Downregulating AKT Activity Research Articles

Carboxyl-terminal modulator protein regulates Akt signaling during skeletal muscle atrophy in vitro and a mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive neuromuscular disease involving motor neuron death, paralysis and, ultimately, respiratory failure. Motor neuron dysfunction leads to target skeletal muscle atrophy involving dysregulation of downstream cell survival, growth and metabolic signaling. Decreased Akt activity is linked to muscle atrophy in ALS and is associated with increased atrophy gene expression. Unfortunately, the regulating mechanism of Akt activity in atrophic muscle remains unclear. Recent research indicates a role of carboxyl-terminal modulator protein (CTMP) in Akt-signaling related neurologic dysfunction and skeletal muscle metabolism. CTMP is known to bind and reduce Akt phosphorylation and activation. We hypothesized that CTMP expression might progressively increase in ALS skeletal muscle as the disease progresses, downregulating Akt activity. We found that CTMP protein expression significantly increased in hindlimb skeletal muscle in the mSOD1G93A mouse model of ALS in late stages of the disease (P < 0.05), which negatively correlated with Akt phosphorylation over this period (R2 = −0.77). Co-immunoprecipitation of Akt revealed CTMP binding in pre-symptomatic and end-stage skeletal muscle, suggesting a possible direct role in reduced Akt signaling during disease progression. Inflammatory TNFα and downstream cellular degradation process markers for autophagy, lysosome production, and atrophy significantly increased in a pattern corresponding to increased CTMP expression and reduced Akt phosphorylation. In an in vitro model of skeletal muscle atrophy, differentiated C2C12 cells exhibited reduced Akt activity and decreased FOXO1 phosphorylation, a process known to promote transcription of atrophy genes in skeletal muscle. These results corresponded with increased Atrogin-1 expression compared to healthy control cells (P < 0.05). Transfection with CTMP siRNA significantly increased Akt phosphorylation in atrophic C2C12 cells, corresponding to significantly decreased CTMP expression. In conclusion, this is the first study to provide evidence for a link between elevated CTMP expression, downregulated Akt phosphorylation and muscle atrophy in ALS and clearly demonstrates a direct influence of CTMP on Akt phosphorylation in an in vitro muscle cell atrophy model.

Regulation of AKT Activity by Inhibition of the Pleckstrin Homology Domain-PtdIns(3,4,5)P3 Interaction Using Flavonoids.

The serine-threonine kinase AKT plays a pivotal role in tumor progression and is frequently overactivated in cancer cells; this protein is therefore a critical therapeutic target for cancer intervention. We aimed to identify small molecule inhibitors of the pleckstrin homology (PH) domain of AKT to disrupt binding of phosphatidylinositol-3,4,5-trisphosphate (PIP3), thereby downregulating AKT activity. Liposome pulldown assays coupled with fluorescence spectrometry were used to screen flavonoids for inhibition of the AKT PH-PIP3 interaction. Western blotting was used to determine the effects of the inhibitors on AKT activation in cancer cells, and in silico docking was used for structural analysis and optimization of inhibitor structure. Several flavonoids showing up to 50% inhibition of the AKT PH-PIP3 interaction decreased the level of AKT activation at the cellular level. In addition, the modified flavonoid showed increased inhibitory effects and the approach would be applied to develop anticancer drug candidates. In this study, we provide a rationale for targeting the lipid-binding domain of AKT, rather than the catalytic kinase domain, in anticancer drug development.

The Antiarrhythmic Drug, Amiodarone, Decreases AKT Activity and Sensitizes Human Acute Myeloid Leukemia Cells to Apoptosis by ABT-263

BackgroundSuccessful treatment of leukemia requires new medications to combat drug resistance, but the development of novel therapies is an arduous and risky endeavor. Repurposing currently approved drugs or those already in clinical development to treat other indications is a more practical approach. Moreover, combinatorial therapeutics are often more efficacious than single agent therapeutics because the former can simultaneously target multiple pathways that mitigate tumor aggressiveness and induce cancer cell death. Material and MethodsIn this study, we combined the class III antiarrhythmic agent amiodarone and the BH3 mimetic ABT-263 based on data from a prior drug screen to assess the degree of apoptotic induction in 2 human leukemia cell lines. ResultsThe combination yielded statistically significant increases in apoptosis in both cell lines by downregulating AKT activity and increasing cleaved caspase-3. ConclusionsOverall, our findings suggest that combining K+ channel blockers with prosurvival Bcl-2 family inhibitors is a promising therapeutic approach in treating leukemia.

PDK1 Deficit Impairs the Development of the Dentate Gyrus in Mice.

3-Phosphoinositide-dependent protein kinase-1 (PDK1) is crucial for the development of the dentate gyrus (DG), the first gateway receiving afferent inputs from the entorhinal cortex. However, the role of PDK1 in DG development is unclear. In the present study, by crossing Pdk1fl/fl mice with the Emx1-cre line, we identified that the ablation of PDK1 disrupted the development of DG via decreasing the proliferation, and increasing the differentiation of dentate neural progenitor cells, downregulating AKT activity and upregulating GSK3β signaling. Moreover, PDK1 deletion disrupted the distribution of Reelin+ cells and decreased the level of Reelin mRNA which may contribute to the defective migration of progenitor cells and the disrupted radial glial scaffolds. Furthermore, the inhibition of GSK3β activity partially restored the decreased proliferation of primary neural stem cells in vitro. Taken together, our data indicated that the ablation of PDK1 affected the proliferation and differentiation of dentate neural progenitor cells in mice.

Hot topic: Ceramide inhibits insulin sensitivity in primary bovine adipocytes

In nonruminants, the sphingolipid ceramide inhibits insulin sensitivity by inactivating protein kinase B (AKT) within the insulin-signaling pathway. We have established that ceramide accrual develops with impaired systemic insulin action in ruminants during the transition from gestation to lactation, dietary palmitic acid supplementation, or controlled nutrient restriction. We hypothesized that ceramide promotes AKT inactivation and antagonizes insulin sensitivity in primary bovine adipocytes. Stromal-vascular cells were grown from bovine adipose tissue explants and cultured in differentiation media. To modify ceramide supply, we treated differentiated adipocytes with (1) myriocin, an inhibitor of de novo ceramide synthesis, or (2) cell-permeable C2:0-ceramide. Insulin-stimulated AKT activation (i.e., phosphorylation) and 2-deoxy-D-[3H]-glucose (2DOG) uptake were measured. Treatment of adipocytes with myriocin consistently decreased concentrations of ceramide, monohexosylceramide, and lactosylceramide. The insulin-stimulated ratio of phosphorylated AKT to total AKT was increased with myriocin but decreased with C2:0-ceramide. Moreover, adipocyte insulin-stimulated 2DOG uptake was decreased with C2:0-ceramide and increased with myriocin. We conclude that ceramide inhibits insulin-stimulated glucose uptake by downregulating AKT activation in primary bovine adipocytes.

Antitumor activity of selenium compounds and its underlying mechanism in human oral squamous cell carcinoma cells: A preliminary study

ObjectiveOral cancer is an aggressive disease that infiltrates the adjacent tissues and frequently metastasizes to lymph nodes in the neck. Currently, no chemotherapy effectively prevents its metastasis. Selenium compounds have been recently scrutinized as chemotherapeutic agents for various cancers. In this study, we aimed to investigate the antitumor activity of selenium compounds and elucidate the underlying inhibitory mechanism of these agents in oral cancer cells. MethodsThe growth inhibitory effects of selenium compounds (sodium selenite, selenomethionine, and Se-methylselenocysteine) on human oral squamous cell carcinoma (HOSCC) cell lines (HSC-3, HSC-4, and SAS) were evaluated by MTT assay. Selenite-induced apoptosis, caspase activity, and endoplasmic reticulum (ER) stress in HSC-3 cells were evaluated by flow cytometry and western blot. Effects of selenite on Akt expression in HSC-3 cells were evaluated by ELISA and western blot. ResultsSelenium compounds significantly inhibited cell growth and induced apoptosis in HOSCC cell lines. HSC-3 cells, in particular, were highly sensitive to selenite. In selenite-treated HSC-3 cells, caspase-3, 8, and 9 were conspicuously activated; pretreatment with pan-caspase inhibitor or caspase-12 inhibitor dramatically reduced selenite-induced apoptosis; ER stress markers, caspase-12 and eIF-2α, were highly activated, but Akt activation in the Akt/phosphoinositide-3-kinase pathway was downregulated. ConclusionOur findings indicate that selenite induces apoptosis in HOSCC cell lines in a caspase-dependent manner through mitochondrial, death-receptor, and ER stress pathways. In addition, selenite could exhibit antitumor activity by downregulating Akt activation, which plays an important role in cell growth and chemotherapy resistance.

Abstract 5253: Oral administration of Withaferin-A effectively suppresses prostate carcinogenesis in PTEN-Knockout mice

Abstract Androgen ablation therapy alone or in combination with radiation therapy is the mainstay for prostate cancer (CaP), which is initially effective in de-bulking the tumor volume, however, eventually these patients will progress to a castration-resistant prostate cancer (CRPC), which requires more aggressive chemotherapies. Treating CRPC with second generation androgen -ablation based therapies like enzalutamide and abiraterone exhibit only a short window of therapeutic benefit resulting in chemoresistance. Hence, there is an immediate need for identification of novel targets to eradicate CRPC effectively. We and others have reported targeting AKT activation could efficiently suppress the growth of CRPC cells; in fact a number of clinical trials have shown some promise that AKT could be an attractive target for CRPC. In fact, clinical studies from our lab have suggested that AKT activation predominantly occurs in Gleason stage specific manner suggesting identification of novel drugs to inhibit AKT activation is imperative. Our earlier finding suggests that Withaferin-A (WA), an herbal molecule effectively inhibits the growth of CRPC cells by downregulating AKT activation and its downstream pro-survival events. Present study investigated whether oral administration of WA may inhibit tumor development in Pten conditional knockout mice [(Pten-loxp/loxp:PB-Cre4+) (Pten-KO)] which will be a unique and appropriate preclinical model to study the AKT driven prostate tumor. Oral administration of WA for 45 weeks effectively prevented the tumor growth without any significant signs of toxicity to organs in Pten-KO mice. Vehicle and WA (5mg/kg body weight) were orally given up to 45 weeks. Gross pathological studies suggested a significant inhibition of growth and micro metastasis in WA-treated mice as compared to the vehicle treated mice. On microscopic examination of the prostatic tissue, we found that the WA-treated tumors showed more necrosis than the control group and some of the tumors were more differentiated than the controls in the same group. All the organs were completely submitted for histological evaluation. None of the WA-treated mice organs showed any metastatic lesion on the other hand, we found discrete metastasis to lungs in the control tumors. Our ongoing immunohistochemistry analysis may corroborate our in vitro findings that down regulation of AKT and epithelial-to-mesenchymal transition (EMT) markers such as β-catenin, snail, and vimentin in WA treated tumors as compared to the control mice. Overall, these results provide important scientific evidence in support of AKT signaling as a target to inhibit CRPC as well as metastatic CRPC. Citation Format: Chendil Damodaran, Suman Suman, Trinath P. Das, Houda Alatassi, Murali K. Ankem. Oral administration of Withaferin-A effectively suppresses prostate carcinogenesis in PTEN-Knockout mice. [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 5253.

Xist reduction in breast cancer upregulates AKT phosphorylation via HDAC3-mediated repression of PHLPP1 expression.

Long noncoding RNAs (lncRNAs) dysregulated in cancer potentially play oncogenic or tumor-suppressive roles. While the X inactivate-specific transcript (Xist) lncRNA is important for X-chromosome inactivation in female cells, very little is known about the role of Xist in human breast cancer in modulating cellular pathway(s). Here, we show that Xist expression is significantly reduced in breast tumor samples and cancer cell lines. Xist knockdown or overexpression resulted in increased or decreased levels, respectively, of AKT phosphorylation and cell viability. Further studies revealed an inverse correlation between Xist and phospho-AKT levels in breast cancer samples. Additionally, Xist knockdown-elicited increase of cell viability was attenuated by AKT inhibitor. These results suggest that Xist negatively regulates cell viability via inhibition of AKT activation. Interestingly, decreased Xist expression in breast cancer samples was associated with reduced levels of Jpx RNA, an lncRNA that positively regulates Xist promoter activity. Accordingly, Jpx knockdown enhanced AKT activation and cell viability. We also demonstrate that knockdown of Xist or SPEN, an intermediator protein to link Xist, SMRT co-repressor and HDAC3 complexes for X-chromosome inactivation, decreased expression of PHLPP1, a phosphatase to remove AKT phosphorylation, via increased HDAC3 recruitment to the PHLPP1 promoter, correlating with increased AKT phosphorylation. Our findings elucidate the tumor suppressor role of Xist in breast cancer and provide the molecular basis of Xist in downregulating AKT activation.

Triptolide has anticancer and chemosensitization effects by down-regulating Akt activation through the MDM2/REST pathway in human breast cancer.

Triptolide has been shown to exhibit anticancer activity. However, its mechanism of action is not clearly defined. Herein we report a novel signaling pathway, MDM2/Akt, is involved in the anticancer mechanism of triptolide. We observed that triptolide inhibits MDM2 expression in human breast cancer cells with either wild-type or mutant p53. This MDM2 inhibition resulted in decreased Akt activation. More specifically, triptolide interfered with the interaction between MDM2 and the transcription factor REST to increase expression of the regulatory subunit of PI3-kinase p85 and consequently inhibit Akt activation. We further showed that, regardless of p53 status, triptolide inhibited proliferation, induced apoptosis, and caused G1 phase cell cycle arrest. Triptolide also enhanced the cytotoxic effect of doxorubicin. MDM2 inhibition plays a causative role in these effects. The inhibitory effect of triptolide on MDM2-mediated Akt activation was eliminated with MDM2 overexpression. MDM2-overexpressing tumor cells, in turn, were less susceptible to the anticancer and chemosensitization effects of triptolide than control cells. Triptolide also exhibited anticancer and chemosensitization effects in nude mouse xenograft model. When it was administered to tumor-bearing nude mice, triptolide inhibited tumor growth and enhanced the antitumor effects of doxorubicin. In summary, triptolide has anticancer and chemosensitization effects by down-regulating Akt activation through the MDM2/REST pathway in human breast cancer. Our study helps to elucidate the p53-independent regulatory function of MDM2 in Akt signaling, offering a novel view of the mechanism by which triptolide functions as an anticancer agent.

Paris saponin I inhibits proliferation and promotes apoptosis through down-regulating AKT activity in human non-small-cell lung cancer cells and inhibiting ERK expression in human small-cell lung cancer cells

PSI regulated AKT activity in NSCLC and inhibited ERK expression in SCLC.

N-methylhemeanthidine chloride, a novel Amaryllidaceae alkaloid, inhibits pancreatic cancer cell proliferation via down-regulating AKT activation

We previously reported the isolation of a novel Amaryllidaceae alkaloid, N-methylhemeanthidine chloride (NMHC), from Zephyranthes candida, which exhibits potent cytotoxicity in a spectrum of tumor cells. However, the mechanism of action remains unclear. Using multiple cell lines derived from human pancreatic cancer, one of the most mortal and refractory human malignancies, we further studied the NMHC-mediated cytotoxicity and found that it induced drastic cytotoxicity in pancreatic cancer cells whereas an insignificant effect on a noncancerous cell line. The NMHC-mediated growth inhibition was more severe than the first-line chemotherapeutic agent gemcitabine, leading to cell cycle arrest, apoptotic death and decreased glycolysis. NMHC exerted its function through down-regulating AKT activation, and the ectopic expression of activated AKT rescued the growth inhibition. Consistently, NMHC injections in a pancreatic cancer xenograft model manifested the anti-tumor effect in vivo. Engrafted tumor cells underwent AKT attenuation and apoptotic death upon treatments. As such, we here demonstrate the AKT inhibition may be one of the mechanisms by which NMHC decreases tumor cell survival rate in vitro and in vivo. Our data thereby suggest that NMHC holds great promise as a potent chemotherapeutic agent against pancreatic cancer and sheds new light on obtaining such agents from natural products toward therapeutic purposes.

Regulation of hematopoietic stem cells using protein transduction domain–fused Polycomb

The Polycomb-group complex is a chromatin regulatory factor that is classified into two different complexes: Polycomb repressive complex 1 and 2. Components of Polycomb repressive complex 1 are involved in the self-renewal of hematopoietic stem cells. Bmi1, one of these components, maintains the immaturity of neural and cancer stem cells as well as that of hematopoietic stem cells. We constructed recombinant protein transduction domain (PTD)-Polycomb proteins and transduced them into murine bone marrow (BM) cells. We designed and fused the PTD-protein transduction domain to three proteins (i.e., green fluorescent protein, Bmi1, and Mel18). Murine BM cells were incubated for 48 h and each PTD-Polycomb protein was added. Then, we analyzed the function of hematopoiesis using the colony assay and transplantation. BM cells exposed to PTD-Bmi1 showed an increased number of colonies. In contrast, BM cells exposed to PTD-Mel18 or to both proteins showed a decreased number of colonies. Hematopoietic cells derived from PTD-Bmi1-transduced BM cells were significantly increased in the peripheral blood at 6 weeks after transplantation. Moreover, 80% of mice transplanted with PTD-Bmi1-transduced BM cells died at 8 to 24 weeks after transplantation. However, only a few early deaths were observed in the mice transplanted with BM cells exposed to both PTD-Bmi1 and PTD-Mel18. We expect that hematopoietic stem cells could proliferate after transduction with PTD-Bmi1, but this may generate undesirable effects, e.g., tumorigenesis. Thus, Bmi1 and Mel18 have opposing functions and are present in distinct complexes.

Cell death is induced by ciglitazone, a peroxisome proliferator-activated receptor γ (PPARγ) agonist, independently of PPARγ in human glioma cells

Peroxisome proliferator-activated receptor γ (PPARγ) regulates multiple signaling pathways, and its agonists induce apoptosis in various cancer cells. However, their role in cell death is unclear. In this study, the relationship between ciglitazone (CGZ) and PPARγ in CGZ-induced cell death was examined. At concentrations of greater than 30μM, CGZ, a synthetic PPARγ agonist, activated caspase-3 and induced apoptosis in T98G cells. Treatment of T98G cells with less than 30μM CGZ effectively induced cell death after pretreatment with 30μM of the PPARγ antagonist GW9662, although GW9662 alone did not induce cell death. This cell death was also observed when cells were co-treated with CGZ and GW9662, but was not observed when cells were treated with CGZ prior to GW9662. In cells in which PPARγ was down-regulated cells by siRNA, lower concentrations of CGZ (<30μM) were sufficient to induce cell death, although higher concentrations of CGZ (⩾30μM) were required to induce cell death in control T98G cells, indicating that CGZ effectively induces cell death in T98G cells independently of PPARγ. Treatment with GW9662 followed by CGZ resulted in a down-regulation of Akt activity and the loss of mitochondrial membrane potential (MMP), which was accompanied by a decrease in Bcl-2 expression and an increase in Bid cleavage. These data suggest that CGZ is capable of inducing apoptotic cell death independently of PPARγ in glioma cells, by down-regulating Akt activity and inducing MMP collapse.

Four-and-a-half-LIM protein 1 down-regulates estrogen receptor α activity through repression of AKT phosphorylation in human breast cancer cell

The Four-and-a-half LIM protein 1 (FHL-1) is a member of LIM-only protein family. It plays important roles in proliferation and apoptosis regulation of certain hepatocellular carcinoma and human breast cancer. Estrogen receptor α (ERα) is involved in the development and progression of human breast cancer. IGF/PI3K/AKT signaling pathway also plays certain roles in the program and regulation of human breast cancer and ovary cancer. However, the biological function of FHL-1 in regulation of human breast cancer and in the cross-talk of estrogen and IGF signaling pathway remains largely unknown. In this paper, we show that FHL-1 protein interacts with ERα and AKT. FHL-1 represses the translation and transcription of estrogen receptor-responsive genes through down-regulating AKT activation. In addition, FHL-1 is not only an ERα-interacting co-regulation protein, but also decreases the phosphorylation of AKT and ERα. Depression of endogenous FHL-1 by FHL-1 targeted small interfering RNA enhances the expression of these proteins and phosphorylation of AKT and ERα. These data suggest that FHL-1 may regulate ER signaling function through regulation of AKT activation besides the physical and functional interaction with ERα. By establishing a linkage role of the FHL-1 between the estrogen ERα signaling pathway and IGF/PI3K/AKT signaling pathway, this study identifies that FHL-1 proteins may be a useful molecular target for human breast cancer therapy.

Mel-18 Acts as a Tumor Suppressor by Repressing Bmi-1 Expression and Down-regulating Akt Activity in Breast Cancer Cells

The Bmi-1 oncogene is overexpressed in a number of malignancies including breast cancer. In addition to Bmi-1, mammalian cells also express four other polycomb group (PcG) proteins that are closely related to Bmi-1. Virtually nothing is known about the role of these PcG proteins in oncogenesis. We have recently reported that Mel-18, a Bmi-1-related PcG protein, negatively regulates Bmi-1 expression, and that its expression negatively correlates with Bmi-1 in proliferating and senescing human fibroblasts. Here, we report that the expression of Bmi-1 and Mel-18 inversely correlates in a number of breast cancer cell lines and in a significant number of breast tumor samples. Overexpression of Mel-18 results in repression of Bmi-1 and reduction of the transformed phenotype in malignant breast cancer cells. Furthermore, the repression of Bmi-1 by Mel-18 is accompanied by the reduction of Akt/protein kinase B (PKB) activity in breast cancer cells. Similarly, Bmi-1 knockdown using RNA interference approach results in down-regulation of Akt/PKB activity and reduction in transformed phenotype of MCF7 cells. Importantly, we show that overexpression of constitutively active Akt overrides tumor-suppressive effect of Mel-18 overexpression and the knockdown of Bmi-1 expression. Thus, our studies suggest that Mel-18 and Bmi-1 may regulate the Akt pathway in breast cancer cells, and that Mel-18 functions as a tumor suppressor by repressing the expression of Bmi-1 and consequently down-regulating Akt activity.

Antiestrogenic effect of 20S‐protopanaxadiol and its synergy with tamoxifen on breast cancer cells

20S-protopanaxadiol (aPPD) is a major gastrointestinal metabolic product of ginsenosides. The latter share structural similarity with steroids and are the main pharmacologically active component in ginseng. The authors investigated the interaction between aPPD and estrogen receptors (ER) in human breast adenocarcinoma MCF-7 cells through receptor binding assay, ER-induced gene expression, and cell proliferation both in vitro and in vivo. aPPD, but not its close analog ginsenosides, competed with the [(3)H]-17-beta estradiol (E2) for ER with IC(50) at 26.3 microM. aPPD alone weakly induced luciferase reporter-gene expression controlled by an estrogen-regulated element, which was completely blocked by tamoxifen. aPPD alone, or in synergy with tamoxifen, blocked E2-induced transcriptional activation. aPPD also inhibited colony formation of endometrial cancer cells. aPPD potently inhibited estrogen-stimulated MCF-7 cell proliferation and synergistically enhanced the cytotoxicity of tamoxifen on both ER+ MCF-7 and ER- MDA-MB231 cells. Furthermore, aPPD, but not tamoxifen, inhibited Akt phosphorylation. Growth of MCF-7 xenograft tumor supplemented with E2 was completely inhibited in animals treated with aPPD, tamoxifen, or aPPD plus tamoxifen. These results suggested that aPPD inhibits estrogen-stimulated gene expression and cell proliferation in ER-positive breast cancer cells. In addition, aPPD synergistically enhances cytotoxicity of tamoxifen in an ER-independent fashion, probably by down-regulating Akt activity.