 g L^-1) treatments. Control-3 was a corresponding control for capsaicin + dihydrocapsaicin treatment (Cap 400 μM + DCap 80 μM) (5:1). SA-beta-gal activities were normalized with respective relative fluorescence unit (RFU) values obtained with CyQUANT cell proliferation assay. Values are mean ± SD; n = 4; p ≤ 0.05 (*, ^, + or ! ); p ≤ 0.01 (**, ^^, ++ or !!); * or ** as compared to control-1; ^ or ^^ as compared to Cap 400 μM; + or ++ as compared to DCap 400 μM; ! or !! as compared to Cap 400 μM + DCap 80 μM.

Fig 6. Mechanism of ghost pepper mediated cell death in human renal adenocarcinoma cells.

Ghost pepper induce apoptosis by regulating the expression of key proteins involved in several cellular pathways, molecular mechanisms and metabolism. Enzymatic assays and global proteomic analysis suggest that Ghost pepper induced apoptosis in human renal adenocarcinoma cells was mediated through intrinsic and extrinsic apoptotic pathways, Ras, Rb/E2F, p53, TGF-beta, WNT-beta catenin, and calcium induced cell death pathways. Broadly two types of protein responses were noticed within each pathway. One type of proteins over expressed while other type were down regulated by ghost pepper treatment. These imbalances favored apoptosis rather than cell proliferation in ghost pepper treatment. Besides these pathways, ghost pepper also induced changes in methylation, acetylation, genome stability, cell cycle check points, carbohydrate, protein and other metabolism.