Capsaicin Is a Novel Blocker of Constitutive and Interleukin-6–Inducible STAT3 Activation

Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide) is a principal pungent ingredient of hot red and chili peppers that belong to the plant genus Capsicum (Solanaceae). In addition to alleviating neuropathic pain and itching in humans (1–4), capsaicin has exhibited anticancer effects in animal models, suppressing carcinogenesis of the skin (5), colon (6), lung (7), tongue (8), and prostate (9, 10). The mechanism by which this vanilloid mediates its anticarcinogenic effects is not understood but has been shown to alter the metabolism of carcinogens such as aflatoxin B1 (11) and the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (12, 13). In culture, capsaicin has been found to selectively suppress the growth of various human tumor cells (14, 15), including leukemic (16–18), gastric (19), hepatic (20), glioma (21), and prostate cells (9). How capsaicin mediates its anticancer effects is not fully understood, but the roles of NADH oxidase activity (14), proteasome (9), cyclooxygenases (22), c-Jun NH₂-terminal kinase (23), nuclear factor-κB (24), peroxisome proliferator-activated receptor γ (25, 26), peroxynitrite (21), and mitochondrial respiration (27) have been implicated. Its immunosuppressive effects have been linked to its ability to suppress nuclear factor-κB activation (28).

Members of the signal transducer and activator of transcription (STAT) family of transcription factors regulate the expression of gene products involved in cell survival, proliferation, chemoresistance, and angiogenesis (29, 30). The activation of STATs involves the phosphorylation of a critical tyrosine residue by Janus-activated kinases (JAK), or the Src family kinases, leading to dimerization of STAT monomers, nuclear translocation, and binding to specific DNA response elements in the promoters of target genes. Among the STATs, STAT3 is perhaps the most intimately linked to tumorigenesis (31). Although STAT3 is activated by interleukin-6 (IL-6), epidermal growth factor, and other growth factors, constitutive activation of STAT3 has been discovered in a wide variety of tumors (31–34).

Because of the critical role of STAT3 in tumor cell survival, proliferation, and angiogenesis and its expression in various tumor cells, we hypothesized that capsaicin must mediate its effects in part through the suppression of the STAT3 pathway. We found that capsaicin does indeed suppress both constitutive and inducible STAT3 activation. This correlated with down-regulation of expression of cell survival, proliferative, and angiogenic gene products, leading to suppression of proliferation, induction of apoptosis, and enhancement of apoptosis induced by Velcade and thalidomide.

Materials. Capsaicin (molecular weight, 305.42) was purchased from Tocris Cookson. A 50 mmol/L stock solution of capsaicin was prepared in DMSO and then further diluted in cell culture medium. Hoechst 33342, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide, Tris, glycine, NaCl, SDS, and bovine serum albumin were purchased from Sigma-Aldrich. RPMI 1640, fetal bovine serum, 0.4% trypan blue vital stain, and antibiotic-antimycotic mixture were obtained from Life Technologies. Rabbit polyclonal antibodies to STAT3 and STAT5 and mouse monoclonal antibodies against phosphorylated STAT3 (Tyr⁷⁰⁵) and phosphorylated STAT5, phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2), ERK, Bcl-2, Bcl-xL, cyclin D1, survivin, procaspase-3, and poly(ADP-ribose) polymerase (PARP) were obtained from Santa Cruz Biotechnology. Goat anti-rabbit horseradish peroxidase conjugate was purchased from Bio-Rad. Anti–vascular endothelial growth factor (VEGF) was purchased from NeoMarkers. Antibodies to phospho-specific src (Tyr⁴¹⁶), src, phospho-specific JAK1 (Tyr^1022/1023), and JAK1 were purchased from Cell Signaling Technology. Goat anti-mouse horseradish peroxidase was purchased from Transduction Laboratories (Lexington, KY), and goat anti-rabbit Alexa 594 was purchased from Molecular Probes. Bacteria-derived recombinant human IL-6 was kindly provided by Novartis Pharmaceuticals. Velcade (PS-341) was obtained from Millennium. Thalidomide was obtained from Tocris Cookson.

Cell lines. The human multiple myeloma cell lines U266 and MM.1S were obtained from the American Type Culture Collection. Cell line U266 (ATCC TIB-196) is a plasmacytoma of B-cell origin and is known to produce monoclonal antibodies and IL-6 (35, 36). The MM.1S cell line, established from the peripheral blood cells of a patient with IgA myeloma, secretes light chain, is negative for the presence of the EBV genome, and expresses leukocyte antigen DR, plasma cell Ag-1, and T9 and T10 antigens (37). U266 and MM.1S cells were cultured in RPMI 1640 containing 1× antibiotic-antimycotic solution with 10% fetal bovine serum. Human myeloid leukemia (KBM-5) cells were obtained from the American Type Culture Collection and cultured in Iscove's modified Dulbecco's medium with 15% fetal bovine serum containing 1× antibiotic-antimycotic solution.

Western blot analysis. For detection of STAT proteins, capsaicin-treated whole-cell extracts were lysed in lysis buffer [20 mmol/L Tris (pH 7.4), 250 mmol/L NaCl, 2 mmol/L EDTA (pH 8), 0.1% Triton X-100, 0.01 mg/mL aprotinin, 0.005 mg/mL leupeptin, 0.4 mmol/L phenylmethylsulfonyl fluoride, and 4 mmol/L NaVO₄]. Lysates were then centrifuged at 14,000 rpm for 10 min to remove insoluble material and resolved on a 7.5% SDS gel. After electrophoresis, the proteins were electrotransferred to a nitrocellulose membrane, blocked with 5% nonfat milk, and probed with anti-STAT antibodies (1:1,000) overnight at 4°C. The blot was washed, exposed to horseradish peroxidase–conjugated secondary antibodies for 1 h, and finally examined by enhanced chemiluminescence (Amersham Pharmacia Biotech).

To detect the expression of STAT3-regulated proteins and caspase-3, U266 cells (2 × 10⁶ per mL) were treated with 50 μmol/L capsaicin for the indicated times. The cells were then washed and extracted by incubation for 30 min on ice in 0.05 mL of buffer containing 20 mmol/L HEPES (pH 7.4), 2 mmol/L EDTA, 250 mmol/L NaCl, 0.1% NP40, 2 μg/mL leupeptin, 2 μg/mL aprotinin, 1 mmol/L phenylmethylsulfonyl fluoride, 0.5 μg/mL benzamidine, 1 mmol/L DTT, and 1 mmol/L sodium vanadate. The lysate was centrifuged, and the supernatant was collected. Whole-cell protein extract (50 μg) was resolved on 12% SDS-PAGE; electrotransferred onto a nitrocellulose membrane; blotted with antibodies against survivin, Bcl-2, Bcl-xl, cyclin D1, VEGF, caspase-3; and then detected by enhanced chemiluminescence (Amersham Pharmacia Biotech).

Electrophoretic mobility shift assay for STAT3-DNA binding. STAT3-DNA binding was analyzed by electrophoretic mobility shift assay using a ³²P-labeled high-affinity sis-inducible element probe as previously described (38). Briefly, nuclear extracts were prepared from capsaicin treated cells and incubated with a high-affinity sis-inducible element probe (5′-CTTCATTTCCCGTAAATCCCTAAAGCT-3′ and 5′-AGCTTTAGGGATTTACGGGAAATGA-3′). DNA-protein complex formed was separated from free oligonucleotide on 5% native polyacrylamide gels. The dried gels were visualized, and the radioactive bands were quantitated with a Storm 820 and Imagequant software (Amersham).

Immunocytochemistry for STAT3 localization. Capsaicin-treated multiple myeloma cells were plated on a glass slide by centrifugation using a Cytospin 4 (Thermoshendon), air-dried for 1 h at room temperature, and fixed with cold acetone. After a brief washing in PBS, slides were blocked with 5% normal goat serum for 1 h and then incubated with rabbit polyclonal anti-human STAT3 antibody (dilution, 1:100). After overnight incubation, the slides were washed and then incubated with goat anti-rabbit IgG-Alexa 594 (1:100) for 1 h and counterstained for nuclei with Hoechst (50 ng/mL) for 5 min. Stained slides were mounted with mounting medium (Sigma-Aldrich) and analyzed under an epifluorescence microscope (Labophot-2, Nikon). Pictures were captured using a Photometrics Coolsnap CF color camera (Nikon) and MetaMorph version 4.6.5 software (Universal Imaging).

Antiproliferative assay. The antiproliferative effects of capsaicin against U266 cells was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide dye uptake method as described earlier (39).

Flow cytometric analysis. To determine the effect of capsaicin on the cell cycle, U266 cells were first synchronized by serum starvation and then exposed to capsaicin for the indicated time intervals. Thereafter cells were washed, fixed with 70% ethanol, and incubated for 30 min at 37°C with 0.1% RNase A in PBS. Cells were then washed again, resuspended, and stained in PBS containing 25 μg/mL propidium iodide for 30 min at room temperature. Cell distribution across the cell cycle was analyzed with a FACSCalibur flow cytometer (Becton Dickinson).

Immunoblot analysis of PARP degradation. Ursolic acid–induced apoptosis was examined by proteolytic cleavage of PARP. Briefly, cells (2 × 10⁶ per mL) were treated with 50 μmol/L capsaicin for indicated time points at 37°C. The cells were then washed and extracted by incubation for 30 min on ice in 0.05 mL buffer containing 20 mmol/L HEPES (pH 7.4), 2 mmol/L EDTA, 250 mmol/L NaCl, 0.1% NP40, 2 μg/mL leupeptin, 2 μg/mL aprotinin, 1 mmol/L phenylmethylsulfonyl fluoride, 0.5 μg/mL benzamidine, 1 mmol/L DTT, and 1 mmol/L sodium vanadate. The lysate was centrifuged, and the supernatant was collected. Cell extract protein (50 μg) was resolved on 7.5% SDS-PAGE, electrotransferred onto a nitrocellulose membrane, blotted with anti-PARP antibody, and then detected by enhanced chemiluminescence (Amersham Pharmacia Biotech).

Live/Dead assay. Apoptosis of cells was also determined by the Live/Dead assay (Molecular Probes), which measures intracellular esterase activity and plasma membrane integrity, as described previously (40). Briefly, 1 × 10⁶ cells were incubated with capsaicin/Velcade/thalidomide alone or in combination for 24 h at 37°C. Cells were stained with the Live/Dead reagent (5 μmol/L ethidium homodimer, 5 μmol/L calcein-AM) and then incubated at 37°C for 30 min. Cells were analyzed under a fluorescence microscope (Labophot-2).

Animals. Male athymic nu/nu mice (4 weeks old) were obtained from the breeding colony of the Department of Experimental Radiation Oncology at The University of Texas M. D. Anderson Cancer Center. The animals were housed in the standard mice plexiglass cages (five per cage) in a room maintained at constant temperature and humidity under 12-h light and darkness cycle and fed with regular autoclave chow diet with water ad libitum. None of the mice exhibited any lesions, and all were tested pathogen free. Before initiating the experiment, we acclimatized all mice to a pulverized diet for 3 days. Our experimental protocol was reviewed and approved by the Institutional Animal Care and Use Committee at The University of Texas M. D. Anderson Cancer Center.

Subcutaneous implantation of U266 cells. U266 cells harvested from subconfluent cultures were washed once in serum-free medium and resuspended in PBS. Cell suspensions consisting of single cells, with >90% viability, were used for the injections. Mice were anesthetized with ketamine-xylazine solution, and 2 × 10⁶ cells in 100 μL (sterile PBS and Matrigel, 1:1) were injected into the left flank of mice, s.c. using a 25-gauge needle and a calibrated push button-controlled dispensing device (Hamilton Syringe Co.). To prevent leakage, a cotton swab was held cautiously for 1 min over the site of injection.

Experimental protocol. After 1 week of implantation, tumor diameters were measured using a vernier caliper. The mice were randomized into the following treatment groups (n = 5) based on the initial tumor volume: (A) untreated control (DMSO, 100 μL) and (B) capsaicin (1 mg/kg in 100 μL). The control group was injected with DMSO and treated group with capsaicin thrice per week, i.p. The treatment was continued up to 3 weeks from the date of randomization. The tumor volume was measured once a week using the formula V = 0.5236 × d₁² × d₂, where d₁ is the shortest diameter, and d₂ is the longest diameter. The mice were sacrificed 25 days after the start of treatment. The tumors were carefully excised, and the diameters were measured for final tumor volume calculation. The final tumor volume was measured using the formula V = 4/3πr³, where r is the mean of the three dimensions (length, width, and depth). The tumor volumes were compared among two groups using Student's t test, with P < 0.05 considered as significant.

To further define the antitumor effects of capsaicin, we investigated the effect of capsaicin on both constitutively active and IL-6–inducible STAT3 phosphorylation in multiple myeloma cells in the present report. We also evaluated the effect of capsaicin on various mediators of cellular proliferation, cell survival, and apoptosis. The structure of capsaicin is shown in Fig. 1A. The dose and duration of capsaicin used for the STAT3 experiments had no effect on cell viability (data not shown).

Capsaicin inhibits constitutive STAT3 phosphorylation in multiple myeloma cells. There are reports from our laboratory and others that multiple myeloma U266 cells express constitutively active STAT3 (31, 33, 34). Whether capsaicin can modulate the constitutive STAT3 activation in these cells was investigated. Western blot analysis with specific antibodies to phosphorylated STAT3 indicated that capsaicin inhibited constitutively active STAT3 in U-266 cells in a dose-dependent (Fig. 1B1) and time-dependent (Fig. 1B2) manner. This inhibition appeared as early as 1 h, with complete abrogation at 4 h after 50 μmol/L capsaicin treatment. Capsaicin treatment did not alter the overall expression of STAT3 protein.

We also investigated whether capsaicin affects the activation of other STAT proteins in U266 cells. We found that besides STAT3, U266 cells expressed constitutively active STAT5 and capsaicin altered neither the levels of constitutively phosphorylated STAT5 nor the expression of STAT5 proteins (data not shown).

Capsaicin inhibits DNA binding of STAT3 in multiple myeloma cells. The translocation of STAT3 to the nucleus leads to a specific DNA binding, which in turn regulates gene transcription (38). We examined whether capsaicin suppresses the DNA-binding activity of STAT3. Electrophoretic mobility shift assay analysis of nuclear extracts prepared from U266 cells showed that capsaicin decreased STAT3 DNA-binding activity in a dose-dependent (Fig. 1C1) and time-dependent (Fig. 1C2) manner. Supershift analysis indicated that the binding of STAT3 to the DNA was blocked by anti-Stat3 antibody and by cold competitor oligonucleotide, thus confirming that the protein/DNA complex observed actually contained STAT3. No constitutive activation of STAT3 could be detected in human myeloid KBM-5 cells (Fig. 1C3). These results show that capsaicin abrogates the DNA-binding ability of STAT3.

Capsaicin depletes the nuclear pool of STAT3 in multiple myeloma cells. Under resting conditions and in the non-phosphorylated state, STAT3 is retained in the cytoplasm. It translocates to the nucleus when phosphorylated (29, 30). Phosphorylation induces STAT3 dimerization, thus permitting its translocation into the nucleus. To determine whether capsaicin affects nuclear pools of STAT3, capsaicin-treated and untreated cells were plated on a glass slide, immunostained with antibody to STAT3, and then visualized by using the Alexa 594–conjugated second antibody. Figure 1D clearly shows that capsaicin depleted the nuclear pool of STAT3 in U266 cells.

Capsaicin inhibits inducible STAT3 phosphorylation in human multiple myeloma cells. Because IL-6 is a growth factor for multiple myeloma cells and induces STAT3 phosphorylation (35, 41), we determined whether capsaicin could inhibit IL-6–induced STAT3 phosphorylation. MM.1S cells were pretreated with capsaicin for different time intervals and then stimulated with IL-6 for 15 min. As shown in Fig. 2A, IL-6–induced STAT3 phosphorylation was blocked by capsaicin in a time-dependent manner. Exposure of the cells to capsaicin for 4 h was sufficient to completely suppress IL-6–induced STAT3 phosphorylation.

Capsaicin-induced inhibition of STAT3 phosphorylation is reversible in human multiple myeloma cells. We further examined whether capsaicin-induced inhibition of STAT3 phosphorylation is reversible. U266 cells were first treated for 60 min with capsaicin, and then the cells were washed twice with PBS to remove capsaicin. The cells were then cultured in fresh medium for various durations, and the levels of phosphorylated STAT3 were measured. Capsaicin induced the suppression of STAT3 phosphorylation (Fig. 2B), and the removal of capsaicin resulted in a gradual increase in phosphorylated STAT3 (Fig. 2C). The reversal was complete by 24 h and did not involve changes in STAT3 protein levels (Fig. 2C, bottom).

Capsaicin suppresses constitutive activation of JAK1. STAT3 has been reported to be activated by soluble tyrosine kinases of the Janus family (JAK; ref. 30). We examined whether capsaicin affects constitutive activation of JAK1 in U266 cells. We found that capsaicin suppressed the constitutive phosphorylation of JAK1 in a time-dependent manner (Fig. 3A). The levels of non-phosphorylated JAK1 remained unchanged under the same conditions (Fig. 3A, bottom).

Capsaicin suppresses constitutive activation of c-Src. STAT3 has also been reported to be activated by soluble tyrosine kinases of the Src kinase families (34). Hence, we determined the effect of capsaicin on constitutive activation of Src kinase in U266 cells. We found that capsaicin suppressed the constitutive phosphorylation of c-Src kinase (Fig. 3B). The levels of non-phosphorylated c-Src kinase remained unchanged under the same conditions.

Capsaicin does not affect constitutive activation of ERK. Apart from tyrosine phosphorylation, STAT3 also undergoes phosphorylation at serine residues (30) through the mitogen-activated protein kinase/RAS pathway (42). We therefore investigated whether capsaicin modulates constitutive activation of ERK1/2 kinases in U266 cells. We found that capsaicin did not affect the constitutive phosphorylation of ERK1/2 kinase in U266 cells (Fig. 3C). The levels of non-phosphorylated ERK1/2 remained unchanged under the same conditions.

Tyrosine phosphatases are involved in capsaicin-induced inhibition of STAT3 activation. Because protein tyrosine phosphatases (PTP) have also been implicated in STAT3 activation (43), we determined whether capsaicin-induced inhibition of STAT3 tyrosine phosphorylation could be due to activation of a protein tyrosine phosphatase (PTPase). Treatment of U266 cells with the broad-acting tyrosine phosphatase inhibitor sodium pervanadate reversed the capsaicin-induced inhibition of STAT3 activation (Fig. 3D). This suggests that tyrosine phosphatases are involved in capsaicin-induced inhibition of STAT3 activation.

Capsaicin down-regulates the expression of cyclin D1, Bcl-2, Bcl-xL, survivin, and VEGF. STAT3 activation has been shown to regulate the expression of various gene products involved in cell survival, proliferation, and angiogenesis (34). We investigated whether the expression of the cell cycle regulator protein cyclin D1; the antiapoptotic proteins Bcl-2, Bcl-xL, and survivin; and the angiogenic gene product VEGF, all reported to be regulated by STAT3 (29–31, 44), was modulated by capsaicin treatment. Capsaicin treatment down-regulated expression of these proteins in a time-dependent manner, with maximum suppression observed at ∼24 h (Fig. 4).

Capsaicin inhibits the proliferation of multiple myeloma cells. Because capsaicin suppressed the activation of STAT3 and STAT3-regulated gene products, we examined whether it modulates the proliferation of cells. We found that capsaicin suppressed the increase in proliferation of U266 cells in a dose-dependent manner (Fig. 5A).

Capsaicin causes the accumulation of the cells in G₁ phase. Because D-type cyclins are required for the progression of cells from the G₁ phase of the cell cycle to S phase (45), and because we observed a rapid decline in levels of cyclin D1 in the capsaicin-treated cells, we determined the effect of capsaicin on cell cycle phase distribution. We found that treatment with capsaicin caused significant accumulation of the cell population in G₁ phase (Fig. 5B).

Capsaicin activates caspase-3 and causes PARP cleavage. Whether suppression of constitutively active STAT3 in U266 cells by capsaicin leads to apoptosis was also investigated. Cell extracts prepared from capsaicin-treated cells showed a time-dependent activation of caspase-3 (Fig. 5C). Activation of downstream caspases led to the cleavage of a 116-kDa PARP protein into an 87-kDa fragment (Fig. 5D). These results clearly suggest that capsaicin induces caspase-3–dependent apoptosis in U266 cells.

Capsaicin potentiates the apoptotic effect of thalidomide and Velcade in multiple myeloma cells. Velcade, an inhibitor of proteasome, and thalidomide, which inhibits tumor necrosis factor expression, have been approved for the treatment of patients with multiple myeloma (46, 47). However, prolonged exposure to these agents is associated with toxicity and development of chemoresistance. One of the mechanisms by which these agents confer resistance is due to the up-regulation of different antiapoptotic proteins (48–50). The ability of capsaicin to down-regulate the expression of antiapoptotic proteins, such as Bcl-2, Bcl-xL, and survivin, as described above prompted us to determine whether capsaicin can also lower the apoptotic threshold for these cytotoxic drugs. U266 cells were treated with capsaicin together with either thalidomide or Velcade and then examined the cells for apoptosis using the Live/Dead assay, which determines plasma membrane stability using esterase staining. As shown in Fig. 6A and B, capsaicin significantly enhanced the apoptotic effect of thalidomide from 15% to 60% and that of Velcade from 20% to 80%.

Thalidomide and Velcade enhance the effect of capsaicin in suppressing STAT3 activation. Whether potentiation of the apoptotic effects of capsaicin by thalidomide and Velcade was due to down-regulation of STAT3 activation was examined. To investigate this, U266 cells were exposed to sub-optimal doses of apsaicin, thalidomide, and Velcade and then examined for STAT3 phosphorylation. As shown in Fig. 6, thalidomide (C1) and Velcade (C2) potentiated the effect of capsaicin in down-regulating constitutive STAT3 activation.

Capsaicin inhibits the growth of human multiple myeloma in vivo. Our in vitro experiments have shown that capsaicin is a potent inhibitor of STAT3 and antiapoptotic proteins in multiple myeloma cells. Next, we analyzed whether capsaicin can inhibit the growth of human multiple myeloma tumor in nude mice. Male nude mice were injected s.c. with U266 cells and treated with capsaicin administered i.p. for 3 weeks (thrice per week). The results showed that capsaicin significantly suppressed the tumor growth in vivo (Fig. 6D). At the end of treatment, we also noticed that two of five mice in the treatment group had barely detectable tumors. Our composite results show that capsaicin is an effective antitumor agent for multiple myeloma cells both under in vitro and in vivo conditions.

The goal of this study was to determine whether the vanilloid capsaicin exerts its anticancer effects through the abrogation of the STAT3 signaling pathway in multiple myeloma cells. We found that capsaicin inhibited the constitutive and IL-6–inducible STAT3 activation in human multiple myeloma cells in parallel with the suppression of JAK1 and c-Src activation. However, capsaicin had no effect on the activation of Erk1/2. We also provided evidence for the role of PTP. Capsaicin also down-regulated the expression of STAT3-regulated gene products, including cyclin D1, survivin, Bcl-2, Bcl-xL, and VEGF. It induced the inhibition of proliferation, accumulation of cells in G₁-G₀ phase, and apoptosis, and it significantly potentiated the apoptotic effects of Velcade and thalidomide in multiple myeloma cells. Capsaicin also inhibited the growth of human multiple myeloma in vivo.

This is the first report to identify capsaicin as a novel blocker of the STAT3 pathway. We found that capsaicin could suppress both constitutive and inducible STAT3 activation, and that these effects were specific to STAT3, as capsaicin had no effect on STAT5 phosphorylation. We also observed that capsaicin suppressed nuclear translocation and DNA-binding activity of STAT3. Unlike curcumin, which has been shown to decrease STAT3 expression after 24 h of treatment (51), capsaicin had no effect on the total levels of STAT3. Besides multiple myeloma cells, other forms of cancer, including head and neck cancers (52), hepatocellular carcinoma (53), lymphomas, and leukemia (54), also express constitutively active STAT3. The suppression of constitutively active STAT3 in multiple myeloma cells raises the possibility that this novel STAT3 inhibitor might also inhibit constitutively activated STAT3 in other types of cancer cells.

The effects of capsaicin on STAT3 phosphorylation correlated with the suppression of upstream protein tyrosine kinases JAK1 and c-Src. It has been reported that all Src-transformed cell lines have persistently activated STAT3, and that dominant-negative STAT3 blocks transformation (55, 56). Interestingly, capsaicin had no affect on the activation of ERK1/2, suggesting that this kinase is not directly involved. The lack of effect of capsaicin on ERK1/2 in the current study agrees with the findings of Macho et al. (57), who reported that capsaicin and related vanilloid compounds did not affect ERK activity.

We also found evidence that the capsaicin-induced inhibition of STAT3 activation involves a PTP. Numerous PTPs have been implicated in STAT3 signaling, including SHP-1, SHP-2, TC-PTP, PTEN, PTP-1D, CD45, PTP-ε, and low molecular weight PTP (58–66). The type of PTP involved in down-regulation of STAT3 phosphorylation is not clear. Loss of SHP1 has been shown to enhance JAK3/STAT3 signaling in ALK-positive anaplastic large-cell lymphoma (43). It is possible that capsaicin mediates its effects through activation of SHP-1, although this PTP is ubiquitously expressed in both hematopoietic and nonhematopoietic cells (58). However, capsaicin-induced apoptosis has been reported to be regulated by the protein phosphatase calcineurin (67).

STAT3 phosphorylation plays a critical role in the transformation and proliferation of tumor cells (32, 34). We found for the first time that capsaicin suppresses the expression of several STAT3-regulated proteins, including proliferative (cyclin D1), antiapoptotic (survivin, Bcl-2, and Bcl-xL), and angiogenic (VEGF) gene products. The down-regulation of cyclin D1 expression by capsaicin correlated with suppression of proliferation and accumulation of cells in G₁ phase of cell cycle. Expression of Bcl-xL is regulated by STAT3 and is overexpressed in multiple myeloma cells (68). Bcl-xL can also block cell death induced by a variety of chemotherapeutic agents, in parallel with an increase in chemoresistance (69).

The down-regulation of the expression of Bcl-2, Bcl-xL, and survivin is likely linked with the ability of capsaicin to induce apoptosis in multiple myeloma cells. Consistent with our observations, dominant-negative STAT3 has also been shown to induce apoptosis in cells with constitutively active STAT3 (70). Additionally, in view of the reported role of STAT3 in repressing p53 expression and function (71), inhibition of constitutive STAT3 activity by capsaicin could induce antitumor effects by enhancing p53 function as shown by a previous report (19). We also observed that capsaicin down-regulates the expression of VEGF in multiple myeloma cells. Our results differ from those of Patel et al. (72), who reported that capsaicin treatment induced VEGF expression by enhancing hypoxia-inducible factor-1α activity in human malignant melanoma cells. Whether this difference in results is due to cell type, dose, or time of exposure to capsaicin is unclear.

Constitutively active STAT3 has been implicated in the induction of resistance to apoptosis (70), possibly through the expression of Bcl-2 and cyclin D1 (73, 74). Recently, a proteasome inhibitor (PS341, also called Velcade) and a tumor necrosis factor inhibitor (thalidomide) were approved for the treatment of multiple myeloma (46, 47). We found for the first time that capsaicin potentiates the apoptotic effect of Velcade and thalidomide in multiple myeloma cells. How does capsaicin potentiates the effect of these drugs? This potentiation of apoptosis could be due to the down-regulation of antiapoptotic proteins, such as Bcl-2, Bcl-xL, and survivin, by capsaicin that contributes to resistance to these cytotoxic drugs in multiple myeloma patients. Additionally, we found that capsaicin when combined with thalidomide and Velcade was more effective in suppressing STAT3 activation than when used alone.

Whether these in vitro observations with capsaicin has any relevance to that in vivo was also investigated. Our results show for the first time that capsaicin significantly suppressed multiple myeloma tumor growth in nude mice. In summary, safety information from preclinical studies described and those reported previously (7–9); the ability of capsaicin to suppress constitutive and inducible STAT-3 activation; down-regulate the expression of cyclin D1, Bcl-2, Bcl-xL, survivin, and VEGF; inhibit cell proliferation; and potentiate the effects of Velcade and thalidomide provide a sound basis for pursuing the use of capsaicin further, either alone or in combination with other agents, to enhance treatment efficacy, reduce toxicity, and overcome the chemoresistance of relapsed or refractory multiple myeloma.

We thank Michael S. Worley for carefully editing the manuscript and providing valuable comments.