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Original Articles

Capsaicin Regulates Mitochondrial Fission to Promote Melanoma Cell Apoptosis

Ma, Jing-Jing; Yang, Yu-Qi; Guo, Sen; Wang, Hui-Na; Yi, Xiu-Li; Zhao, Tao; Liu, Lin; Yue, Qiao; Liu, Yu; Shi, Qiong; Gao, Tian-Wen; Guo, Wei-Nan; Li, Chun-Ying

Author Information
International Journal of Dermatology and Venereology: September 2021 - Volume 4 - Issue 3 - p 140-151
doi: 10.1097/JD9.0000000000000124

Abstract

Introduction

Malignant melanoma is an aggressive form of skin cancer and accounts for approximately 80% of skin cancer-related deaths.1 Although both immunotherapy and therapy that targets mitogen-activated protein kinase inhibitors have improved the overall survival of patients with melanoma, the average survival rates remain poor because of disease recurrence and resistance to currently available therapies.2 Therefore, identification of new therapeutic targets and agents for melanoma treatment is urgently needed.

Capsaicin (8-methyl-N-vanillyl-6-noneamide) (CPS), a major component of the red pepper, is a high-affinity agonist of the transient receptor potential vanilloid 1 (TRPV1) receptor, which belongs to the TRPV family.3 TRPV6 is a more recently identified receptor of CPS.4 Once activated by CPS, these TRPV channels preferentially lead to augmented permeability of Ca2+ flux, initiating intracellular Ca2+-dependent signaling cascades.5 CPS is widely used in clinical practice for the treatment of pain and inflammation caused by various diseases.6 Moreover, CPS can potently repress the growth of various cancers through the induction of apoptosis by TRPV-dependent or -independent pathways.7 The tumor-suppressive role of CPS in melanoma has been proven both in vitro and in vivo.8-9 Intriguingly, CPS does not have a prominent effect on the viability of normal cells,4,10 making it an attractive agent for cancer therapy. However, the mechanism underlying its anti-tumor capacity in melanoma has not been fully clarified.

The mitochondrion is the crucial cytoplasmic organelle for maintaining homeostasis within eukaryotic cells; thus, targeting the mitochondria is a promising therapeutic strategy. Mitochondrial structure and function are dynamically regulated by two counteracting processes: fusion and fission.11 Accumulated evidence has revealed that mitochondrial fission can be induced by various stresses and is tightly correlated with cell death via the regulation of mitochondrial function.12-13 Although previous studies have demonstrated some mechanisms underlying the anti-cancer effect of CPS in cancers, whether mitochondrial fission is implicated in this effect remains unclear.

In the present study, we investigated the effects of CPS treatment on the vitality of melanoma cells, and its underline mechanism. Next, we investigated whether CPS treatment leads to mitochondrial dysfunction in melanoma cells. Finally, the implication of mitochondrial fission in the process of CPS-induced melanoma cell apoptosis was measured.

Material and methods

Cell culture and reagents

The human melanoma cell lines A2058 and WM35 were purchased from American Type Culture Collection (Manassas, VA, USA). Experiments were performed on cells passaged for no more than 6 months. The A2058 cell line was cultured in DMEM/F12 (Hyclone Laboratories/GE Healthcare, Chicago, IL, USA) supplemented with 10% fetal bovine serum (Gibco/Thermo Fisher Scientific, Waltham, MA, USA). The WM35 cell line was cultured in RPMI-1640 (Hyclone Laboratories/GE Healthcare) supplemented with 10% fetal bovine serum at 37 °C in the presence of 5% carbon dioxide. Both of these melanoma cell lines were authenticated by short tandem repeat fingerprinting by Beijing Microread Genetics Co., Ltd. in 2016 and tested for mycoplasma contamination. An immortalized normal human epidermal melanocyte cell line (PIG1) (a gift from Dr. Caroline Le Poole, Loyola University Chicago, Maywood, IL, USA) and human primary melanocytes (HPMs) isolated from human foreskin specimens obtained during surgical circumcision were cultured in Medium 254 (Cascade Biologics/Invitrogen, Portland, OR, USA) supplemented with human melanocyte growth supplement (Cascade Biologics/Invitrogen), 5% fetal bovine serum (Invitrogen, San Diego, CA, USA), and a penicillin–streptomycin antibiotic mix at 37 °C in the presence of 5% carbon dioxide.14

CPS and dimethyl sulfoxide were purchased from Sigma-Aldrich (St. Louis, MO, USA). Z-VAD-FMK (a pan-caspase inhibitor) and ruthenium red (RR) (a TRPV antagonist) were purchased from R&D Systems (Minneapolis, MN, USA). N-Acetyl-L-cysteine (NAC, an antioxidant) was purchased from Beyotime (Shanghai, China).

Cell vitality analysis

Cell vitality was monitored using a cell counting kit-8 (CCK-8) assay according to the manufacturer's protocol (Beyotime). In brief, melanoma cells were initially cultured at a density of 5 × 103 cells per well in 96-well plates overnight. The cells were then incubated for indicated times with different treatments at 37 °C. Next, CCK-8 solution was added to each culture well and incubated for 20 minutes at 37 °C. The absorbance at 450 nm was then measured with a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). All experiments were performed in triplicate, and the absorbance measured in treated cells was calculated as the percentage of the absorbance in untreated control cells.

Western blotting analysis

Cells that underwent the indicated treatment were washed once with ice-cold phosphate-buffered saline (PBS), and total proteins were extracted with RIPA Lysis Buffer containing the protease inhibitor phenylmethanesulfonyl fluoride (Beyotime). The homogenate was incubated on ice for 10 minutes and centrifuged at 10,000 g for 15 minutes to pellet large cellular debris. Protein concentrations were measured with a BCA Protein Assay Kit (Beyotime). Protein samples were denatured in 5 × sodium dodecyl sulfate (SDS) loading buffer by heating them at 100 °C for 5 minutes before use.

For the western blotting analysis, 30-μg protein samples were electrophoresed through SDS-polyacrylamide gels in Tris-glycine SDS running buffer. The separated proteins were electroblotted onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). After the membranes had been blocked in 5% nonfat milk at room temperature for 1 hour, they were incubated with the following primary antibodies diluted at 1:1000 overnight at 4 °C: dynamin-related protein 1 (DRP1) (ab56788; Abcam Australia, Melbourne, Victoria, Australia), phospho-DRP1 (pDRP1) (Ser616) (#3455; Cell Signaling Technology, Danvers, MA, USA), pDRP1 (Ser637) (#4867; Cell Signaling Technology), mitofusin 1 (MFN1) (13798-1-AP; Proteintech Group, Rosemont, IL, USA), MFN2 (12186-1-AP; Proteintech Group), fission 1 (Fis1) (10956-1-AP; Proteintech Group), optic atrophy 1 (OPA1) (#67589; Cell Signaling Technology), and cleaved caspase-3 (#9661; Cell Signaling Technology). As an internal standard between the samples, β-actin (CW0096 M; CWBio, Beijing, China) was used at a dilution of 1:5,000. The specific protein–antibody complex was detected using horseradish peroxidase-conjugated immunoglobulin G (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) diluted at 1:5,000 for 2 hours at room temperature. Detection of the chemiluminescence reaction was carried out with an enhanced chemiluminescence kit (Pierce/Thermo Fisher Scientific). The Western blot data were quantified by analyzing the band intensity with Image Lab software, Version 5.2.1 (Bio-Rad Laboratories).

Annexin V-FITC/propidium iodide apoptosis assay

To quantify apoptotic and necrotic death, melanoma cells were plated in 12-well plates at a density of 1 × 104 cells per well overnight, and the cells were then exposed to different treatments for indicated times. The cells were then collected and washed once with PBS. Apoptosis assay was conducted using an Annexin V-FITC Apoptosis Detection Kit (7Sea Biotech, Shanghai, China) according to the manufacturer's instructions. The numbers of viable, apoptotic, and necrotic cells were quantified by flow cytometry (Beckman Coulter, Brea, CA, USA), and the analysis was performed with Expo32 software (Beckman Coulter). Each treatment was repeated in triplicate.

Measurement of intracellular reactive oxygen species (ROS)

The production of ROS was monitored by flow cytometry using CM-H2DCFDA as the label. After undergoing the indicated treatments, the cells were loaded with CM-H2DCFDA (Life Technologies, Carlsbad, CA, USA) at 37 °C for 30 minutes and then incubated with culture medium for another 5 minutes. The intracellular ROS level was analyzed by flow cytometry (Beckman Coulter) at an excitation wavelength of 488 nm and emission wavelength of 530 nm.

Measurement of mitochondrial membrane potential (MMP)

The MMP (Δψm) was evaluated by a JC-1 staining assay. Briefly, cells that had undergone the indicated treatments were collected and resuspended in 0.5 mL of fresh culture medium containing 10 μmol/L JC-1 (Life Technologies) at 37 °C for 30 minutes followed by analysis with flow cytometry (Beckman Coulter). The MMP was quantified by the ratio of red to green fluorescence staining signals.

Measurement of intracellular adenosine triphosphate (ATP) level

The intracellular ATP level was measured using an ATP Assay Kit (Beyotime) according to the manufacturer's instructions. In brief, lysed cells were added to ATP detection buffer, and the ATP level was measured with a luminometer (Bio-Rad Laboratories). The measurement was performed three times for each sample. The ATP concentration was calculated from the luminescence value according to the standard curve.

Mitochondrial network imaging by confocal microscopy

The fluorescent dye MitoTracker Green FM (Invitrogen) was used to monitor mitochondrial morphology in living cells according to the manufacturer's instructions. In brief, the cells were loaded with MitoTracker Green FM at 37 °C in serum-free media for 30 minutes, the media was exchanged for fresh media, and the cells were viewed with an Olympus FV1000/ES confocal microscope (Olympus Corporation, Tokyo, Japan).

Mitochondrial morphology can also be observed by immunofluorescence staining of TOM20.15 Cells were fixed in 4% paraformaldehyde for 10 minutes, washed twice with PBS, permeated with 0.1% Triton X-100, and blocked with goat serum for 1 hour. The cells were then incubated with TOM20 antibody (1:200; Proteintech) overnight at 4 °C. After being washed with PBS, the cells were incubated with secondary antibodies (FITC-tagged goat anti-rabbit, 1:400) for 1 hour. The cells were then washed with PBS and further incubated with DAPI (1:1,000; Dako, Glostrup, Denmark) for 15 minutes. Fluorescent images were obtained with an FV1000/ES confocal microscope (Olympus).

Mitochondrial fission is indicated by a dramatic increase in mitochondria fragmentation. Therefore, the length of mitochondria, including fragmented, intermediate, and elongated mitochondria, was measured using ImageJ software (National Institutes of Health, Bethesda, MD, USA) to analyze the extent of mitochondrial fission or fusion.16

Statistical analyses

One-way analysis of variance and the Bonferroni method were used to detect differences among multiple groups. The unpaired two-tailed Student t test was used for comparisons between two groups. These analyses were performed with GraphPad Prism software, Version 7.0 (GraphPad Software, San Diego, CA, USA). Differences with P < 0.05, P < 0.01, or P < 0.001 were considered statistically significant. Data are presented as mean ± standard deviation for at least three independent experiments.

Results

CPS induces apoptosis of melanoma cells through TRPV channels and caspase cascade

We first examined the effect of CPS on melanoma cells and elucidated the involvement of TRPV channels. We treated melanoma cells (A2058 and WM35 cell lines) and melanocytes (HPMs and PIG1 cell line) with different concentrations of CPS for 16 hours. The results showed that the cell vitality of the two melanoma cell lines was significantly reduced when the concentration of CPS was ≥120 μmol/L (For A2058 cells: 0 vs. 120 μmol/L: [100% ± 0%] vs. [51.02% ± 6.40%], P < 0.05; For WM35 cells: 0 vs. 120 μmol/L: [100% ± 0%] vs. [51.80% ± 3.45%], P < 0.05), while CPS showed less impact on HPMs and the PIG1 cell line (Supplementary Fig. 1, https://links.lww.com/JD9/A2). These findings suggest that melanoma cells are more sensitive to CPS than are melanocytes. Additionally, the vitality of all of these cells decreased as the concentration of CPS increased (Supplementary Fig. 2, https://links.lww.com/JP9/A12), indicating a dose-dependent effect of CPS on the vitality of melanoma cells and melanocytes.

Pretreatment with RR, a widely used TRPV channel antagonist, reversed the inhibitory effect of CPS on cell viability (For A2058 cells: CPS vs. CPS+RR: [41.61% ± 2.11%] vs. [65.22% ± 3.78%], P < 0.01; For WM35 cells: CPS vs. CPS+RR: [26.95% ± 3.98%] vs. [43.28% ± 4.00%], P < 0.05) (Fig. 1A). Consistent with this, the flow cytometry analysis revealed that while CPS prominently promoted cell apoptosis in the two melanoma cell lines (For A2058 cells: Control vs. CPS: [1.53% ± 0.49%] vs. [9.7% ± 0.46%], P < 0.05; For WM35 cells: Control vs. CPS: [5.33% ± 0.94%] vs. [20.13% ± 0.88%], P < 0.01), pretreatment with RR protected the melanoma cells against CPS-induced cell apoptosis (For A2058 cells: CPS vs. CPS+RR: [9.70% ± 0.46%] vs. [5.40% ± 0.50%], P < 0.05; For WM35 cells: CPS vs. CPS+RR: [20.13% ± 0.88%] vs. [7.03% ± 1.13%], P < 0.01) (Fig. 1B and 1C). RR treatment alone had no effects on cell vitality or apoptosis (data not shown). Western blotting analysis showed that CPS promoted the expression of cleaved caspase 3, whereas pretreatment with RR reduced its expression in a dose-dependent manner (Fig. 1D). Notably, we used Z-VAD-FMK, a pan-caspase inhibitor and specific cell apoptosis inhibitor, to ensure that the CPS-induced cell death was mostly due to cell apoptosis and caspase signal-dependent (CPS vs. CPS+Z-VAD-FMK: [16.85% ± 0.55%] vs. [4.95% ± 0.65%], P < 0.01) (Fig. 1E and 1F). Collectively, these results demonstrate that CPS treatment promotes apoptosis of melanoma cells through TRPV channels and the caspase cascade.

Figure 1
Figure 1:
RR, a TRPV antagonist, blocks CPS-induced apoptosis and caspase activation. A2058 and WM35 cells were pretreated with RR (20 μmol/L) for 1 hour and then incubated with or without CPS (120 μmol/L) for 16 hours. (A) The cell viability was determined by a CCK-8 assay. (B and C) Cell apoptosis was analyzed with flow cytometry. (D) The protein level of cleaved caspase 3 in A2058 cells was measured after the indicated treatment. β-Actin was detected as a loading control. (E and F) A2058 cells were pretreated with Z-VAD-FMK (20 μmol/L) for 1 hour and then incubated with or without CPS (120 μmol/L) for 16 hours. Cell apoptosis was analyzed by flow cytometry. The data are presented as mean ± standard deviation of three independent experiments. P < 0.05, ∗∗ P < 0.01. CCK-8: cell counting kit-8; CPS: capsaicin; RR: ruthenium red; TRPV: transient receptor potential vanilloid 1.

CPS-triggered ROS generation is mediated by TRPV channels and contributes to cell apoptosis

A previous study showed that CPS-induced tumor cell apoptosis is associated with the generation of ROS.17 Therefore, we analyzed the production of intracellular ROS in melanoma cells after CPS treatment. We found that the level of ROS significantly increased as the concentration of CPS gradually increased in both cell lines (For A2058 cells: 0 vs. 140 μmol/L: [1.00 ± 0] vs. [1.70 ± 0.08], P < 0.05; For WM35 cells: 0 vs. 140 μmol/L: [1.00 ± 0] vs. [1.60 ± 0.28], P < 0.05) (Fig. 2A and 2B).

Figure 2
Figure 2:
CPS increases intracellular ROS generation through TRPV channels. (A and B) A2058 and WM35 cells were treated with various concentrations of CPS for 16 hours, and the cellular ROS levels were detected. (C and D) A2058 and WM35 cells were pretreated with RR (20 μmol/L) for 1 hour and then incubated with or without CPS (120 μmol/L) for 16 hours. The intracellular ROS levels were analyzed by flow cytometry. The data are presented as mean ± standard deviation of three independent experiments. P < 0.05, ∗∗ P < 0.01. CPS: capsaicin; RR: ruthenium red; TRPV: transient receptor potential vanilloid 1.

To explore whether CPS-triggered ROS generation is mediated by TRPV channels, we pretreated A2058 and WM35 cells with RR followed by CPS treatment and found that the generation of intracellular ROS was prominently diminished (For A2058 cells: CPS vs. CPS+RR: [2.34 ± 0.30] vs. [1.34 ± 0.12], P < 0.05; For WM35 cells: CPS vs. CPS+RR: [2.25 ± 0.25] vs. [1.65 ± 0.13], P < 0.05) (Fig. 2C and 2D). This finding indicates that TRPV channels mediated CPS-triggered intracellular ROS production. RR treatment alone had no effect on ROS generation (data not shown).

We further investigated the role of ROS in CPS-induced apoptosis of melanoma cells. The cells were pretreated with different concentrations of the ROS scavenger NAC followed by CPS treatment. The results showed that the viability of melanoma cells was significantly suppressed with CPS treatment alone while it gradually recovered in the presence of NAC (For A2058 cells: CPS vs. CPS+4 mmol/L: [46.44% ± 5.81%] vs. [84.75% ± 10.90%], P < 0.05; For WM35 cells: CPS vs. CPS+4 mol/L: [40.77% ± 7.11%] vs. [95.34% ± 16.95%], P < 0.01) (Fig. 3A and 3B). Similarly, while CPS treatment alone obviously induced cell apoptosis of A2058 and WM35 cells, NAC pretreatment prominently abrogated this effect (For A2058 cells: CPS vs. CPS+NAC: [22.65% ± 2.05%] vs. [8.70% ± 2.20%], P < 0.05; For WM35 cells: CPS vs. CPS+NAC: [16.05% ± 3.32%] vs. [7.78% ± 0.50%], P < 0.05) (Fig. 3C and 3D). NAC treatment alone had no effect on cell vitality or apoptosis (data not shown). Therefore, the generation of intracellular ROS contributed to CPS-induced apoptosis of melanoma cells.

Figure 3
Figure 3:
ROS contributes to CPS-induced apoptosis of melanoma cells. (A and B) A2058 and WM35 cells were pretreated with various concentrations of NAC for 1 hour and then incubated with or without CPS (120 μmol/L) for 16 hours. The cell viability was determined by CCK-8 assay. (C and D) A2058 and WM35 cells were pretreated with NAC (4 mmol/L) for 1 hour and then incubated with or without CPS (120 μmol/L) for 16 hours. Cell apoptosis was analyzed by flow cytometry. The data are presented as mean ± standard deviation of three independent experiments. P < 0.05, ∗∗ P < 0.01. CCK-8: cell counting Kit-8; CPS: capsaicin; NAC: N-acetyl-L-cysteine; ROS: reactive oxygen species.

CPS treatment leads to TRPV channel-dependent MMP dissipation and ATP reduction in melanoma cells

Because mitochondrion is the main source of intracellular ROS,18 we were interested in determining whether CPS treatment influences mitochondrial function and thereby regulates cell apoptosis. Through the flow cytometry assay, we found that CPS treatment prominently induced dissipation of the MMP (Control vs. CPS: [1.00 ± 0] vs. [0.61 ± 0.08], P < 0.05) (Fig. 4A and 4B), which has been regarded as a characteristic of mitochondrial dysfunction and is highly correlated with the generation of ROS and induction of apoptosis.19 Moreover, pretreatment with RR abolished the CPS-induced dissipation of the MMP (CPS vs. CPS+RR: [0.61 ± 0.08] vs. [1.10 ± 0.11], P < 0.05) (Fig. 4A and 4B). RR treatment alone had no effects on MMP of melanoma cells (data not shown).

Figure 4
Figure 4:
CPS triggers MMP dissipation and ATP reduction through TRPV channels. (A and B) A2058 cells were pretreated with RR (20 μmol/L) for 1 hour and then incubated with or without CPS (120 μmol/L) for 16 hours. The MMP level was examined by JC-1 staining. (A) The scatter plot of the flow cytometry analysis shows the distribution of JC-1 aggregates (red) and the JC-1 monomer (green) cell population. (B) The histogram calculated the relative ratio of red to green fluorescence. (C) A2058 and WM35 cells were treated with various concentrations of CPS for 16 hours, and the ATP levels were detected. (D) A2058 and WM35 cells were pretreated with RR (20 μmol/L) for 1 hour and then incubated with or without CPS (120 μmol/L) for 16 hours; the ATP levels were then detected. The data are presented as mean ± standard deviation of three independent experiments. P < 0.05, ∗∗ P < 0.01. ATP: adenosine triphosphate; CPS: capsaicin; MMP: mitochondrial membrane potential; RR: ruthenium red; TRPV: transient receptor potential vanilloid 1.

Further, we examined the alteration of intracellular ATP to reveal the functional status of the mitochondria. Consistent with the trend of the MMP, CPS treatment prominently suppressed the intracellular ATP level, and this suppression was reversed by RR treatment (For A2058 cells: Control vs. CPS: [1.00 ± 0.00] vs. [0.57 ± 0.01], P < 0.05; CPS vs. CPS+RR: [0.57 ± 0.01] vs. [1.12 ± 0.12], P < 0.05; For WM35 cells: Control vs. CPS: [1.00 ± 0.00] vs. [0.05 ± 0.02], P < 0.01; CPS vs. CPS+RR: [0.05 ± 0.02] vs. [0.35% ± 0.06%], P < 0.05) (Fig. 4C and 4D). RR treatment alone had no effect on the ATP level (data not shown). Collectively, these results demonstrate that CPS treatment results in mitochondrial dysfunction characterized by the generation of ROS, dissipation of the MMP, and down-regulation of intracellular ATP in a TRPV-dependent manner.

Mitochondrial fission connects CPS treatment to mitochondrial dysfunction

During the process of apoptosis, mitochondrial fission is an initial step that occurs before MMP dissipation and caspase activation.20 Therefore, we speculated that CPS treatment may induce the alteration of the mitochondrial dynamics and thereby promote cell apoptosis. To this end, we employed immunofluorescence staining using the mitochondrial dye MitoTracker Green or TOM20 to reveal the alterations of the mitochondrial dynamics. We found that CPS treatment led to dramatic mitochondrial fission in both the A2058 and WM35 melanoma cell lines as indicated by a dramatic increase in mitochondrial fragmentation (For A2058 cells: Control vs. CPS: [6.35% ± 3.83%] vs. [72.10% ± 5.05%], P < 0.001; For WM35 cells: Control vs. CPS: [2.5% ± 2.5%] vs. [77.21% ± 6.20%], P < 0.001) (Fig. 5A–D, Supplementary Fig. 2, https://links.lww.com/JP9/A12). However, we did not observe significant mitochondrial fission in HPMs or the PIG1 cell line treated with CPS (Supplementary Fig. 3, https://links.lww.com/JD9/A14). We then found that pretreatment with RR obviously suppressed CPS-induced mitochondrial fission (For A2058 cells: CPS vs. CPS+RR: [72.10% ± 5.05%] vs. [15.63% ± 5.98%], P < 0.01; For WM35 cells: CPS vs. CPS+RR: [77.21% ± 6.20%] vs. [12.57% ± 5.61%], P < 0.01) (Fig. 5A–D), illustrating the involvement of the TRPV channels.

Figure 5
Figure 5:
CPS promotes mitochondrial fission in a TRPV channel-dependent manner. (A–D) A2058 and WM35 cells were pretreated with RR (20 μmol/L) for 1 hour and then incubated with or without CPS (120 μmol/L) for 16 hours. The mitochondrial network was displayed with MitoTracker Green FM staining. (A and C) Representative confocal microscopic images of the mitochondrial network are shown. (B and D) The proportion of cells (n = 100 cells for each sample) with fragmented, intermediate, and elongated mitochondria was quantified. (E and F) A2058 and WM35 cells were treated with various concentrations of CPS for 16 hours, and mitochondrial dynamics-related protein levels were examined by western blotting assay. The data are presented as mean ± standard deviation of three independent experiments. P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. CPS: capsaicin; RR: ruthenium red; TRPV: transient receptor potential vanilloid 1; DRP: dynamin-related protein 1; p-DRP: phosphorylated-DRP; Fis1: fission1; MFN1: mitofusin 1; OPA1: optic atrophy 1.

Mitochondrial dynamics is governed by a series of regulators, including DRP1, Fis1, MFN1, MFN2, and OPA1. Among them, Fis1 and DRP1 facilitate mitochondrial fission while OPA1, MFN1, and MFN2 promote mitochondrial fusion.11 Notably, phosphorylation of DRP1 at S616 can promote its activity to facilitate mitochondrial fission, while phosphorylation of DRP1 at S637 suppresses its activity to inhibit mitochondrial fission.21 Our western blotting analysis revealed that while the expressions of DRP1, Fis1, OPA1, MFN1, and MFN2 were not significantly altered, the phosphorylation of DRP1 at S616 was prominently increased (For A2058 cells: 0 vs. 100 vs. 120 μmol/L: [1.00 ± 0] vs. [2.50 ± 0.13] vs. [3.78 ± 0.22], P < 0.01; For WM35 cells: 0 vs. 100 vs. 120 μM: [1.00 ± 0] vs. [1.86 ± 0.15] vs. [1.95 ± 0.15], P < 0.05) (Fig. 5E and 5F). However, we did not observe an alteration of the phosphorylation of DRP1 at S637 (Fig. 5E and 5F). This result indicates that increased phosphorylation of DRP1 at S616 may be responsible for CPS-induced mitochondrial fission.

Discussion

In the present study, we found that CPS treatment significantly inhibited the vitality of melanoma cells whereas it exerted less impact on normal melanocytes, indicating that melanoma cells are more sensitive to CPS than are normal melanocytes. This makes CPS an attractive agent for melanoma therapy. We also proved that CPS induced apoptosis of melanoma cells through TRPV channels and the caspase cascade. Moreover, we proved that intracellular ROS generation contributed to CPS-induced melanoma cell apoptosis. We also showed that mitochondrial dysfunction, including dissipation of the MMP and downregulation of intracellular ATP, occurred after CPS treatment. Our data further revealed that mitochondrial fission connected CPS treatment to mitochondrial dysfunction in melanoma cells. Collectively, our findings demonstrate the role of mitochondrial fission and its related mitochondrial dysfunction in mediating the therapeutic effect of CPS on melanoma (Fig. 6).

Figure 6
Figure 6:
Proposed model of the mechanism underlying the anti-tumor effect of CPS in melanoma. Once activated by CPS, TRPV channels preferentially lead to prominent Ca2+ influx into melanoma cells, which triggers extensive mitochondrial fission, mitochondrial dysfunction including MMP dissipation, ATP reduction, and ROS generation. All of these effects finally result in melanoma cell apoptosis. ATP: adenosine triphosphate; MMP: mitochondrial membrane potential; ROS: reactive oxygen species; TRPV: transient receptor potential vanilloid 1.

The suppressive role of CPS on the growth of melanoma has been proven,8-9 but the underlying mechanisms are not well characterized. CPS reportedly inhibits the growth of melanoma via inhibition of NADH oxidase activity in the plasma membrane.22 Our recent study showed that CPS treatment inhibited melanoma growth by activating p53 and inducing cell apoptosis.8 The present study emphasized the involvement of the alteration of mitochondrial dynamics in CPS-induced apoptosis of melanoma cells. While mitochondrial fission may play an important oncogenic role during tumorigenesis and progression of melanomas,23-24 excessive mitochondrial fragmentation leads to defective cellular metabolism, oxidative stress, mitochondrial dysfunction, and even cell death.21 Importantly, we proved for the first time that mitochondrial fission connected CPS treatment to cell apoptosis in cancer cells. Consistent with a previous study demonstrating that CPS enhanced Ca2+-dependent mitochondrial fission through TRPV1 in dorsal root ganglia,25 our results showed that CPS-induced mitochondrial fission in melanoma is also TRPV channel-dependent, suggesting that calcium signaling plays a role in regulating the mitochondrial dynamics of melanoma cells. The two melanoma cell lines A2058 and WM35 used in the present study are metastatic and primary melanoma cell lines, respectively. We compared the effects of CPS on the cell vitality, cell apoptosis, mitochondrial fission, and intracellular ROS and ATP levels of these two melanoma cell lines. The results showed that the effects of CPS on these parameters were comparable between the two melanoma cell lines with the exception of the ATP level, which decreased more significantly in the WM35 than A2058 cell line (Supplementary Fig. 4, https://links.lww.com/JD9/A15).

Mitochondrial fission is highly correlated with cell death via the regulation of mitochondrial function.13 Our results showed that impaired mitochondrial function, characterized by dissipation of the MMP and reduction of ATP, occurred after CPS treatment through TRPV channels. This is consistent with some previous studies showing that CPS could induce MMP dissipation in urothelial cancer cells26 and glioma cells.27 Following MMP dissipation, cytochrome c is released from the mitochondria to the cytoplasm and activates caspase cascade-dependent apoptosis.19 Therefore, dissipation of the MMP is correlated with CPS-induced activation of the caspase cascade in melanoma.

Massive mitochondrial fragmentation increases ROS production.18 As expected, we observed increased intracellular ROS generation after CPS treatment. This increase contributed to CPS-induced apoptosis of melanoma cells and was dependent upon TRPV channels, indicating that intracellular Ca2+ influx may mediate ROS generation. Indeed, TRPV1 activation and sustained increase of intracellular Ca2+ facilitate the production of ROS and trigger cell apoptosis.10 Notably, research has shown that nitric oxide can also mediate CPS-induced apoptosis in A375 melanoma cells,28 suggesting the pivotal role of different types of ROS in the inhibitory effect of CPS on melanoma growth.

The bioavailability of CPS has been studied in rats, nude mice, and humans.29 In humans, 5 g of capsaicinoids (primarily comprising CPS, dihydrocapsaicin, nordihydrocapsaicin, homohydrocapsaicin, homodihydrocapsaicin, and nonivamide) was administered orally. The half-life of CPS in the blood was found to be about 25 minutes, and the peak plasma concentration of CPS was 2.5 ng/mL (approximately 8.2 nmol/L) at 45 minutes.30 Although the concentration of CPS used in the present study was high, CPS can be applied locally via direct injection into a tumor as described in our previous study, in which CPS was intratumorally administered and significantly inhibited melanoma growth in vivo.8

In conclusion, our data highlight the importance of mitochondrial fission and resultant mitochondrial dysfunction, which are induced through TRPV channels, in mediating CPS-induced apoptosis of melanoma cells. Our findings reveal novel mechanisms for understanding the anti-tumor activity of CPS, which may be a clinically useful agent for cancer therapy.

Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China (Nos. 81625020, 81402736, and 81902791) and the Key Research and Development Program of Shaanxi Province (No. 2019sf-079).

References

[1]. Pal HC, Hunt KM, Diamond A, et al. Phytochemicals for the management of melanoma. Mini Rev Med Chem 2016;16 (12):953–979. doi: 10.2174/1389557516666160211120157.
[2]. Lin WM, Fisher DE. Signaling and immune regulation in melanoma development and responses to therapy. Ann Rev Pathol 2017;12:75–102. doi:10.1146/annurev-pathol-052016-100208.
[3]. Santoni G, Farfariello V. TRP channels and cancer: new targets for diagnosis and chemotherapy. Endocr Metab Immune Disord Drug Targets 2011;11 (1):54–67. doi:10.2174/187153011794982068.
[4]. Lau JK, Brown KC, Dom AM, et al. Capsaicin induces apoptosis in human small cell lung cancer via the TRPV6 receptor and the calpain pathway. Apoptosis 2014;19 (8):1190–1201. doi:10.1007/s10495-014-1007-y.
[5]. Tsagareli MG, Nozadze I. An overview on transient receptor potential channels superfamily. Behav Pharmacol 2020;31 (5):413–434. doi:10.1097/FBP.0000000000000524.
[6]. Fattori V, Hohmann MS, Rossaneis AC, et al. Capsaicin: current understanding of its mechanisms and therapy of pain and other pre-clinical and clinical uses. Molecules 2016;21 (7):844. doi:10.3390/molecules21070844.
[7]. Bley K, Boorman G, Mohammad B, et al. A comprehensive review of the carcinogenic and anticarcinogenic potential of capsaicin. Toxicol Pathol 2012;40 (6):847–873. doi:10.1177/0192623312444471.
[8]. Yang Y, Guo W, Ma J, et al. Downregulated TRPV1 expression contributes to melanoma growth via the calcineurin-ATF3-p53 pathway. J Invest Dermatol 2018;138 (10):2205–2215. doi:10.1016/j.jid.2018.03.1510.
[9]. Chu H, Li M, Wang X. Capsaicin induces apoptosis and autophagy in human melanoma cells. Oncol Lett 2019;17 (6):4827–4834. doi:10.3892/ol.2019.10206.
[10]. Xie L, Xiang GH, Tang T, et al. Capsaicin and dihydrocapsaicin induce apoptosis in human glioma cells via ROS and Ca2+mediated mitochondrial pathway. Mol Med Rep 2016;14 (5):4198–4208. doi:10.3892/mmr.2016.5784.
[11]. Chan DC. Fusion and fission: interlinked processes critical for mitochondrial health. Annu Rev Genet 2012;46:265–287. doi:10.1146/annurev-genet-110410-132529.
[12]. Suliman HB, Piantadosi CA. Mitochondrial quality control as a therapeutic target. Pharmacol Rev 2016;68 (1):20–48. doi:10.1124/pr.115.011502.
[13]. Montessuit S, Somasekharan SP, Terrones O, et al. Membrane remodeling induced by the dynamin-related protein Drp1 stimulates Bax oligomerization. Cell 2010;142 (6):889–901. doi:10.1016/j.cell.2010.08.017.
[14]. Jian Z, Li K, Liu L, et al. Heme oxygenase-1 protects human melanocytes from H2O2-induced oxidative stress via the Nrf2-ARE pathway. J Invest Dermatol 2011;131 (7):1420–1427. doi:10.1038/jid.2011.56.
[15]. Liang X, Wang S, Wang L, et al. Mitophagy inhibitor liensinine suppresses doxorubicin-induced cardiotoxicity through inhibition of Drp1-mediated maladaptive mitochondrial fission. Pharmacol Res 2020;157:104846. doi:10.1016/j.phrs.2020.104846.
[16]. Yi X, Guo W, Shi Q, et al. SIRT3-dependent mitochondrial dynamics remodeling contributes to oxidative stress-induced melanocyte degeneration in vitiligo. Theranostics 2019;9 (6):1614–1633. doi:10.7150/thno.30398.
[17]. Lan CC, Wu CS, Yu HS. Solar-simulated radiation and heat treatment induced metalloproteinase-1 expression in cultured dermal fibroblasts via distinct pathways: implications on reduction of sun-associated aging. J Dermatol Sci 2013;72 (3):290–295. doi:10.1016/j.jdermsci.2013.07.015.
[18]. Kim ES, Park SJ, Goh MJ, et al. Mitochondrial dynamics regulate melanogenesis through proteasomal degradation of MITF via ROS-ERK activation. Pigment Cell Melanoma Res 2014;27 (6):1051–1062. doi:10.1111/pcmr.12298.
[19]. Zong WX, Rabinowitz JD, White E. Mitochondria and cancer. Mol Cell 2016;61 (5):667–676. doi:10.1016/j.molcel.2016.02.011.
[20]. Youle RJ, Karbowski M. Mitochondrial fission in apoptosis. Nat Rev Mol Cell Biol 2005;6 (8):657–663. https://doi.org/10.1038/nrm1697.
[21]. Singh S, Sharma S. Dynamin-related protein-1 as potential therapeutic target in various diseases. Inflammopharmacology 2017;25 (4):383–392. doi:10.1007/s10787-017-0347-y.
[22]. Pal HC, Hunt KM, Diamond A, et al. Phytochemicals for the Management of Melanoma. Mini Rev Med Chem 2016;16 (12):953–979. doi: 10.2174/1389557516666160211120157.
[23]. Soares CD, de Lima Morais TM. Expression of mitochondrial dynamics markers during melanoma progression: Comparative study of head and neck cutaneous and mucosal melanomas. J Oral Pathol Med 2019;48 (5):373–381. doi:10.1111/jop.12855.
[24]. Soares CD, Morais TML, Carlos R, et al. Prognostic importance of mitochondrial markers in mucosal and cutaneous head and neck melanomas. Hum Pathol 2019;85:279–289. doi:10.1016/j.humpath.2018.11.009.
[25]. Chiang H, Ohno N, Hsieh YL, et al. Mitochondrial fission augments capsaicin-induced axonal degeneration. Acta Neuropathol 2015;129 (1):81–96. doi:10.1007/s00401-014-1354-3.
[26]. Caprodossi S, Amantini C, Nabissi M, et al. Capsaicin promotes a more aggressive gene expression phenotype and invasiveness in null-TRPV1 urothelial cancer cells. Carcinogenesis 2011;32 (5):686–694. doi:10.1093/carcin/bgr025.
[27]. Xie L, Xiang GH, Tang T, et al. Capsaicin and dihydrocapsaicin induce apoptosis in human glioma cells via ROS and Ca2+ mediated mitochondrial pathway. Mol Med Rep 2016;14 (5):4198–4208. doi: 10.3892/mmr.2016.5784.
[28]. Kim MY. Nitric oxide triggers apoptosis in A375 human melanoma cells treated with capsaicin and resveratrol. Mol Med Rep 2012;5 (2):585–591. doi:10.3892/mmr.2011.688.
[29]. Rollyson WD, Stover CA, Brown KC, et al. Bioavailability of capsaicin and its implications for drug delivery. J Control Release 2014;196:96–105. doi:10.1016/j.jconrel.2014.09.027.
[30]. Gaikwad S, Srivastava SK. Role of Phytochemicals in Perturbation of Redox Homeostasis in Cancer. Antioxidants (Basel) 2021;10 (1):83. doi:10.3390/antiox10010083.
Keywords:

capsaicin; melanoma; mitochondrial dysfunction; mitochondrial fission; TRPV channels

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