Cannabinoid receptor agonist induced apoptosis of human prostate cancer cells LNCaP proceeds through activation of MAPKs and G1 cell cycle arrest
Authors: Sami Sarfaraz, Farrukh Afaq, Vaqar M. Adhami, Arshi Malik, Hasan Mukhtar
Journal: Cancer Research (2006)

Proc Amer Assoc Cancer Res, Volume 47, 2006

4616 

Developing novel treatment options for prostate cancer has become an important goal. In recent years cannabinoids and their derivatives are drawing attention because of their diverse pharmacological activities such as cell growth inhibition, anti-inflammatory effects and tumor regression. Recently we have shown that expression levels of both cannabinoid receptors CB1 and CB2 are higher in human prostate cancer cells than in normal prostate epithelial cells and treatment of LNCaP cells with WIN-55,212-2 (a mixed CB1/CB2 agonist) resulted in inhibition of cell growth and induction of apoptosis. Based on these data we suggested that WIN-55,212-2 or other non-habit forming cannabinoid receptor agonists could be developed as novel therapeutic agents for the treatment of prostate cancer (Cancer Res. 65:1635-41, 2005). This study was conducted to understand the mechanistic basis of cannabinoid receptor-agonist induced cell growth inhibition and apoptosis. LNCaP cells were treated with WIN-55,212-2 (1-10 μM) for 24 h after which they were processed for cell cycle analysis, western blot analysis and immunofluorescence. Treatment of cells with WIN-55,212-2 was found to result in i) an arrest of the cells in the G/G1 phase of the cell cycle; ii) up regulation of ERK1/2, JNK1/2, p38; and iii) inhibition of PI3k/Akt pathways. To define the involvement of regulatory proteins operative in the G/G1 phase of the cell cycle, we next determined the effects of WIN-55,212-2 treatment of cells on cyclin kinase inhibitor (cki)-cyclin-cyclin dependent kinase (cdk) machinery. We observed that WIN-55,212-2 (1-10 μM) treatment resulted in i) an induction of p27/KIP1; ii) down-regulation of cyclin D1, D2, E; and iii) decrease in the expression of cdk -2, -4, and -6. Since retinoblastoma (pRb) regulates the progression of the cell cycle by binding to and inhibiting the E2F transcription factors and their heterodimeric partners DP-1 and DP-2, we next determined the effect of WIN-55,212-2 treatment on (pRb)-E2F/DP pathway. Western blot analysis showed a decrease in the protein expression of i) pRb; ii) E2F (1 through 4); and iii) DP1 and DP2. We also observed that WIN-55,212-2 treatment of cells resulted in a dose-dependent increase in Bax/Bcl-2 ratio in such a way that favors apoptosis. The induction of apoptosis proceeded through down regulation of caspases 3, 6, 7, and 9 and cleavage of PARP. Taken together, our data suggests the involvement of two distinct pathways through which WIN-55,212-2 induces apoptosis of LNCaP cells. In the first pathway, activation of ERK1/2 leads to cell cycle dysregulation and arrest and in the second pathway up regulation of Bax/Bcl2 ratio and activation of caspases results in an induction of apoptosis. We reiterate our suggestion that cannabinoid receptor agonists should be considered as novel agents for the management of prostate cancer.