Single agent of proteasome inhibitor resulted in significant responses in leukemic cells and the combination of proteasome inhibitors and other chemotherapeutic drugs enhanced its antitumoral efficacy [3, 33–37]. Initially, the experiments were planned to test whether resveratrol could sensitized K562 cells to the anticancer actions of proteasome inhibitors. To our surprise, resveratrol did not promote, but rather attenuated the apoptotic effects of MG132 in cultured K562 cells. We further extended our investigation using a panel of leukemic cells and found that resveratrol also attenuated the cytotoxic actions of MG132 in NB4, U937, Raji and Daudi cells. Furthermore, resveratrol also compromised the apoptotic effects of other three structurally different proteasome inhibitors, PSI, epoxomicin and lactacystin. This was consistent with the previous study that resveratrol exerted its protective effects against proteasome inhibitor-induced cellular damages in human skeletal myotubes . Consistent with our previous report, in the current study, we found that resveratrol per se did not cause obvious apoptosis when less than 100 μM concentration was used within 24 h. Chakraborty PK et al. reported that treatment with 40 μM resveratrol for 48 h induced apoptosis of K562 cells . The different effects of resveratrol on apoptosis of K562 cells might be ascribed to different period of exposure. Alternatively, Chakraborty PK et al. used subG1 fractions represented as apoptotic cells , while in the current study, we used Annexin V/PI double staining followed by flow cytometry to detect apoptotic cells. The different methods used in these studies might contribute to the different apoptotic actions of resveratrol. The higher cytoprotective effect of resveratrol on cytotoxic actions of proteasome inhibitors was observed when it was used at 5-20 μM concentration. We observed that 50-100 μM resveratrol was slightly cytotoxic for K562 cells, which could explain why this concentration exerted a lower cytoprotective action compared with 20 μM resveratrol. Even this, when cells were concurrently incubated with 100 μM of resveratrol, the apoptosis observed after exposure to MG132 was significantly lower than the one observed in the cells exposed to MG132 alone, indicating that even when 100 μM resveratrol could induce a certain degree of cytotoxicity in these cells, at the same time exerted a cytoprotective action against cytotoxicity-mediated by proteasome inhibition. Resveratrol was reported to be abundant in grapes, blueberries and peanuts. In grapes, its highest concentration was in the skin (50-100 μg per gram), thereby making red wines (but not white wines) the richest dietary source . In plasma, it bound with lipoproteins and albumin which facilitated its carrier-mediated cellular uptake . In experimental animals, resveratrol was rapidly metabolized by the liver and its plasma half-life remained quite low , however, in human, about 70% of orally administered resveratrol (25 mg) was absorbed with a peak plasma level of ~2 μM and a half-life of ~10 h . In the current study, we found that 5 μM of resveratrol could antagonize the cytotoxic effects of proteasome inhibitors. Therefore, concurrent intake of resveratrol products should be discreet.
Arrest at G1/S transition appeared to be a general property of cells that switched to a nonproliferative phenotype [16, 17, 44]. Compared with nonproliferating, quiescent cells, proliferating cells were much more sensitive to cytotoxicity induced by proteasome inhibitors [14, 15]. In the current study, we found that combination of resveratrol and MG132 significantly increased proportion of cells in G1 fraction, therefore, protective effects of resveratrol against proteasome inhibition might be the result of blocking cell cycle progression at the G1/S transition and thus preventing the cells from proliferation.
Proteasome inhibitor-induced apoptosis generally was accompanied by the accumulation of p27Kip1, a universal CDK-cyclin inhibitor responsible for cell cycle arrest at G1/S transition . A rather broad spectrum of effects were ascribed to elevated levels of p27Kip1 protein ranging from proapoptotic functions in various systems to survival-promoting properties in others. Conflicting observations were also reported regarding the role of p27Kip1 in apoptosis induced by proteasome inhibitors. As overexpression of p27Kip1 in various tumor cell lines was sufficient to induce apoptosis in various cancer cell lines [46, 47], it had therefore been deduced that cytotoxicity induced by proteasome inhibitors could be due to the uncoordinated upregulation of p27Kip1 [45, 48, 49]. These pro-apoptotic properties were also consistent with the notion that p27Kip1 exerted the task of a tumor suppressor gene. In contrast to these observations, the cytotoxic effects of proteasome inhibitors in general appeared to be selective for proliferating cells, but quiescent cells generally with high levels of p27Kip1 in nucleus seemed to be protected [14, 15]. For example, primary endothelial cells which became contact inhibited upon reaching confluence displayed a remarkable degree of resistance against apoptosis induced by proteasome inhibitors in the presence of increased steady state levels of p27Kip1, when compared with their proliferating counterparts . Similar observations were also observed in different cancer cell lines engineered to overexpress p27Kip1 [50–52]. Likewise, inducible overexpression of p27Kip1 protected K562 cells against induction of apoptosis by proteasome inhibitors . Since proliferation and differentiation were usually mutually exclusive, it was not surprised that cell cycle arrest at G1/S transition and p27Kip1 was also involved in the differentiation of erythroid precursors [53, 54]. Thus, induction of cell differentiation via accumulation of p27Kip1 and G1/S arrest might also contribute to the protective roles of resveratrol against proteasome inhibition-mediated cytotoxicity.
A major consequence of the anti-apoptotic properties of p27Kip1 appeared that high levels of p27Kip1 in tumor cells might not be always good news for cancer patients: high levels of active p27Kip1 within tumor cells might indicate that although less aggressive and more slowly growing, this tumor might be more difficult to be attacked by treatment with proteasome inhibitors or other chemotherapeutic drugs.
In conclusion, the present study demonstrated that resveratrol had the potential to negate the therapeutic efficacy of proteasome inhibitors in leukemic cells and suggested that intake of resveratrol-related products might be contraindicated for patients undergoing treatment with proteasome inhibitors. Considering the widespread use of resveratrol among cancer patients, further investigations should be necessary to elucidate the in vivo significance of these findings, which in turn might inform the need for dietary advice on the consumption of resveratrol during chemotherapy with proteasome inhibitors.