Nicotine Exposure Augments Renal Toxicity of 5-azacytidine Through p66shc: Prevention by Resveratrol

Abstract

Abstract. Background Studies demonstrated that nephrotoxicity is a frequent side-effect of various anticancer agents that hampers their clinical use (1). Previously we reported that the anticancer agent 5-aza-cytidine (AZA) elicits oxidative stress-mediated toxicity in cultured renal proximal tubule cells via transcriptional upregulation of the prooxidant p66shc gene and consequent increase in mitochondrial reactive oxygen species (ROS) release (2). Studies revealed that smoking augments severity and progression of renal diseases in humans (3) and in various animal models (4-6). However, there are no data available on whether smoking could affect renal toxicity of anticancer agents.Smoking exerts its adverse effects via nicotine (NIC)-induced oxidative stress (7). We reported that NIC transcriptionally activates and Serine 36 phosphorylates p66shc that results in its mitochondrial translocation and therein cytochrome c binding and consequent excess ROS production (8) similar to AZA (2). Hence, it is plausible that NIC exposure exacerbates renal toxicity of AZA by further augmenting p66shc expression and resultant ROS production.Resveratrol (RES) is a powerful dietary antioxidant (9) that has been shown to protect the kidney from cigarette smoke-associated injury in rats (10). RES exerts antioxidant effect -at least partly -through activation of the Nrf2/HO-1 axis (11,12). Whether beneficial effect of RES on NIC+AZA-exposed renal cells are due to activation of Nrf2/HO-1 and/or down-regulation of p66shc is unknown.Accordingly, we hypothesized that (i) NIC exposure additively augments AZA-dependent induction of p66shc transcription, thus, exacerbates AZA-associated injury and (ii) RES attenuates NIC+AZA-mediated injury by suppressing p66shc transcription and/or activation of HO-1 in cultured renal proximal tubule cells. Materials and MethodsCell culture and treatment. The rat proximal tubule cell line (NRK52E) was maintained in 5% CO 2 at 37˚C in DMEM with 10% FBS as recommended. A set of cultures was treated with 200 μM nicotine (NIC; Sigma-Aldrich, St. Louis, MO, USA) or 100 nM 5-aza-cytidine (AZA; Sigma-Aldrich) as described earlier in mouse proximal tubule cells (2). Some cultures were co-treated with NIC+AZA. Resveratrol (RES; Selleckchem, Houston, TX, USA) was applied in 20 μM concentration overnight prior to NIC+AZA treatment.Determination of cell injury. Extent of cell injury was determined by LDH release using the fluorescent "Cytotox-One Homogeneous Membrane Integrity" kit (Promega, Madison, WI) as described earlier (13). Briefly, cells grown in 96-well-plates were transfected and treated as described in the appropriate section and 24 h later LDH content of the supernatants were determined and compared to total 4075

Role of adaptor protein p66Shc in renal pathologies

“…Whereas previous reports have indicated links of nicotine exposure to increased renal damage via p66Shc-mediated oxidative stress (1,45), this study demonstrated further increased damage induced to the renal tubules due to the combination of 5AZA and nicotine. These data suggest both a mechanism to a phenomenon that has been plaguing physicians trying to treat cancer patients (39,83) and also proposes that the antioxidant resveratrol could be used a therapeutic treatment to decrease the p66Shc-induced oxidative stress induced both by the anti-cancer treatment as well as nicotine exposure (6).…”

“…It has been observed that the epigenetic modifying anticancer drugs trichostatin A (TSA) and 5-aza-cytidine (5AZA) induce renal tubular injury in part by increasing mitochondrial ROS production (69) and that this ROS corresponds to increased expression and mitochondrial localization of p66Shc (2,107). Interestingly, in a recent study by Arany et al (6), it was observed that nicotine exposure augments the renal toxic effects of 5AZA. Whereas previous reports have indicated links of nicotine exposure to increased renal damage via p66Shc-mediated oxidative stress (1,45), this study demonstrated further increased damage induced to the renal tubules due to the combination of 5AZA and nicotine.…”

Molecular Mechanisms of Oxidative Stress in Acute Kidney Injury: Targeting the Loci by Resveratrol

“…This mostly transpires as the Ser36 phosphorylated p66shc does not translocate to the mitochondria, which indicates its pro-oxidant function; however, it activates the ARE, and, consequently, a set of antioxidant genes that harbor the ARE in their promoter. RSV activates the ARE via Nrf2, which induces the HO-1 promoter and, hence, offers protection to the cell via the cytoplasmic p66shc/Nrf2/HO-1 axis [ 197 ].…”