Lycopene enhances the anti-proliferative and pro-apoptotic effects of capsaicin in prostate cancer in vitro

Background: Increasing evidence suggests dietary factors influence the development and progression of prostate cancer (PCa). The chemopreventive properties of lycopene, the antioxidant found in tomatoes, have been extensively studied for PCa; however, the effect of lycopene, as a single agent, remains unclear. Studies that are more promising, involve using lycopene in combination with other dietary agents. Capsaicin, the active compound in chilli peppers, is reported to exert potent anti-cancer effects in both in vitro and in vivo PCa models. We have investigated whether lycopene enhances the anti-proliferative and apoptotic effects of capsaicin in vitro. Methods: Using the MTS Cell Proliferation Assay we assessed the anti-proliferative effect of capsaicin alone, or in combination with lycopene in human PCa cell lines (LNCaP, PC3, PC3-AR2, DU145) and prostate stromal cells. Flow cytometry and Western Blotting techniques were used to assess potential mechanisms of interaction. Results: Capsaicin exhibited anti-proliferative and pro-apoptotic effects, which were significantly enhanced with the addition of lycopene to PCa cells. The greatest effects were observed in androgen-sensitive cell lines. Detailed mechanistic studies revealed this combination may be interrupting the androgen-signaling pathways; independent of TRP-V1 signaling. Cell cycle analysis revealed that capsaicin and lycopene induce a cell cycle arrest, corresponding to alterations in tumor-suppressor proteins. Treating LNCaP cells with capsaicin and lycopene also altered proteins involved the apoptosis signaling pathway including, cleaved PARP, Caspase-3 and Bax/Bcl-xL expression. Conclusions: Our results suggest that lycopene enhances the anti-cancer properties of capsaicin. Understanding micronutrient interactions may help improve current chemoprevention strategies.


Introduction
Prostate cancer remains the most commonly diagnosed cancer in men living in the Western world [1]. There is increasing evidence that dietary factors play a role in the development and progression of prostate cancer. It is estimated that at least 30% of all prostate cancer patients use complementary and alternative medicine, which includes the consumption of micronutrient supplements [2]. Many dietary agents have been studied for the protective effects on prostate cancer [3,4]. Although a clear understanding of how these micronutrients interact with each other is largely unknown, many micronutrient supplements often contain a variety of dietary agents. Hence, studies examining the interaction of these dietary agents are essential for more effective chemopreventive regimens. Lycopene, a potent antioxidant and carotenoid found in high concentrations in tomatoes, is one of the most extensively studied micronutrients for the chemoprevention of prostate cancer [5,6]. Despite numerous studies, the protective effect of lycopene, as a single agent, on prostate cancer remains unclear [7]. In 2004, Hantz et al. [8] reported that lycopene induced apoptosis in androgen-sensitive prostate cancer cells, mediated through the disruption of the mitochondrial membrane and the generation of reactive oxygen species (ROS). Subsequent studies however have been unable to successfully replicate these findings [7]. More recent studies suggest the protective effects of lycopene to be most apparent in combination studies. Recently, our group has found that combining lycopene with vitamin E and selenium resulted in significant inhibition of the growth and development of prostate tumours in the Lady transgenic mouse model compared to vitamin E and selenium alone, demonstrating that lycopene was necessary for effective combination regimens [9]. Lycopene has also been found to enhance the effect of other therapies [10].
Capsaicin has recently emerged as a potent anti-cancer agent, exhibiting anti-proliferative and pro-apoptotic properties in several different prostate cancer model systems [11]. It is widely consumed as a food additive throughout the world, particularly in areas with a low incidence of prostate cancer, such as, South East Asia. The use of capsaicin in vitro has been reported to induce apoptosis through the generation of ROS, dissipation of the mitochondrial inner transmembrane potential and downstream activation of the caspase-3 cascade [12][13][14][15][16]. It also demonstrates efficacy in vivo by suppressing the growth of prostatic tumour xenografts in nude mice [13]. A recent case study reported by Jankovic et al., [17] describes a 66-year-old patient with prostate cancer who experienced significant improvement in his PSA levels upon daily consumption of capsaicin. Cessation of the capsaicin was followed by a sharp increase in his PSA levels (challenge/re-challenge effect) and PSA trends suggesting a dose-response relationship [17].
In our present study, we have investigated for the first time, whether lycopene enhances the effect of capsaicin using an in vitro prostate cancer model. Through proliferation studies, flow cytometry and protein analysis techniques, we have found that the anti-proliferative and pro-apoptotic effects of capsaicin are greater when it is combined with lycopene. Our in vitro analysis suggests that these anti-proliferative effects may be mediated through androgen-signalling pathways, independent of the capsaicin receptor, TRP-V1.

Chemicals
Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide) was obtained from Tocris (Bristol, UK) and Capsazepine (CZP) was obtained from Santa Cruz Biotechnology (CA, USA). Both capsaicin and CZP were dissolved in dimethyl sulfoxide (DMSO, Sigma, USA) to create a stock concentration of 0.1M and stored at -20°C. Lycopene was obtained from Sigma (USA) and was dissolved in tetrahydrofuran (THF) to create a 0.01M stock solution. Lycopene was stored in a dark area at -80°C. Stock solutions of capsaicin (10-200µM), capsazepine (10µM) and lycopene (10µM) were diluted in appropriate medium and prepared fresh daily prior for treatment. All compounds were prepared and stored with minimal exposure to light to avoid oxidation. All other chemicals were purchased from Sigma unless otherwise specified.

Cell proliferation assay
Proliferation was assessed using the CyQuant MTS cell proliferation assay (Molecular Probes, OR, USA). Cells were plated in 96-well micro-titre plates, at a density of 500 (PrSC) or 5000 (LNCaP, PC3, PC3AR2, DU145) cells per well. Cells were left to adhere for 24 hours and then treated accordingly. Initial standardization was carried out using a range of doses of capsaicin from 0µM-200µM, or lycopene 0µM -100µM (lycopene data not shown). Combination studies were carried out using 100µM concentration of capsaicin, and 10µM of lycopene. For antagonist studies using CZP and/or capsaicin, cells were treated with a range of doses of capsaicin (0-100 µM) or with or without CZP (10 µM); treatments were applied at the same time. All experiments were carried out in triplicates and repeated a minimum of three times. Control wells were treated with vehicle alone (DMSO -0.02% and THF -0.01%). Cells were then incubated for 24 hours post treatment. At the end of the treatment period, 20µL of tetrazolium dye was added to each well and cells incubates for 2 hours. Absorbance was measured using a 96-well plate reader at 490nm.

Flow cytometry
Cell cycle arrest pattern and alterations in the percentage of cells in the S phase enumeration were determined in LNCaP cells by flow cytometry on cells labelled with antibromodeoxyuridine (BrdU) fluorescein isothiocyanate (FITC) and counterstained with propidium iodide (PI

Statistical analysis
Data are presented as the mean ± S.D. of three experiments or more. Statistical comparisons among groups were made with a Student's t test or the one-way analysis of variance (ANOVA) test. Results were considered significant at the 5% critical level (p < 0.05).

Treating cells with a combination of capsaicin and lycopene reduces cell proliferation
To determine whether the anti-proliferative effect of capsaicin could be enhanced by lycopene, we treated cells with capsaicin (100µM) along with a low-dose lycopene (10µM) (Figure 1b-

Capsaicin and lycopene in combination alters the percentage of cells in the S-phase of the cell cycle
To determine the minimal concentration of capsaicin and lycopene that resulted in a significant reduction in the percentage of cells in the S-phase of the cell cycle, flow cytometric analysis was performed on LNCaP cells with a low dose of lycopene (10 µM) and increasing concentrations of capsaicin (0-100 µM). Lycopene treatment alone was not effective at altering the percentage of cells in the S-phase. Low concentrations of capsaicin (0-10 µM) did not reduce the percentage of cells in the S-phase of the cell cycle. At a concentration of 50 µM, the combination of capsaicin and lycopene was effective at reducing the percentage of cells from the S-phase at a 10 µM concentration of lycopene, greater than either agent alone. Similar effects were seen at 100 µM concentration of capsaicin. Concentrations of capsaicin greater than 100µM resulted in a significant reduction in the reduction (p<0.05) in the percentage of cells in the S-phase compared to control. At these concentrations, there were no significant differences between the capsaicin alone or in combination with lycopene. At these concentrations the combination of capsaicin and lycopene reduced percentage of cells in the S-phase greater than either agent alone (Figure 2).

Treatment with capsaicin and lycopene reduce Androgen Receptor (AR) and Prostate Specific Antigen (PSA) expression in LNCaP Cells
To better understand the effect of capsaicin and/or lycopene on androgen-sensitive cells, we examined the expression levels of AR and PSA. Capsaicin treatment alone caused a marked reduction in AR expression in LNCaP cells. Treatment of LNCaP cells with lycopene monotherapy did not affect AR or PSA expression. Interestingly, treating cells with a combination of capsaicin and lycopene resulted in a reduced expression of PSA and AR, greater than either individual

A combination of capsaicin and lycopene promote apoptosis in androgen-sensitive cells
Based on our cell cycle profiles obtained by flow cytometry, we examined proteins implicated in the apoptotic pathway using Western Blot analysis. Lycopene treatment alone, did not significantly alter the apoptotic pathway. We found a time-dependent increase in the expression of cleaved PARP (Poly ADP ribose polymerase) to a greater extent than either agent alone, maximal at 24 hours (Figure 4a). At the 24 hour time point, we found an increase in the BAX:BCL-xL ratio and reduction in total Caspase 3 in androgen-sensitive cell lines, LNCaP and PC3AR2; this effect was not found in the androgen-resistant, PC3, cell line. (Figure 4b). These findings suggest that lycopene is enhancing the pro-apoptotic effect of capsaicin.

The TRP-V1 antagonist, capsazepine (CZP), does not inhibit the anti-proliferative effects of capsaicin
Using the TRP-V1 antagonist, CZP, we assessed whether the anti-proliferative effects of capsaicin could be reversed. We observed no significant changes in proliferation with CZP and capsaicin, indicating that capsaicin's effects were not mediated through the TRP-V1 receptor (data not shown), suggesting an alternative mechanism for proliferation.

Discussion
In our study, we report for the first time that lycopene, a micronutrient well studied for prostate cancer prevention, enhances the anti-proliferative and pro-apoptotic effects of capsaicin in prostate cancer cells. Previous in vivo reports published by our group, have shown that lycopene in combination with antioxidants can significantly reduce the incidence and progression of prostate tumors in the Lady transgenic mouse model [9]. Lycopene has also been described as a necessary component of combinatorial studies. Recently, Tang et al., have reported the enhancing effect of lycopene in combination with other chemotherapeutic agents, including docetaxel [10]. The lack of consensus in the literature about the antiproliferative effects of lycopene as a single agent has yet to be resolved. Few studies report that lycopene reduces cell proliferation in vitro; however, several others have been unable to successfully translate these findings in related preclinical/clinical model systems [5,7]. In our present study, we have demonstrated that lycopene, as a single agent, does not have the potential to reduce the proliferation in several prostate cancer cell lines, except in the androgensensitive, LNCaP cells. This differential effect of lycopene on androgen-sensitive cell lines has been previously described [18]. Cell cycle distribution analysis on LNCaP cells revealed that treatment with lycopene resulted in a non-significant reduction of the percentage of cells in the S-phase. However, when combined with capsaicin, this reduction in the percentage of cells in the S-phase was significantly greater than either monotherapy, indicating that cell-cycle arrest induced by lycopene may augment the anti-proliferative effects induced by capsaicin. Alterations in protein expression, particularly a reduction in PCNA and the up-regulation of the tumour suppressor proteins, p21 cip1 and p27 kip1 support these findings. Suggesting that the inhibition of the cell cycle is one possible way in which lycopene is making cell more susceptible to the inhibitory effects of capsaicin. Reduction of p21 cip1 and p27 kip1 , by the combination of capsaicin and lycopene suggest that alternative anti-proliferative mechanisms and/or apoptotic mechanisms may play a role in understanding how these micronutrients are acting in combination [19].
Androgen signalling plays a key role in prostate cancer growth and development [20]. Some of the most promising chemopreventive agents for prostate cancer include the 5-alpha reductase inhibitors, which block the conversion of testosterone to the more potent dihydrotestosterone (DHT), thereby inhibiting prostate cancer growth [21]. The inhibition or activation of the AR causes alterations in the growth and proliferation of prostate cancer cells [22]. Our findings suggest that capsaicin and lycopene may reduce proliferation through the inhibition of androgen signalling. The effect of capsaicin on androgen signalling has been previously reported [12,13]. Studies carried out by Mori et al. [12], have found that capsaicin can cause a significant down-regulation in the expression of androgen-receptor and PSA in androgen-sensitive cells. In our study, we have successfully replicated these findings and demonstrated that We have compared both androgen-sensitive andinsensitive cell lines and determined androgen-sensitive cells are most responsive to the anti-proliferative effects of capsaicin and lycopene. In particular, we have compared the PC3 and PC3 AR2 (PC3 AR2 cells are PC3 cells transfected with a full length AR) cell lines. We demonstrated that the incorporation of a functional AR to PC3 cells causes cells to have an increased sensitivity to the anti-proliferative effects of capsaicin and lycopene, comparable to the LNCaP cell line, suggesting that a functional AR is an important determinant for inhibition of prostate cancer cell proliferation through the use of capsaicin and/or lycopene. Secondly, we have demonstrated that capsaicin and lycopene can reduce protein expression levels of AR and PSA. We have observed that treatment with a combination of lycopene and capsaicin reduces the expression of AR, this effect being significantly greater than either agent alone, indicating that these compounds may be acting through the androgen-signalling pathway. It is likely that the inhibition of PSA is mediated at least partly through the down-regulation of the AR. Whether the down-regulation of PSA is enhanced by lycopene, is not clear from our studies. Although the combination of capsaicin and lycopene enhance the down-regulation of AR, does not further reduce PSA, suggesting that PSA may be mediated through an androgen-independent manner.
The role of the transient receptor vanilloid-1 receptor (TRP-V1), commonly referred to as the capsaicin receptor, in prostate cancer chemoprevention has not been clearly elucidated. In our studies, we have found that the capsaicin antagonist, capsazepine, does not reverse the antiproliferative effects of capsaicin. Furthermore, there is not significant difference in the expression of this receptor, suggesting that the effects of capsaicin are not TRP-V1 dependent.
Accumulating data clearly indicate that the induction of apoptosis is an important event for the chemoprevention of prostate cancer by naturally occurring dietary agents [23]. Capsaicin has been reported to induce apoptosis through a number of pathways in prostate cancer cells [12][13][14][15][16]. In our present studies, we have found that the apoptotic and anti-proliferative effects of capsaicin are enhanced by treatment with lycopene. Several studies have found that capsaicin alone, can up-regulate the generation of ROS and induce apoptosis. We demonstrate that a combination of capsaicin and lycopene induce apoptosis through alterations in the Bax:Bcl-xl ratio with activation of the caspase-3 cascade in androgen sensitive cell lines, and not the androgen-insensitive cell lines figure 5. It is likely that these effects are downstream of ROS generation and mitochondrial destabilization, which have been previously reported in studies using capsaicin and lycopene independently in vitro [8,16].
The dose of capsaicin and lycopene used in our studies were selected based on dose-standardization studies, and previously reported studies [13,14]. These concentrations do not reflect the physiological concentration of capsaicin, a major caveat of our study. There is a lack of data detailing the physiological serum concentration of capsaicin and amount of capsaicin in the prostate tissue; however, we are currently investigating whether these concentrations are physiologically relevant using pre-clinical models. Based on our in vitro findings, the effect of capsaicin should be examined in androgen-sensitive xenograft / transgenic model. Previous studies have found that capsaicin alone can reduce the growth of androgen-resistant, PC-3, tumors. Replicating these findings using an androgensensitive model (i.e. LNCaP) with the administration of a combination of capsaicin and lycopene will provide a better understanding of this potential novel therapeutic strategy against AR-dependent prostate cancer.

Conclusions
In summary, combination studies using dietary agents are promising new approaches to developing effective chemoprevention strategies. Decoding the exact mechanism of action of each agent alone and in combination is the key to a better understanding of the effect of micronutrients on prostate cancer. In our study, we have described for the first time, the chemopreventive interaction of capsaicin and lycopene, two promising dietary agents. Through understanding relationships of these dietary agents, we can postulate the anti-cancer effects of existing widely consumed supplements and aid in the development of supplements that can prevent or even delay the progression of prostate cancer.