Wutou decoction attenuates rheumatoid arthritis by modulating the Ahr/LOC101928120/SHC1 pathway.

CONTEXT: Wutou decoction (WTD) is a Chinese herbal formula alleviating rheumatoid arthritis (RA). SHC adaptor protein 1 (SHC1) regulates apoptosis, inflammation, and the production of reactive oxygen species (ROS). The LOC101928120 gene is located near the SHC1 gene. Bioinformatics analysis showed that the long non-coding RNA LOC101928120 binds to histone deacetylase HDAC1 that might regulate SHC1 expression. The LOC101928120 gene might be targeted by the transcriptional factor Aryl hydrocarbon receptor (Ahr). OBJECTIVE: This study determines the involvement of the Ahr/LOC101928120/SHC1 pathway in WTD alleviation of RA. MATERIALS AND METHODS: Wistar rats were injected with complete Freund's adjuvant in the hind footpad to construe the RA model. WTD (9.8 g/kg/day) was administered intragastrically for 15 days. The CHON-001 chondrocyte cells were treated with IL-1ß (10 ng/mL) alone or in combination with WTD (1 µg/mL). A RNA pull-down assay was performed to determine the interaction between LOC101928120 and HDAC1. Ahr targeting the LOC101928120 gene was detected using luciferase reporter and chromatin immunoprecipitation assays. RESULTS: WTD alleviated the swelling of the hind paw in rats with RA and suppressed the chondrocyte apoptosis and ROS production caused by IL-1ß. WTD decreased SHC1 but increased LOC101928120 in IL-1ß-treated chondrocytes. SHC1 knockdown and LOC101928120 overexpression also showed the protection. However, LOC101928120 knockdown attenuated the protective effects of WTD. WTD stimulated Ahr, which promoted LOC101928120 transcription. LOC101928120 recruited HDAC1 to the promoter region of the SHC1 gene, thereby decreasing SHC1. DISCUSSION AND CONCLUSION: This study revealed a new mechanism by which WTD alleviates RA by modulating the Ahr/LOC101928120/SHC1 pathway.

Curcumin reverses diabetic nephropathy in streptozotocin-induced diabetes in rats by inhibition of PKCß/p66Shc axis and activation of FOXO-3a.

This study investigated if the nephroprotective effect of Curcumin in streptozotocin-induced type 1 diabetes mellitus (DM) in rats involves downregulation/inhibition of p66Shc and examined the underlying mechanisms. Rats were divided into 4 groups (n = 12/group) as control, control + Curcumin (100 mg/kg), T1DM, and T1DM + Curcumin. Curcumin was administered orally to control or diabetic rats for 12 weeks daily. As compared to diabetic rats, Curcumin didn't affect either plasma glucose or insulin levels but significantly reduced serum levels of urea, blood urea nitrogen, and creatinine, and concurrently reduced albumin/protein urea and increased creatinine clearance. It also prevented the damage in renal tubules and mitochondria, mesangial cell expansion, the thickness of the basement membrane. Mechanistically, Curcumin reduced mRNA and protein levels of collagen I/III and transforming growth factor- ß-1 (TGF-ß1), reduced inflammatory cytokines levels, improved markers of mitochondrial function, and suppressed the release of cytochrome-c and the activation of caspase-3. In the kidneys of both control and diabetic rats, Curcumin reduced the levels of reactive oxygen species (ROS), increased mRNA levels of manganese superoxide dismutase (MnSOD) and gamma-glutamyl ligase, increased glutathione (GSH) and protein levels of Bcl-2 and MnSOD, and increased the nuclear levels of nuclear factor2 (Nrf2) and FOXO-3a. Besides, Curcumin reduced the nuclear activity of the nuclear factor-kappa B (NF-κB), downregulated protein kinase CßII (PKCßII), NADPH oxidase, and p66Shc, and decreased the activation of p66Shc. In conclusion, Curcumin prevents kidney damage in diabetic rats by activating Nrf2, inhibiting Nf-κB, suppressing NADPH oxidase, and downregulating/inhibiting PKCßII/p66Shc axis.

Resolvin D1 Prevents the Impairment in the Retention Memory and Hippocampal Damage in Rats Fed a Corn Oil-Based High Fat Diet by Upregulation of Nrf2 and Downregulation and Inactivation of p66Shc.

This study investigated the effect of a high-fat diet rich in corn oil (CO-HFD) on the memory retention and hippocampal oxidative stress, inflammation, and apoptosis in rats, and examined if the underlying mechanisms involve modulating Resolvin D1 (RvD1) levels and activation of p66Shc. Also, we tested if co-administration of RvD1 could prevent these neural adverse effects induced by CO-HFD. Adult male Wistar rats were divided into 4 groups (n = 18/each) as control fed standard diet (STD) (3.82 kcal/g), STD + RvD1 (0.2 µg/Kg, i.p/twice/week), CO-HFD (5.4 kcal/g), and CO-HFD + RvD1. All treatments were conducted for 8 weeks. With normal fasting glucose levels, CO-HFD induced hyperlipidemia, hyperinsulinemia, increased HOMA-IRI and reduced the rats' memory retention. In parallel, CO-HFD increased levels of reactive oxygen species (ROS), malondialdehyde (MDA), cytoplasmic cytochrome-c, and cleaved caspase-3 and significantly decreased levels of glutathione (GSH), Bcl-2, and manganese superoxide dismutase (MnSOD) in rats' hippocampi. Besides, CO-HFD significantly reduced hippocampal levels of docosahexaenoic acid (DHA) and RvD1, as well as total protein levels of Nrf2 and significantly increased nuclear protein levels of p-NF-κB. Concomitantly, CO-HFD increased hippocampal protein levels of p-JNK, p53, p66Shc, p-p66Shc, and NADPH oxidase. However, without altering plasma and serum levels of glucose, insulin, and lipids, co-administration of RvD1 to CO-HFD completely reversed all these events. It also resulted in similar effects in the STD fed-rats. In conclusion, CO-HFD impairs memory function and induces hippocampal damage by reducing levels of RvD1 and activation of JNK/p53/p66Shc/NADPH oxidase, effects that are prevented by co-administration of RvD1.

MiR-124 attenuates doxorubicin-induced cardiac injury via inhibiting p66Shc-mediated oxidative stress.

Previous studies showed that miR-124 had a protective role by reducing oxidant stress and preventing cell apoptosis and autophagy. However, its role in doxorubicin-induced cardiomyopathy was less known. In our study, we confirmed increased ROS and decreased expression of miR-124 in doxorubicin-treated heart tissues and primary cardiomyocytes. The oxidative stress and cell apoptosis were alleviated by overexpressing miR-124, characterized by decreased activity of MDA and increased activity of SOD. While inhibiting miR-124 generated opposed effects. Mechanistically, our bioinformatic prediction and luciferase assay confirmed that miR-124 inhibited the expression of p66Shc, a proapoptotic signaling pathway. Our results suggested that miR-124 was hopeful to become a therapeutic target in doxorubicin-related cardiomyopathy.

Idebenone is a cytoprotective insulin sensitizer whose mechanism is Shc inhibition.

When insulin binds insulin receptor, IRS1 signaling is stimulated to trigger the maximal insulin response. p52Shc protein competes directly with IRS1, thus damping and diverting maximal insulin response. Genetic reduction of p52Shc minimizes competition with IRS1, and improves insulin signaling and glucose control in mice, and improves pathophysiological consequences of hyperglycemia. Given the multiple benefits of Shc reduction in vivo, we investigated whether any of 1680 drugs used in humans may function as Shc inhibitors, and thus potentially serve as novel anti-diabetics. Of the 1680, 30 insulin sensitizers were identified by screening in vitro, and of these 30 we demonstrated that 7 bound Shc protein. Of the 7 drugs, idebenone dose-dependently bound Shc protein in the 50-100 nM range, and induced insulin sensitivity and cytoprotection in this same 100 nM range that clinically dosed idebenone reaches in human plasma. By contrast we observe mitochondrial effects of idebenone in the 5,000 nM range that are not reached in human dosing. Multiple assays of target engagement demonstrate that idebenone physically interacts with Shc protein. Idebenone sensitizes mice to insulin in two different mouse models of prediabetes. Genetic depletion of idebenone's target eliminates idebenone's ability to insulin-sensitize in vivo. Thus, idebenone is the first-in-class member of a novel category of insulin-sensitizing and cytoprotective agents, the Shc inhibitors. Idebenone is an approved drug and could be considered for other indications such as type 2 diabetes and fatty liver disease, in which insulin resistance occurs.

p66Shc regulates migration of castration-resistant prostate cancer cells.

Metastatic castration-resistant (CR) prostate cancer (PCa) is a lethal disease for which no effective treatment is currently available. p66Shc is an oxidase previously shown to promote androgen-independent cell growth through generation of reactive oxygen species (ROS) and is elevated in clinical PCa and multiple CR PCa cell lines. We hypothesize p66Shc also increases the migratory activity of PCa cells through ROS and investigate the associated mechanism. Using the transwell assay, our study reveals that the level of p66Shc protein correlates with cell migratory ability across several PCa cell lines. Furthermore, we show hydrogen peroxide treatment induces migration of PCa cells that express low levels of p66Shc in a dose-dependent manner, while antioxidants inhibit migration. Conversely, PCa cells that express high levels of endogenous p66Shc or by cDNA transfection possess increased cell migration which is mitigated upon p66Shc shRNA transfection or expression of oxidase-deficient dominant-negative p66Shc W134F mutant. Protein microarray and immunoblot analyses reveal multiple proteins, including ErbB-2, AKT, mTOR, ERK, FOXM1, PYK2 and Rac1, are activated in p66Shc-elevated cells. Their involvement in PCa migration was examined using respective small-molecule inhibitors. The role of Rac1 was further validated using cDNA transfection and, significantly, p66Shc is found to promote lamellipodia formation through Rac1 activation. In summary, the results of our current studies clearly indicate p66Shc also regulates PCa cell migration through ROS-mediated activation of migration-associated proteins, notably Rac1.

The implication of p66shc in oxidative stress induced by deltamethrin.

Deltamethrin (DLT) is effective against a broad spectrum of insects. Exposure to DLT has been demonstrated to cause oxidative stress. However, the mechanism of oxidative stress induced by DLT is little known. Groups of rats were gavaged with DLT once daily for 7 days at six dosages: 0, 2, 5, 10, 20, 40 mg/kg. The intensity of neurotoxicity and liver dysfunction caused by DLT were significantly increased in a dose-dependent manner. We found that DLT caused the increase of cytosolic superoxide in tissues. Western blot analysis showed that both the expression of p66shc and Ser36 phosphorylated p66shc, which were involved in ROS generation, were increased in tissues treated with DLT. Further investigation showed that DLT treatment resulted in the increase of intracellular ROS accompanied with elevated p66shc expression in different cell lines. And treatment of cells with DLT induced p66shc phosphorylation at Ser36 and the translocation of p66shc from cytoplasm to mitochondria. Moreover, the overexpression of wildtype p66shc caused the increase of DLT-mediated ROS level in SH-SY5Y cells, but cells overexpressing p66shcSer36Ala mutant plasmid had the opposite effect. And p66shc suppression by siRNA blunted DLT-mediated ROS generation. Taken together, our findings indicated p66shc mediated DLT-induced oxidative stress, which may be partly responsible for toxic effects.

Modulation of mitochondrial dysfunction-related oxidative stress in fibroblasts of patients with Leigh syndrome by inhibition of prooxidative p66Shc pathway.

The mitochondrial respiratory chain, and in particular, complex I, is a major source of reactive oxygen species (ROS) in cells. Elevated levels of ROS are associated with an imbalance between the rate of ROS formation and the capacity of the antioxidant defense system. Increased ROS production may lead to oxidation of DNA, lipids and proteins and thus can affect fundamental cellular processes. The aim of this study was to investigate the magnitude of intracellular oxidative stress in fibroblasts of patients with Leigh syndrome with defined mutations in complex I. Moreover, we hypothesized that activation of the p66Shc protein (phosphorylation of p66Shc at Ser36 by PKCß), being part of the oxidative stress response pathway, is partially responsible for the increased ROS production in cells with dysfunctional complex I. Characterization of bioenergetic parameters and ROS production showed that the cellular model of Leigh syndrome is described by increased intracellular oxidative stress and oxidative damage to DNA and proteins, which correlate with increased p66Shc phosphorylation at Ser36. Treatment of patients' fibroblasts with hispidin (an inhibitor of the protein kinase PKCß), in addition to decreasing ROS production and intracellular oxidative stress, resulted in restoration of complex I activity.

Probucol ameliorates renal injury in diabetic nephropathy by inhibiting the expression of the redox enzyme p66Shc.

AIMS: Probucol is an anti-hyperlipidemic agent and a potent antioxidant drug that can delay progression of diabetic nephropathy (DN) and reverses renal oxidative stress in diabetic animal models; however, the mechanisms underlying these effects remain unclear. p66Shc is a newly recognized mediator of mitochondrial ROS production in renal cells under high-glucose (HG) ambience. We previously showed that p66Shc can serve as a biomarker for renal oxidative injury in DN patients and that p66Shc up-regulation is correlated with renal damage in vivo and in vitro. Here, we determined whether probucol ameliorates renal injury in DN by inhibiting p66Shc expression. RESULTS: We found that the expression of SIRT1, Ac-H3 and p66Shc in kidneys of DN patients was altered. Also, probucol reduced the levels of serum creatinine, urine protein and LDL-c and attenuated renal oxidative injury and fibrosis in STZ induced diabetic mice. In addition, probucol reversed p-AMPK, SIRT1, Ac-H3 and p66Shc expression. Correlation analyses showed that p66Shc expression was correlated with p-AMPK and Sirt1 expression and severity of renal injury. In vitro pretreatment of HK-2 cells with p-AMPK and SIRT1 siRNA negated the beneficial effects of probucol. Furthermore, we noted that probucol activates p-AMPK and Sirt1 and inhibits p66shc mRNA transcription by facilitating the binding of Sirt1 to the p66Shc promoter and modulation of Ac-H3 expression in HK-2 cells under HG ambience. INNOVATION AND CONCLUSION: Our results suggest for the first time that probucol ameliorates renal damage in DN by epigenetically suppressing p66Shc expression via the AMPK-SIRT1-AcH3 pathway.

Inhibition of p66Shc-mediated mitochondrial apoptosis via targeting prolyl-isomerase Pin1 attenuates intestinal ischemia/reperfusion injury in rats.

Intestinal epithelial oxidative stress and apoptosis constitute key pathogenic mechanisms underlying intestinal ischemia/reperfusion (I/R) injury. We previously reported that the adaptor 66 kDa isoform of the adaptor molecule ShcA (p66Shc)-mediated pro-apoptotic pathway was activated after intestinal I/R. However, the upstream regulators of the p66Shc pathway involved in intestinal I/R remain to be fully identified. Here, we focused on the role of a prolyl-isomerase, peptidyl-prolyl cis-trans isomerase (Pin1), in the regulation of p66Shc activity during intestinal I/R. Intestinal I/R was induced in rats by superior mesenteric artery (SMA) occlusion. Juglone (Pin1 inhibitor) or vehicle was injected intraperitoneally before I/R challenge. Caco-2 cells were exposed to hypoxia/reoxygenation (H/R) in vitro to simulate an in vivo I/R model. We found that p66Shc was significantly up-regulated in the I/R intestine and that this up-regulation resulted in the accumulation of intestinal mitochondrial reactive oxygen species (ROS) and massive epithelial apoptosis. Moreover, intestinal I/R resulted in elevated protein expression and enzyme activity of Pin1 as well as increased interaction between Pin1 and p66Shc. This Pin1 activation was responsible for the translocation of p66Shc to the mitochondria during intestinal I/R, as Pin1 suppression by juglone or siRNA markedly blunted p66Shc mitochondrial translocation and the subsequent ROS generation and cellular apoptosis. Additionally, Pin1 inhibition alleviated gut damage and secondary lung injury, leading to improvement of survival after I/R. Collectively, our findings demonstrate for the first time that Pin1 inhibition protects against intestinal I/R injury, which could be partially attributed to the p66Shc-mediated mitochondrial apoptosis pathway. This may represent a novel prophylactic target for intestinal I/R injury.

p66shc-mediated toxicity of high-dose α-tocopherol in renal proximal tubule cells.

α-Tocopherol (TOC) is a widely used supplement known for its role as an antioxidant. Previously, we have shown that TOC elicits adaptive responses by upregulating the ERK/CREB/HO-1 pathway, which depends on its concentration in cultured renal proximal tubule cells (RPTCs). This suggests that high-dose TOC (hTOC) may elicit adverse effects via inflicting oxidative stress. Since the pro-oxidant p66shc is a major mediator of oxidant injury in various models of renal toxicants, we tested the hypothesis that hTOC elicits renal toxicity through activation of p66shc and consequent oxidative stress. RPTCs (NRK52E) were treated with high-dose TOC (hTOC; 400 nM) in cells where expression or mitochondrial cytochrome c-binding of p66shc was manipulated by genetic means. Intracellular production of reactive oxygen species (ROS), mitochondrial depolarization, and cell viability was also determined. Additionally, activation of the pro-survival ERK/CREB/HO-1 signaling and the p66shc promoter was determined via reporter luciferase assays. hTOC decreased cell viability via increasing ROS-dependent mitochondrial depolarization and suppressing the pro-survival ERK/CREB/HO-1 pathway via transcriptional activation of p66shc. Conversely, either knockdown of p66shc, mutation of its mitochondrial cytochrome c-binding site, or overexpression of ERK or HO-1 ameliorated adverse effects of hTOC and restored the pro-survival signaling. The pro-oxidant p66shc plays dual role in toxicity of high-dose TOC: it provokes oxidative stress and suppresses adaptive responses.

Gambogic Acid Inhibits Malignant Melanoma Cell Proliferation Through Mitochondrial p66shc/ROS-p53/Bax-Mediated Apoptosis.

BACKGROUND/AIMS: Malignant melanoma has high metastatic potential, is highly resistant to chemotherapy, and has a poor survival rate. Gambogic acid (GA), a polyprenylated xanthone extracted from a traditional Chinese medicinal herb, has been proven to exhibit antitumor activity. The present study aimed to investigate the signaling pathways that mediated GA-induced inhibition of human malignant skin melanoma proliferation. METHODS: The study was conducted using A375 cells and the corresponding tumor transplanted in nude mice. RESULTS: Incubation of A375 cells with 1-10 µg/ml GA decreased cell viability and increased apoptosis. GA concentration-dependently increased p66shc expression and intracellular ROS levels. GA also decreased the oxygen consumption rate and the mitochondrial membrane potential (MMP) in A375 cells. Experimental inhibition of p66shc by siRNA suppressed GA-induced increase of ROS, decrease of oxygen consumption rate, MMP and cell viability, whilst suppressing GA-induced increase of apoptosis. GA concentration-dependently upregulated p53 and Bax expression in A375 cells. GA also increased p53-TA-luciferase activity and p53-binding to Bax promoter, which was inhibited by Sip53. Experimental inhibition of p53 with Sip53 blocked GA-induced decrease of the oxygen consumption rate and cell viability, and blocked the increase of apoptosis. In tumor-bearing nude mice, GA notably inhibited tumor growth, and this action was suppressed by N-acetylcysteine (NAC), a potent antioxidant, and by PFT-α, a p53 inhibitor. In A375 tumors transplanted in nude mice, GA increased both p66shc and p53 expression. NAC and PFT-α treatment did not significantly affect p66shc expression in tumors grown in mice treated with GA. In contrast, both NAC and PFT-α treatment inhibited GA-induced p53 expression in mouse tumors. CONCLUSION: Results provided novel preclinical insights into the chemotherapeutic use of GA by highlighting the importance of p66shc/ROS-p53/Bax pathways in the antitumor effect of GA in malignant melanoma.