Control of stability of cyclin D1 by quinone reductase 2 in CWR22Rv1 prostate cancer cells

Abstract

Aberrant expression of cyclin D1, frequently observed in human malignant disorders, has been linked to the control of G(1)→S cell cycle phase transition and development and progression in carcinogenesis. Cyclin D1 level changes are partially controlled by GSK-3β-dependent phosphorylation at threonine-286 (Thr286), which targets cyclin D1 for ubiquitination and proteolytic degradation. In our continuing studies on the mechanism of prostate cancer prevention by resveratrol, focusing on the role of its recently discovered target protein, quinone reductase 2 (NQO2), we generated NQO2 knockdown CWR22Rv1 using short hairpin RNA (shRNA)-mediated gene silencing approach. We found that, compared with cells expressing NQO2 (shRNA08), NQO2 knockdown cells (shRNA25) displayed slower proliferation and G(1) phase cell accumulation. Immunoblot analyses revealed a significant decrease in phosphorylation of retinoblastoma Rb and cyclin D1 in shRNA25 compared with shRNA08. Moreover, shRNA25 cells showed a 37% decrease in chymotrypsin-like proteasome activity. An increase in AKT activity was also observed in shRNA25, supported by a ∼1.5-fold elevation in phosphorylation and ∼50% reduction/deactivation of GSK-3α/β at Ser21/9, which were accompanied by a decrease in phosphorylation of cyclin D1 at T286. NQO2 knockdown cells also showed attenuation of resveratrol-induced downregulation of cyclin D1. Our results indicate a hitherto unreported role of NQO2 in the control of AKT/GSK-3β/cyclin D1 and highlight the involvement of NQO2 in degradation of cyclin D1, as part of mechanism of chemoprevention by resveratrol.

MicroRNA-221 Induces Cell Survival and Cisplatin Resistance through PI3K/Akt Pathway in Human Osteosarcoma

“…Furthermore, expression of p27, which has previously been described as a miR-221 target [20], [21] and as a Akt pathway downstream gene [31], was reduced after miR-221 over-expression in our model system (Figure 5B, row6). However, expression of p57, which was previously identified as a functional target of miR-221 [46], showed little change with miR-221 overexpression and downexpression (Figure 5B, row7). These results further indicate that the PTEN/Akt pathway is a major target of miR-221 and largely mediates miR-221 function.…”

Biochemical and Cellular Evidence Demonstrating AKT-1 as a Binding Partner for Resveratrol Targeting Protein NQO2

“…Based on our observation that NQO2 attenuates AKT activity [19], we hypothesize that resveratrol, alone or in complex with NQO2, may serve as a novel modulator of endogenous AKT by fine tuning the control of AKT activation, activity and function. This hypothesis was evaluated using the UCSF’s DOCK6.3 approach.…”

“…In our previous studies we found that NQO2 is involved in GSK-3β-mediated cyclin D1 degradation as well as in control of AKT activity [19]. In the current study, we have expanded these studies using leads revealed by computer modeling analysis.…”

“…NQO2 is an oxidoreductive enzyme that utilizes dihydronicotinamide riboside (NRH) as a co-substrate for converting quinones to hydroquinones, and is traditionally considered as a phase II detoxification enzyme. NQO2 has been identified by us as a resveratrol target protein [18] and its participation in chemoprevention by resveratrol is supported by our recent studies showing that resveratrol mediates NQO2-dependent cyclin D1 degradation in CWR22Rv1 CaP cells [19]. AKT phosphorylates glycogen synthase kinase 3 (GSK-3α/β) at serine 21/9 (Ser21/9).…”

“…CWR22Rv1 cells containing stably expressed shRNA-mediated NQO2-knockdown were established using procedures described [19]. Single colonies were picked for propagation and expansion.…”