Regulation of Akt/FOXO3a/GSK-3beta/AR signaling network by isoflavone in prostate cancer cells

We have previously shown that genistein could inhibit Akt activation and down-regulate AR (androgen receptor) and PSA (prostate-specific antigen) expression in prostate cancer (PCa) cells. However, pure genistein showed increased lymph node metastasis in an animal model, but such an adverse effect was not seen with isoflavone, suggesting that further mechanistic studies are needed for elucidating the role of isoflavone in PCa. It is known that FOXO3a and GSK-3beta, targets of Akt, regulate cell proliferation and apoptosis. Moreover, FOXO3a, GSK-3beta, and Src are AR regulators and regulate transactivation of AR, mediating the development and progression of PCa. Therefore, we investigated the molecular effects of isoflavone on the Akt/FOXO3a/GSK-3beta/AR signaling network in hormone-sensitive LNCaP and hormone-insensitive C4-2B PCa cells. We found that isoflavone inhibited the phosphorylation of Akt and FOXO3a, regulated the phosphorylation of Src, and increased the expression of GSK-3beta, leading to the down-regulation of AR and its target gene PSA. We also found that isoflavone inhibited AR nuclear translocation and promoted FOXO3a translocation to the nucleus. By electrophoretic mobility shift assay and chromatin immunoprecipitation assay, we found that isoflavone inhibited FOXO3a binding to the promoter of AR and increased FOXO3a binding to the p27(KIP1) promoter, resulting in the alteration of AR and p27(KIP1) expression, the inhibition of cell proliferation, and the induction of apoptosis in both androgen-sensitive and -insensitive PCa cells. These results suggest that isoflavone-induced inhibition of cell proliferation and induction of apoptosis are partly mediated through the regulation of the Akt/FOXO3a/GSK-3beta/AR signaling network. In conclusion, our data suggest that isoflavone could be useful for the prevention and/or treatment of PCa.     

Regulation of GSK-3 beta in the proliferation and apoptosis of human thyrocytes investigated using a GSK-3 beta-targeting RNAi adenovirus expression vector: involvement the Wnt/beta-catenin pathway

Disorders in the proliferation and apoptosis of thyrocytes may induce goitre, adenoma and carcinoma in the thyroid. The Wnt/beta-catenin pathway has been demonstrated to be involved in the regulation of cell proliferation, differentiation and apoptosis in various cell lines. The regulatory mechanism on the proliferation and differentiation of thyrocytes is not well characterized. In the present study, a GSK-3beta-targeting RNA interference (RNAi) adenovirus vector was constructed and delivered to primary human thyrocytes. Results showed that the expression of beta-catenin protein in primary human thyrocytes was increased after GSK-3beta-targeting RNAi adenovirus infection, the proliferation of primary human thyrocytes was significantly stimulated using Bromodeoxyuridine (BrdU) assay, while cell apoptosis was slightly affected which was observed through flow cytometry. It is concluded that the Wnt/beta-catenin pathway plays a significant role in the regulation of the proliferation of primary human thyrocytes.     

GSK-3beta inhibition enhances sorafenib-induced apoptosis in melanoma cell lines

Glycogen synthase kinase-3beta (GSK-3beta) can participate in the induction of apoptosis or, alternatively, provide a survival signal that minimizes cellular injury. We previously demonstrated that the multikinase inhibitor sorafenib induces apoptosis in melanoma cell lines. In this report, we show that sorafenib activates GSK-3beta in multiple subcellular compartments and that this activation undermines the lethality of the drug. Pharmacologic inhibition and/or down-modulation of the kinase enhances sorafenib-induced apoptosis as determined by propidium iodide staining and by assessing the mitochondrial release of apoptosis-inducing factor and Smac/DIABLO. Conversely, the forced expression of a constitutively active form of the enzyme (GSK-3beta(S9A)) protects the cells from the apoptotic effects of the drug. This protective effect is associated with a marked increase in basal levels of Bcl-2, Bcl-x(L), and survivin and a diminution in the degree to which these anti-apoptotic proteins are down-modulated by sorafenib exposure. Sorafenib down-modulates the pro-apoptotic Bcl-2 family member Noxa in cells with high constitutive GSK-3beta activity. Pharmacologic inhibition of GSK-3beta prevents the disappearance of Noxa induced by sorafenib and enhances the down-modulation of Mcl-1. Down-modulation of Noxa largely eliminates the enhancing effect of GSK-3 inhibition on sorafenib-induced apoptosis. These data provide a strong rationale for the use of GSK-3beta inhibitors as adjuncts to sorafenib treatment and suggest that preservation of Noxa may contribute to their efficacy.     

Regulation of dishevelled and beta-catenin in rat skeletal muscle: an alternative exercise-induced GSK-3beta signaling pathway

beta-catenin is a multifunctional protein involved in cell-cell adhesion and the Wnt signaling pathway. beta-Catenin is activated upon its dephosphorylation, an event triggered by Dishevelled (Dvl)-mediated phosphorylation and deactivation of glycogen synthase kinase-3beta (GSK-3beta). In skeletal muscle, both insulin and exercise decrease GSK-3beta activity, and we tested the hypothesis that these two stimuli regulate beta-catenin. Immunoblotting demonstrated that Dvl, Axin, GSK-3beta, and beta-catenin proteins are expressed in rat red and white gastrocnemius muscles. Treadmill running exercise in vivo significantly decreased beta-catenin phosphorylation in both muscle types, with complete dephosphorylation being elicited by maximal exercise. beta-Catenin dephosphorylation was intensity dependent, as dephosphorylation was highly correlated with muscle glycogen depletion during exercise (r(2) = 0.84, P < 0.001). beta-Catenin dephosphorylation was accompanied by increases in GSK-3beta Ser(9) phosphorylation and Dvl-GSK-3beta association. In contrast to exercise, maximal insulin treatment (1 U/kg body wt) had no effect on skeletal muscle beta-catenin phosphorylation or Dvl-GSK-3beta interaction. In conclusion, exercise in vivo, but not insulin, increases the association between Dvl and GSK-3beta in skeletal muscle, an event paralleled by beta-catenin dephosphorylation.     

Concomitant degradation of beta-catenin and GSK-3 beta potently contributes to glutamate-induced neurotoxicity in rat hippocampal slice cultures

Increasingly, published evidence links glutamate with the pathogenesis of Alzheimer's disease. We investigated the molecular mechanism underlying glutamate-induced neurotoxicity in hippocampus, which is primarily linked to cognitive dysfunction in Alzheimer's disease. Acute exposure of rat hippocampal slices to glutamate significantly induced cell death, as determined by media lactate dehydrogenase levels and PI staining. Moreover, this was accompanied by Ca²⁺ influx and calpain-1 activation, as confirmed by the proteolytic pattern of spectrin. Notably, glutamate-induced calpain-1 activation decreased the level of β-catenin, and this process appeared to be independent of glycogen synthase kinase 3beta (GSK-3β), since glutamate also led to loss of GSK-3β. Calpeptin, a calpain inhibitor, attenuated the glutamate-mediated degradations of spectrin, synaptophysin, and β-catenin except GSK-3β and modestly increased cell survival. In contrast, the NMDA receptor antagonist 2-amino-5-phosphonopentanoic acid (APV) effectively reduced all glutamate-evoked responses, i.e., the breakdowns of spectrin, synaptophysin, β-catenin and GSK-3β, and cell death. Pharmacological studies and in vitro calpain-1 proteolysis confirmed that in the glutamate-treated hippocampus, calpain-1-mediated decrease of β-catenin could occur independently of GSK-3β and of proteasome, and that GSK-3β degradation is independent of calpain-1. These findings together provide the first direct evidence that glutamate promotes the down-regulations of β-catenin and GSK-3β, which potently contribute to neurotoxicity in hippocampus during excitotoxic cell death, and a molecular basis for the protection afforded by calpeptin and APV from the neurotoxic effect of glutamate.     

Axin, a negative regulator of the Wnt signaling pathway, forms a complex with GSK-3beta and beta-catenin and promotes GSK-3beta-dependent phosphorylation of beta-catenin.

Glycogen synthase kinase-3 (GSK-3) mediates epidermal growth factor, insulin and Wnt signals to various downstream events such as glycogen metabolism, gene expression, proliferation and differentiation. We have isolated here a GSK-3beta-interacting protein from a rat brain cDNA library using a yeast two-hybrid method. This protein consists of 832 amino acids and possesses Regulators of G protein Signaling (RGS) and dishevelled (Dsh) homologous domains in its N- and C-terminal regions, respectively. The predicted amino acid sequence of this GSK-3beta-interacting protein shows 94% identity with mouse Axin, which recently has been identified as a negative regulator of the Wnt signaling pathway; therefore, we termed this protein rAxin (rat Axin). rAxin interacted directly with, and was phosphorylated by, GSK-3beta. rAxin also interacted directly with the armadillo repeats of beta-catenin. The binding site of rAxin for GSK-3beta was distinct from the beta-catenin-binding site, and these three proteins formed a ternary complex. Furthermore, rAxin promoted GSK-3beta-dependent phosphorylation of beta-catenin. These results suggest that rAxin negatively regulates the Wnt signaling pathway by interacting with GSK-3beta and beta-catenin and mediating the signal from GSK-3beta to beta-catenin.     

Zingerone suppresses cell proliferation via inducing cellular apoptosis and inhibition of the PI3K/AKT/mTOR signaling pathway in human prostate cancer PC‐3 cells

Prostate cancer (PCa) is both the foremost and second cause of cancer death in the male population. Patients with hormone‐dependent PCa are initially sensitive to androgen‐deprivation therapy, later the cancer progress to a hormone‐independent state and fails to respond and progress to the metastatic stage, where the cells gain the ability to escape cell death and develop resistance to current therapies, thereby leading to migration, invasion, and metastasis of cancer. Many clinical trials using nutraceuticals on cancer using human subjects have also been extensively studied, these studies confirm the efficacy of drugs tested in in vitro and in vivo preclinical models. Among various dietary phytochemicals, ginger is commonly used in the diet and possesses many active principles that act against cancer. Among various active principles, zingerone is a key active phenolic compound present in Zingiber officinale (Ginger), it has potent antioxidant property and it acts against carcinogens. The present study evaluated the efficacy of zingerone at different doses on the PCa cell line regarding apoptosis, upstream signing molecules such as Akt/mTOR, and migration metastasis. A cell viability assay using MTT was performed to estimate the percentage of viability of zingerone‐treated PC‐3 cells. The mitochondrial membrane potential, intracellular reactive oxygen species, and apoptosis induction in the zingerone‐treated PC‐3 cells were studied by using different fluorescence staining techniques. The expression patterns of PI3K, AKT, p‐AKT, mTOR, and p‐mTOR were investigated through the Western blot analysis assay. Zingerone induces apoptosis and alters Akt/mTOR molecules; it also inhibits cell adhesion and migration of PCa cells. From the present study, it is concluded that zingerone effectively induces apoptosis and inhibits cancer signaling, thereby acting as a potent drug against PCa.     

Down-regulation of Notch1 by gamma-secretase inhibition contributes to cell growth inhibition and apoptosis in ovarian cancer cells A2780

The release of Notch intracellular domain (NICD) is mediated by γ-secretase. γ-Secretase inhibitors have been shown to be potent inhibitors of NICD. We hypothesized that Notch1 is acting as an oncogene in ovarian cancer and that inhibition of Notch1 would lead to inhibition of cell growth and apoptotic cell death in ovarian cancer cells. In this study, expressions of Notch1 and hes1 in four human ovarian cancer (A2780, SKOV3, HO-8910, and HO-8910PM), and one ovarian surface (IOSE 144) cell lines were detected by Western blot and quantitative real-time RT-PCR. The effects of γ-secretase inhibition (N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester, DAPT) were measured by MTT assay, flow cytometry, ELISA and colony-forming assay. Our results showed that Notch1 and hes1 were found in all the four human ovarian cancer and IOSE 144 cell lines, and they were significantly higher in ovarian cancer cells A2780 compared to another four ovarian cells. Down-regulation of Notch1 expression by DAPT was able to substantially inhibit cell growth, induce G1 cell cycle arrest and induce cell apoptosis in A2780 in dose- and time-dependent manner. In addition, hes1 was found to be down-regulated in dose- and time-dependent manner by DAPT in A2780. These results demonstrate that treatment with DAPT leads to growth inhibition and apoptosis of A2780 cells in dose- and time-dependent manner. These findings also support the conclusion that blocking of the Notch1 activity by γ-secretase inhibitors represents a potentially attractive strategy of targeted therapy for ovarian cancer.     

Targeting bone remodeling by isoflavone and 3,3'-diindolylmethane in the context of prostate cancer bone metastasis

Prostate cancer (PCa) bone metastases have long been believed to be osteoblastic because of bone remodeling leading to the formation of new bone. However, recent studies have shown increased osteolytic activity in the beginning stages of PCa bone metastases, suggesting that targeting both osteolytic and osteoblastic mediators would likely inhibit bone remodeling and PCa bone metastasis. In this study, we found that PCa cells could stimulate differentiation of osteoclasts and osteoblasts through the up-regulation of RANKL, RUNX2 and osteopontin, promoting bone remodeling. Interestingly, we found that formulated isoflavone and 3,3'-diindolylmethane (BR-DIM) were able to inhibit the differentiation of osteoclasts and osteoblasts through the inhibition of cell signal transduction in RANKL, osteoblastic, and PCa cell signaling. Moreover, we found that isoflavone and BR-DIM down-regulated the expression of miR-92a, which is known to be associated with RANKL signaling, EMT and cancer progression. By pathway and network analysis, we also observed the regulatory effects of isoflavone and BR-DIM on multiple signaling pathways such as AR/PSA, NKX3-1/Akt/p27, MITF, etc. Therefore, isoflavone and BR-DIM with their multi-targeted effects could be useful for the prevention of PCa progression, especially by attenuating bone metastasis mechanisms.     

Pigment epithelium derived factor regulates human Sost/Sclerostin and other osteocyte gene expression via the receptor and induction of Erk/GSK-3beta/beta-catenin signaling

Mutations in Serpinf1 gene which encodes pigment epithelium derived factor (PEDF) lead to osteogenesis imperfecta type VI whose hallmark is defective mineralization. We reported that PEDF suppressed expression of Sost/Sclerostin and other osteocyte related genes in mineralizing osteoblast cultures and suggested that this could be part of the mechanisms by which PEDF regulates matrix mineralization (Li et al. J Cellular Phys. 2014). We have used a long-term differentiated mineralizing osteoblast culture (LTD) to define mechanisms by which PEDF regulates osteocyte gene expression. LTD cultures were established by culturing human osteoblasts in an osteogenic medium for 4?months followed by analysis of osteocytes related genes and encoded proteins. LTD cells synthesized Sclerostin, matrix extracellular phosphoglycoprotein (MEPE) and dentin matrix protein (DMP-1) and their synthesis was reduced by treatment with PEDF. Treatment of the cultures with PEDF induced phosphorylation of Erk and glycogen synthase kinase 3-beta (GSK-3β), and accumulation of nonphosphorylated β-catenin. Inhibition of Erk activation and neutralizing antibodies to the pigment epithelium derived receptor (PEDF-R) suppressed GSK-3β phosphorylation and accumulation of nonphosphorylated β-catenin in presence of PEDF. Topflash assays demonstrated that PEDF activated luciferase reporter activity and this activity was inhibited by treatment with Erk inhibitor or neutralizing antibodies to PEDF-R. Dickkopf-related protein 1 treatment of the cells in presence of PEDF had minimal effect suggesting that GSK-3β phosphorylation and accumulation of nonphosphorylayted β-catenin may not involve LRP5/6 in osteocytes. Taken together, the data demonstrate that PEDF regulates osteocyte gene expression through its receptor and possible involvement of Erk/GSK-3β/β-catenin signaling pathway.     

CSNK2B contributes to colorectal cancer cell proliferation by activating the mTOR signaling

The function of Casein kinase 2 beta (CSNK2B) in human malignancies has drawn increasing attention in recent years. However, its role in colorectal cancer (CRC) remains unclear. In the present study, we aimed to explore the expression and biological functions of CSNK2B in CRC. Public gene expression microarray data from online database and immunohistochemistry analysis demonstrated that CSNK2B was highly expressed in CRC tissues than in normal tissues. In vitro and in vivo cellular functional experiments showed that increased CSNK2B expression promoted CRC cell viability and tumorigenesis of CRC. Further western blots and rescue experiments confirmed that CSNK2B promoted CRC cell proliferation mainly by activating the mTOR signaling pathway. These findings identified CSNK2B as a novel oncogene contributing to the development of CRC.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12079-021-00619-1.     

DPP3/CDK1 contributes to the progression of colorectal cancer through regulating cell proliferation,cell apoptosis,and cell migration

At present, colorectal cancer (CRC) has become a serious threat to human health in the world. Dipeptidyl peptidase 3 (DPP3) is a zinc-dependent hydrolase that may be involved in several physiological processes. However, whether DPP3 affects the development and progression of CRC remains a mystery. This study is the first to demonstrate the role of DPP3 in CRC. Firstly, the results of immunohistochemistry analysis showed the upregulation of DPP3 in CRC tissues compared with normal tissues, which is statistically analyzed to be positively correlated with lymphatic metastasis, pathological stage, positive number of lymph nodes. Moreover, the high expression of DPP3 predicts poor prognosis in CRC patients. In addition, the results of cell dysfunction experiments clarified that the downregulation of DPP3 significantly inhibited cell proliferation, colony formation, cell migration, and promoted apoptosis in vitro. DPP3 depletion could induce cell apoptosis by upregulating the expression of BID, BIM, Caspase3, Caspase8, HSP60, p21, p27, p53, and SMAC. In addition, downregulation of DPP3 can reduce tumorigenicity of CRC cells in vivo. Furthermore, CDK1 is determined to be a downstream target of DPP3-mediated regulation of CRC by RNA-seq, qPCR, and WB. The interaction between DPP3 and CDK1 shows mutual regulation. Specifically, downregulation of DPP3 can accentuate the effects of CDK1 knockdown on the function of CRC cells. Overexpression of CDK1 alleviates the inhibitory effects of DPP3 knockdown in CRC cells. In summary, DPP3 has oncogene-like functions in the development and progression of CRC by targeting CDK1, which may be an effective molecular target for the prognosis and treatment of CRC.Subject terms: Cancer, Diseases     

Regulation of circGOLPH3 and its binding protein CBX7 on the proliferation and apoptosis of prostate cancer cells

To clarify the mechanism of circGOLPH3 regulation on prostate cancer cells, we performed an overexpression and interference circGOLPH3 assay in prostate cancer cells PC-3 and then evaluated cellular viability, proliferation, cell cycle, and apoptosis of prostate cancer cells by MTT, CCK8, Edu stain, TUNEL stain, and flow cytometry. Binding proteins of CircGOLPH3 were identified by RNA pull-down, mass spectrometry, and RNA-binding protein immunoprecipitation (RIP) assays. The expressions of CircGOLPH3 and CBX7 were measured by qRT-PCR. The results showed that after overexpression of circGOLPH3, the proliferative capacity and the viability of PC-3cells were significantly improved, whereas apoptosis was inhibited. CircGOLPH3 could bind to the CBX7 protein that was highly expressed in the PC-3 cell. Additionally, a functional test on CBX7 showed that the CBX7 overexpression notably improved the proliferative capacity and the viability of PC-3 cells and decreased cellular apoptosis, which was consistent with the effects of circGOLPH3. The validated the present study that circGOLPH3 and its binding protein CBX7 can promote prostate cancer cell proliferation and inhibit apoptosis.