Ana María Sánchez

Induction of apoptosis in prostate tumor PC-3 cells and inhibition of xenograft prostate tumor growth by the vanilloid capsaicin

Capsaicin, the pungent ingredient of hot chilli pepper, has been recently shown to induce apoptosis in several cell lines through a not well known mechanism. Here, we investigated the role of the vanilloid capsaicin in the death regulation of the human cancer androgen-resistant cell line PC-3. Capsaicin inhibited the growth of PC-3 with an IC50 of 20 μM cells and induced cell apoptosis, as assessed by flow cytometry and nuclei staining with DAPI. Capsaicin induced apoptosis in prostate cells by a mechanism involving reactive oxygen species generation, dissipation of the mitochondrial inner transmembrane potential (ΔΨm) and activation of caspase 3. Capsaicin-induced apoptosis was not reduced by the antagonist capsazepine in a dose range from 0.1 μM to 20 μM, suggesting a receptor-independent mechanism. To study the in vivo effects of capsaicinoids, PC-3 cells were grown as xenografts in nude mice. Subcutaneous injection of either capsaicin or capsazepine (5 mg/kg body weight) in nude mice suppressed PC-3 tumor growth in all tumors investigated and induced apoptosis of tumor cells. Our data show a role for capsaicin against androgen-independent prostate cancer cells in vitro and in vivo and suggest that capsaicin is a promising anti-tumor agent in hormone-refractory prostate cancer, which shows resistance to many chemotherapeutic agents.

Apoptosis induced by capsaicin in prostate PC-3 cells involves ceramide accumulation, neutral sphingomyelinase, and JNK activation

Numerous studies have recently focused on the anticarcinogenic, antimutagenic, or chemopreventive activities of the main pungent component of red pepper, capsaicin (N-vanillyl-8-methyl-1-nonenamide). We have previously shown that, in the androgen-independent prostate cancer PC-3 cells, capsaicin inhibits cell growth and induces apoptosis through reactive oxygen species (ROS) generation [Apoptosis 11 (2006) 89–99]. In the present study, we investigated the signaling pathways involved in the antiproliferative effect of capsaicin. Here, we report that capsaicin apoptotic effect was mediated by ceramide generation which occurred by sphingomyelin hydrolysis. Using siRNA, we demonstrated that N-SMase expression is required for the effect of capsaicin on prostate cell viability. We then investigated the role of MAP kinase cascades, extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK, in the antiproliferative effect of capsaicin, and we confirmed that capsaicin could activate ERK and JNK but not p38 MAPK. Pharmacological inhibition of JNK kinase, as well as inhibition of ROS by the reducing agent N-acetylcysteine, prevented ceramide accumulation and capsaicin-induced cell death. However, inhibition of ceramide accumulation by the SMase inhibitor D609 did not modify JNK activation. These data reveal JNK as an upstream regulator of ceramide production. Capsaicin-promoted activation of ERK was prevented with all the inhibitors tested. We conclude that capsaicin induces apoptosis in PC-3 cells via ROS generation, JNK activation, ceramide accumulation, and second, ERK activation.

Activation of phosphoinositide 3-kinase/PKB pathway by CB1 and CB2 cannabinoid receptors expressed in prostate PC-3 cells. Involvement in Raf-1 stimulation and NGF induction

Cannabinoids exert a variety of physiological and pharmacological responses in humans through interaction with specific cannabinoid receptors. Cannabinoid receptors described to date belong to the seven-transmembrane-domain receptor superfamily and are coupled through the inhibitory G(i) protein to adenylyl cyclase inhibition. However, downstream signal transduction mechanisms triggered by cannabinoids are poorly understood. We examined here the involvement of the phosphoinositide 3-kinase (PI3K)/PKB pathway in the mechanism of action of cannabinoids in human prostate epithelial PC-3 cells. Cannabinoid receptors CB(1) and CB(2) are expressed in these cells, as shown by RT-PCR, Western blot and immunofluorescence techniques. Treatment of PC-3 cells with either Delta(9)-tetrahydrocannabinol (THC), the major psychoactive ingredient of marijuana, or R-(+)-methanandamide (MET), an analogue of the endogenous cannabinoid anandamide, increased phosphorylation of PKB in Thr308 and Ser473. The stimulation of PKB induced by cannabinoids was blocked by the two cannabinoid receptor antagonists, SR 141716 and SR 144528, and by the PI3K inhibitor LY 294002. These results indicate that activation of cannabinoid receptors in PC-3 cells stimulate the PI3K/PKB pathway. We further investigated the involvement of Raf-1/Erk activation in the mechanism of action of cannabinoid receptors. THC and MET induced translocation of Raf-1 to the membrane and phosphorylation of p44/42 Erk kinase, which was reversed by cannabinoid receptor antagonists and PI3K inhibitor. These results point to a sequential connection between cannabinoid receptors/PI3K/PKB pathway and Raf-1/Erk in prostate PC-3 cells. We also show that this pathway is involved in the mechanism of NGF induction exerted by cannabinoids in PC-3 cells.

Induction of the endoplasmic reticulum stress protein GADD153/CHOP by capsaicin in prostate PC-3 cells: A microarray study

The effect of capsaicin, main pungent ingredient of hot chilli peppers, in the gene expression profile of human prostate PC-3 cancer cells has been analyzed using a microarray approach. We identified 10 genes that were down-regulated and five genes that were induced upon capsaicin treatment. The data obtained from microarray analysis were then validated using quantitative real-time PCR assays and Western blot analysis. The most remarkable change was the up-regulation of GADD153/CHOP, an endoplasmic reticulum stress-regulated gene. Activation of GADD153/CHOP protein was corroborated by immunofluorescence and Western blot. We then tested the contribution of GADD153/CHOP to protection against capsaicin-induced cell death using RNA interference. Blockage of GADD153/CHOP expression by small interfering RNA, significantly reduced capsaicin-induced cell death in PC-3 cells. Taken together, these results suggested that capsaicin induces the antiproliferative effect through a mechanism facilitated by ER stress in prostate PC-3 cells.

Spisulosine (ES-285) induces prostate tumor PC-3 and LNCaP cell death by de novo synthesis of ceramide and PKCζ activation

During the past decades, intense attention has been focused on the anti-tumor properties of marine compounds which some of them have been revealed as potent apoptotic inducers. In the present work, we studied the mechanism of action of a new compound, Spisulosine (ES-285), isolated from the sea mollusc Spisula polynyma, in the prostate tumor PC-3 and LNCaP cell lines. Spisulosine inhibited cell proliferation with an IC50 of 1 microM in both cell lines, although it was more effective in the androgen-independent PC-3 cells. The anti-proliferative effect induced by Spisulosine in prostate cells was independent of peroxisome proliferator activated receptor gamma (PPARgamma) and phosphatidylinositol 3-kinase/(PI3K/Akt), Jun N-terminal kinase (JNK), p38 or classical protein kinase C (PKCs) pathways, as it was inferred from the results obtained with specific inhibitors of these routes. However, Spisulosine treatment of prostate cells induced an increase in the intracellular ceramide levels, that was totally blocked by the ceramide synthase inhibitor Fumonisin B1, indicating that the ceramide accumulation came from the de novo biosynthesis. Spisulosine also induced in both PC-3 and LNCaP cells, an activation of the atypical PKC isoform, PKCzeta, which is one of the target proteins of ceramide. These results indicate that the marine compound Spisulosine inhibits the growth of the prostate PC-3 and LNCaP cells through intracellular ceramide accumulation and PKCzeta activation.

Enhancement of androgen receptor expression induced by (R)‐methanandamide in prostate LNCaP cells

It has been recently shown that cannabinoids may regulate the growth of many cell types. In the present work we examined the effect of the anandamide analogue (R)‐methanandamide (MET) on androgen‐dependent prostate LNCaP cell growth. We found that 0.1 μM MET had a mitogenic effect measured by [3H]thymidine incorporation into DNA. The effect exerted by MET was blocked by the cannabinoid receptor antagonists SR141716 (SR1) and SR144528 (SR2) as well as by the phosphoinositide 3‐kinase (PI3K) inhibitor LY294002, suggesting an involvement of cannabinoid receptors and the PI3K pathway in the mechanism of MET action. MET treatment of LNCaP cells also induced an up‐regulation of androgen receptor expression that was blocked by the two cannabinoid receptor antagonists SR1 and SR2. These results show for the first time that cannabinoids may modify androgen receptor expression in an androgen‐dependent cell line and by this mechanism could regulate prostate cell growth.

Characterization of an anandamide degradation system in prostate epithelial PC-3 cells: synthesis of new transporter inhibitors as tools for this study

The response of anandamide is terminated by a carrier‐mediated transport followed by degradation catalyzed by the cloned enzyme fatty acid amidohydrolase (FAAH). In this study, we provide biochemical data showing an anandamide uptake process and the expression of FAAH in human prostate. Anandamide was accumulated in PC‐3 cells by a saturable and temperature‐dependent process. Kinetic studies of anandamide uptake, determined in the presence of cannabinoid and vanilloid antagonists, revealed apparent parameters of KM=4.7±0.2 μM and Vmax=3.3±0.3 pmol min−1 (106 cells)−1. The accumulation of anandamide was moderately inhibited by previously characterized anandamide transporter inhibitors (AM404, UCM707 and VDM11) but was unaffected by inhibitors of other lipid transport systems (phloretin or verapamil) and moderately affected by the FAAH inhibitor methyl arachidonyl fluorophosphonate. The presence of FAAH in human prostate epithelial PC‐3 cells was confirmed by analyzing its expression by Western blot and measuring FAAH activity. To further study the structural requirements of the putative carrier, we synthesized a series of structurally different compounds 1–8 and evaluated their capacity as uptake inhibitors. They showed different inhibitory capacity in PC‐3 cells, with (9Z,12Z)‐N‐(fur‐3‐ylmethyl)octadeca‐9,12‐dienamide (4, UCM119) being the most efficacious, with maximal inhibition and IC50 values of 49% and 11.3±0.5 μM, respectively. In conclusion, PC‐3 cells possess a complete inactivation system for anandamide formed by an uptake process and the enzyme FAAH. These results suggest a possible physiological function of anandamide in the prostate, reinforcing the role of endocannabinoid system as a neuroendocrine modulator.