B2G-2

The present study aimed to determine whether grape seed extract (GSE) procyanidin mix, and its active constituent procyanidin B2 3,3″‐di‐O‐gallate (B2G2) have the potential to target cancer stem cells (CSCs) in prostate cancer (PCa). The CSC populations were isolated and purified based on CD44+‐α2β1high surface markers in PCa cell lines LNCaP, C4‐2B, 22Rv1, PC3, and DU145, and then subjected to prostasphere formation assays in the absence or presence of GSE or B2G2. Results indicated that at lower doses (<15 μg) , the GSE procyanidin mix produced activity in unsorted prostate cancer antigen (PCA) cells, but not in sorted; however, multiple treatments with low dose GSE over a course of time inhibited sphere formation by sorted PCA CSCs. Importantly, B2G2 demonstrated significant potential to target both unsorted and sorted CSCs at lower doses. As formation of spheroids, under specific in vitro conditions, is a measure of stemness, these results indicated the potential of both GSE and B2G2 to target the self‐renewal of CSC in PCa cell lines, though B2G2 was more potent in its efficacy. Subsequent mechanistic studies revealed that both GSE procyanidins and B2G2 strongly decreased the constitutive as well as Jagged1 (Notch1 ligand)‐induced activated Notch1 pathway. In totality, these in vitro studies warrant extensive dose‐profiling‐based assessments in vivo settings to conclusively determine the impact on CSC pool kinetics on the efficacy of both GSE and B2G2 to target PCa growth as well as tumor relapse.

Neoplastic cells exhibit higher oxidative stress compared to normal cells; however, antioxidants based clinical trials have mostly failed. Another attractive therapeutic approach is to further increase the oxidative stress in cancer cells leading to cell death. Herein, we show that Procyanidin B2 3,3″‐di‐O‐gallate (B2G2), the most active constituent of grape seed extract, treatment causes cell death in human prostate cancer (PCa) cells (LNCaP and 22Rv1) via increasing the reactive oxygen species (ROS) generation. Mechanistically, B2G2 treatment decreased the mitochondrial electron transport chain complex III activity leading to enhanced mitochondrial superoxide generation and decreased ATP production in LNCaP cells. Additional molecular studies revealed that B2G2‐induced cell death was mediated mainly through ROS‐induced sustained activation of ERK1/2, which was due to inhibition of MAP kinase phosphatase (MKP) activity as over‐expression of MKP3 in LNCaP cells conferred significant protection against B2G2‐induced cell death. Along with ERK1/2, AMP‐activated protein kinase α (AMPKα) was also activated by B2G2 treatment, and pre‐treatment with AMPKα inhibitor compound C significantly reversed the cytotoxic effects of B2G2 in LNCaP cells. Furthermore, pre‐treatment of MKP3 over‐expressing LNCaP cells with compound C further reduced the B2G2‐induced cell death, suggesting the involvement of AMPKα along with MKP3 and ERK1/2 in the biological effects of B2G2. Together, these results for the first time identified that oxidative stress and MKP3 inhibition play a critical role in B2G2‐induced cell death in PCa cells through sustained activation of both ERK1/2 and AMPKα. These results offer a unique opportunity to control this deadly malignancy through B2G2 use.