Sánchez et al. Cancer Cell Int (2019) 19:54 https://doi.org/10.1186/s12935-019-0769-2 Cancer Cell International

PRIMARY RESEARCH Open Access Combination of the natural product capsaicin and docetaxel synergistically kills human prostate cancer cells through the metabolic regulator AMP‑activated kinase Belén G. Sánchez1, Alicia Bort1, Pedro A. Mateos‑Gómez1, Nieves Rodríguez‑Henche1 and Inés Díaz‑Laviada1,2*

Abstract Background: Current chemotherapy for castration-resistant prostate cancer is established on taxane-based com‑ pounds like docetaxel. However, eventually, the development of toxic side efects and resistance limits the thera‑ peutic beneft being the major concern in the treatment of prostate cancer. Combination therapies in many cases, enhance drug efcacy and delay the appearance of undesired efects, representing an important option for the treat‑ ment of castration-resistant prostate cancer. In this study, we tested the efcacy of the combination of docetaxel and capsaicin, the pungent ingredient of hot chili peppers, on prostate cancer cells proliferation. Methods: 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. Results: Docetaxel and capsaicin synergistically inhibited the growth of LNCaP and PC3 cells, with a combina‑ tion 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 efect 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, phar‑ macological 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 syner‑ gistic anti-proliferative efect may be attributed to two independent efects: Inhibition of the PI3K/Akt/mTOR signal‑ ing pathway by one side, and AMPK activation by the other. In vivo experiments confrmed the synergistic efects of docetaxel and capsaicin in reducing the tumor growth of PC3 cells.

*Correspondence: [email protected] 1 Department of Systems Biology, Biochemistry and Molecular Biology Unit, School of Medicine and Health Sciences, Alcala University, Alcalá de Henares, Ctra A‑2 Km 32., 28871 Madrid, Spain Full list of author information is available at the end of the article

© The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​mmons​.org/ publi​cdoma​in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Sánchez et al. Cancer Cell Int (2019) 19:54 Page 2 of 14

Conclusion: Combination of docetaxel and capsaicin represents a therapeutically relevant approach for the treat‑ ment of Prostate Cancer. Keywords: Docetaxel, Capsaicin, AMPK, PC3 cells, LNCaP cells, Prostate cancer

Background sorafenib [10]. Yet, the mechanisms underlying the cap- Prostate cancer (PCa) is the most prevalent malignancy saicin-mediated inhibition of cell proliferation and drug in men worldwide, and the second leading cause of can- sensitization are divers and poorly understood. Labo- cer related deaths [1, 2]. Environmental factors such as ratory data supports the notion that dietary capsaicin hypercaloric diets, sedentary life, increasing life expec- has anti-obesity role by increasing energy expenditure, tancy and modifed diagnostic techniques contribute enhancing fat oxidation, decreasing adipogenesis and to the increase in prostate cancer incidence. For locally suppressing appetite [11]. Although a molecular mecha- advanced and metastatic cancers androgen deprivation nism has not been clarifed, all these functions may be therapy is the standard of care. Despite initial disease regulated by the AMP-activated kinase (AMPK). regression, most men eventually progress to castration- Te cellular metabolic sensor AMPK has emerged resistant prostate cancer (CRPC) with no response to as a key therapeutic target for many cancers. Besides hormonal therapy and a lethal outcome. Currently, doc- its role in energy homeostasis, AMPK blocks cell cycle, etaxel is the frst-line chemotherapeutic agent available induces apoptosis, regulates autophagy and suppresses to patients with this lethal form of the disease, but the the anabolic processes required for rapid cell growth [12]. survival of patients remains limited by the occurrence Moreover, pharmacological activation of AMPK by the of dose-dependent adverse efects and acquired resist- antidiabetic drug metformin, has been demonstrated to ance. Mechanisms underpinning resistance development sensitize cancer cells to cytotoxic therapy [13]. AMPK is include overexpression of multidrug efux pumps, muta- a heterotrimeric protein consisting of a catalytic α sub- tion of β-tubulin, and activation of signaling proteins unit, and regulatory β and γ subunits. It is activated by as MAPK or Akt [3]. Docetaxel resistance is a clinical AMP binding to the γ subunit as well as by phosphoryla- problem since it is the main therapy for CRPC. Moreo- tion of the Tr172 residue of α subunit mainly by LKB1 ver, newer chemotherapeutic drugs developed to treat kinase although other upstream kinases have also been docetaxel resistant patients carry signifcant hemato- described [14]. logical toxicities [3]. Terefore, approaches to improve In this study we evaluated the ability of CAP to inhibit taxane-based chemotherapy are urgently required [4]. prostate cancer cell proliferation. We found that CAP Tus, it is of highly clinical signifcance to identify agents synergizes with docetaxel to potently block cell growth that when combined with the current chemotherapeutic in vitro and tumor growth in vivo by a mechanism involv- drugs allow to decrease the doses without reducing their ing activation of AMPK. efectiveness as well as to avoid and/or to overcome drug resistance. Terefore, combination therapy, a treatment Materials and methods modality that combines two or more therapeutic agents, Materials is becoming a cornerstone of cancer therapy [5]. Capsaicin (CAP) and Ddocetaxel (DTX) were purchased Over the past few years, many anti-cancer drugs have to TOCRIS (Bristol, UK). dorsomorphin and STO-609 been identifed from natural nutritional compounds. were purchased to Sigma (St. Louis, USA). Primary Capsaicin (CAP), the spicy ingredient of hot chili pep- antibodies anti-pAMPKα1-thr172, pACC-ser79, pAkt- pers, exhibit anti-neoplastic activity in many cancer ser473, pmTOR, pS6, pLKB1 and the antibodies against cell lines as well as in vivo [6]. In addition, recent data the corresponding total forms were obtained from Cell indicate that CAP sensitizes cells to chemotherapeutic Signaling Technology (Danvers, MA, USA). Peroxidase agents. For instance, the combination of CAP and cam- labeled secondary anti-mouse IgG was from Sigma (St. phothecin increases apoptosis in small cell lung cancer Louis, MO, USA) and anti-rabbit IgG was from Calbio- [7]. In cholangocarcinoma, CAP increases sensitivity to chem (San Diego, USA). 5-fuorouracil and the mixture of both compounds inhib- its tumor growth with greater efcacy than 5-fuorouracil Cell culture alone [8]. In human prostate cancer cells CAP combined PC3 and LNCaP human prostate cancer cell lines were with brassinin enhances apoptotic and anti-metastatic obtained from American Type Culture Collection (ATCC efects [9]. We have shown that, in hepatocellular carci- CRL-1435 and ATCC CRL-1740 respectively) (Rock- noma cells, CAP increases the antiproliferative efects of ville, MD, USA). Cells were routinely grown in RPMI Sánchez et al. Cancer Cell Int (2019) 19:54 Page 3 of 14

1640 medium supplemented with 100 IU/ml penicillin USA), Akt selective siRNA duplexes (Cell Signaling G sodium, 100 μg/ml streptomycin sulfate, 0.25 μg/ml Technology, Danvers, MA, USA) or control scrambled amphotericin B (Invitrogen, Paisley, UK) and 10% fetal RNA (Invitrogen, Carlsbad, CA) for 72 h according to bovine serum. For treatment experiments, cells were manufacturer’s protocols. At 72 h after transfection, the plated and grown 48 h, the medium was then replaced medium was removed and replaced for RPMI containing with serum-free RPMI 1640 for 24 h and then incubated 10% fetal bovine serum. At dedicated time points after with diferent treatments for the indicated times. Cells transfection, cells were used for MTT cell viability assays were used at passages 4–20. or Western blot.

Cell viability assay (MTT) PTEN transfections Cell viability was measured by MTT (3-(4,5-dimethylth- Te plasmid encoding full-length human PTEN was iazol-2-yl)-2,5-diphenyltetrazolium bromide) Cell Pro- provided by Jaewhan Song (Addgene plasmid # 78777; liferation assay (Sigma, St. Louis, MO, USA) 24 h after http://n2t.net/addge​ne:78777​; RRID: Addgene_78777 exposure to treatments. In brief, a total of 5000 cells/well [15], Addgene Watertown, MA, USA). PC3 cells were were seeded into 12-well plate in a fnal volume of 1 ml. seeded in 6 or 12 well plates with complete medium and After treatments, 100 µl MTT solution (5 mg/ml in PBS) transfected with 4 μg of PTEN recombinant plasmid was added to the medium and cells were incubated at (pcDNA3-FLAG PTEN), using 5 μl of Lipofectamine 37 °C for 4 h. Ten, the supernatant was discarded and 3000 (Termofsher, Waltham, MA, USA) and Opti- dimethyl sulfoxide was added to dissolve the formazan MEM. After 48 h of transfection, the medium was crystals. Treatments were carried out in triplicate. Te replaced by another without serum and the diferent optical density in each well was evaluated by measure- treatments were administered. Subsequently, cell viability ment of absorbance at 490 and 650 nm using an iMark™ was assessed by MTT and protein expression was ana- Absorbance Reader from Bio-Rad (Richmond, CA, USA). lyzed by Western blot using anti-FLAG antibodies (Flag M2 antibody, Sigma, Saint Louis, MO, USA). Western blot analysis Cells were lysed in a lysis bufer (50 mM Tris pH 7.4, Anti‑tumor activity in mouse tumor models 0.8 M NaCl, 5 mM MgCl­ 2, 0.1% Triton X-100) containing Four-week-old athymic nude-Foxn1 (nu/nu) mice were Protease Inhibitor and Phosphatase inhibitor Cocktail purchased from Envigo RMS (Barcelona, Spain) and (Roche, Diagnostics; Mannheim, Germany), incubated housed in a laminar air-fow cabinet under pathogen- on ice for 15 min and cleared by microcentrifugation. free conditions on a 12-h light/dark schedule at 21–23 °C Protein concentrations were measured by BioRad™ and 40–60% humidity with access to food pellets and tap protein assay kit (Richmond, CA, USA). Cell proteins water ad libitum. 4 animals were housed by cage. G Power extracts (20 μg) were separated by sodium dodecyl sul- analysis was used to calculate sample size [16], according fate–polyacrylamide gel electrophoresis (SDS-PAGE) to our previous data and experience and considering two and then transferred onto a PVDF membrane. Tereaf- tails efect and a signifcance level of 5%. Prostate tumors ter, nonspecifc binding was blocked with 5% of BSA in were induced in athymic mice by subcutaneal injection 6 6 TTBS for 1 h at room temperature. Membranes were of 5 × 10 PC-3 cells or 5 × 10 LNCaP cells, according to then incubated overnight at 4 °C with primary antibod- the experiment. When tumors reached 70 mm3 the mice ies. After washing in TTBS, membranes were incubated were randomly divided into four experimental groups of with peroxidase-conjugated anti-mouse or anti-rabbit 6 animals each, and the following treatments were started secondary antibodies (1:2000) for 2 h at room tempera- by daily i.p. injection: Vehicle (DMSO), 2 mg/kg capsai- ture. Te immune complex was visualized with an ECL cin (CAP), 10 mg/kg docetaxel (DTX) or 2 mg/kg cap- system (Cell Signaling Technology). saicin + 10 mg/kg docetaxel (CAP + DTX). Tumor sizes were measured every day and calculated using the for- siRNA transfections 3 2 mula V ­(mm ) = 1/2(Length × Width ). At the end of the Cells were transfected in 1 ml OptiMEM (Invitrogen, study, the mice were sacrifced by placing them in a ­CO2 Carlsbad, CA, USA) containing 4 µg lipofectamine gas-flled chamber, and the excised tumors were recov- iMax (Invitrogen, Carlsbad, CA), with 100 nM AMPK ered and weighted. specifc siRNA duplexes (5′-CCCAUA​ UUA​ UUU​ GCG​ ​ UGU​AdTdT-3′ and 5′-UAC​ACG​CCA​AAU​AAU​AUG​ Combined drug analysis GGdTdT-3′), LKB1 selective siRNA duplexes (5′-GUA​ Drug interaction was determined using the combination CUU​CUG​UCA​GCU​GAU​UdTdT-3′ and 5′-AAU​CAG​ index (CI)-isobologram equation that allows quantitative CUG​ACA​GAA​GUA​CdTdT-3′) (Sigma, St. Louis, MO, determination of drug interactions, where CI < 1 implied Sánchez et al. Cancer Cell Int (2019) 19:54 Page 4 of 14

of Akt, mTOR and S6 phosphorylation only in PC3 cells synergism, CI = 1 additive, and CI > 1 implied antagonism [17, 18]. ­Compusyn© version 1.0 software (ComboSyn, and that CAP diminished Akt, mTOR and S6 phospho- Inc. Paramus, NJ, USA) was used to generate the dose– rylation both in PC3 and LNCaP cells at 1 h and 24 h response curves, dose–efect analysis, and CI-efect plot. (Fig. 2). However, co-treatment with DTX and CAP leaded to a higher inhibition of Akt phosphorylation in Statistical analysis LNCaP cells and suppressed Akt phosphorylation in PC3 Te statistical analysis of the results was performed using cells, both at 1 h and 24 h (Fig. 2). Likewise, the phospho- a two-way ANOVA and Dunnett’s multiple comparisons rylation of mTOR and S6 were also signifcantly inhibited test or Tukey’s multiple comparisons test. Te results in the co-treated PC3 cells (Fig. 2). In LNCaP cells the combined treatment also caused inhibition of mTOR and were reported as mean ± SEM or SD as indicated in fg- ure caption, of at least three independent experiments S6 phosphorylation but the efect was weaker than that of PC3 cells even at 24 h, indicating that the combined Data were considered signifcant when p ≤ 0.05. treatment is more efcient in the castration resistant Results prostate PC3 cells (Fig. 2). ANOVA analysis revealed that Capsaicin and docetaxel synergistically inhibit prostate the inhibitory efect produced by DTX + CAP on Akt, cancer cells growth mTOR and S6 phosphorylation was diferent from that To assess the antiproliferative efect of docetaxel (DTX) produced by DTX or CAP given singly. Moreover, the and capsaicin (CAP), prostate cancer LNCaP and inhibitory efect on Akt, mTOR and S6 at 24 h in the co- PC3 cells were incubated with increasing doses of DTX, treated cells was higher than the sum of DTX and CAP CAP or the combination of both compounds and cell via- individual efects and combination index was lesser than bility was determined by MTT. As shown in Fig. 1, both 1, indicating synergism (Fig. 2). Tose results suggest that DTX and CAP singly reduced cell viability of LNCaP and CAP and DTX synergistically block the PI3K route which PC3 cells. CAP alone dose-dependently inhibited cell could underly the antiproliferative efect of DTX and viability from 40 µM in LNCaP cells and from 20 μM in CAP combination on prostate cancer cells. PC3 cells. It is worthy to note that 50% inhibitory con- To further corroborate this notion, we knocked down centration (IC50) was much higher for DTX (300 μM) Akt by siRNA and determined cell viability by MTT. As than for CAP (72 μM) in PC3 cells. When DTX and CAP shown in Fig. 3a, Akt pathway blockage abolished DTX were combined a dramatic reduction on cell viability was inhibitory efect while enhanced CAP inhibitory efect observed. We tested a constant CAP:DTX ratio (2:1) to on PC3 cell viability suggesting that DTX needs Akt to create isobolograms using CompuSyn­ © software and exert its antiproliferative efect. To additionally explore to calculate the combination index (CI). CI allows the this pathway, we overexpressed the phosphatase PTEN quantifcation of synergism or antagonism for two drugs in PC3 cells by transient transfection with a plasmid con- where CI of 1 indicates an additive efect, whereas a CI < 1 taining a fag tagged human PTEN. As shown in Fig. 3b, or CI > 1 indicates synergism or antagonism, respectively. overexpression of PTEN in PC3 cells did not prevent the Combination-index showed a potent synergy of cell kill- inhibitory efect of CAP and DTX + CAP on Akt, mTOR ing at four of the fve combinations used in LNCaP cells and S6 phosphorylation but hindered the inhibitory and at the fve combinations used for PC3 cells. Likewise, efect on cell viability (Fig. 3c). Moreover, the synergistic isobologram for the combination of docetaxel and cap- antiproliferative efect of the combined treatment was saicin showed that all combination data points in PC3 lost at four of the fve combinations tested (Fig. 3c). Tese cells fall on the lower left, indicating synergism (Fig. 1). fndings indicate that activation of the PI3K/Akt pathway In LNCaP the synergic efect was evident in four combi- is involved in the synergistic antiproliferative efect elic- nations, but it was less strong that in PC3 cells. Ten, for ited by the combination of DTX and CAP in the castra- further experiments we chose the combination of DTX tion resistant prostate PC3 cells. 40 μM + CAP 80 μM as it had a high synergistic efect but a moderate cytotoxic efect. AMPK activation is involved in the antiproliferative efect of CAP DTX Akt is a major component of the PI3K signaling path- + way which is constitutively active in PC3 and LNCaP Clinical data indicate that AMPK activation by Met- cells due to a PTEN deletion. Activation of the PI3K axe formin, an antidiabetic biguanide, improve the survival of is involved in many cell functions that induce cell growth. castration-resistant prostate cancer patients through sen- To corroborate the antiproliferative efect of the combi- sitization to chemotherapy [19]. To test whether AMPK nation of DTX and CAP, we evaluated the phosphoryla- activation was associated with the synergistic efect of tion of Akt and its downstream efectors mTOR and S6. CAP and DTX, we determined levels of phosphoryl- Results indicated that DTX induced a modest inhibition ated AMPK in the Tr172 of the α catalytic subunit Sánchez et al. Cancer Cell Int (2019) 19:54 Page 5 of 14

120 LNCaP DTX CAP DTX + CAP 100 * ‡ * 80 * * * * * * 60 * 40 * Cell viability (%) ‡ 20 * ‡ * * 0 DTX, µM 12345- 10 20 40 60 806 CAP, µM - 20 40 80 120 160 DTX + CAP, µM - 10+20 20+40 40+80 60+120 80+160

10µM DTX + 20µM CAP DTX CAP Effect CI 20µM DTX + 40µM CAP µM µM % 40µM DTX + 80µM CAP 10 20 34 0.490 60µM DTX + 120µM CAP 80µM DTX + 160µM CAP 20 40 30 1.156

40 80 65 0.889 Docetaxel

60 120 95 0.649

80 160 95 0.865

Capsaicin

120 PC-3 DTX CAP 100 * DTX + CAP * * * * * * 80 * ‡ * * * 60 *

40 ‡

Cell viability (%) * ‡ 20 * * 0 DTX, µM - 5 10 20 40 80 CAP, µM - 10 20 40 80 160 DTX + CAP, µM - 5+10 10+20 20+40 40+80 80+160

5µM DTX + 10µM CAP DTX CAP Effect CI 10µM DTX + 20µM CAP µM µM % 20µM DTX + 40µM CAP 5 10 19 0.669 40µM DTX + 80µM CAP 80µM DTX +160µM CAP 10 20 25 0.911

20 40 43 0.809 Docetaxel

40 80 78 0.418

80 160 90 0.388

Capsaicin Fig. 1 Synergistic antiproliferative efect of capsaicin and docetaxel on prostate tumor cells. LNCaP cells (upper panel) or PC3 cells (lower panel) were incubated with increasing doses of docetaxel (DTX), capsaicin (CAP) or the combination of both compounds for 24 h and cell viability was evaluated by MTT. Data represent the mean (n 3) SD. *p < 0.0001 signifcant diference between treated and control cells by two-way ANOVA = ± and Dunnett’s multiple comparisons test; ‡p < 0.0001 indicates signifcant interaction between DTX and CAP treatment. Data were analyzed with CompuSyn software to calculate the combination index (CI) Sánchez et al. Cancer Cell Int (2019) 19:54 Page 6 of 14

LNCaP 1h PC3 1h pAkt pAkt

Akt Akt

pmTOR pmTOR

mTOR mTOR

pS6 pS6

S6 S6

-tubulin -tubulin

DTX,40 M - + - + DTX,40 M - + - + CAP, 80 M - - + + CAP, 80 M - - + + 1.2 1.2 pAKT/AKT pAkt/Akt pmTOR/mTOR pmTOR/mTOR 1.0 pS6/S6 1 * * pS6/S6 * lues lues * ‡ 0.8 0.8 va * va * * * * * * * 0.6 0.6 *

0.4 0.4 ‡ *

0.2 Densitometric 0.2 Densitometric

0.0 0 ControlControlD DTXTX CAPCAPD DTXTX + +CAP CAP ControlContro lDDTXXT CAPCA PDDTXXT+CAP +CAP LNCaP 24h PC3 24h pAkt pAkt

Akt Akt

pmTOR pmTOR

mTOR mTOR

pS6 pS6

S6 S6

-tubulin -tubulin

DTX,40 M - + - + DTX,40 M - + - + CAP, 80 M - - + + CAP, 80 M - - + +

1.2 1.2 pAKT/AKT pAkt/Akt pmTOR/mTOR pmTOR/mTOR pS6/S6 1 * pS6/S6 1.0 * *

* lues * lues * 0.8 * * va

va 0.8 * * ‡ 0.6 0.6 * * ‡ * ‡ * 0.4 0.4 * ‡ Densitometric Densitometric 0.2 0.2 *

0.0 0 Control DTX CAP DTX +CAP ControControl lDDTXTX CACAPPD TXDTX ++CAP CA P ControlDXT CAP DXT+CAP LNCaP (24 h) PC3 (24 h)

Control DTX CAP DTX+CAP CI Control DTX CAP DTX+CAP CI

pAkt 1 0 ± 0.06 31 ± 0.01 58 ± 0.10 0.152 pAkt 1 14 ± 0.02 37 ± .0.3 60 ± 0.3 0.199

pmTOR 1 13 ± 0.04 22 ± 0.05 43 ± 0.01 0.272 pmTOR 1 18 ± 0.2 28 ± 0.2 56 ± 0.3 0.190

pS6 1 0 ± 0.01 23 ± 0.10 30 ± 0.12 0.485 pS6 1 8 ± 0.3 41 ± 0.1 62 ± 0.1 0.270 Fig. 2 Combination of docetaxel and capsaicin efectively inhibits Akt/mTOR pathway. LNCaP or PC3 cells were incubated with 40 μM docetaxel (DTX), 80 μM capsaicin (CAP) or the combination of both during 1 h or 24 h and levels of phospho-Akt, phospho-mTOR, phospho-S6 and their corresponding total forms were determined by Western blot. β-Tubulin serves as a loading control. The densitometric analyses of bands represented as the mean SD of three diferent experiments are shown below the plots. *p < 0.05 signifcant diference between treated and control cells by ± two-way ANOVA and Dunnett’s multiple comparisons test; and ‡p < 0.05 indicate signifcant interaction between DTX and CAP treatment Sánchez et al. Cancer Cell Int (2019) 19:54 Page 7 of 14

a siC siAkt 120 # siC Akt siAKT 100 pAkt * 80 * ‡ * pmTOR # 60 * 40 ‡ pS6 # Cell viability (%) * -tubulin 20 0 DTX, 40 M - + - + - + - + ControlDTX CAPDTX + CAP CAP, 80 M - - + + - - + + PC3 PTEN+ 1h PC3 PTEN+ 24h b pAkt pAkt

Akt Akt pAkt pmTOR pmTOR Flag mTOR mTOR -tubulin pS6 pS6

S6 S6

-tubulin -tubulin DTX,40 M - + - + DTX,40 M - + - + CAP, 80 M - - + + CAP, 80 M - - + + 1.2 1.2 pAKT/AKT pAKT/AKT pmTOR/mTOR pmTOR/mTOR 1.0 pS6/S6 1.0 pS6/S6 * lues * lues 0.8 0.8 va * * va *

0.6 * 0.6 * * * ‡ 0.4 ‡ 0.4 * ‡ * Densitometric Densitometric * * * 0.2 0.2 *

0.0 0.0 Control DTX +CAP Control DTX DTX +CAP Control DTX CACAPPD TX + CAP ControlDTX CAPCA PDTX + CAP PC3 PTEN+ DTX c 120 CAP DTX + CAP

) 100

(% * *

y * 80 * * * 60

Cell viabilit * ‡ 40 * * * 20

0 DTX, µM -05 +5 10 10 10 + 20 20 20 + 40 40 40 + 80 80 80 + 160 CAP, µM - 10 20 40 80 160 DTX + CAP, µM - 5+10 10+20 20+40 40+80 80+160

5µM DTX + 10µM CAP DTX CAP Effect CI 10µM DTX + 20µM CAP µM µM % 20µM DTX + 40µM CAP 5 10 17 1.946 40µM DTX + 80µM CAP 80µM DTX +160µM CAP 10 20 16 1.946

20 40 31 1.772 Docetaxel

40 80 70 0.847

80 160 80 1.094

Capsaicin Sánchez et al. Cancer Cell Int (2019) 19:54 Page 8 of 14

(See fgure on previous page.) Fig. 3 Targetting PI3K/Akt pathway hampers the inhibitory efect of docetaxel and abrogates synergy. a Akt was knocked down in PC3 cells by transfection with selective siRNA. Levels of phospho-Akt, phospho-mTOR, phospho-S6 and their corresponding total forms determined by Western blot is shown on the left. Cell viability by MTT is shown on the right. b PTEN phosphatase was overexpressed in PC3 by transient transfection with a plasmid containing full-length human PTEN and Flag and levels of phospho-Akt, phospho-mTOR, phospho-S6 and their corresponding total forms were determined by Western blot. The densitometric analyses of bands is shown below. c Cell viability and isobologram of PC3 cells transfected with PTEN plasmid and treated with DTX, CAP or both. *p < 0.05 signifcant diference between treated and control cells by two-way ANOVA and Dunnett’s multiple comparisons test; ‡p < 0.05 indicates signifcant interaction between DTX and CAP treatment; #p < 0.05 signifcant diferent between siAKT and siC by two-way ANOVA and Sidak’s multiple comparisons test

and phosphorylation of its well-known substrate Acetyl siRNA did not prevent CAP-promoted Akt inhibition. CoA carboxylase (ACC). As shown in Fig. 4a, when PC3 Interestingly, mTOR phosphorylation was signifcantly cells were incubated with CAP an increase in pAMPK increased in LKB1 depleted cells (Fig. 5c) according to as well as of pACC was observed, both at 1 h and 24 h. the well-established inhibition of mTOR by the LKB1/ Te co-treatment with DTX and CAP produced a higher AMPK axe independently of Akt [20, 21]. Tese results increase in AMPK and ACC phosphorylation indicating indicate that AMPK activation is not the only responsible a strong AMPK activation. We next determined the infu- for Akt inhibition since appreciable efect of capsaicin on ence of the AMPK activation on cell viability. To that end, Akt inhibition could be still seen in AMPK-depleted cells, we use the AMPK inhibitor Dorsomorphin (DORSO, suggesting independent pathways. also known as compound C) or we knocked down AMPK or its upstream kinase LKB1 with selective siRNA. As In vivo anti‑tumor study shown in Fig. 4b, AMPK inhibitor signifcantly prevented To evaluate the therapeutic efcacy of the combination the cell viability decrease in co-treated cells (Fig. 4b). of docetaxel and capsaicin in vivo, xenograft LNCaP Moreover, AMPK knockdown as well as LKB1 knock- and PC3 tumors were induced in nude mice by s.c. down by selective siRNA prevented the synergy between fank injection. When tumors reached 70 mm3 volume, capsaicin and docetaxel (Fig. 4b). Tese results indicate the animals were assigned randomly to various groups that targeting AMPK impairs the antiproliferative efect (n = 6) and injected intraperitoneally with capsaicin or of the combination of DTX and CAP suggesting a role for docetaxel alone or with the combination of both com- AMPK in the CAP-induced sensitization to DTX. pounds. LNCaP tumors growth very slowly according to the less aggressive behavior of this androgen-sen- sitive cell line (Fig. 6a). Treatment of LNCaP tumors Capsaicin inhibits Akt independently of AMPK activation with DTX or CAP singly or in combination moderately Recent studies show that AMPK and Akt display antago- reduced tumor growth. In PC3 tumors, docetaxel treat- nistic roles in cellular homeostasis. Furthermore, AMPK ment signifcantly decreased the growth from day six and AKT can phosphorylate each other modulating their and DTX + CAP co-treatment could achieve a signif- respective activities. To explore the cross-talk between cantly potent antitumor efcacy (Fig. 6a). It is worthy AMPK and Akt in PC3 cells, AMPK was either pharma- to note that the combination of docetaxel and capsai- cologically inhibited with DORSO or genetically silenced cin showed greater antitumor activity than either agent by siRNA, and Akt phosphorylation was determined by alone and that this efect was synergistic from day 3, as Western blotting. As shown in Fig. 5, AMPK targeting deduced from the combination index (Additional fle 1: by DORSO or by interfering siRNA, did not modify Akt Fig. S1). By the end of the 15 days treatments, the PC3 inhibition induced by CAP alone or by the combination tumor volume from mice treated with 2 mg/kg CAP of DTX and CAP. Likewise, knocking down LKB1 with was 76% of the controls, that from mice treated with

(See fgure on next page.) Fig. 4 Combination of docetaxel and capsaicin activates AMPK. a PC3 cells were incubated with 40 μM docetaxel (DTX), 80 μM capsaicin (CAP) or the combination of both during 1 h or 24 h and levels of phospho-AMPK, phospho-ACC and their corresponding total forms were determined by Western blot. β-Tubulin serves as a loading control. The densitometric analyses of bands is shown on the right. b Efect of the AMPK inhibitor dorsomorphin (DORSO, 5 μM), of AMPK silencing by siRNA or LKB1 silencing by siRNA on cell viability. Data are presented as the mean SD of ± three diferent experiments. *p < 0.05 signifcant diference between treated and control cells; #p < 0.05 signifcant diference between non-silenced and silenced cells by two-way ANOVA and Dunnett’s multiple comparisons test; ‡p < 0.05 indicates a signifcant interaction between DTX and CAP treatment Sánchez et al. Cancer Cell Int (2019) 19:54 Page 9 of 14

a 2 pAMPK/AMPK 1h ‡

pAMPK s pACC/ACC *

1.5 * AMPK value * * pACC 1 * ACC

0.5 β-tubulin Densitometric

DTX,40μM - + - + 0 - - ++ CAP, 80μM ControControllDDTXTXCCAAPPDDTX+CAXT+CAPP 2 pAMPK/AMPK 24h * * pACC/ACC pAMPK s * * AMPK alue 1.5 cv pACC 1 ACC

β-tubulin Densitometri 0.5

DTX,40μM - + - + - - 0 CAP, 80μM ++ Control DTXCAP DTX+CAP

- DORSO b 120 siC + DORSO 120 siAMPK 100 100 %) %) * ‡ 80 # 80 # y( * * # * ‡ 60 60 * # * viabilit viabilit y(

40 ll 40 Cell Ce 20 20

0 0 Control DTXCAP DTX+CAP ControlDTX CAPDTX + CAP

120 siC siLKB1 100 #

# *

%) ‡ 80 * # y( * ‡ * 60 * viabilit

ll 40 Ce 20

0 ControlDTX CAPDTX + CAP Sánchez et al. Cancer Cell Int (2019) 19:54 Page 10 of 14

(See fgure on next page.) Fig. 5 Akt/mTOR inhibition by capsaicin is independent of AMPK activation. Efect of the AMPK inhibitor dorsomorphin (DORSO, 5μM), or AMPK silencing by siRNA or LKB1 silencing by siRNA on Akt/mTOR signaling pathway. a PC3 cells were incubated with 40 μM docetaxel (DTX), 80 μM capsaicin (CAP) or the combination of both in the presence or not of 5 μM Dorsomorphin (DORSO) during 1 h. b PC3 cells were transfected with sicontrol (siC) or selective siAMPK for 72 h and then treated with 40 μM docetaxel (DTX), 80 μM capsaicin (CAP) or the combination of both during 1 h. c PC3 cells were transfected with sicontrol or selective siLKB1 for 72 h and then treated with 40 μM docetaxel (DTX), 80 μM capsaicin (CAP) or the combination of both during 1 h. Levels of proteins were determined by Western blot. β-Tubulin serves as a loading control. The densitometric analyses of bands represented as the mean SD of three diferent experiments are shown on the right. *p < 0.05 signifcant diference between ± treated and control cells by two-way ANOVA and Dunnett’s multiple comparisons test; #p < 0.05 signifcant diference between non-silenced and silenced cells by two-way ANOVA and Dunnett’s multiple comparisons test; ‡p < 0.05 indicates signifcant interaction between DTX and CAP treatment

10 mg/kg DTX was 55% and the tumor volume from reported with other compounds used in combination mice co-treated with DTX + CAP was 22% of the con- with docetaxel [24–26]. Our results show that the com- trols (Fig. 6a). Likewise, the tumors that were treated bination of docetaxel and capsaicin caused a strong with combination therapy had signifcantly lower decrease in the levels of pAkt, pmTOR and pS6 and that wet weight than the tumors in the mice treated with targeting this pathway abolishes the cell viability inhibi- either docetaxel or capsaicin alone (Fig. 6a). No signif- tion induced by docetaxel and the synergistic efect. cant change in mice weights was observed during the Recent data indicate that capsaicin displays synergism treatment, indicating that no general toxicity occurred with diverse conventional drugs as camptothecin [7], by the treatments (Additional fle 2: Table S1 and Addi- pirarubicin [27], brassinin [9] and resveratrol in several tional fle 3: Table S2). tumor cell lines [28]. Nevertheless, the molecular mech- Finally, we tested whether AMPK activity was upreg- anisms involved in this synergistic efect continue to be ulated in the PC3 tumors of nude mice. Figure 6b shows largely elusive. Our results show that combination of that, in line with the in vitro results, the AMPK activity CAP and DTX increases AMPKα catalytic subunit phos- in the PC3 tumors treated with the combination of CAP phorylation in Tr179 and the phosphorylation of its and DTX was substantially higher than in those treated downstream substrate ACC. Pharmacological inhibition with capsaicin alone or docetaxel alone. In addition, of AMPK as well as AMPK or LKB1 knocking down by Akt phosphorylation was nearly detected in co-treated siRNA abrogates the capsaicin-dependent inhibition of tumors and was less than in those tumors treated with cell growth and hampers the synergistic efect, indicat- CAP or DTX singly (Fig. 6b). Tese results indicate that ing that AMPK activation by capsaicin is critical for the the combination of capsaicin and docetaxel synergis- antiproliferative efect. Moreover, we propose that the tically reduces PC3 tumor growth in vivo and is very Akt/mTOR axe inhibition by the co-administration was efective for this model of castration resistant prostate independent of AMPK activation, since AMPK knock- cancer. ing down and inhibition did not have efect on capsa- icin-induced Akt downregulation. Tese results are in Discussion agreement with the notion that synergy implies multiple Docetaxel is considered the most promising anticancer sites of action by defnition [29]. Terefore, docetaxel drug for prostate cancer treatment. However, the quick and capsaicin, by regulating two independent pathways, emergence of resistance and systemic toxicity diminished potentiate each other and synergistically inhibit prostate its efcacy. Combination therapy represents a promis- cell viability. In line with our results, it has been shown ing therapeutic strategy to overcome toxicity by reduc- that combined treatment of the AKT inhibitor perifosine tion of the efective dose. Several promising agents are and the AMPK activator AICAR, markedly suppressed emerging with a potential role in docetaxel-based com- prostate PC3 cell growth compared to either treatment binations based on efcacy and manageable toxicity. Pre- alone which indicates that concurrent modulation of clinical fndings suggest that combining such innovative AKT and AMPK is more efective than either alone in strategies with traditional treatments ofers new ben- prostate cancer therapy [30]. Terefore, the co-adminis- efts improving treatment outcome [22, 23]. In this study tration of capsaicin and docetaxel might trigger two sign- we evaluated the efectiveness of combining docetaxel aling pathways that together produce a synergic efect and the natural compound capsaicin to reduce prostate that mediate cancer cell death and growth inhibition. tumor growth. We found that the combination of both To further investigate the synergistic antitumor compounds exhibited synergistic antitumor efect both efect of the combination of docetaxel and capsaicin in vitro and in vivo. Similar results have been previously we induced xenograft tumors in nude mice which were treated with CAP, DTX or their combination. According Sánchez et al. Cancer Cell Int (2019) 19:54 Page 11 of 14

a pAMPK

pAkt/Akt AMPK pmTOR/mTOR 1.2 pAkt/Akt + DORSO pACC pmTOR/mTOR + DORSO 1 ACC 0.8 values pAkt 0.6 * * Akt * 0.4 ‡ ‡ *

pmTOR Densitometric 0.2 * *

mTOR 0 ControlDXT CAP DXT+CAP -Tub DTX, 40 M - + - + - + - + CAP, 80 M - - + + - - + + DORSO, 5 M - - - - + + + + b siC siAMPK 1.2 siC siAMPK AMPK pAKT/AKT 1.0 * * # pmTOR/mTOR pAKT # # 0.8 values * * # AKT 0.6 * * pmTOR 0.4 ‡ ‡ * 0.2 * * * mTOR Densitometric 0.0 -Tubulin

DTX,40 M - + - + - + - + CAP, 80 M - - + + - - + + c siC siLKB1

LKB1 siC siLKB1

1.6 pAKT/AKT pAMPK # pmTOR/mTOR # 1.2 AMPK # values 0.8 * pACC * * ‡ # ‡ * * 0.4 * * ACC ‡ ‡ *

Densitometric * pAKT 0.0

pmTOR

-Tub DTX, 40 M - + - + - + - + CAP, 80 M - - + + - - + + Sánchez et al. Cancer Cell Int (2019) 19:54 Page 12 of 14

a Control 1,000 240 LNCaP Control PC3 DTX DTXDXT 220 CAP CAP DTX+CAP 800 DXT+CAP 200

180 600 160

140 * *

Tumor volume (%) * Tumor volume (%) 400 * * 120 * * 100 200 * * * * * * * * * 80 *

00 00 123456789101112 123456789101112131415 Days Days 100

1000 80

60 500 weight (mg) 40 * * weight (mg)

20 * * Tumor Tumor 0 0 Control DTX CAP DTX+CAP Control DTX CAP DTX+CAP b

pLKB1 LKB1

pAMPK

AMPK

pACC

ACC

pAkt

Akt

pmTOR

mTOR

-Tubulin DTX - - + + - - + + CAP - - - - + + + + Fig. 6 In vivo anti-tumor activity of Docetaxel and Capsaicin combination. Athymic nude mice were injected s.c. in the right fank with LNCaP or PC3 cells. When tumors reached a 70 mm3 size, mice were daily treated by i.p. injection with vehicle (control), 10 mg/kg Docetaxel (DTX), 2 mg/ kg Capsaicin (CAP) or both compounds (DTX CAP). Tumor volumes were measured daily. a LNCaP tumor growth curve (left) or PC3 tumor + growth curve (right) after administration of vehicle (diamonds), DTX (circles), CAP (squares) or both compounds (triangles). Results represent the mean SEM of six mice in each group. *p < 0.05 signifcant diference between treated and control mice by two-way ANOVA and Tukey’s multiple ± comparisons test. Below is shown the tumor weight at the end of the treatment. b Immunoblot analysis of the regulatory signalling pathways in the PC3 dissected tumors Sánchez et al. Cancer Cell Int (2019) 19:54 Page 13 of 14

to published data regarding capsaicin bioavailability reading of the manuscript. NRH contributed with design of methodology. All authors read and approved the fnal manuscript. and absorption [31], for in vivo studies we used a dose of capsaicin equivalent to that used with cells (consider- Author details 1 ing a mice blood volume of 2.5 ml and an average mice Department of Systems Biology, Biochemistry and Molecular Biology Unit, School of Medicine and Health Sciences, Alcala University, Alcalá de Henares, weight of 30 g, 80 µM is equivalent to 2 mg/kg). On the Ctra A‑2 Km 32., 28871 Madrid, Spain. 2 Chemical Research Institute “Andrés M. other hand, DTX has low bioavailability mainly due to its del Río” (IQAR), Alcalá University, Alcalá de Henares, 28871 Madrid, Spain. poor aqueous solubility and its transportation in blood Acknowledgements by binding to plasma proteins such as lipoproteins, albu- Authors would like to thank J.M. Orellana for technical assistance in animal min and α1 acid glycoprotein. Terefore, in vivo doses of welfare. docetaxel are usually higher than that used in cells. Tus, Competing interests we choose a docetaxel dose of 10 mg/kg which is a com- The authors declare that they have no competing interests. mon used dose in the in vivo studies [32–34]. In LNCaP tumors CAP or DTX singly administered or in combina- Availability of data and materials The dataset supporting the conclusions of this article is available in the Men‑ tion, had little efect on tumor growth. However, in PC3 deley repository. https​://doi.org/10.17632​/48pfm​wbcb5​. tumors, DTX and CAP signifcantly decreased tumor growth and the DTX CAP combination had stronger Consent for publication + Not applicable. anti-tumor activity that either compound singly admin- istered. Co-treatment induced a robust AMPK activa- Ethics approval and consent to participate tion and Akt/mTOR axe inhibition in the PC3 prostate Animal experiments have followed the ARRIVE guidelines and have been carried out in accordance with the U.K. Animals (Scientifc Procedures) Act, tumors. Terefore, we demonstrated that the proposed 1986 and associated guidelines, EU Directive 2010/63/EU for animal experi‑ combination of docetaxel and capsaicin potently inhib- ments. The procedure was approved by Alcalá University Ethical Commission ited the growth of castration resistant prostate cancer and by the Ethical Commission of the Comunidad de Madrid (procedure PROEX 241/15). All animal studies were conducted in accordance with the cells in vitro and in vivo. Spanish institutional regulation (RD 53/2013) for the housing, care and use of experimental animals and met the European Community directives regulating Conclusion animal research. Recommendations made by the United Kingdom coordinat‑ ing Committee on Cancer Research (UKCCCR) have been kept carefully. To In conclusion, these fndings indicate that docetaxel assess the welfare of animals a panel of 10 indicators were recorded each day. and capsaicin co-administration represents a thera- When adverse efects, pain or distress were appreciated in the animals (score peutically relevant strategy to improve docetaxel of 15 out of 40) the humane endpoint was applied. chemotherapy in prostate cancer patients. Te fact that Funding AMPK activation is in the underpinning mechanism The authors would like to thank Fundación Tatiana Pérez de Guzmán (Grant that sensitizes prostate cells to docetaxel suggests that No. Patrocinio 2013-001 and Grant Patrocinio 2019-001) for fnancial support into their research. impact metabolism could be a new option to modulate chemotherapeutic drugs efect. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub‑ lished maps and institutional afliations. Additional fles Received: 29 October 2018 Accepted: 28 February 2019

Additional fle 1: Figure S1. Isobologram and combination index (CI) of the combined treatment (DTX CAP) inhibitory efect on LNCaP and PC3 xenograft tumor growth. + Additional fle 2: Table S1. LNCaP xenografts-wearing mice weights References during the treatment. 1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2017. CA Cancer J Clin. 2017;67(1):7–30. Additional fle 3: Table S2. PC3 xenografts-wearing mice weighs during 2. Torre LA, Siegel RL, Ward EM, Jemal A. Global cancer incidence and the treatment. mortality rates and trends–an update. Cancer Epidemiol Biomarkers Prev. 2016;25(1):16–27. 3. Ganju A, Yallapu MM, Khan S, Behrman SW, Chauhan SC, Jaggi M. Nano‑ Abbreviations ways to overcome docetaxel resistance in prostate cancer. Drug Resist ACC​: acetyl CoA carboxylase; AMPK: AMP-activated kinase; CI: combination Updat. 2014;17(1–2):13–23. index; CAP: capsaicin; DORSO: dorsomorphin; DTX: docetaxel; LKB1: liver 4. Kroon J, Kooijman S, Cho NJ, Storm G, van der Pluijm G. Improving kinase B. taxane-based chemotherapy in castration-resistant prostate cancer. Trends Pharmacol Sci. 2016;37(6):451–62. Authors’ contributions 5. Bayat Mokhtari R, Homayouni TS, Baluch N, Morgatskaya E, Kumar S, IDL conceived and supervised the study, wrote the manuscript, and secured Das B, Yeger H. Combination therapy in combating cancer. Oncotarget. funding. BSG and AB designed and performed experiments. BSG, validate and 2017;8(23):38022–43. performed formal analysis of data. PMG contributed with design of meth‑ 6. Diaz-Laviada I, Rodriguez-Henche N. The potential antitumor efects of odology, acquisition, analysis, interpretation of data for the work and critical capsaicin. Prog Drug Res. 2014;68:181–208. Sánchez et al. Cancer Cell Int (2019) 19:54 Page 14 of 14

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