FOXO3a Protein Research Articles

The Ketone Metabolite β-Hydroxybutyrate Attenuates Oxidative Stress in Spinal Cord Injury by Suppression of Class I Histone Deacetylases.

The ketone metabolite β-hydroxybutyrate (βOHB), is reported to be neuroprotective after spinal cord injury (SCI) in rats, but the underlying mechanism remains unknown. The present study aims to investigate effects of βOHB on suppression of oxidative stress and inhibition of class I histone deacetylases (HDACs) in in vivo and in vitro models. Rats were fed with ketogenic diet (KD) or standard diet (SD) for 3 weeks. A C5 hemi-contusion injury was applied to these animals on the 14th day of experiment, and spinal cord samples were harvested on the 1st, 3rd and 7th days after SCI, respectively. The blood ketone levels were significantly higher in the KD groups. KD reduced oxidative stress markers and reactive oxygen species (ROS) products, downregulated the expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX)2 and NOX4, and upregulated the expression of forkhead box group O (FOXO)3a, mitochondrial superoxide dismutase (MnSOD), and catalase after SCI. The in vitro study, performed on PC12 cells, indicated that βOHB inhibited H2O2-induced ROS production, decreased NOX2 and NOX4 protein levels, and upregulated FOXO3a, MnSOD, and catalase levels in a dose-dependent manner, which was consistent with the in vivo results. The ketone metabolite βOHB inhibited HDAC1, HDAC2, and HDAC3 activity, but not HDAC8 in SCI rats and PC12 cells. Depletion of HDAC1 or HDAC2 with small interfering RNA (siRNA) attenuated H2O2-induced ROS production and protein carbonylation and elevated FOXO3a protein levels, meanwhile reducing NOX2 and NOX4 protein expression in PC12 cells. Our results indicate that the ketone metabolite βOHB attenuates oxidative stress in SCI by inhibition of class I HDACs, and selected suppression of HDAC1 or HDAC2 regulates FOXO3a, NOX2, and NOX4 expression. Therefore, the ketone metabolite βOHB may be a novel promising therapeutic agent for SCI.

Knockdown of IL-8 Provoked Premature Senescence of Placenta-Derived Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) have shown promise for use in cell therapy, and due to their tumor tropism can serve as vehicles for delivering therapeutic agents to tumor sites. Because interleukin-8 (IL-8) is known to mediate the protumor effect of MSCs, elimination of IL-8 secretion by MSCs may enhance their safety for use in cancer gene therapy. However, little is known concerning the effect of endogenously secreted IL-8 on MSCs. We performed studies using placenta-derived MSCs (PMSCs) to determine whether knockdown of IL-8 would influence their biological activity. We first verified that IL-8 and its membrane receptor CXCR2, but not CXCR1, were highly expressed in PMSCs. We then employed lentivirus-mediated small hairpin RNA interference to generate stable IL-8-silenced PMSCs, which displayed a variety of characteristic senescent phenotypes. We observed that at day 9 post-transfection, IL-8-silenced PMSCs had become larger and displayed a more flattened appearance when compared with their controls. Moreover, their proliferation, colony forming unit-fibroblast formation, adipogenic and osteogenic differentiation, and immunosuppressive potentials were significantly impaired. Enhanced senescence-associated β-galactosidase (SA-β-gal) activity and specific global gene expression profiles confirmed that IL-8 silencing evoked the senescence process in PMSCs. Increased levels of p-Akt and decreased levels of FOXO3a protein expression suggested that reactive oxygen species played a role in the initiation and maintenance of senescence in IL-8-silenced PMSCs. Notably, the majority of CXCR2 ligands were downregulated in presenescent IL-8-silenced PMSCs but upregulated in senescent cells, indicating an antagonistic pleiotropy of the IL-8/CXCR2 signaling pathway in PMSCs. This effect may promote the proliferation of young cells and accelerate senescence of old cells.

Circulating microRNA-194 regulates human melanoma cells via PI3K/AKT/FoxO3a and p53/p21 signaling pathway.

In the present study, we analyzed the role of microRNA-194 circulating regulated human melanoma cell growth. We found that microRNA-194 expression was markedly suppressed in human melanoma patients, compared with negative control group. Next, disease-free survival (DFS) and overall survival (OS) of high expression in human melanoma patients was higher than those of low expression in human melanoma patients. MicroRNA-194 overexpression inhibited cell proliferation, induced apoptosis, increased caspase-3/−9 activities and promoted Bax/Bcl-2 of human melanoma cells. Furthermore, microRNA-194 overexpression also suppressed PI3K/AKT/FoxO3a signaling pathway and induced p53/p21 signaling pathway. PI3K inhibitor, suppressed PI3K, phosphorylation-AKT, FoxO3a protein expression and increased the effects of microRNA-194 overexpression on cell growth, apoptosis, caspase-3/−9 activities and Bax/Bcl-2 protein expression of human melanoma cells through the induction of p53/p21 signaling pathway. Taken together, these data indicate that circulating microRNA-194 regulated human melanoma cells via PI3K/AKT/FoxO3a and p53/p21 signaling pathway.

Expression of PH Domain Leucine-rich Repeat Protein Phosphatase, Forkhead Homeobox Type O 3a and RAD51, and their Relationships with Clinicopathologic Features and Prognosis in Ovarian Serous Adenocarcinoma.

Background:Ovarian serous adenocarcinoma can be divided into low- and high-grade tumors, which exhibit substantial differences in pathogenesis, clinicopathology, and prognosis. This study aimed to investigate the differences in the PH domain leucine-rich repeat protein phosphatase (PHLPP), forkhead homeobox type O 3a (FoxO3a), and RAD51 protein expressions, and their associations with prognosis in patients with low- and high-grade ovarian serous adenocarcinomas.Methods:The PHLPP, FoxO3a, and RAD51 protein expressions were examined in 94 high- and 26 low-grade ovarian serous adenocarcinomas by immunohistochemistry. The differences in expression and their relationships with pathological features and prognosis were analyzed.Results:In high-grade serous adenocarcinomas, the positive rates of PHLPP and FoxO3a were 24.5% and 26.6%, while in low-grade tumors, they were 23.1% and 26.9%, respectively (P < 0.05 vs. the control specimens; low- vs. high-grade: P > 0.05). The positive rates of RAD51 were 70.2% and 65.4% in high- and low-grade serous adenocarcinomas, respectively (P < 0.05 vs. the control specimens; low- vs. high-grade: P > 0.05). Meanwhile, in high-grade tumors, Stage III/IV tumors and lymph node and omental metastases were significantly associated with lower PHLPP and FoxO3a and higher RAD51 expression. The 5-year survival rates of patients with PHLPP- and FoxO3a-positive high-grade tumors (43.5% and 36.0%) were significantly higher than in patients with PHLPP-negative tumors (5.6% and 7.2%, respectively; P < 0.05). Similarly, the 5-year survival rate of RAD51-positive patients (3.0%) was significantly lower than in negative patients (42.9%; P < 0.05). In low-grade tumors, the PHLPP, FoxO3a, and RAD51 expressions were not significantly correlated with lymph node metastasis, omental metastasis, Federation of Gynecology and Obstetrics stage, or prognosis.Conclusions:Abnormal PHLPP, FoxO3a, and RAD51 protein expressions may be involved in the development of high- and low-grade ovarian serous adenocarcinomas, suggesting common molecular pathways. Decreased PHLPP and FoxO3a and increased RAD51 protein expression may be important molecular markers for poor prognosis, and RAD51 may be an independent prognosis factor, of high-grade, but not low-grade, ovarian serous adenocarcinomas.

N-3 polyunsaturated fatty acids alleviate high glucose-mediated dysfunction of endothelial progenitor cells and prevent ischemic injuries both in vitro and in vivo

Hyperglycemia is associated with a reduced number of endothelial progenitor cells (EPCs) that impairs vascular function. Circulating EPCs play important roles in postnatal neovasculogenesis and the prevention of ischemic injury. Frequent consumption of fish oil (FO) that is abundant with eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA) is reportedly associated with an alleviation of diabetic complications and a lowered incidence of cardiovascular disease. The aim of this study was to examine whether N-3 polyunsaturated fatty acids such as EPA and DHA would reverse the high glucose-mediated dysfunction of EPCs in vitro and thereby prevent the ischemic injury that occurs under the hyperglycemic conditions in Type 2 diabetes (T2D) db−/− mice. The results demonstrate that EPA and DHA alleviate high glucose-mediated impairment of tubular formation in EPCs through a rescue of neovasculogenic capability. The molecular mechanisms underlying the effects of EPA and DHA include the activation of the extracellular signal-regulated kinase 1/2, Akt/endothelial nitric oxide synthase (eNOS) and AMP-activated kinase (AMPK) signaling cascades as well as the phosphorylation of the downstream FOXO3a protein in EPCs. Moreover, EPA and DHA up-regulate the expression of c-kit, erythroid 2-related factor and heme oxygenase-1 proteins. Daily consumption of FO at dosages of 4% and 6% (wt/wt) significantly increased the level of bone marrow-derived and circulating EPCs, induced a recovery of blood flow and prevented ischemic injuries in a T2D db−/− mouse model. The effects of FO consumption were exerted the activation of Akt/eNOS and AMPK signaling cascades without any effect on the plasma VEGF level in vivo.

Cytosolic Extract of Human Adipose Stem Cells Reverses the Amyloid Beta-Induced Mitochondrial Apoptosis via P53/Foxo3a Pathway.

Human adipose stem cells (hASC) have therapeutic potential for the treatment of neurodegenerative disorders. Mitochondrial dysfunction is frequently observed in most neurodegenerative disorders, including Alzheimer’s disease. We explored the therapeutic potential of hASC cytosolic extracts to attenuate neuronal death induced by mitochondrial dysfunction in an Alzheimer’s disease (AD) in vitro models. Amyloid beta (Aβ) was used to induce cytotoxity in an immortal hippocampal cell line (HT22) and neuronal stem cells from the brain of TG2576 transgenic mice were also used to test the protective role of hASC cytosolic extracts. Cell viability and flow cytometry results demonstrated that the hASC extract prevents the toxicity and apoptosis in AD in vitro models. Moreover, JC-1 and MitoSoxRed staining followed by fluorescence microscopy and flow cytometry results showed that the hASC extract ameliorated the effect of Aβ-induced mitochondrial oxidative stress and reduced the mitochondrial membrane potential. Western blot result showed that hASC extract modulated mitochondria-associated proteins, such as Bax and Bcl2, and down-regulated cleaved caspase-3. In addition, hASC extract decreased Aβ generation and reversed up-regulated p53 and foxo3a protein level in AD in vitro model cell derived from TG2576 mice. Taken together, these findings implicate a protective role of the hASC extract in the Aβ-induced mitochondrial apoptosis via regulation of P53/foxo3a pathway, providing insight into the molecular mechanisms of hASC extract and a therapeutic strategy to ameliorate neuronal death induced by Aβ.

Targeting miR155 restores chemotherapy sensitivity in drug-resistant myeloma cell-line RPMI8226/DOX cells

目的分析miR155异常表达在多发性骨髓瘤（MM）化疗耐药机制中的作用，探讨靶向抑制miR155表达对耐阿霉素MM细胞株RPMI8226/DOX耐药性的影响，进一步分析其作用机制。方法通过浓度梯度递增法建立耐阿霉素MM细胞系RPMI8226/DOX；采用实时荧光定量PCR法检测RPMI8226/DOX细胞和MM敏感细胞株RPMI8226/S miR155基因表达，Western blot检测FOXO3a、BCL-2蛋白表达。在RPMI8226/DOX细胞中分别转染miR155抑制物和模拟物，通过实时荧光定量PCR法检测miR155抑制物和模拟物的转染效率，应用CCK-8法检测转染后细胞对阿霉素的敏感性。在靶向抑制物干预MM细胞后，Western blot分析FOXO3a、BCL-2通路蛋白表达的变化。结果①RPMI8226/DOX细胞miR155相对表达量为RPMI8226/S细胞的（26.860±2.340）倍，BCL-2蛋白表达上调，FOXO3a蛋白表达下调。②靶向抑制miR155表达72 h后，转染抑制率为64.57%，miR155基因表达下调，FOXO3a蛋白表达上调，BCL-2蛋白表达下调；RPMI8226/DOX细胞部分恢复了对阿霉素的敏感性，逆转耐药倍数为2.518。结论miR155异常表达与MM的化疗耐药形成相关，靶向抑制miR155表达可以通过影响FOXO3a蛋白表达恢复MM耐药细胞对化疗药物的敏感性。

PP2A Balances Glucose Metabolism and Foxo Activation to Maintain Cellular Redox Homeostasis in Acute Lymphoblastic Leukemia

Introduction: Protein phosphatase 2A (PP2A) is a Ser/Thr phosphatase negatively regulates a diverse set of signaling pathways promoting tumor growth. While PP2A functions as tumor suppressor in multiple types of cancer, here, we demonstrate an unexpected pro-survival role of PP2A in pre-B acute lymphoblastic leukemia (ALL) cells. By calibrating PI3K-AKT-mTOR signaling strength, PP2A regulates glycolysis rate and thereby balances energy demands against anti-oxidant protection of pre-B ALL cells. In addition, PP2A reinstates activity of FOXO factors by dephosphorylation and thereby enables the anti-oxidant function of FOXO1 and FOXO3. Consistent with previous studies, we find PP2A is dispensable for the survival of myeloid leukemia cells which indicates a lineage-specific role of PP2A. Combined with genetic study and small molecule inhibitor, we verify that regulation in patient-derived xenografts and highlight PP2A as a therapeutic target in pre-B ALL.Results: Consistent with a divergent role of PP2A in pre-B ALL (compared to CML), we found that high mRNA levels of PP2A subunits at the time of diagnosis predict poor outcome of children (COG P9906; n=207) and adults (ECOG 2993; n=215) with ALL. Consistent with these findings, mutations in PP2A subunits are extremely rare in B cell malignancies but relatively common in solid tumors and myeloid malignancies (COSMIC). We therefore, studied the function of PP2A in a genetic mouse model for Cre-induced deletion of Ppp2r1a in BCR-ABL1 (Ph+) ALL. Inducible activation of Cre reduced protein expression of the targeted PP2A subunit A and the catalytic subunit C, which leads to near-complete loss of PP2A phosphatase activity. Conversely, Cre-mediated deletion increased phosphorylation levels of FoxO1, FoxO3a, p70S6K and S6 ribosomal protein, which indicated elevated PI3K-Akt-mTOR signaling. Acute deletion of Ppp2r1afl/fl in B cell-lineage ALL cells dramatically affected survival and colony formation, both of which could be rescued by overexpression of wildtype PP2A. PI3K and mTOR inhibitors also have rescue effect on PP2A deficient ALL cells in growth-competition assay. Luciferase-labeled PP2A-deleted ALL cells showed reduced cell growth and leukemia progression after being transplanted into recipient mice. However, Cre-mediated deletion had no deleterious effects in a Ppp2r1afl/fl CML model. This lineage-specific role of PP2A was verified by inducible CEBPα expression to reprogram B cell lineage ALL cells into myeloid cells.Interestingly, inducible deletion of PP2A caused profound imbalances of glucose metabolism in Ph+ ALL but not in CML cells. Upon PP2A-deletion, ALL cells showed higher glycolytic flux shunted into lactate rather than NADPH production. By employing glucose flux metabolic profiling assay using [1,2-13C2]-D-glucose tracer, we found elevated glycolysis and repressed pentose phosphate pathway (PPP) flux in PP2A-deleted pre-B ALL cells. Lower NADPH/NADP ratio and higher reactive oxygen species level in PP2A-deleted pre-B ALL cells, together with decreased anti-oxidant gene expression, increased DNA damage, including H2AX phosphorylation and p53 expression. The mechanistic role of ROS downstream of PP2A was supported by a strong rescue effect of overexpression of the antioxidant catalase in PP2A-deleted cells. The unexpected role of PP2A in Ph+ ALL was further validated by CRISPR-Cas9 mediated disruption of PPP2R1A in ALL xenografts derived from three patients. In addition, a PP2A specific inhibitor LB-100 (in clinical trial for solid tumors) was employed to pharmacologically inhibit PP2A activity. Low micromolar concentrations of LB-100 induced cell death in patient-derived ALL xenografts in parallel with ROS-accumulation and increased S6 and H2AX phosphorylation.Conclusion: Here we revealed an unexpected role of PP2A in maintaining redox homeostasis in pre-B ALL cells. By regulating AKT-mTOR signaling, PP2A keeps the balance of glycolysis and PPP to meet the energy demands of pre-B ALL cells and avoids extreme levels of oxidative stress. We confirmed this pro-survival role of PP2A in both genetic mouse ALL model and human Ph+ ALL-patients derived leukemia cells. These findings highlight PP2A as a therapeutic target and suggest that agents like the PP2A inhibitor LB-100 may be of interest for pre-clinical development and testing. DisclosuresNo relevant conflicts of interest to declare.

Neuroprotective effects of miR-27a against traumatic brain injury via suppressing FoxO3a-mediated neuronal autophagy

MicroRNA-27a (miR-27a) has been reported to be a brain-specific miRNA and aberrantly expressed in the brain suffered from traumatic brain injury (TBI). The present study is designed to investigate the potential role and molecular mechanism of miR-27a in the pathogenesis of TBI. The level of miR-27a in brain was manipulated by intracerebroventricular injection of lentiviral-encoding miR-27a before TBI induction. Real-time PCR was used to detected miR-27a and Forkhead box O3a (FoxO3a) levels in the hippocampus. Then, we evaluated the impact of miR-27a overexpression on neurological function, brain edema, lesion volume and neuronal autophagy after TBI. The blinding of miR-27a to the 3′UTR of FoxO3a mRNA and its effects on FoxO3a translation were analyzed by luciferase reporter assay and Western blot. The downregulation of miR-27a and the increase in FoxO3a level were observed in the hippocampus post-TBI. Overexpression of miR-27a significantly attenuated neurological deficits and brain injury, especially suppressed autophagic activation after TBI. Furthermore, we identified that miR-27a directly targeted the FoxO3a 3′UTR region to reduced FoxO3a protein expression. Knockdown of FoxO3a significantly reversed high levels of autophagy-related genes induced by TBI. Taken together, Overexpression of miR-27a may protect against brain injury via suppressing FoxO3a-mediated neuronal autophagy following TBI.

FOXO3a Expression Regulated by ERK Signaling is Inversely Correlated With Y-Box Binding Protein-1 Expression in Prostate Cancer.

FOXO3a is a member of the forkhead O transcription factors. FOXO3a induces the factors that contribute to cell cycle arrest and is considered a tumor suppressor in several malignant tumors. Y-box binding protein-1 (YB-1) is a multifunctional protein whose high expression is correlated with poor prognoses in various malignant tumors. In the current study, we investigated the relationship between FOXO3a and YB-1 to validate their functional roles in prostate cancer. Western blotting and cytotoxicity assays were conducted in prostate cancer cells, LNCaP, and 22Rv1 cells. We also evaluated the protein expressions of FOXO3a and YB-1 in human prostate cancer tissues, using radical prostatectomy specimens. Then, we investigated the correlations between protein expressions and clinicopathologic parameters. We found that both FOXO3a and YB-1 proteins were phosphorylated by ERK signaling, resulting in FOXO3a inactivation and YB-1 activation in LNCaP and 22Rv1 cells. Inversely, inhibition of MEK or treatment with metformin activated FOXO3a through inactivation of ERK signaling and suppressed the viability of LNCaP and 22Rv1 cells in a dose-dependent manner. In immunohistochemical analysis, FOXO3a nuclear expression was inversely correlated with YB-1 nuclear expression (P < 0.0001). Furthermore, high FOXO3a nuclear expression was inversely correlated with a higher Gleason grade (P < 0.0001) and higher preoperative PSA (P = 0.0437). These results showed that in prostate cancer, FOXO3a, and YB-1 play inverse reciprocal roles as a tumor-suppressor gene and oncogene, respectively, through their master regulator ERK. Prostate 77:145-153, 2017. © 2016 Wiley Periodicals, Inc.

FoxO3a mediates glioma cell invasion by regulating MMP9 expression.

The role of FoxO3a in glioma progression is poorly defined. Herein, we show that silencing FoxO3a in U251 cells leads to decreased invasive migration and proliferation, while ectopic expression of FoxO3a in U87 cells with weak endogenous FoxO3a protein levels enhances invasion and proliferation. To further investigate the mechanism by which FoxO3a promotes invasion, we detected key members of the matrix metalloproteinases (MMPs) that are associated with invasion. Our findings showed that, among these MMP members, only FoxO3a induced MMP9 expression, and MMP9 overexpression reversed the effect that silencing FoxO3a had on the attenuation of cell invasion. Taken together, these data link FoxO3a to the promotion of metastasis in glioma cells.

Restoring miR122 in human stem-like hepatocarcinoma cells, prompts tumor dormancy through Smad-independent TGF-β pathway.

miR122 is the prevalent miRNA in adult healthy liver and it is responsible for liver stem cell differentiation towards hepatocyte lineage. Its expression is frequently lost in hepatocellular carcinoma (HCC). We studied the effects of restoring miR122 expression in a distinctive cell line derived from human HCC-BCLC9 cells-with a solid stem-like cell profile, high tumor initiating ability and undetectable miR122 expression. We generated a stable BCLC9 cell line that expresses miR122 (BCLC9-miR122). Restitution of miR122 in BCLC9 cells, decreases cell proliferation rate and reduces significantly tumor size in vivo. BCLC9-miR122 cells down-regulate expression of MYC, KLF4, FOXM1, AKT2 and AKT3 genes and up-regulate FOXO1 and FOXO3A gene expression. In addition, miR122 transfected cells decreased AKT2 kinase activation while decreased FOXO1 and FOXO3A protein inactivation. Reduction in tumor size in BCLC9-miR122 associated with an increase in p38MAPK protein expression and activation leading to a low phospho-ERK1/2 to phospho-p38 ratio. Treatment of miR122 positive cells with an inhibitor of TGFBR1 activation, abolished tumor dormancy program and recovered cell proliferation rate through a Smad-independent TGF-β response.HCC stem-like cells can be directed towards cell differentiation and tumor dormancy by restoring miR122 expression. We demonstrate, for the first time, that dormancy program is achieved through a Smad-independent TGF-β pathway. Reestablishing miR122 expression is a promising therapeutic strategy that would work concurrently reducing tumor aggressiveness and decreasing disease recurrence.

Blunting Autoantigen-induced FOXO3a Protein Phosphorylation and Degradation Is a Novel Pathway of Glucocorticoids for the Treatment of Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease affecting multiple organs. Glucocorticoids (GCs), the potent anti-inflammatory drugs, remain as a cornerstone in the treatment for SLE; nevertheless, their clinical efficacy is compromised by the side effects of long term treatment and resistance. To improve the therapeutic efficacy of GCs in SLE, it is important to further decipher the molecular mechanisms of how GCs exert their anti-inflammatory effects. In this investigation, FOXO3a was identified as a molecule that was down-regulated in the course of SLE. Of interest, GC treatment was found to rescue FOXO3a expression both in SLE mice and in SLE patients. Gain- and loss-of-function studies demonstrated that FOXO3a played a crucial role in GC treatment of SLE via inhibiting inflammatory responses. Further studies showed that the up-regulation of FOXO3a by GCs relied on the suppression of pI3K/AKT-mediated FOXO3a phosphorylation and the arrest of FOXO3a in the nucleus. Finally, our data revealed that FOXO3a was critical for GC-mediated inhibition of NF-κB activity, which might involve its interaction with NF-κB p65 protein. Collectively, these data indicated that FOXO3a played an important role in GC treatment of SLE by suppressing pro-inflammatory response, and targeting FOXO3a might provide a novel therapeutic strategy against SLE.

Abstract 3507: Rhamnetin enhances anti-proliferative and apoptotic effects on prostate cancer cells

Abstract Dysregulation of oxidative stress plays important role in both tumor development and response to anticancer drug treatment. Increased oxidative stress induces reactive oxygen species (ROS) which stimulate genes viz. FoxO3a and Klotho. Klotho modulates stress-induced senescence, whereas forkhead transcription factor; FoxO3a is known to involve in antioxidant, anti-proliferative and tumor suppressor functions. We have previously demonstrated that FoxO3a is deregulated in human prostate adenocarcinoma and in transgenic adenocarcinoma of the mouse prostate (TRAMP) model that mimics progressive forms of human disease. Here we demonstrate the potential involvement of Klotho protein in prostate cancer progression and using a natural agent; rhamnetin as anticancer in TRAMP mice modulated Klotho and FoxO3a signaling by altering ROS to inhibit prostate cancer. Per-oral feeding of rhamnetin at 10- and 20-μg/mouse/day (gavaged in 0.2 ml vehicle containing 0.5% methyl cellulose and 0.025% Tween 20) to TRAMP mice for six days per week for 16 weeks, starting at 8 weeks of age, exhibited marked reduction in tumor growth. Rhamnetin intake led to increased antioxidant potentials, which was evident from increased levels of klotho, Nrf2 and with significantly increased expression of Shc66 in the dorso-lateral prostate. Reports suggested that secretory klotho binds to multiple FGFRs (fibroblast growth factor receptors) with different affinity. However, TRAMP mice exhibited significantly increased FGFR1 expression in dorso-lateral prostates, which were reduced significantly after rhamnetin feeding to TRAMP mice. Furthermore, inhibited FGFR1 expression resulted in reduced phospho-Akt (Ser-473) and ERK1/2 phosphorylation in rhamnetin fed mice than control. This modulated signaling cascade decreased nuclear presence of p-Akt (Ser-473), which restricted FoxO3a phosphorylation and led to nuclear retention in dorso-lateral prostate. Increased nuclear presence of FoxO3a elevated transcriptional activity and expression of FoxO-dependent apoptotic protein, Bim. Moreover, rhamnetin feeding to TRAMP mice reduced Alix expression, which further helped in accelerating apoptotic machinery. Similarly rhamnetin treatment to human prostate cancer LNCaP and PC-3 cells also resulted in the restoration of Klotho and FoxO3a in the nucleus. Increase nuclear presence of Klotho and FoxO3a protein may be a decisive factor for increase resistance to oxidative stress and inhibited prostate cancer progression. Our finding suggests that rhamnetin may be developed as a preventive agent in prostate cancer. Citation Format: Riddhi Patel, Rebecca Pakradooni, Christine Oak, Natarajan Bhaskaran, Sanjeev Shukla. Rhamnetin enhances anti-proliferative and apoptotic effects on prostate cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3507.

Abstract 3520: Solidago virga-aurea extract induced apoptosis of breast cancer cells through FOXO3 mediated bim expression

Abstract Solidago virga-aurea (European goldenrod or woundwort) is an herbaceous perennial plant of the family Asteraceae. It is used for medical system with astringent, diuretic, antiseptic and homeopathic properties. And the studies about Solidago virga-aurea are in progress for the other functions. Recently, we found that Solidago virga-aurea extract induces apoptosis of breast cancer cells. MTT assay showed that cellular viability was decreased in Solidago virga-aurea extract treated MDA-MB-231 and MCF-7 breast cancer cell lines by dose and time dependent manner. And apoptotic protein, especially bim, was increased in Solidago virga-aurea extract treated MCF-7 cell line. However, anti-apoptotic protein, Bcl-xL, does not changed by Solidago virga-aurea extract. In order to find out positive regulator of bim expression, we tested whether FOXO (forkhead-box transcription factor, group O) 3a and AMPK (5’ AMP-activated protein kinase) protein expression. In immunoblot analysis showed that FOXO3a expression level was dramatically increased in Solidago virga-aurea treated MCF-7 cells without changes of AMPK expression. Moreover, immunostaining showed that Solidago virga-aurea induces co-localization of FOXO3a and bim proteins in MCF-7 and MB-231 cells. Taken together, Solidago virga-aurea induces apoptosis by FOXO3a mediated up-regulation of bim protein. The mechanisms of Solidago virga-aurea mediated FOXO3a regulation is not fully elucidated yet, but further studies about this molecular mechanism between two proteins will give some ideas for cancer therapies for certain breast cancer patients. Citation Format: Haesung Kim, Chea Ha Kim, Jeong-Hyeon Kim, Jeong-Min Lee, Jeong-Ho Jeon, Jae-Yong Lee. Solidago virga-aurea extract induced apoptosis of breast cancer cells through FOXO3 mediated bim expression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3520.

Abstract 4646: Synergistic antitumor effect of selinexor, a selective inhibitor of nuclear export (SINE) compound and trastuzumab in a mouse model of breast cancer

Abstract Introduction: Selinexor (sel) is an oral, first-in-class SINE compound that binds to the primary nuclear exporter XPO1/CRM1. XPO1 exports include major tumor suppressor proteins (TSPs) leading to their inactivation. Inhibition of XPO1 results in nuclear retention of TSPs and restores their normal functions. We have previously shown that HER2+ breast cancer (BC) cells are highly sensitive to sel (SKBR3 and MCF7 IC50 0.21 and 0.073 μM, respectively). Here we tested the hypothesis that inhibition of HER2 signaling with trastuzumab (tras) synergizes with sel as both mediators (PI3K-AKT and Ras-Raf-MEK pathways) and effectors (FOXO3A, MDM2) of the HER2 pathway are among direct cargos of XPO1. Methods: The effects of sel and tras alone or in combination on cell viability were tested on MCF7 and BT474 HER2+ BC cells using MTT assays. Total RNA and whole protein cell lysates were extracted and analyzed by qPCR and by immunoblots. In-vivo, BT474 cells were used to derive a xenograft mouse model. Mice were treated with sub-therapeutic doses of sel and tras alone or in combination and with the therapeutic dose of sel. Tumor Growth (TG) was monitored for 60 days. Xenografts were harvested and analyzed by immunohistochemestry (IHC). Results: Sel-tras combination was highly effective in-vitro and in-vivo. In MTT assays, both sel and tras demonstrated low IC50 values (nanomolar) and when combined, they synergistically inhibit proliferation. In-vivo, the combination resulted in significant survival benefit and enhanced TG inhibition of 88% compared to the monotherapy groups 57% (sel) and 25% (tras). Interestingly, an increase in pro-survival cytoplasmic FOXO3A protein was observed in the tras-treated group however, in the combination group, FOXO3A, which is an XPO1cargo, was restricted to the nucleus where it can trigger apoptosis. In addition, a synergistic increase in P27 protein, the G1 phase arrest regulator, was observed in the sel-tras-treated group. While in the monotherapy groups p-ERK was not modulated, in the combination group an increase in cytoplasmic p-ERK was observed, suggesting an affect on its downstream regulators of apoptosis. Indeed, apoptosis was evident in the combination samples by increased IHC staining to Cleaved caspase3 and Apoptag. In addition, an increase in PARP protein was observed in the tras-treated group however, in the combination group, PARP was dramatically reduced. We have previously shown that selinxor modulates DNA Damage Response (DDR) proteins suggesting that one mechanism by which the combination enhances cancer cell death is by accumulation of DNA damage that cannot be resolved. Conclusion: Sel-tras combination is a novel candidate therapy to HER2+ BC modulating DDR and apoptosis. It synergizes to induce survival benefit and TG inhibition. These data provide rational support for study of sel-tras combination in clinical trials. Citation Format: Sivan Elloul, Hua Hua Chang, Boris Klebanov, Trinayan Kashyap, Maxwell Werman, Margaret Lee, Yosef Landesman, Sharon Shacham, Michael Kauffman, Sharon Y. Friedlander. Synergistic antitumor effect of selinexor, a selective inhibitor of nuclear export (SINE) compound and trastuzumab in a mouse model of breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4646.

Vitexin attenuates acute doxorubicin cardiotoxicity in rats via the suppression of oxidative stress, inflammation and apoptosis and the activation of FOXO3a.

Doxorubicin (DOX) is one of the most effective chemotherapeutic drugs. However, its clinical use has been hampered due to the development of cardiotoxicity. Vitexin, which is the active ingredient of hawthorn leaf extract, has various biological activities, including antioxidant and anti-inflammatory actions. The present study aimed to investigate whether vitexin was able to protect against DOX-induced acute cardiotoxicity in model rats and the mechanisms of this protective effect were assessed. Adult Sprague-Dawley rats were randomly assigned into the control (saline only), model (DOX only) and vitexin-treated (DOX plus vitexin) groups. Rats in the model and vitexin-treated groups were injected with DOX (2 mg/kg; i.p.) once a week for 4 weeks. Rats in the vitexin-treated group were administered 30 mg/kg oral vitexin once daily at doses for 4 weeks. The levels of lactate dehydrogenase, creatine kinase isoenzyme-MB, malondialdehyde, superoxide dismutase, catalase and myeloperoxidase were assessed using assay kits. The levels of inflammatory mediators, including tumor necrosis factor-α, interleukin (IL)-1β, IL-6, nuclear factor (NF)-κB, and caspase-3, were assayed by the enzyme-linked immunosorbent assay method. Western blot analysis was performed to assess the protein expression levels of p-FOXO3a. Vitexin pretreatment significantly protected against DOX-induced myocardial damage, which was characterized by increased serum creatine kinase isoenzyme-MB and lactate dehydrogenase. Vitexin significantly ameliorated oxidative stress injury evoked by DOX, demonstrated by the inhibition of lipid peroxidation and the elevation of antioxidant enzyme activities. Furthermore, DOX provoked inflammatory responses by increasing the expression levels of IL-1β, IL-6, NF-κB and tumor necrosis factor-α, whereas vitexin pretreatment significantly inhibited these inflammatory responses. Notably, DOX induced apoptotic tissue damage by increasing caspase-3 activity, whereas vitexin administration was able to decrease caspase-3 activity. In addition, vitexin induced elevated FOXO3a protein expression levels in the vitexin-treated group. In conclusion, the findings of the present study suggested that vitexin possesses cardioprotective action against DOX-induced cardiotoxicity by suppressing oxidative stress, inflammation and apoptotic signals.

Protein kinase C dependent development of dendritic cells.

Abstract Dendritic cells (DC) are professional antigen presenting cells that are important activators of adaptive immune responses and play a role in the activation of CD8+ T cells that target tumor cells. Our lab has also previously shown that protein kinase C (PKC) βII is essential for DC differentiation and that tumor derived factors, particularly IL-6, inhibit DC differentiation by repressing the expression of PKCβII through a Stat3 dependent mechanism. However, the pathways activated downstream of PKCβII during DC differentiation have yet to be fully characterized. To study this, we used the K562 cell line, which differentiates into DCs with addition of PMA, a known chemical activator of conventional PKC isoenzymes. We have also shown previously that PMA specifically activates PKCβII in K562 cells. Addition of PMA to K562 cells showed the activation of the ERK1/2 and NFκB pathways, including both the canonical and non-canonical NFκB pathways. Chemical inhibition of either the ERK1/2 or NFκB pathway yielded immature DCs with a reduced capacity to stimulate T-cell proliferation when incubated with PMA. Inhibition of ERK1/2 also showed a change in levels of RelB, a NFκB transcription factor, with PMA treatment and during PMA induced DC differentiation. Our lab has also shown that matured DCs reduce Foxo3a protein levels with the stimulation of CD80/CD86 through an Akt dependent mechanism. Inhibition of either the ERK1/2 or NFκB pathways showed an increase in Foxo3a levels, indicating a role for the ERK1/2 and NFκB pathways in Foxo3a regulation during differentiation into mature DCs outside of Akt. Taken together, these results show an integral role for both the ERK and NFκB pathways downstream of PKCβII activation during DC differentiation.

Aqueous Extracts of Toona sinensis Leaves Inhibit Renal Carcinoma Cell Growth and Migration Through JAK2/stat3, Akt, MEK/ERK, and mTOR/HIF-2α Pathways

ABSTRACTToona sinensis (TS) is a type of deciduous tree, which is distributed widely in Asia and used as a traditional herb medicine. Previously, we demonstrated that aqueous extracts of TS leaves (TSL-1) induce apoptosis in two clear types of human renal carcinoma cells (ccRCC) via mitochondria-dependent pathway. In this study, we further investigated the more detailed mechanism of TSL-1-induced antitumor effects on ccRCCs. TSL-1 treatment arrested ccRCC cells in G0/G1 phase through the decrease of cyclin D1, cyclin-dependent kinase (CDK)2, and CDK4 as well as induction of p53 and FOXO3a protein expressions. On the other hand, the inhibitory effects of TSL-1 on migration were also observed in 786-O and A-498 cells. Mechanically, we presented that TSL-1 could suppress cell cycle progression and motility via inhibiting the phosphorylation of JAK2/stat3, Akt, MEK/ERK, and mTOR in a concentration- and time-dependent manner. Moreover, we found that TSL-1 inhibited p21, HIF-2α, c-Myc, VEGF, and MMP9 protein expressions in both cell lines. In conclusion, these findings suggested that TS-induced apoptosis and its antimigration activity in ccRCC cells were accompanied by inactivation of several oncogenic pathways.

Sirt1 deficiency protects cochlear cells and delays the early onset of age-related hearing loss in C57BL/6 mice

Hearing gradually declines with age in both animals and humans, and this condition is known as age-related hearing loss (AHL). Here, we investigated the effects of deficiency of Sirt1, a member of the mammalian sirtuin family, on age-related cochlear pathology and associated hearing loss in C57BL/6 mice, a mouse model of early-onset AHL. Sirt1 deficiency reduced age-related oxidative damage of cochlear hair cells and spiral ganglion neurons and delayed the early onset of AHL. In cultured mouse inner ear cell lines, Sirt1 knockdown increased cell viability under oxidative stress conditions, induced nuclear translocation of Foxo3a, and increased acetylation status of Foxo3a. This resulted in increased activity of the antioxidant enzyme catalase. In young wild-type mice, both Sirt1 and Foxo3a proteins resided in the cytoplasm of the supporting cells within the organ of Corti of the cochlea. Therefore, our findings suggest that SIRT1 promotes early-onset AHL through suppressing FOXO3a-mediated oxidative stress resistance in the cochlea of C57BL/6 mice.