Since inappropriate regulation of NF-κB/p65/RelA correlates with prostate cancer progression, therefore, as a first step to determine the constitutive activation of NF-κB, we analyzed the expression of NF-κB/p65 and IκBα in various human prostate cancer cells. As shown in , protein expression of NF-κB/p65 and IκBα was higher in androgen-refractory human prostate cancer DU145 and PC-3 cells compared to androgen-responsive LNCaP cells. Since NF-κB activation requires IκBα phosphorylation at Ser32/36 followed by ubiquitination and degradation, we determined the phosphorylated levels of IκBα in LNCaP, DU145 and PC-3 cells. IκBα is more highly phosphorylated in androgen-refractory DU145 and PC-3 cells than in androgen-responsive LNCaP cells. A modest decrease in the levels of IKKα was observed in DU145 and PC-3 cells compared to LNCaP cells (). Next we determined DNA binding for NF-κB/p65, which was higher in DU145 and PC-3 cells compared to LNCaP cells (). It is well recognized that degradation of phosphorylated IκB by the 26 S proteasome regulates the DNA binding of NF-κB subunits therefore; next we used MG132, a pharmacological inhibitor of proteasome activity, to determine the effect of treatment in these cells. Previous study has demonstrated that use of proteasomal inhibitor MG132 blocks degradation of phosphorylated IκBα [30
]. Exposure of LNCaP, DU145 and PC-3 cells to 20 μM MG132 for 6 and 12 h decreased the rate of IκBα phosphorylation, similar to published observations (). Exposure of cells to MG132 for 12 h resulted in accumulation of phosphorylated IκBα protein in all three cell lines; however, the rate of p-IκBα accumulation was faster in PC-3 cells whereas the final amount of the p-IκBα protein was higher in DU145 and PC-3 cells compared to LNCaP cells, as evidenced by time-dependent studies (). MG132 treatment significantly inhibited the DNA binding activity of NF-κB/p65 at 12 h in all three prostate cancer cell lines (). Based on the rapid turnover of IκBα in PC-3 cells we considered this cell line for further studies with BetA.
Constitutive NF-κB/p65 activation in human prostate cancer LNCaP, DU145 and PC-3 cells
Our next aim was to investigate whether BetA treatment imparts anti-proliferative effects against androgen-refractory human prostate carcinoma PC-3 cells. BetA is a naturally occurring pentacyclic triterpenoid which has shown to possess anti-viral, anti-inflammatory and anticancer properties (). Exposure of PC-3 cells to 20 μM concentration of BetA for 24 h resulted in loss of adhesion to substrate, and altered morphology, consistent with apoptosis (). Furthermore, BetA treatment (1 to 40 μM) resulted in a dose-dependent inhibition of cell growth, as compared to vehicle-treated controls. Exposure to BetA at the highest doses of 40 μM resulted in 91.2% to 96.7% decrease in cell viability between 24 to 72 h. BetA treatment for 24 h resulted in dose-dependent reduction in cell viability which ranged from 2.9% to 91.2% in PC-3 cells at 1 to 40 μM concentrations. BetA treatment also resulted in time-dependent inhibition of cell growth. This effect was more pronounced at 48 and 72 h post-treatment with BetA (). We next investigated whether BetA-mediated loss of cell viability in human prostate cancer PC-3 cells is a result of apoptosis. To demonstrate these effects we performed cell death detection by ELISA. Compared to vehicle treated controls, 1.4, 2.5 and 4.8 fold increases in induction of apoptosis were observed with BetA in PC-3 cells, following 24 h of treatment (). Since BetA treatment to PC-3 cells resulted in decreased cell survival and induction of apoptosis therefore, next we studied its effect on NF-κB signaling.
Effect of Betulinic acid on cell survival and apoptosis in human prostate carcinoma PC-3 cells
To examine the effect of BetA on NF-κB constitutive activation, PC-3 cells were treated with 10 and 20 μM BetA for 12, 24 and 48 h. As shown in , BetA treated cells showed exclusive cytoplasmic accumulation of NF-κB/p65 protein inhibiting its translocation into the nucleus in time and dose dependent manner. This effect correlated with significant dose and time dependent increase in the levels of total IκBα. To further determine whether inhibition of BetA on constitutive activation of NF-κB was caused by inhibition of IκBα degradation, we examined the cells for p-IκBα status in the cytoplasm. The level of serine 32-phosphorylated IκBα (p-IκBα) exhibited a dose and time dependent decrease in the phospho-protein expression after BetA treatment. Because phosphorylation of IκBα is mediated by IKKs, we next determined whether the inhibitory effect of BetA on IκBα phosphorylation is mediated via IKKα. BetA treatment markedly decreased the protein level of IKKα over 48 h. This finding raised the possibility that the primary target of BetA in the regulation of the IκB/NF-κB pathway might be regulated via IKKα. Furthermore, we examined the effect of BetA on DNA binding activities of NF-κB by EMSA assay in nuclear extracts of PC-3 cells. Treatment with 20 μM BetA for 24 h resulted in a decrease of NF-κB DNA binding (). Taken together, these experiments demonstrate that BetA inhibits constitutive activation of NF-κB in human prostate cancer PC-3 cells.
Effect of Betulinic acid on NF-κB signaling pathway in human prostate carcinoma PC-3cells
Since BetA prevents the entry of NF-κB/p65 into the nucleus, it is plausible that it might induce alterations in survival factors such as Bcl-2; therefore we next studied the time and dose dependent effects of BetA on the constitutive protein levels of Bax and Bcl-2 in PC-3 cells. The time-dependent treatment with 10 and 20 μM concentration of BetA resulted in the increase accumulation of Bax protein, which was observed up to 48 h post-treatment compared to the basal levels. In sharp contrast, the protein expression of Bcl-2 was significantly decreased by BetA treatment in a dose and time dependent fashion (). This resulted in a significant time and dose dependent shift in the ratio of Bax to Bcl-2 after BetA treatment which correlated with the cleavage of poly(ADP)ribose polymerase, indicative of induction of the apoptotic process ().
Effect of Betulinic acid on Bcl-2 family member proteins and apoptosis in human prostate cancer PC-3 cells
Since cytokine stimulation leads to NF-κB activation, we assessed the effect of BetA on TNFα-induced activation of NF-κB. PC-3 cells were serum starved and pretreated with 20 μM BetA for 16 h followed by TNFα (10 ng/ml) treatment for 30 min. Vehicle treated controls and cultures exposed only to TNFα were used as controls. Cytoplasmic and nuclear extracts were prepared from these cultures, and nuclear extracts were analyzed by EMSA for NF-κB DNA binding activity. Compared to vehicle treated control, treatment of cells with TNFα resulted in a significant increase in NF-κB/p65 DNA binding activity. Pre-treatment of cells with BetA significantly reduced NF-κB/p65 DNA binding activity, suggesting that BetA inhibits TNFα-induced NF-κB activation (). Further investigation on cytoplasmic extracts from this experiment, for total IκBα and p-IκBα, revealed that treatment with TNFα resulted in an increase in total IκBα, which was further increased after pretreatment with BetA. However, TNFα-induced p-IκBα levels were strongly inhibited by treatment with BetA ().
Effect of Betulinic acid on TNFα-induced NF-κB activation and apoptosis in human prostate cancer PC-3 cells
Although we have shown by EMSA that BetA blocks NF-κB activation, however, DNA binding does not always correlate with NF-κB-dependent gene transcription, suggesting there are additional regulatory steps. To determine the effect of BetA on TNFα-induced NF-κB-dependent reporter gene expression, we transiently transfected the cells with the NF-κB-regulated reporter construct, incubated them with 20 μM BetA for 16 h, and then stimulated the cells with 10 ng/ml TNFα for 30 min. A 4.4-fold increase in NF-κB-Luc activity over the vector control was observed upon stimulation with TNFα, and BetA completely suppressed the TNFα-induced stimulation (). These results demonstrate that BetA also represses NF-κB-dependent reporter gene expression induced by TNFα.
Since TNFα-induced NF-κB activity is the major factor in PC-3 cells that make them resistant to apoptosis, we tested if BetA is effective in sensitizing these cells to TNFα-induced apoptosis, possibly by virtue of its ability to inhibit NF-κB activity. As observed by annexin V-PI staining, TNFα treatment of PC-3 cells resulted in apoptotic cell death rates that were comparable to those observed in vehicle treated controls; BetA exposure alone resulted in a 33% increase in rate of apoptotic cell death over controls. However, pre-treatment of cells with BetA followed by TNFα treatment resulted in a significant increase (44%; p<0.001) in the percentage of apoptotic cells (). These observations clearly suggest that BetA has the potential to sensitize highly resistant PC-3 cells to TNFα-induced apoptosis and that this effect of BetA may be mediated via inhibition of TNFα-induced NF-κB activation in these cells.