Capsaicin Exerts Therapeutic Effects by targeting tNOX-SIRT1 Axis and Augmenting ROS-Dependent Cytotoxic Autophagy in Melanoma Cancer Cells


 Background: Although considered a rare form of skin cancer, malignant melanoma has steadily increased internationally and is a main cause of cancer-associated death worldwide. The treatment options for malignant melanoma are very limited. Accumulating data suggest that the natural compound, capsaicin, exhibits preferential anticancer properties to act as a nutraceutical agent. Here, we explored the underlying molecular events involved in the inhibitory effects of capsaicin on the growth of melanoma cells.Methods: The cellular thermal shift assay (CETSA) and isothermal dose response fingerprint (ITDRFCETSA) were utilized to validate the binding of capsaicin with the tumor-associated NADH oxidase, tNOX (ENOX2) in melanoma cells. We also assessed the cellular impact of capsaicin-targeting of tNOX on A375 cells by flow cytometry and protein analysis. The essential role of tNOX in tumor- and melanoma-growth limiting abilities of capsaicin was evaluated in C57BL/6 mice.Results: Our data show that capsaicin directly targets cellular tNOX to inhibit its enzymatic activity and enhance protein degradation capacity. The inhibition of tNOX by capsaicin is accompanied by the attenuation of SIRT1, a NAD+-dependent deacetylase that enhances ULK1 acetylation to induce ROS-dependent autophagy in melanoma cells. Capsaicin treatment of mice implanted with melanoma cancer cells suppressed tumor growth by down-regulating tNOX and SIRT1, which was also seen in an in vivo xenograft study with tNOX-depleted melanoma cells. Conclusions: Together, our findings suggest that tNOX expression is important for the growth of melanoma cancer cells both in vitro and in vivo, and that inhibition of the tNOX-SIRT1 axis contributes to inducting cytotoxic ROS-dependent autophagy in melanoma cells.


Background
Malignant melanoma accounts for approximately 5% of skin cancers; however, its high metastatic potential means that it accounts for > 75% of all skin cancer deaths in the United States, with particularly high mortality seen in those diagnosed at a later stage [1,2]. Unfortunately, acquired drug resistance largely restricts the e cacy of chemotherapy in melanoma, resulting in disease progression and a poor long-term survival rate [3][4][5]. Accumulated evidence demonstrates that phytochemicals, such as capsaicin (8-methyl-N-vanillyl-6-nonenamide; the active component in chili peppers), can exhibit superior toxicity toward cancer cells over non-cancerous cells and may offer bene ts as nutraceutical agents [6][7][8][9][10][11]. Indeed, capsaicin has long been known to suppress the growth of human and mouse melanoma cells [12][13][14][15][16][17]. However, we current lack direct and physical evidence linking the speci c target(s) of capsaicin to its superior cytotoxicity. Thus, it is worth investigating novel cellular target(s) for capsaicin that have therapeutic potential, and assessing the relevant mechanism(s) of action in melanoma.
In previous work, we explored the distinct cellular impacts of capsaicin in different systems and identi ed a tumor-associated NADH oxidase (tNOX; ENOX2) that is hormone-and growth factor-stimulated in non-Apoptosis was measured using an Annexin V-FITC Apoptosis Detection Kit (BD Pharmingen, San Jose, CA, USA). Cells cultured in 6-cm dishes were trypsinized and collected by centrifugation. The cell pellet was washed, resuspended in 1x binding buffer and stained with annexin V-FITC ( uorescein isothiocyanate), as recommended by the manufacturer. Cells were also stained with propidium iodide (PI) to detect necrosis or late apoptosis. The distribution of viable (FITC/PI double-negative), early apoptotic (FITC-positive), late apoptotic (FITC/PI double-positive) and necrotic (PI-positive/FITC-negative) cells was analyzed using a Beckman Coulter FC500 ow cytometer. The results are expressed as a percentage of total cells.

Autophagy determination
Autophagosomes-acidic intracellular compartments that mediate the degradation of cytoplasmic materials during autophagy-were visualized by staining with Acridine Orange (AO; Sigma Chemical Co.).
After incubation, cells were washed with PBS and stained with 2 mg/ml AO for 10 min at 37°C. AOstained cells were then washed, trypsinized, and analyzed using a Beckman Coulter FC500. The results are expressed as a percentage of total cells.

Reverse transcriptase-polymerase chain reaction (RT-PCR)
Total RNA from melanoma cancer cells was isolated using the TRIzol reagent (Gibco, Carlsbad, CA, USA).

Measurement of reactive oxygen species (ROS)
Oxidative stress was determined by measuring the level of hydrogen peroxide (H 2 O 2 ) generated in the cells, as assessed by 5-(6)-carboxy-2',7'-dichlorodihydro uorescein diacetate (carboxy-H 2 DCFDA) staining. The nonpolar, nonionic H 2 -DCFDA is cell permeable and is hydrolyzed to non uorescent H 2 -DCF by intracellular esterases. In the presence of peroxide, H 2 -DCF is rapidly oxidized to highly uorescent DCF. In brief, at the end of test compound treatment, cells (2 × 10 5 ) were washed with PBS and incubated with 5 mM H 2 DCFDA in binding buffer for 30 min. The cells were then collected by trypsinization and centrifugation, washed with PBS, centrifuged at 200 × g for 5 min and analyzed immediately using a Beckman Coulter FC500 ow cytometer.

SIRT1 activity measurement
The cellular SIRT1 activity was measured using a SIRT1 Activity Assay Kit (Fluorometric)(Abcam Inc. Cambridge, MA, USA). Brie y, 1 ´ 10 5 cells (either untreated or capsaicin-treated for 24 h) were washed, harvested and resuspended in 1 mL of lysis buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 15 mM MgCl 2 , 250 mM sucrose, 0.5% NP-40, 0.1 mM EGTA). Cell lysates were sonicated 4 times for 5 seconds each on ice, and were centrifuged for 10 min at 4°C. The supernatant was then used for the determination of SIRT1 activity according to the manufacturer's instructions.
Cellular target identi cation of capsaicin by cellular thermal shift assay (CETSA) Intracellular tNOX as cellular target of capsaicin was established by CETSA. Samples were prepared from control and capsaicin-exposed cells. For each set, 2 × 10 7 cells were seeded in a 10-cm cultured dish.
After 24 h of culture, the cells were pretreated with 10 mM MG132 for 1 h, washed with PBS, treated with trypsin, and collected. Samples were centrifuged at 12,000 rpm for 3 min at room temperature, the pellets were gently resuspended with 1 mL of PBS, and the samples were centrifuged at 7,500 rpm for 3 min at room temperature. The pellets were resuspended with 1 mL of PBS containing 20 mM Tris-HCl pH 7.4, 100 mM NaCl, 5mM EDTA, 2 mM phenylmethylsulfonyl uoride (PMSF), 10 ng/ml leupeptin, and 10 mg/ml aprotinin. The samples were transferred to Eppendorf tubes and subjected to three freeze-thaw cycles; for each cycle, they were exposed to liquid nitrogen for 3 min, placed in a heating block at 37°C for 3 min, and vortexed brie y. For the experimental sample set, capsaicin was added to a nal concentration of 2 mM; for the control sample set, the same volume of vesicle solvent was added. The samples were heated at 37°C for 1 h and dispensed to 100 ml aliquots. Pairs consisting of one control aliquot and one experimental aliquot were heated at 40°C, 43°C, 46°C, 49°C, 52°C, 55°C, 58°C, or 61°C for 3 min. Insoluble proteins were separated by centrifugation at 12,000 rpm for 30 min at 4°C, and the supernatants with soluble proteins were used for SDS-PAGE and Western blot analysis using antisera to tNOX [31, 33]. bactin was used as the control.
The procedure for establishing an isothermal dose-response ngerprint (ITDRF CETSA ) was similar to that of the CETSA melting curve experiments as described above. Cells were seeded in 60 mm cultured dishes. After 24 h of culture, the cells were pretreated with 10 mM MG132 and exposed to different nal concentrations of 0.001, 0.01, 0.05, 0.25, 0.5, 1, 10, 100, 200 mM capsaicin for 1 h, washed with PBS, treated with trypsin, and collected with centrifugation at 12,000 rpm for 2 min at room temperature. The pellets were gently resuspended with 1 mL of PBS, and then centrifuged at 7,500 rpm for 3 min at room temperature, and resuspended with PBS containing 20 mM Tris-HCl pH 7.4, 100 mM NaCl, 5mM EDTA, 2 mM phenylmethylsulfonyl uoride (PMSF), 10 ng/ml leupeptin, and 10 mg/ml aprotinin, and then subjected to three freeze-thaw cycles; for each cycle, they were exposed to liquid nitrogen for 3 min, placed in a heating block at 25°C for 3 min, and vortexed brie y. The samples were then heated at 54°C for 3 min and cooled for 3 min at room temperature. Insoluble proteins were separated by centrifugation at 12,000 rpm for 30 min at 4°C, and the supernatants with soluble proteins were used for SDS-PAGE and Western blot analysis using antisera to tNOX. b-actin was used as the control.

Immunoblotting and immunoprecipitation
Cell extracts were prepared in lysis buffer (20 mM Tris-HCl pH 7.4, 100 mM NaCl, 5 mM EDTA, 2 mM phenylmethylsulfonyl uoride (PMSF), 10 ng/ml leupeptin, 10 mg/ml aprotinin). Volumes of extract containing equal amounts of proteins (40 µg) were applied to SDS-PAGE gels, and resolved proteins were transferred to PVDF membranes (Schleicher & Schuell, Keene, NH, USA). The membranes were blocked, washed, and probed with primary antibody. After washing to remove unbound primary antibody, membranes were incubated with horseradish peroxidase-conjugated secondary antibody for 2 hours. The blots were washed again and developed using enhanced chemiluminescence (ECL) reagents, according to the manufacturer's protocol (Amersham Biosciences, Piscataway, NJ, USA).
For immunoprecipitation, protein extracts from 100 mm dishes were incubated with 60 ml of Protein G Agarose Beads (for rabbit antibodies) for 1 h at 4°C in rotation for pre-clearing. The ULK1 antibody or control IgG were incubated onto beads in 500 ml of PBS-Tween 20 1% for overnight in rotation at 4°C. Beads were precipitated by centrifugation at 3000 rpm, 2 min at 4°C and 80 ml of supernatants were collected for input total lysates. Beads were washed three times with lysis buffer and samples were prepared for Western blotting analysis.

In vivo xenograft studies
Speci c pathogen free (SPF) female C57BL/6 mice were purchased from the National Laboratory Animal Center (Taipei, Taiwan). The animal use protocols were approved by the Institutional Animal Care and Use Committee of National Chung Hsing University (Taichung, Taiwan). Mice were subcutaneously inoculated with 100 ml of a B16F10 cell mixture containing 2 × 10 5 live cells in PBS. The tumor-bearing mice were randomly divided into two groups (n = 4 per group) when the tumor mass reached an average diameter of 0.5-1 cm. Group 1 mice were intratumorally injected with vehicle buffer (1.12% ethanol in 1X PBS) as untreated control (VC group). Group 2 mice were intratumorally treated with 200 mg capsaicin in vehicle buffer (Cap group). Intratumoral therapy was performed two times at an interval of one week. The tumor size was recorded every 2-3 days and the tumor volume was calculated using the formula: length × width 2 × 0.5). Mice were euthanized at 4 days after the nal treatment. The signi cance of differences in tumor size was determined by one-tailed Student's t test.
To con rm the contribution of tNOX to tumor growth, 1 ´ 10 6 B16F10 mouse melanoma cells transfected with either synthesized scramble (control-i) or tNOX (tNOX-i) shRNA were used for inoculation. Eightweek-old female C57BL/6 mice were randomly divided into two groups and subcutaneously inoculated with tumor cells at the right ank: Group-1 mice (n=3) were injected with control-i cells as a control group and group-2 mice (n=3) were injected with tNOX-i cells. Fourteen days after tumor cell injection, mice were euthanized, the tumor mass was quickly removed, and the mass was weighed.

Statistical analyses
All in vitro experiments were performed at least three times and all in vivo experiments were conducted with at least three animals. The results of each experiment are expressed as the means ± SEs. Betweengroup comparisons were performed using one-way analysis of variance (ANOVA) followed by an appropriate post-hoc test. A value of p<0.05 was considered to indicate a statistically signi cant difference.

Results
Capsaicin suppresses the growth of mouse and human melanoma cells We rst set out to determine the effect of capsaicin on the growth of melanoma cells, utilizing both mouse (B16F10) and human (A375) cells by dynamically monitoring cell impedance, and displayed the results as cell index (CI) values [34,35]. Our results suggested that capsaicin attenuated the growth of these two cell lines with comparable degrees of cytotoxicity at concentrations above 10 µM (Fig. 1A). We observed a slight rise in the proliferation of these cancer cell lines when exposed to 10 µM capsaicin, which was similar to a previous reported nding [21]. Consistent results were also obtained with a cell viability assay, which indicated that capsaicin induced dose-and time-dependent decreases in melanoma cell viability, except at 10 µM for the A375 cells (Fig. 1B). However, when we used a colony-forming assay to examine the longer-term effect, we found that capsaicin markedly diminished A375 cell proliferation at 10 and 50 µM, and completely blocked cell proliferation at 100 and 200 µM (Fig. 1C).

Capsaicin Induces Ros-dependent Autophagy But Not Apoptosis
Given that capsaicin has long been recognized as a nutraceutical agent for its apoptotic activity, we next validated the cellular outcomes in melanoma cells exposed to capsaicin. However, our results suggested that capsaicin was unable to induce apoptosis in A375 cells even at 200 µM ( Fig. 2A). Instead, it appeared to induce autophagy in a concentration-and time-dependent fashion (Fig. 2B). Pretreatment with the autophagy inhibitor 3-methyladenine (3-MA), signi cantly increased capsaicin-induced apoptosis at 100 and 200 µM (Fig. 2C), suggesting that capsaicin-induced autophagy was inhibitory to apoptosis in our system. Protein analysis showed that capsaicin-induced autophagy involved the up-regulation of the autophagy-related proteins Beclin 1, Atg5, Atg7, and p62, and the level of cleaved LC-3 II, and that this was accompanied by a decrease in the activation/phosphorylation of mTOR and Akt (Fig. 2D). On the contrary, apoptosis markers were unchanged by capsaicin exposure, including cleaved/activated caspases, the levels of the pro-apoptotic proteins Bak, Bax, p53, and the cleavage of PARP (Fig. 2E).
Capsaicin directly binds to tNOX, as indicated by cellular thermal shift assay (CETSA) Capsaicin has long been known to suppress plasma membrane NADH oxidase (a.k.a tNOX; ENOX2) and the growth of human and mouse melanoma cells [12]. The previous study mainly correlated the inhibition of NADH oxidase activity with attenuation of cancer cell growth, but did not reveal the molecular events contributing to this capsaicin-mediated suppression. To shed light on the action mechanism of capsaicin, we next examined whether there is a direct interaction between capsaicin and tNOX to regulate autophagy in melanoma cells. Given that the interaction between a ligand and its target protein enhances its heat resistance, a cellular thermal shift assay (CETSA) was conducted to examine the direct targeting between capsaicin and target proteins. Interestingly, in the presence of capsaicin, tNOX apparently precipitated at higher temperatures compared with that seen in control A375 cells (Fig. 4A). The relative tNOX intensity observed at all temperatures tested was signi cantly higher in capsaicin-exposed cell compared to controls (Fig. 4B). Moreover, the melting temperature values (T m ; the temperature at which 50% of proteins are unfolded and rapidly precipitated by heat) derived from the plot of thermal melting curves showed that the addition of capsaicin increased the parameter by 6.1 degrees, from 46.5°C (control) to 52.6°C (exposed to capsaicin) (Fig. 4C), suggesting that there is direct binding between capsaicin and tNOX. We also examined the dose-response relationship between capsaicin and the heat stability of tNOX; this experiment was performed at 54 °C, as most tNOX proteins would be denatured and precipitated unless they were thermally stabilized by binding to the ligand, capsaicin. Indeed, capsaicin dose-dependently increased the stability of tNOX, with an OC 50 value of 30 µM for A375 melanoma cells Capsaicin reduces tNOX and SIRT1 expression at the translational and transcriptional levels Based on our nding that capsaicin can directly bind to cellular tNOX protein in melanoma cells, we next examined the cellular impact of this interaction. We found that capsaicin concentration-dependently downregulated tNOX in A375 cells, and that this was seen at both transcriptional and translational levels ( Fig. 5A). Pretreatment with the protein-synthesis inhibitor, revealed that the half-life of tNOX in A375 cells was signi cantly reduced by 200 µM capsaicin starting at 6 h (Fig. 5B). ROS signaling seemed to play a key role in capsaicin-mediated tNOX downregulation as evidenced by the marked enhancement of tNOX stability seen in cultures pretreated with NAC (Fig. 5C). Treatment with the lysosome inhibitor, chloroquine (CQ), profoundly enhanced tNOX expression in A375 cells exposed to capsaicin (Fig. 5D), indicating that lysosomal degradation might be involved in capsaicin-induced tNOX downregulation. Pretreatment with the proteasome inhibitor, MG132, markedly reversed tNOX expression in A375 cells exposed to capsaicin (Fig. 5E), indicating that capsaicin-induced tNOX downregulation might also be through proteasomal degradation. We further examined the effect of tNOX depletion in A375 cells subjected to RNA interference, and found that tNOX knockdown signi cantly enhanced the autophagy induced by 100 and 200 µM capsaicin, but did not induce spontaneous autophagy in the absence of capsaicin (Fig. 5F).
Capsaicin induces autophagy by suppressing tNOX-SIRT1 deacetylase activity to enhance ULK1 acetylation Previous studies linked the inhibition of tNOX activity with the cancer cell growth-attenuating activity of capsaicin [6,12], and inhibition of tNOX activity was reported to decrease the intracellular NAD + concentration [22]. Thus, we next questioned whether a NAD + -dependent SIRT1 deacetylase could act as a tNOX-mediated NAD + generation-regulated switch for autophagy. We found that capsaicin attenuated SIRT1 expression at both transcriptional and translational levels in A375 cells (Fig. 6A). Furthermore, 100 and 200 µM capsaicin signi cantly inhibited cellular SIRT1 deacetylase activity in A375 cells, as assessed using a uorometric SIRT1 activity assay (Fig. 6B). We next focused on the autophagy-related pathway, using immunoprecipitation to reveal that ULK1 acetylation was augmented in our system, possibly through capsaicin-inhibited SIRT1 activity/expression (Fig. 6C). Interestingly, our data suggested that the capsaicin-induced suppression of tNOX expression was accompanied by a decrease in SIRT1 expression in A375 cells (Fig. 6D), as reported previously in other cancer cell types [22,23]. Other cell death regulation-related downstream targets of SIRT1 were also examined: We found that p53 acetylation was increased, providing further evidence that capsaicin induced autophagy, not apoptosis, in our system ( Fig. 6D).

Capsaicin-induced inhibition of tNOX or depletion of tNOX limit tumor growth in vivo
To determine whether capsaicin exerts an inhibitory activity on melanoma cancer in vivo, mice were inoculated with B16F10 cells. Tumor-bearing mice with average tumor diameters of around 0.5-1 cm were randomly divided into two groups and intratumorally injected with either 200 µg capsaicin in vehicle buffer or buffer only. Mice were terminated at Day 10 and we found that capsaicin effectively lessened the tumor size (Fig. 7A). Furthermore, protein analysis of the tumor tissues from the control and capsaicin-treated groups revealed that capsaicin treatment effectively reduced the expression of tNOX/SIRT1 expression (Fig. 7B). We next explored the involvement of tNOX in melanoma xenograft experiments utilizing RNA interference (RNAi) technology in B16F10 cells. tNOX-speci c shRNA effectively attenuated tNOX expression in B16F10 cells compared to the controls (Fig. 7C). The siRNAtransfected melanoma cells were injected into randomized C57BL/6 mice for an in vivo study. At the end of experiments, we found that average tumor mass grown in mice with tNOX-speci c shRNA-transfected cells was marked reduced compared to that of the control group, suggesting that tNOX depletion decreased the ability of melanoma cells to induce tumor growth in vivo (Fig. 7C). Together, our ndings suggest that capsaicin exerts its anticancer properties by directly targeting and enhancing the degradation of tNOX to inhibit NADH oxidation and SIRT1 deacetylase activity. In melanoma cells, through enhancement of ULK acetylation, capsaicin triggers cytotoxic autophagy dependent of ROS signaling.

Discussion
Capsaicin can be considered a nutraceutical, which is de ned as any food component that has medical bene t, such as in the prevention and treatment of disease [39,40]. An emerging body of evidence suggests that capsaicin suppresses the growth of melanoma cells. This has been reported to act through induction of both apoptosis and autophagy [10], downregulation of Bcl-2 with enhanced apoptosis [15], downregulation of IL-8 with reduced cell proliferation [13], and suppression of B16F10 cell migration via PI3K/Akt/Rac1 pathway [16]. Unsurprisingly, capsaicin has been used as a prototype for the design and synthesis of analogs aimed at inducing selective apoptosis in melanoma cells [41]. Capsaicin has also been shown to have a synergistic effect when combined with other drugs: It has been shown to further delay tumor growth in a dose-dependent fashion in mice with lung (LLC), bladder (MBT-2), and melanoma (B16F10) cancers [42]. Consistent with the previous reports, we herein demonstrate that capsaicin reduces the growth of melanoma cells in vitro and in vivo and further show for the rst time that this occur via inhibition of the tNOX-SIRT1 axis and the subsequent induction of cytotoxic autophagy. The therapeutic e ciency of capsaicin was not ideal in our animal studies, but our further experiments revealed that a NAD + -dependent SIRT1 deacetylase activity appears to act as an orchestrated switch under regulation of tNOX-mediated NAD + generation to trigger cytotoxic autophagy via enhanced ULK1 acetylation. Thus, capsaicin may be considered as a novel candidate drug with speci c intercellular protein targets (tNOX and SIRT1) for melanoma treatment.
Recent efforts to quantify the engagement between an unlabeled drug and a cellular protein target have shed lights on the action mechanisms of drugs. An example of such a technique is CETSA, which relies on the observation that ligand binding enhances the thermal stability of a target protein [26,27]. The preferential cytotoxicity of capsaicin had been long linked with tNOX in melanoma cells [12], but we previously lacked any direct physical evidence supporting their undeviating engagement. Here, we used CETSA to reveal that capsaicin enhanced the thermal stabilization of tNOX through direct binding in melanoma cells. In other studies, we have used this approach to show that intracellular tNOX engages directly with capsaicin in other cell types, including T24 bladder cancer cells [24] and oral cancer cells [43]. Here, we show that this direct binding in melanoma cells in uenced the degradation of tNOX and its function as a NADH oxidase to suppress SIRT1 deacetylase, which increased the acetylation of ULK1 to activate autophagy. Similar to our ndings, other studies have reported that capsaicin exhibits autophagic activity that coexists or cooperates with apoptosis to enhance cell death [44,45].
An array of diverse signaling pathways have been linked to the apoptotic/autophagic activity of capsaicin. Among them, oxidative stress signaling has often being highlighted. Given that ROS are generated mainly through the electron transport chain in mitochondria, many reports have emphasized the correlation between mitochondrial disruption and capsaicin-induced apoptosis in cancer cells [46][47][48][49]. Compared to the ROS-dependent apoptotic activity of capsaicin, however, the ability of capsaicin to induce autophagy is relatively less common in the literature. Mostly, capsaicin-mediated has been proposed to promote survival and/or inhibit apoptosis under stressful conditions, regardless of the cancer status of the cells [22,36,50,51]. Very recently, we investigated the interplay between apoptosis and autophagy in capsaicin-treated oral cancer cells with either functional or mutant p53 and found that capsaicin induced signi cant cytotoxicity via autophagy but not apoptosis in p53-functional oral cancer cells [43]. Similarly, in this present study, we demonstrated that capsaicin triggers cytotoxic autophagy in A375 cells, which bears wild-type p53 gene status. Interestingly, capsaicin treatment of p53-mutated cells triggered both autophagy and apoptosis, with autophagy occurring rst. In that case, capsaicin-induced autophagy suppressed apoptosis in the early stage, but thereafter facilitated apoptosis at a later stage, suggesting a complicated crosstalk between these two pathways mediated by capsaicin [43].
In sum, we herein used CETSA to show that tNOX directly engages with capsaicin to mediate a pathway that leads to cytotoxic autophagy of melanoma cells through enhanced ULK1 acetylation. Our experimental ndings and the results of our animal studies collectively suggest that tNOX could be a useful therapeutic drug target, and that capsaicin could be further developed as an anticancer therapeutic.                       Capsaicin-induced tNOX downregulation is associated with proteasomal and lysosomal degradation in  Representative images are shown. Values (means ± SDs) are from at least three independent experiments (**p < 0.01, *** p < 0.001). (F) RNA interference-mediated tNOX depletion was conducted in A375 cells. These cells were exposed to different concentrations of capsaicin or ethanol for 24 h, and the percentage of autophagic cells was determined by ow cytometry. The presented values (mean ± SDs) represent at least three independent experiments (* p <0.05 or *** p <0.001 for tNOX-depleted cells vs. controls or capsaicin-treated vs. controls).

Figure 6
Capsaicin inhibits SIRT1 expression and enzymatic activity in A375 cells. (A) Capsaicin signi cantly induced SIRT1 downregulation at the translational level analyzed by Western blotting and at the transcriptional level determined by RT-PCR. (B) Capsaicin suppressed cellular SIRT1 activity analyzed by a SIRT1 Activity Assay Kit (Fluorometric) with control or capsaicin-exposed A375 cell lysates. Values (mean ± SDs) are from three independent experiments (*** p <0.001). (C) The lysates of A375 cells were immunoprecipitated with nonimmune IgG or an antibody against ULK1, and the bound proteins were detected by Western blotting with pan-acetylated lysine or ULK1 antibodies. (D) A375 cells were exposed to different concentrations of capsaicin or ethanol for 24 hours. Aliquots of cell lysates were separated by SDS-PAGE and analyzed for protein expression by Western blotting. β-actin was used as an internal control to monitor for equal loading. Representative images are shown from at least three independent experiments.  SDS-PAGE and analyzed for protein expression by Western blotting. β-actin was used as an internal control to monitor for equal loading. Representative images are shown from at least three independent experiments.

Figure 6
Capsaicin inhibits SIRT1 expression and enzymatic activity in A375 cells. (A) Capsaicin signi cantly induced SIRT1 downregulation at the translational level analyzed by Western blotting and at the transcriptional level determined by RT-PCR. (B) Capsaicin suppressed cellular SIRT1 activity analyzed by a SIRT1 Activity Assay Kit (Fluorometric) with control or capsaicin-exposed A375 cell lysates. Values (mean ± SDs) are from three independent experiments (*** p <0.001). (C) The lysates of A375 cells were immunoprecipitated with nonimmune IgG or an antibody against ULK1, and the bound proteins were detected by Western blotting with pan-acetylated lysine or ULK1 antibodies. (D) A375 cells were exposed to different concentrations of capsaicin or ethanol for 24 hours. Aliquots of cell lysates were separated by SDS-PAGE and analyzed for protein expression by Western blotting. β-actin was used as an internal control to monitor for equal loading. Representative images are shown from at least three independent experiments.

Figure 6
Capsaicin inhibits SIRT1 expression and enzymatic activity in A375 cells. (A) Capsaicin signi cantly induced SIRT1 downregulation at the translational level analyzed by Western blotting and at the transcriptional level determined by RT-PCR. (B) Capsaicin suppressed cellular SIRT1 activity analyzed by a SIRT1 Activity Assay Kit (Fluorometric) with control or capsaicin-exposed A375 cell lysates. Values (mean ± SDs) are from three independent experiments (*** p <0.001). (C) The lysates of A375 cells were immunoprecipitated with nonimmune IgG or an antibody against ULK1, and the bound proteins were detected by Western blotting with pan-acetylated lysine or ULK1 antibodies. (D) A375 cells were exposed to different concentrations of capsaicin or ethanol for 24 hours. Aliquots of cell lysates were separated by SDS-PAGE and analyzed for protein expression by Western blotting. β-actin was used as an internal control to monitor for equal loading. Representative images are shown from at least three independent experiments.

Figure 7
In vivo veri cation for capsaicin-reduced tNOX and tNOX-depletion suppresses melanoma tumor growth.  In vivo veri cation for capsaicin-reduced tNOX and tNOX-depletion suppresses melanoma tumor growth.  In vivo veri cation for capsaicin-reduced tNOX and tNOX-depletion suppresses melanoma tumor growth.  In vivo veri cation for capsaicin-reduced tNOX and tNOX-depletion suppresses melanoma tumor growth.  In vivo veri cation for capsaicin-reduced tNOX and tNOX-depletion suppresses melanoma tumor growth.