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

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.


Cell proliferation assay
Cell proliferation was measured with the CyQUANT cell proliferation assay kit by using a green fluorescent dye, CyQUANT GR dye, which exhibits strong fluorescence enhancement when binding to cellular nucleic acids (Life Technologies, Grand Island, NY). A total of 500 live cells were added to each well containing 200μL culture media in a 96-well flat black-bottomed plate (Greiner, Monroe, NC). After 24H (hours after treatment), culture media was replaced with freshly prepared control or treatment media. At 24, 48 and 72H after treatment, media was gently removed, and plates were kept at -80˚C until further analysis. Before assay, plates were thawed for 0.5H at room temperature. In total, 200μL CyQuant GR solution (1x) was added in each well, mixed well with a multichannel pipette, and fluorescence was measured at 480/520 nm (ex/em) in a microplate reader (Synergy HT, BioTek Instruments, Winooski, VT).

Polycaspase assay
Apoptosis was detected with the FAM FLICA Polycaspase assay kit (ImmunoChemistry Technologies, Bloomington, MN) with the green fluorescent inhibitor probe FAM-VAD-FMK that labels active caspase enzymes in living cells. Cells were cultured on phenol red-free media in T25 flasks. Cells at about 90% confluence were tested with various treatments. Staurosporine (6μM) (ImmunoChemistry Technologies) was used as a positive control. At 0.5, 1, 2 and 4H, floating cells with media were collected in a 15-mL disposable centrifuge tube and centrifuged at 5000g for 5 min at room temperature. After discarding the supernatant, cells were mixed with 600μL of 1x apoptosis wash buffer. Remaining adhered cells on the flask were lifted with trypsin, centrifuged, and mixed with the previously collected cell suspension after discarding the supernatant. FAM-FLICA poly caspase inhibitor reagent (1x) was mixed with 500μL cell suspension and incubated at 37˚C for 1 h with intermittent shaking. During this incubation period, a portion of the remaining cells was used for counting the cells with a hemacytometer with trypan blue (0.04%). After incubation, 2mL wash buffer was added to cells, centrifuged, and supernatant was discarded. Again 2mL of wash buffer was added to cells and incubated at 37˚C for 10min to remove excess FAM-FLICA reagent. Cells were centrifuged and the supernatant was discarded. Finally, cells were suspended in 500μL wash buffer and kept on ice. In total, 100μL cell suspension was used for estimating poly-caspase activity in 96-well flat blackbottom plates. Fluorescence was measured in the microplate reader at 488/520nm (ex/em), and the measured RFU values were normalized to total number of cells.
used it for CyQuant cell proliferation assay. From the remaining solution, 200μL was mixed with an equal amount of 2x reaction buffer containing SA-beta-gal substrate and incubated at 37˚C for 1 h in the dark. After incubation, the solution was mixed by vortex, and a 200-μL solution was mixed with 800μL stop solution. From this, a 200-μL solution was used to determine SA-beta-gal activity in 96-well flat black-bottom plates. Fluorescence was measured in the microplate reader at 360/465 nm (ex/em), and RFU values were normalized to those obtained from CyQuant cell proliferation assay.

Shotgun proteomics
Global proteomic profile for control and Ghost pepper (3 g L -1 ) treated cells at 6H were analyzed by a shotgun proteomic approach with tandem mass spectrometry. AT 6H, all the cells including floating cells were washed three times with PBS, flash frozen in liquid nitrogen and sent to BioProximity, LLC, Chantilly, VA on dry ice for shotgun proteomic analysis. Details of the proteomic analysis are given below:

Protein denaturation and digestion
Samples were prepared for digestion with the filter-assisted sample preparation method [14]. Briefly, samples were suspended in 8M urea, 50 mM Tris-HCl, pH 7.6, 3 mM DTT, sonicated briefly, and incubated in a Thermo-Mixer at 40 o C, 1000 RPM for 20 min. Samples were centrifuged and the supernatant was transferred to a 30-kD MWCO device (Millipore) and centrifuged at 13,000 g for 30 min. The buffer for the remaining sample was exchanged with 8M urea, 100 mM Tris-HCl, pH 7.6, then alkylated with 15mM iodoacetamide. The urea concentration was reduced to 2 M. Samples were digested overnight with trypsin at a ratio of enzyme to substrate of 1:100 at 37˚C in a Thermo-Mixer at 1000 RPM. Digested peptides were collected by centrifugation.

Peptide desalting
A portion of the digested peptides, about 20μg, was desalted with use of C18 stop-and-go extraction (STAGE) tips [15]. Briefly, for each sample, a C18 STAGE tip was activated with methanol, and then conditioned with 60% acetonitrile and 0.5% acetic acid, followed by 2% acetonitrile and 0.5% acetic acid. Samples were loaded onto the tips and desalted with 0.5% acetic acid. Peptides were eluted with 60% acetonitrile, 0.5% acetic acid and lyophilized in a SpeedVac (Thermo Savant) to near dryness, approximately 2h.

Liquid chromatography-tandem mass spectrometry
Each digestion mixture was analyzed by ultra-HPLC-MS/MS. LC involved the Easy-nLC 1000 UHPLC system (Thermo). The mobile phase A was 97.5% MilliQ water, 2% acetonitrile, and 0.5% acetic acid and mobile phase B was 99.5% acetonitrile and 0.5% acetic acid. The 240-min LC gradient ran from 0% to 35% B over 210 min, then to 80% B for the remaining 30 min. Samples were loaded directly onto the column. The column was 50 cm x 75 μm ID and packed with 2 micron C18 media (Thermo Easy Spray PepMap). The LC was interfaced to a quadrupole-Orbitrap mass spectrometer (Q-Exactive, Thermo Fisher) via nano-electrospray ionization with a source via an integrated column heater (Thermo Easy Spray source). The column was heated to 50˚C. An electrospray voltage of 2.2 kV was applied. The mass spectrometer was programmed to acquire, by data-dependent acquisition, tandem mass spectra from the top 20 ions in the full scan from 400-1200 m/z. Dynamic exclusion was set at 15s, singly-charged ions were excluded, isolation width was 1.6 Da, full MS resolution was 70,000 and MS/MS resolution 17,500. Normalized collision energy was set to 25, automatic gain control to 2e5, max fill MS to 20 MS, max fill MS/MS to 60 MS and underfill ratio to 0.1%.

Data processing and library searching
Mass spectrometer RAW data files were converted to MGF format by use of msconvert [16]. Detailed search parameters are printed in the search output XML files. Briefly, all searches required 10ppm precursor mass tolerance, 0.02 Da fragment mass tolerance, strict tryptic cleavage, 0 or 1 missed cleavages, fixed modification of cysteine alkylation, variable modification of methionine oxidation and expectation value scores � 0.01. MGF files in the human sequence library were searched with use of X!!Tandem [17] with both the native [18] and kscore [19] scoring algorithms and by OMSSA [20]. All searches were performed with Amazon Web Services-based cluster computed instances with the Proteome Cluster interface. XML output files were parsed and non-redundant protein sets were determined by use of Proteome Cluster [21]. MS1-based peak areas were calculated by use of XCMS [22]. Proteins were required to have � 1 unique peptides across the analyzed samples with e-scores � 0.001.

Statistical analysis
Unless otherwise mentioned, all the experiments were conducted with 4 replicates, and data are presented as mean ± SD. Statistical analysis was performed using SAS software (University Edition, Cary, NC) using BASE SAS, PROC IMPORT, PROC SORT, PROC TRANSPOSE, PROC GLM, PROC PRINT and SAS MACRO programs. Significant differences among means were determined using one-way analysis of variance (ANOVA) followed by Tukey's honestly significant differences multiple-rank test at the p � 0.05 significance level. Proteomic data was analyzed in Microsoft Excel software using paired Student's t-test with two-tailed distribution.

Results
Ghost pepper powder contained a 5:1 ratio of capsaicin to dihydrocapsaicin HPLC analysis revealed that commercially available ghost pepper powder contained 25.80 and 5.07 mg g -1 dry weight (DW) capsaicin and dihydrocapsaicin, respectively. Furthermore, the ratio of capsaicin to dihydrocapsaicin was 5:1 in this Ghost pepper powder ( Fig 1B).

Adenocarcinoma cell proliferation was affected by concentration and duration of treatment
The proportion of adenocarcinoma cells decreased with increasing concentration of Ghost pepper and capsaicinoid treatments at 24, 48 and 72H (Hours After Treatment) (Fig 2). At 24H (Hours After Treatment), capsaicin (400μM), dihydrocapsaicin (400μM), capsaicin (400μM) + dihydrocapsaicin (80μM), and ghost pepper (3 g L-1) decreased the proportion to 36%, 18%, 33% and 20%, respectively, as compared with controls. At 48 and 72H, these values were further reduced to 5% to 15% and 6% to 8%, respectively. Various controls corresponding to different treatment combinations were tested for cell proliferation activity at 24 and 72H (Fig 3). Controls with culture media and different levels of ethanol (control-2 to control-9) did not affect cell proliferation as compared with culture media alone (control-1).

Apoptosis versus senescence
As compared with control(s), all treatments increased polycaspase activity in adenocarcinoma cells at 0.5H (Fig 4). This trend was continued up to 2H. At 4H, as compared to control, all treatments except dihydrocapsaicin (400 μM) maintained high polycaspase activity. Peak polycaspase activity occurred with both dihydrocapsaicin (400 μM) and capsaicin (400 μM) + Effect of ghost pepper on human renal adenocarcinoma cells dihydrocapsaicin (80 μM) at 0.5H. As compared to control-1, peak polycaspase activity occurred with Ghost pepper (3 g L -1 ) and capsaicin (400 μM) at 1 and 2H, respectively. Polycaspase activity rapidly decreased with dihydrocapsaicin followed by Ghost pepper at 4H. Overall, polycaspase activities began to decrease at 4 H. Rapid, early, and late polycaspase response was noticed with dihydrocapsaicin, Ghost pepper and capsaicin, respectively. SAbeta-gal activity with capsaicin (400 μM) treatment was similar to control(s) activity at 0.5 and 1H and thereafter started to decline ( Fig 5). Interestingly, the activity was significantly low in other treatments from 0.5 to 2H. Overall, SA-beta-gal activity started to decrease from 2H with all treatments. Dihydrocapsaicin (400μM) produced the lowest SA-beta-gal activity among all treatments at all treatment times.

Shotgun proteomics revealed a complex network of proteins involved in Ghost pepper-treated cells
By shotgun proteomic approach, we have identified over 10,000 protein groups in each, for about 20,000 protein groups across the control and ghost pepper treated samples which map to about 68,220 protein isoforms at 6H. All the identified proteins exhibited up to several fold difference between treatment and control. Among them, approximately 8.2% (5,577) protein isoforms in Ghost pepper treated cells were significantly different from control. A selective list of proteins that were significantly affected by ghost pepper treatment are presented in Table 1 with experimental evidence. Some of the identified proteins are directly or indirectly related to apoptotic (extrinsic and intrinsic), Ras, Rb/E2F, p53, TGF-beta, WNT-beta catenin, and calcium induced cell death pathways. Several other proteins involved in methylation, acetylation, genome stability, cell cycle check point regulation, carbohydrate, protein and other metabolism and cellular mechanisms were also affected by ghost pepper treatment. Due to space constraints, key proteins and their roles in ghost pepper induced cell death are elaborated under discussion section. In summary, shotgun proteomic analysis revealed that each pathway or cellular mechanism that was affected by ghost pepper had selective upregulated as well as down

Discussion
In this study, we have examined 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. Results of these findings are discussed here in detail.

Ghost pepper and major capsaicinoids had similar effects on adenocarcinoma cell proliferation
Our HPLC analysis of commercial Ghost pepper powder revealed a ratio of 5:1 for major capsaicinoids in terms of capsaicin and dihydrocapsaicin (Fig 1). In general, the ratio of capsaicin to dihydrocapsaicin ranges from 1:1 to 2:1 in chilies [6] depending on the pepper source and method of extraction [10]. All our experiments were designed to reflect the ratio of capsaicin to dihydrocapsaicin in the purchased ghost pepper powder. The ratio of major capsaicinoids in the ethanol extracts of ghost pepper may not be exactly equal to the ratio estimated by HPLC. However, this ratio serves as a reference for cell culture experiments.   Effect of ghost pepper on human renal adenocarcinoma cells Various concentrations of capsaicin, dihydrocapsaicin, capsaicin + dihydrocapsaicin and ghost pepper had the similar effect on adenocarcinoma cell proliferation (Fig 2). Therefore, ghost pepper could exert its effects on cell proliferation via capsaicin and dihydrocapsaicin. However, the role of other minor capsaicinoids of ghost pepper in anticancer properties cannot be ignored. The anticancer effects of capsaicin in vitro were previously found to be both dose-and time-dependent [23]. Bley et al. [12] found that the effects of capsaicin are in the low micromolar range and become maximal at approximately 200 to 300μM. The duration of exposure enhances the potency of capsaicin and its stability under specific experimental conditions. Recent studies have confirmed and extended these observations to additional cell lines and to rodent in vivo xenograft tumor models [12]. Together, capsaicin and dihydrocapsaicin at 5:1 ratio did not show any additive or synergistic effects on cell proliferation compared to either compound alone. We are not sure of specific reasons for this kind of response in human renal adenocarcinoma cells. Data pertaining to cell proliferation in various controls indicate that ethanol, the solvent we used to dissolve capsaicinoids, did not significantly affect the proliferation of human adenocarcinoma cells in vitro (Fig 3).

Apoptosis versus senescence
Ghost pepper and its major capsaicinoids induced early polycaspase responses in human renal adenocarcinoma cells in vitro (Fig 4). The polycaspase FLICA probe, FAM-VAD-FMK, detects apoptosis by recognizing different types of activated caspases in a cell. On the other hand, SAbeta-gal is considered one of the markers of senescence in cells [24]. In this study, SA-beta-gal activity was normal or was downregulated with various treatments in human renal adenocarcinoma cells (Fig 5). The mechanism that directs whether a cell undergo apoptosis or senescence is unknown. These two processes seem to be exclusive [25]. Our results indicate that ghost pepper, capsaicin and dihydrocapsaicin induce apoptosis rather than senescence in human renal cell adenocarcinoma in vitro. Thus, the antiproliferative activity of the treatments we tested could be attributed to their ability to induce apoptosis with various caspases. Capsaicin appears to induce apoptosis in more than 40 different cancer cell lines, mostly human cancer lines [12]. The mechanisms of capsaicin-induced apoptosis have been discussed in detail [1,12]. One or more of these mechanisms are probably responsible for the apoptosis we found. These mechanisms include inhibition of mitochondrial respiration; suppression of plasma membrane NADH-oxidoreductase; significant elevation of intracellular reactive oxygen species production; blocking cell cycle progression and triggering apoptosis by downregulating cyclin D1; and suppression of activation, nuclear translocation and/or DNA binding of STAT [1]. Other mechanisms proposed to play a role in anticancer activities of capsaicin include antioxidant activity, activation of peroxisome proliferator-activated receptor gamma, inhibition of angiogenesis, modulation of lipid metabolism, and/or inhibition of aromatase activity [12]. The mechanisms associated with anticancer activities of capsaicin are complex. Normal or noncancerous cells appear to be significantly less sensitive to the apoptotic or growth inhibitory effects of capsaicin than cancerous cells [12]. Earlier video microscopy studies revealed that dynamic morphologic changes in cell culture take place in less than 2h. An estimated duration of an apoptotic cell death is in between 6 and 24h in vivo. This duration is influenced by type of cells undergoing apoptosis [26].

Ghost pepper modulates a complex network of proteins
Data pertaining to global proteomic analysis further supports the results of cell proliferation and apoptosis (Table 1). Data suggests that 'fold change' of a protein does not alone explain any difference between control and treatment. For this reason, p value (� 0.05) was considered along with fold change value to draw conclusion on a protein. Selective proteins that were significantly affected by ghost pepper are discussed below in detailed.

Intrinsic and extrinsic apoptotic pathways
Proteins that were upregulated or down regulated by ghost pepper treatment in adenocarcinoma cells at 6 H appeared to be late-responsive and/or stably expressed proteins. In general, caspases are activated by pro-apoptotic proteins released from mitochondria which ultimately induce apoptosis [27]. In the present study, polycaspases were induced at 0.5 H, and their expression started to decrease at 4 H (Fig 4). Similarly, cytochrome C (P99999; CYCS) [28] and FADD proteins (Q6LCB0; FADD) [29], which activate different caspases, were also decreased at 6 H in Ghost pepper treated cells as compared to control (Table 1). Apoptosisinducing factor 3 (Q96NN9, AIFM3 or AIFL) when expressed heterologously induce apoptosis in a caspase-dependent manner [30]. Decreased AIFM3 levels in Ghost pepper treatment were also in harmony with caspase levels. Because of this dynamic nature, certain early-responsive proteins may or may not appear at a later time during Ghost-pepper treatment. Furthermore, visual observations under microscope at 6 H indicate that most of the cells exhibited symptoms of apoptosis in Ghost pepper treated cells (personal communications). To date, three pathways viz., extrinsic (death receptor pathway), intrinsic (mitochondrial pathway), and perforin/granzyme pathways, are established for apoptosis. All these three pathways have same terminal execution pathway which involves caspase-3, DNA fragmentation, cytoskeletal and nuclear protein degradation, protein cross linking, formation of apoptotic bodies, and removal of dead cells. The perforin/granzyme pathway of apoptosis is a non-caspase pathway involves single stranded DNA damage [31]. The intrinsic pathway is current target for tumor suppression studies [32], and it is initiated within the cell in response to DNA damage, severe cellular stress, or loss of survival factors in the cells [33]. Up regulation of reactive oxygen species modulator 1 (P60602; ROMO1), mitochondrial superoxide dismutase (Q5TCM1; SOD2), superoxide dismutase-1 (W8Q444; SOD-1), catalase (P04040; CAT), glutathione peroxidase (R4GNE4; GPX4), gamma-glutamyltranspeptidase 1 (P19440; GGT1), and heme oxygenase 2 (I3L276; HMOX2) in cells treated by ghost pepper indirectly suggests that these cells were under severe oxidative stress. Furthermore, over production of other stress response proteins viz., mitochondrial stress-70 protein (Q9UC56; HSPA9), and mitochondrial 60 kDa heat shock protein (C9JL25; HSPD1), also indicates cellular stress in ghost pepper treated cells. B-cell lymphoma 2 (Bcl-2) family of proteins regulate intrinsic pathway by controlling pro-and anti-apoptotic signals in the cell [33]. In the present study, Bcl-2 associated transcription factor 1 (H0YF14; BCLAF1) was significantly over expressed in ghost pepper treatment at 6 H. Bcl-2 associated transcription factor 1 protein is known to interact with several members of the Bcl-2 family of proteins and its overexpression induces apoptosis or cell cycle arrest [34]. Increased tumor suppressor p53-binding protein 1 (Q12888; TP53BP1) was probably involved in DNA-damage signaling pathways in ghost pepper treated cells [35]. As TP53-regulated inhibitor of apoptosis 1 (O43715; TRIAP1) was decreased in treated cells, its role in inhibiting activation of caspase-9 and prevention of apoptosis induction [36] was probably negatively affected by Ghost pepper at 6 H. However, tumor necrosis factor receptor superfamily member 12A (Q9NP84; TNFRSF12A) and TNF receptor-associated factor 2 (Q12933; TRAF2) were still expressed more in treated cells at 6 H. Both of these proteins' roles were implicated in positive regulation of extrinsic apoptotic pathway [37]. Similarly, over expression of janus kinase 1 (Q4LDX3; JAK1) protein in treated cells indicates its role in ghost pepper induced early signaling events related to cytokine receptors [38]. Ceramide, a membrane sphingolipid metabolite, is synthesized de novo by ceramide synthase. It perpetuates cellular stress response and induces apoptosis, terminal differentiation, or cell cycle arrest [39,40]. Ceramide induces apoptosis by activating caspases, especially caspase 3 [41], endonucleases that are responsible for DNA cleavage [42], and by regulating release of cytochrome C [41]. It also plays a role in G0/G1 cell cycle arrest through retinoblastoma gene product [41]. Over expressed ceramide synthase 2 (Q96G23; CERS2) might have played a role in apoptosis and/or cell cycle arrest through ceramide production in ghost pepper treated cells in the present study. Over expressed nitric oxide-associated protein 1 (Q8NC60; NOA1), a large mitochondrial GTPase, might have also supported apoptosis process in treated cells by interacting with complex I of the electron transport chain, and DAP3 (death-associated protein 3), a positive regulator of apoptosis [43]. These findings are further supported by over expression of mitochondrial 28S ribosomal protein S29 (P51398), another name for DAP3, in Ghost pepper treated cells. Furthermore, increased levels of THO complex subunit 1 (Q96FV9 or p84N5; THOC1) in ghost pepper treatment suggest that apoptotic pathway different from those activated by death domain-containing receptors or p53 was active in the treated cells [44].
Down regulation of calponin-2 (Q99439; CNN2) in Ghost pepper treated cells might be associated with morphological changes and detachment of cancer cells [45]. Furthermore, increased levels of the complement C2 of the innate immunity (P06681; C2) appears to be associated with clearance of immune complexes and apoptotic materials [46]. Lysosomal cell death (LCD) is mainly carried out by the lysosomal cathepsin proteases, and their inhibition do not give full proof protection from LCD [47]. Cathepsin mediated LCD is not appears to be a major event in Ghost pepper treated cells at 6 H. This is evident from down regulation of two cathepsins viz., cathepsin L, isoform CRA_b (Q9HBQ7; CTSL) and cathepsin B, isoform CRA_a (A0A024R374; CTSB) in the treated cells.

Ras pathway
Cell growth and proliferation are influenced by extracellular signals. Pathways of cell proliferation are generally initiated by activation of a receptor tyrosine kinase by a growth factor. The Ras pathway is considered as an important one among various cell proliferation pathways. Key components of the Ras pathway are a cascade of serine/threonine kinases, a mitogen-activated protein kinase (MAPK), and transcription factors like FOS and JUN [48]. In the present study, many kinases (tyrosine-protein kinase CSK, P41240, CSK; serine/threonine-protein kinase PLK, B2R841; serine/threonine-protein kinase Nek6, Q9HC98, NEK6; serine threonine kinase 39 isoform B, X5DP03, STK39; SRSF protein kinase 1, H3BLV9, SRPK1; calcium/calmodulindependent protein kinase type IV, Q16566, CAMK4; protein kinase C alpha, B0LPH5, PRKCA; and MAP kinase-activated protein kinase 2, P49137, MAPKAPK2) were over expressed in Ghost pepper treatment (Table 1). A similar trend was observed in Ras specific proteins (RAB7, member RAS oncogene family-like 1 variant, Q53EX5; Ras association domain-containing protein 3, Q86WH2, RASSF3; Ras association domain-containing protein 7, H0YEI0, RASSF7; Ras-related protein Rab-5B, F8VUA5, RAB5B; and Ras-related protein Rap-1b, F5H6R7, RAP1B) in ghost pepper treatment. These finding suggest that the machinery for cell proliferation was very much active while adenocarcinoma cells were undergoing apoptosis in ghost pepper treatment. However, cell proliferation data (Fig 2) confirms the dominant role of apoptosis over cell proliferation.

Rb/E2F pathway
Retinoblastoma (Rb) and p53 family of tumor suppressor genes are considered as some of the important targets for the treatment of drug resistant-cancer patients [49]. Control of Rb/E2F pathway, which regulates initiation of DNA replication, is disrupted in virtually all human cancers. Thymidine kinase which is involved in nucleotide biosynthesis during initiation of DNA replication is under the control of E2F [50]. Increased thymidine kinase (B5BU32; TK1) level is an indication of disrupted Rb/E2F pathway and ongoing cell cycle activity in the dying Ghost pepper treated cells (Table 1). Retinoblastoma-binding protein 5 (Q15291; RBBP5) is another Rb related protein overexpressed in the treated cells. It is an important component required for the activity of methyltransferases involved in histone H3 Lys-4 methylation [51].

P53 pathway
Tumor suppressor p53 protein is a transcriptional regulator. It activates expression of several genes involved in cell death, cell cycle arrest, senescence and DNA-repair. Perhaps, it is inactivated in most cancers [52]. Over expressed 'promyelocytic leukemia gene protein' (Q9UE85; PML) might be an essential component of Ghost pepper induced stress or DNA damage-activated apoptotic pathways (Table 1). PML is known for its role in the pathogenesis of acute promyelocytic leukemia (APL). It is not only involved p53-dependent apoptosis but also in FAS and TNFα-induced apoptosis [53]. Furthermore, cytoplasmic PML is considered as a critical regulator of TGF-beta. Often, TGF-beta signaling is deregulated in cancer [54]. Over expression of 'cell division cycle and apoptosis regulator protein 1' (Q5VUP6; CCAR1 or CARP-1) might have a role in regulation of expression of key cell proliferation-inducing genes, and might have acted as a p53 coactivator. Expression of CARP-1 induces apoptosis but affected by expression of c-Myc or 14-3-3 [55]. Increased levels of cullin7 (Q14999; CUL7), a novel oncogene that promote cell proliferation and invasion by suppressing p53 expression [56], indicates its role against p53-dependent apoptosis ghost pepper treated cells.

TGF-Beta pathway
Transforming growth factor-beta (TGF-beta) signalling has either a tumour-suppressing or tumour-promoting function depending on cellular context [57]. A few TGF-beta pathway associated proteins were elevated in ghost pepper treatment. Among them, 'TGF-beta-activated kinase 1 and MAP3K7-binding protein 2' (Q9NYJ8; TAB2) is a novel adaptor protein known to stimulate TAK1 MAPKKK by linking TAK1 to TRAF6 in the IL-1 signal transduction pathway during the inflammation process (Table 1) [58]. Another TGF-beta pathway related protein was TGF-beta-induced protein ig-h3 (O43219; TGFBI). TGFBI silencing effectively reduces cell proliferation and elevates motility of melanoma cells in vitro [59]. Whereas, in mesothelioma and breast cancer cells, TGFBI suppress cell proliferation, delay G1-S phase transition, and induces death [60]. Another protein that influences TGF-beta pathway is menin (O00632; MEN1). Its suppression antagonizes TGF-beta mediated cell growth inhibition [61]. Its over expression probably having opposite role in ghost pepper treatment.

WNT-beta catenin pathway
In general, wnt signaling pathways offer less specificity for candidate drugs. These pathways include the Wnt/beta-catenin pathway, and beta-catenin-independent pathways (the planar cell polarity (PCP) pathway and Wnt/ Ca2+ pathway) [62]. Among them, transcription factor 7-like 2 (Q9NQB3; TCF7L2 or TCF-4); and catenin beta-1 (B4DGU4; CTNNB1) proteins were over expressed in ghost pepper treatment (Table 1). Perhaps, over expression of TCF-4 and CTNNB1 genes were associated with the activation of MAPK gene in breast cancer cell lines with different degrees of invasiveness [63]. Whereas, chondrocyte apoptosis in osteoarthritis was induced by elevated TCF-4 mRNA expression through NF-κB signaling [64]. On the other hand, a decreased expression levels of catenin alpha-1 (P35221; CTNNA1 or Renal carcinoma antigen NY-REN-13) is commonly seen in gastric carcinoma patients [65].

Genome stability and cell cycle check point regulation
Telomere maintenance is active in many human cancers and in vitro immortalized cell lines by a telomerase-independent pathway called as the Alternative Lengthening of Telomeres (ALT) pathway. Loss of transcriptional regulator ATRX protein (Q7Z2J1; ATRX) and mutations in its gene are the hallmarks of ALT-immortalized cell lines [78]. Gain of ALTRX protein in ghost pepper treated cells is an indication of negative effect of Ghost pepper on immortal behavior of adenocarcinoma cells (Table 1). However, data on SA-beta-gal suggests that these changes might not be sufficient to induce senescence in Ghost pepper treated human renal adenocarcinoma cells (Fig 5). However, increased levels of Menin (O00632), a protein involved in histone methylation, suggests that the activity of hTERT and related telomere maintenance might have suppressed in Ghost pepper treated cells [77]. On the other hand, over expressed telomeric repeat-binding factor 2-interacting protein 1 (Q9NYB0; TERF2IP or TRF2) and histone H3.1t (Q16695; HIST3H3) might have protected telomeres [37,79] to some extent in ghost pepper treated cells. These findings also suggest that there was a competition between maintenance and suppression of maintenance of telomeres in ghost pepper treated cells.

Protein and other metabolism
Several proteins of protein metabolism were also affected by Ghost pepper treatment. Decreased elongation factor 1-alpha 2 protein (Q05639; EEF1A2 or Statin-S1) ( Table 1), a translation factor of protein synthesis, might have upregulated apoptosis pathway proteins (caspase3, BAD, BAX, PUMA) in ghost pepper treatment. These findings are supported by a recent study on prostate cancer tissues. In these tissues, the levels of EEF1A2 and caspase3 were inversely correlated [98]. Proteins associated with protein biosynthesis (mitochondrial aspartate-tRNA ligase, Q6PI48, DARS2; 60S ribosomal protein L3, P39023, RPL3; mitochondrial 28S ribosomal protein S23, Q9Y3D9, MRPS23; mitochondrial elongation factor Ts, F8VS27, TSFM, etc.), fatty acid metabolism (elongation of very long chain fatty acids protein, D6RHI2, ELOVL6; and fatty aldehyde dehydrogenase, P51648, ALDH3A2), DNA metabolism (DNA topoisomerase 2-beta, Q02880, TOP2B; thymidine kinase, B5BU32, TK1; and DNA ligase, B4DTU4, LIG1), were over expressed by ghost pepper treatment in the human kidney adenocarcinoma cells. On the other hand, some of the proteins related to protein biosynthesis (eukaryotic translation initiation factor 3 subunit L, Q6ICD2, EIF3L; elongation factor 1-alpha 2, Q05639, EEF1A2; and methionine aminopeptidase 2, F8VQZ7, METAP2) were under expressed by ghost pepper treatment in human kidney adenocarcinoma cells. Perhaps, various metabolic checkpoints that dictate cell fate in response to metabolic fluctuations and cell death regulation were reviewed recently [99]. Proteins involved in other metabolism and cellular mechanisms were also affected by ghost pepper (see data in Dryad, doi:10.5061/dryad.d0s2gm0). These proteins are not discussed here due to space constraints.

Conclusions
Unlike common chili peppers, ghost pepper contains very high proportions of capsaicin and dihydrocapsaicin. Dose and time dependent reduction of human adenocarcinoma cell proliferation was observed with capsaicin, dihydrocapsaicin and ghost pepper extract in vitro. Reduced cell proliferation with these treatments was ascribed to apoptosis rather than senescence. This was evident from upregulation of early polycaspase activities, and normal or suppressed senescence specific SA-beta-gal activity in the treated cells. In summary, global proteomic analysis revealed 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 (Fig 6). Broadly two types of protein responses were noticed within each pathway. One type of proteins over expressed while other type was downregulated by ghost pepper treatment. These imbalances probably favored apoptosis rather than cell proliferation. Besides these pathways, ghost pepper also induced changes in methylation, acetylation, genome stability, cell cycle check points, carbohydrate, protein and other metabolism. Several other proteins were also affected by ghost pepper in human renal adenocarcinoma cells (see data in Dryad, doi:10.5061/dryad.d0s2gm0). Further in depth studies are required before making any conclusion on ghost pepper for clinical applications. In future, we are planning to conduct similar experiments to understand toxic 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.
https://doi.org/10.1371/journal.pone.0206183.g006 effects of ghost pepper on normal human cells including normal human kidney cells. Furthermore, time-course experiments will help us to identify a short list of candidate proteins for apoptosis in ghost pepper treatment in future.