Combination of the natural product capsaicin and docetaxel synergistically kills human prostate cancer cells through the metabolic regulator AMP-activated kinase
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Current chemotherapy for castration-resistant prostate cancer is established on taxane-based compounds like docetaxel. However, eventually, the development of toxic side effects and resistance limits the therapeutic benefit being the major concern in the treatment of prostate cancer. Combination therapies in many cases, enhance drug efficacy and delay the appearance of undesired effects, representing an important option for the treatment of castration-resistant prostate cancer. In this study, we tested the efficacy of the combination of docetaxel and capsaicin, the pungent ingredient of hot chili peppers, on prostate cancer cells proliferation.
Prostate cancer LNCaP and PC3 cell lines were used in this study. Levels of total and phosphorylated forms of Akt, mTOR, S6, LKB1, AMPK and ACC were determined by Western blot. AMPK, LKB1 and Akt knock down was performed by siRNA. PTEN was overexpressed by transient transfection with plasmids. Xenograft prostate tumors were induced in nude mice and treatments (docetaxel and capsaicin) were administered intraperitoneally. Statistical analyses were performed with GraphPad software. Combination index was calculated with Compusyn software.
Docetaxel and capsaicin synergistically inhibited the growth of LNCaP and PC3 cells, with a combination index lower than 1 for most of the combinations tested. Co-treatment with docetaxel and capsaicin notably decreased Akt and its downstream targets mTOR and S6 phosphorylation. Overexpression of PTEN phosphatase abrogated the synergistic antiproliferative effect of docetaxel and capsaicin. The combined treatment also increased the phosphorylation of AMP-activated kinase (AMPK) and the phosphorylation of its substrate ACC. In addition, pharmacological inhibition of AMPK with dorsomorphin (compound C) as well as knock down by siRNA of AMPK or its upstream kinase LKB1, abolished the synergy of docetaxel and capsaicin. Mechanistically, we showed that the synergistic anti-proliferative effect may be attributed to two independent effects: Inhibition of the PI3K/Akt/mTOR signaling pathway by one side, and AMPK activation by the other. In vivo experiments confirmed the synergistic effects of docetaxel and capsaicin in reducing the tumor growth of PC3 cells.
Combination of docetaxel and capsaicin represents a therapeutically relevant approach for the treatment of Prostate Cancer.
KeywordsDocetaxel Capsaicin AMPK PC3 cells LNCaP cells Prostate cancer
acetyl CoA carboxylase
liver kinase B
Prostate cancer (PCa) is the most prevalent malignancy in men worldwide, and the second leading cause of cancer related deaths [1, 2]. Environmental factors such as hypercaloric diets, sedentary life, increasing life expectancy and modified diagnostic techniques contribute to the increase in prostate cancer incidence. For locally advanced and metastatic cancers androgen deprivation therapy is the standard of care. Despite initial disease regression, most men eventually progress to castration-resistant prostate cancer (CRPC) with no response to hormonal therapy and a lethal outcome. Currently, docetaxel is the first-line chemotherapeutic agent available to patients with this lethal form of the disease, but the survival of patients remains limited by the occurrence of dose-dependent adverse effects and acquired resistance. Mechanisms underpinning resistance development include overexpression of multidrug efflux pumps, mutation of β-tubulin, and activation of signaling proteins as MAPK or Akt . Docetaxel resistance is a clinical problem since it is the main therapy for CRPC. Moreover, newer chemotherapeutic drugs developed to treat docetaxel resistant patients carry significant hematological toxicities . Therefore, approaches to improve taxane-based chemotherapy are urgently required . Thus, it is of highly clinical significance to identify agents that when combined with the current chemotherapeutic drugs allow to decrease the doses without reducing their effectiveness as well as to avoid and/or to overcome drug resistance. Therefore, combination therapy, a treatment modality that combines two or more therapeutic agents, is becoming a cornerstone of cancer therapy .
Over the past few years, many anti-cancer drugs have been identified from natural nutritional compounds. Capsaicin (CAP), the spicy ingredient of hot chili peppers, exhibit anti-neoplastic activity in many cancer cell lines as well as in vivo . In addition, recent data indicate that CAP sensitizes cells to chemotherapeutic agents. For instance, the combination of CAP and camphothecin increases apoptosis in small cell lung cancer . In cholangocarcinoma, CAP increases sensitivity to 5-fluorouracil and the mixture of both compounds inhibits tumor growth with greater efficacy than 5-fluorouracil alone . In human prostate cancer cells CAP combined with brassinin enhances apoptotic and anti-metastatic effects . We have shown that, in hepatocellular carcinoma cells, CAP increases the antiproliferative effects of sorafenib . Yet, the mechanisms underlying the capsaicin-mediated inhibition of cell proliferation and drug sensitization are divers and poorly understood. Laboratory data supports the notion that dietary capsaicin has anti-obesity role by increasing energy expenditure, enhancing fat oxidation, decreasing adipogenesis and suppressing appetite . Although a molecular mechanism has not been clarified, all these functions may be regulated by the AMP-activated kinase (AMPK).
The cellular metabolic sensor AMPK has emerged as a key therapeutic target for many cancers. Besides its role in energy homeostasis, AMPK blocks cell cycle, induces apoptosis, regulates autophagy and suppresses the anabolic processes required for rapid cell growth . Moreover, pharmacological activation of AMPK by the antidiabetic drug metformin, has been demonstrated to sensitize cancer cells to cytotoxic therapy . AMPK is a heterotrimeric protein consisting of a catalytic α subunit, and regulatory β and γ subunits. It is activated by AMP binding to the γ subunit as well as by phosphorylation of the Thr172 residue of α subunit mainly by LKB1 kinase although other upstream kinases have also been described .
In this study we evaluated the ability of CAP to inhibit prostate cancer cell proliferation. We found that CAP synergizes with docetaxel to potently block cell growth in vitro and tumor growth in vivo by a mechanism involving activation of AMPK.
Materials and methods
Capsaicin (CAP) and Ddocetaxel (DTX) were purchased to TOCRIS (Bristol, UK). dorsomorphin and STO-609 were purchased to Sigma (St. Louis, USA). Primary antibodies anti-pAMPKα1-thr172, pACC-ser79, pAkt-ser473, pmTOR, pS6, pLKB1 and the antibodies against the corresponding total forms were obtained from Cell Signaling Technology (Danvers, MA, USA). Peroxidase labeled secondary anti-mouse IgG was from Sigma (St. Louis, MO, USA) and anti-rabbit IgG was from Calbiochem (San Diego, USA).
PC3 and LNCaP human prostate cancer cell lines were obtained from American Type Culture Collection (ATCC CRL-1435 and ATCC CRL-1740 respectively) (Rockville, MD, USA). Cells were routinely grown in RPMI 1640 medium supplemented with 100 IU/ml penicillin G sodium, 100 μg/ml streptomycin sulfate, 0.25 μg/ml amphotericin B (Invitrogen, Paisley, UK) and 10% fetal bovine serum. For treatment experiments, cells were plated and grown 48 h, the medium was then replaced with serum-free RPMI 1640 for 24 h and then incubated with different treatments for the indicated times. Cells were used at passages 4–20.
Cell viability assay (MTT)
Cell viability was measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) Cell Proliferation assay (Sigma, St. Louis, MO, USA) 24 h after exposure to treatments. In brief, a total of 5000 cells/well were seeded into 12-well plate in a final volume of 1 ml. After treatments, 100 µl MTT solution (5 mg/ml in PBS) was added to the medium and cells were incubated at 37 °C for 4 h. Then, the supernatant was discarded and dimethyl sulfoxide was added to dissolve the formazan crystals. Treatments were carried out in triplicate. The optical density in each well was evaluated by measurement of absorbance at 490 and 650 nm using an iMark™ Absorbance Reader from Bio-Rad (Richmond, CA, USA).
Western blot analysis
Cells were lysed in a lysis buffer (50 mM Tris pH 7.4, 0.8 M NaCl, 5 mM MgCl2, 0.1% Triton X-100) containing Protease Inhibitor and Phosphatase inhibitor Cocktail (Roche, Diagnostics; Mannheim, Germany), incubated on ice for 15 min and cleared by microcentrifugation. Protein concentrations were measured by BioRad™ protein assay kit (Richmond, CA, USA). Cell proteins extracts (20 μg) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a PVDF membrane. Thereafter, nonspecific binding was blocked with 5% of BSA in TTBS for 1 h at room temperature. Membranes were then incubated overnight at 4 °C with primary antibodies. After washing in TTBS, membranes were incubated with peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (1:2000) for 2 h at room temperature. The immune complex was visualized with an ECL system (Cell Signaling Technology).
Cells were transfected in 1 ml OptiMEM (Invitrogen, Carlsbad, CA, USA) containing 4 µg lipofectamine iMax (Invitrogen, Carlsbad, CA), with 100 nM AMPK specific siRNA duplexes (5′-CCCAUAUUAUUUGCGUGUAdTdT-3′ and 5′-UACACGCCAAAUAAUAUGGGdTdT-3′), LKB1 selective siRNA duplexes (5′-GUACUUCUGUCAGCUGAUUdTdT-3′ and 5′-AAUCAGCUGACAGAAGUACdTdT-3′) (Sigma, St. Louis, MO, USA), Akt selective siRNA duplexes (Cell Signaling Technology, Danvers, MA, USA) or control scrambled RNA (Invitrogen, Carlsbad, CA) for 72 h according to manufacturer’s protocols. At 72 h after transfection, the medium was removed and replaced for RPMI containing 10% fetal bovine serum. At dedicated time points after transfection, cells were used for MTT cell viability assays or Western blot.
The plasmid encoding full-length human PTEN was provided by Jaewhan Song (Addgene plasmid # 78777; http://n2t.net/addgene:78777; RRID: Addgene_78777 , Addgene Watertown, MA, USA). PC3 cells were seeded in 6 or 12 well plates with complete medium and transfected with 4 μg of PTEN recombinant plasmid (pcDNA3-FLAG PTEN), using 5 μl of Lipofectamine 3000 (Thermofisher, Waltham, MA, USA) and OptiMEM. After 48 h of transfection, the medium was replaced by another without serum and the different treatments were administered. Subsequently, cell viability was assessed by MTT and protein expression was analyzed by Western blot using anti-FLAG antibodies (Flag M2 antibody, Sigma, Saint Louis, MO, USA).
Anti-tumor activity in mouse tumor models
Four-week-old athymic nude-Foxn1 (nu/nu) mice were purchased from Envigo RMS (Barcelona, Spain) and housed in a laminar air-flow cabinet under pathogen-free conditions on a 12-h light/dark schedule at 21–23 °C and 40–60% humidity with access to food pellets and tap water ad libitum. 4 animals were housed by cage. G Power analysis was used to calculate sample size , according to our previous data and experience and considering two tails effect and a significance level of 5%. Prostate tumors were induced in athymic mice by subcutaneal injection of 5 × 106 PC-3 cells or 5 × 106 LNCaP cells, according to the experiment. When tumors reached 70 mm3 the mice were randomly divided into four experimental groups of 6 animals each, and the following treatments were started by daily i.p. injection: Vehicle (DMSO), 2 mg/kg capsaicin (CAP), 10 mg/kg docetaxel (DTX) or 2 mg/kg capsaicin + 10 mg/kg docetaxel (CAP + DTX). Tumor sizes were measured every day and calculated using the formula V (mm3) = 1/2(Length × Width2). At the end of the study, the mice were sacrificed by placing them in a CO2 gas-filled chamber, and the excised tumors were recovered and weighted.
Combined drug analysis
Drug interaction was determined using the combination index (CI)-isobologram equation that allows quantitative determination of drug interactions, where CI < 1 implied synergism, CI = 1 additive, and CI > 1 implied antagonism [17, 18]. Compusyn© version 1.0 software (ComboSyn, Inc. Paramus, NJ, USA) was used to generate the dose–response curves, dose–effect analysis, and CI-effect plot.
The statistical analysis of the results was performed using a two-way ANOVA and Dunnett’s multiple comparisons test or Tukey’s multiple comparisons test. The results were reported as mean ± SEM or SD as indicated in figure caption, of at least three independent experiments Data were considered significant when p ≤ 0.05.
Capsaicin and docetaxel synergistically inhibit prostate cancer cells growth
AMPK activation is involved in the antiproliferative effect of CAP + DTX
Capsaicin inhibits Akt independently of AMPK activation
In vivo anti-tumor study
Finally, we tested whether AMPK activity was upregulated in the PC3 tumors of nude mice. Figure 6b shows that, in line with the in vitro results, the AMPK activity in the PC3 tumors treated with the combination of CAP and DTX was substantially higher than in those treated with capsaicin alone or docetaxel alone. In addition, Akt phosphorylation was nearly detected in co-treated tumors and was less than in those tumors treated with CAP or DTX singly (Fig. 6b). These results indicate that the combination of capsaicin and docetaxel synergistically reduces PC3 tumor growth in vivo and is very effective for this model of castration resistant prostate cancer.
Docetaxel is considered the most promising anticancer drug for prostate cancer treatment. However, the quick emergence of resistance and systemic toxicity diminished its efficacy. Combination therapy represents a promising therapeutic strategy to overcome toxicity by reduction of the effective dose. Several promising agents are emerging with a potential role in docetaxel-based combinations based on efficacy and manageable toxicity. Preclinical findings suggest that combining such innovative strategies with traditional treatments offers new benefits improving treatment outcome [22, 23]. In this study we evaluated the effectiveness of combining docetaxel and the natural compound capsaicin to reduce prostate tumor growth. We found that the combination of both compounds exhibited synergistic antitumor effect both in vitro and in vivo. Similar results have been previously reported with other compounds used in combination with docetaxel [24, 25, 26]. Our results show that the combination of docetaxel and capsaicin caused a strong decrease in the levels of pAkt, pmTOR and pS6 and that targeting this pathway abolishes the cell viability inhibition induced by docetaxel and the synergistic effect.
Recent data indicate that capsaicin displays synergism with diverse conventional drugs as camptothecin , pirarubicin , brassinin  and resveratrol in several tumor cell lines . Nevertheless, the molecular mechanisms involved in this synergistic effect continue to be largely elusive. Our results show that combination of CAP and DTX increases AMPKα catalytic subunit phosphorylation in Thr179 and the phosphorylation of its downstream substrate ACC. Pharmacological inhibition of AMPK as well as AMPK or LKB1 knocking down by siRNA abrogates the capsaicin-dependent inhibition of cell growth and hampers the synergistic effect, indicating that AMPK activation by capsaicin is critical for the antiproliferative effect. Moreover, we propose that the Akt/mTOR axe inhibition by the co-administration was independent of AMPK activation, since AMPK knocking down and inhibition did not have effect on capsaicin-induced Akt downregulation. These results are in agreement with the notion that synergy implies multiple sites of action by definition . Therefore, docetaxel and capsaicin, by regulating two independent pathways, potentiate each other and synergistically inhibit prostate cell viability. In line with our results, it has been shown that combined treatment of the AKT inhibitor perifosine and the AMPK activator AICAR, markedly suppressed prostate PC3 cell growth compared to either treatment alone which indicates that concurrent modulation of AKT and AMPK is more effective than either alone in prostate cancer therapy . Therefore, the co-administration of capsaicin and docetaxel might trigger two signaling pathways that together produce a synergic effect that mediate cancer cell death and growth inhibition.
To further investigate the synergistic antitumor effect of the combination of docetaxel and capsaicin we induced xenograft tumors in nude mice which were treated with CAP, DTX or their combination. According to published data regarding capsaicin bioavailability and absorption , for in vivo studies we used a dose of capsaicin equivalent to that used with cells (considering a mice blood volume of 2.5 ml and an average mice weight of 30 g, 80 µM is equivalent to 2 mg/kg). On the other hand, DTX has low bioavailability mainly due to its poor aqueous solubility and its transportation in blood by binding to plasma proteins such as lipoproteins, albumin and α1 acid glycoprotein. Therefore, in vivo doses of docetaxel are usually higher than that used in cells. Thus, we choose a docetaxel dose of 10 mg/kg which is a common used dose in the in vivo studies [32, 33, 34]. In LNCaP tumors CAP or DTX singly administered or in combination, had little effect on tumor growth. However, in PC3 tumors, DTX and CAP significantly decreased tumor growth and the DTX + CAP combination had stronger anti-tumor activity that either compound singly administered. Co-treatment induced a robust AMPK activation and Akt/mTOR axe inhibition in the PC3 prostate tumors. Therefore, we demonstrated that the proposed combination of docetaxel and capsaicin potently inhibited the growth of castration resistant prostate cancer cells in vitro and in vivo.
In conclusion, these findings indicate that docetaxel and capsaicin co-administration represents a therapeutically relevant strategy to improve docetaxel chemotherapy in prostate cancer patients. The fact that AMPK activation is in the underpinning mechanism that sensitizes prostate cells to docetaxel suggests that impact metabolism could be a new option to modulate chemotherapeutic drugs effect.
IDL conceived and supervised the study, wrote the manuscript, and secured funding. BSG and AB designed and performed experiments. BSG, validate and performed formal analysis of data. PMG contributed with design of methodology, acquisition, analysis, interpretation of data for the work and critical reading of the manuscript. NRH contributed with design of methodology. All authors read and approved the final manuscript.
Authors would like to thank J.M. Orellana for technical assistance in animal welfare.
The authors declare that they have no competing interests.
Availability of data and materials
The dataset supporting the conclusions of this article is available in the Mendeley repository. https://doi.org/10.17632/48pfmwbcb5.
Consent for publication
Ethics approval and consent to participate
Animal experiments have followed the ARRIVE guidelines and have been carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, EU Directive 2010/63/EU for animal experiments. The procedure was approved by Alcalá University Ethical Commission and by the Ethical Commission of the Comunidad de Madrid (procedure PROEX 241/15). All animal studies were conducted in accordance with the Spanish institutional regulation (RD 53/2013) for the housing, care and use of experimental animals and met the European Community directives regulating animal research. Recommendations made by the United Kingdom coordinating Committee on Cancer Research (UKCCCR) have been kept carefully. To assess the welfare of animals a panel of 10 indicators were recorded each day. When adverse effects, pain or distress were appreciated in the animals (score of 15 out of 40) the humane endpoint was applied.
The authors would like to thank Fundación Tatiana Pérez de Guzmán (Grant No. Patrocinio 2013-001 and Grant Patrocinio 2019-001) for financial support into their research.
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- 12.Troncone M, Cargnelli SM, Villani LA, Isfahanian N, Broadfield LA, Zychla L, Wright J, Pond G, Steinberg GR, Tsakiridis T. Targeting metabolism and AMP-activated kinase with metformin to sensitize non-small cell lung cancer (NSCLC) to cytotoxic therapy; translational biology and rationale for current clinical trials. Oncotarget. 2017;8:57733.CrossRefGoogle Scholar
- 32.Nguyen HM, Vessella RL, Morrissey C, Brown LG, Coleman IM, Higano CS, Mostaghel EA, Zhang X, True LD, Lam HM, et al. LuCaP prostate cancer patient-derived xenografts reflect the molecular heterogeneity of advanced disease an–d serve as models for evaluating cancer therapeutics. Prostate. 2017;77(6):654–71.CrossRefGoogle Scholar
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