Transcriptional Up-Regulation of FBXW7 by KCa1.1 K+ Channel Inhibition through the Nrf2 Signaling Pathway in Human Prostate Cancer LNCaP Cell Spheroid Model.

The tumor suppressor gene F-box and WD repeat domain-containing (FBXW) 7 reduces cancer stemness properties by promoting the protein degradation of pluripotent stem cell markers. We recently demonstrated the transcriptional repression of FBXW7 by the three-dimensional (3D) spheroid formation of several cancer cells. In the present study, we found that the transcriptional activity of FBXW7 was promoted by the inhibition of the Ca2+-activated K+ channel, KCa1.1, in a 3D spheroid model of human prostate cancer LNCaP cells through the Akt-Nrf2 signaling pathway. The transcriptional activity of FBXW7 was reduced by the siRNA-mediated inhibition of the CCAAT-enhancer-binding protein C/EBP δ (CEBPD) after the transfection of miR223 mimics in the LNCaP spheroid model, suggesting the transcriptional regulation of FBXW7 through the Akt-Nrf2-CEBPD-miR223 transcriptional axis in the LNCaP spheroid model. Furthermore, the KCa1.1 inhibition-induced activation of FBXW7 reduced (1) KCa1.1 activity and protein levels in the plasma membrane and (2) the protein level of the cancer stem cell (CSC) markers, c-Myc, which is a molecule degraded by FBXW7, in the LNCaP spheroid model, indicating that KCa1.1 inhibition-induced FBXW7 activation suppressed CSC conversion in KCa1.1-positive cancer cells.

Down-Regulation of CYP3A4 by the KCa1.1 Inhibition Is Responsible for Overcoming Resistance to Doxorubicin in Cancer Spheroid Models.

The large-conductance Ca2+-activated K+ channel, KCa1.1, plays a pivotal role in cancer progression, metastasis, and the acquisition of chemoresistance. Previous studies indicated that the pharmacological inhibition of KCa1.1 overcame resistance to doxorubicin (DOX) by down-regulating multidrug resistance-associated proteins in the three-dimensional spheroid models of human prostate cancer LNCaP, osteosarcoma MG-63, and chondrosarcoma SW-1353 cells. Investigations have recently focused on the critical roles of intratumoral, drug-metabolizing cytochrome P450 enzymes (CYPs) in chemoresistance. In the present study, we examined the involvement of CYPs in the acquisition of DOX resistance and its overcoming by inhibiting KCa1.1 in cancer spheroid models. Among the CYP isoforms involved in DOX metabolism, CYP3A4 was up-regulated by spheroid formation and significantly suppressed by the inhibition of KCa1.1 through the transcriptional repression of CCAAT/enhancer-binding protein, CEBPB, which is a downstream transcription factor of the Nrf2 signaling pathway. DOX resistance was overcome by the siRNA-mediated inhibition of CYP3A4 and treatment with the potent CYP3A4 inhibitor, ketoconazole, in cancer spheroid models. The phosphorylation levels of Akt were significantly reduced by inhibiting KCa1.1 in cancer spheroid models, and KCa1.1-induced down-regulation of CYP3A4 was reversed by the treatment with Akt and Nrf2 activators. Collectively, the present results indicate that the up-regulation of CYP3A4 is responsible for the acquisition of DOX resistance in cancer spheroid models, and the inhibition of KCa1.1 overcame DOX resistance by repressing CYP3A4 transcription mainly through the Akt-Nrf2-CEBPB axis.

KCa3.1 regulates cell cycle progression by modulating Ca2+ signaling in murine preosteoblasts.

Osteoblasts synthesize and deposit essential components of the extracellular bone matrix and collagen scaffolds, leading to mineralized bone formation. Therefore, the proliferation of preosteoblasts (precursors of mature osteoblasts) helps in regulating skeletal homeostasis. This study demonstrated that the functional expression of KCa3.1, an intermediate-conductance Ca2+-activated K+ channel, is markedly upregulated in murine preosteoblastic MC3T3-E1 cells in the G0/G1 phase. The enhancement of KCa3.1 is involved in the establishment of more negative membrane potentials in MC3T3-E1 cells. This hyperpolarization can promote intracellular Ca2+ signaling because store-operated Ca2+ channels are activated. Treatment with TRAM-34, a specific KCa3.1 inhibitor, attenuated the cell cycle progression from the G0/G1 phase to the S/G2/M phases. In MC3T3-E1 cells, KCa3.1 significantly promoted the transition from the G1 phase to the S phase. KCa3.1 inhibition also caused G0 phase cell accumulation. Furthermore, TRAM-34 decreased the expression of alkaline phosphatase, bone sialoprotein, and osteocalcin, osteoblast differentiation markers in MC3T3-E1 cells, and inhibited the endochondral ossification of murine metatarsals. These results reveal novel ways by which KCa3.1 activity can strongly modulate osteoblast maturation during bone formation.

Downregulation of IL-8 and IL-10 by the Activation of Ca2+-Activated K+ Channel KCa3.1 in THP-1-Derived M2 Macrophages.

THP-1-differentiated macrophages are useful for investigating the physiological significance of tumor-associated macrophages (TAMs). In the tumor microenvironment (TME), TAMs with the M2-like phenotype play a critical role in promoting cancer progression and metastasis by inhibiting the immune surveillance system. We examined the involvement of Ca2+-activated K+ channel KCa3.1 in TAMs in expressing pro-tumorigenic cytokines and angiogenic growth factors. In THP-1-derived M2 macrophages, the expression levels of IL-8 and IL-10 were significantly decreased by treatment with the selective KCa3.1 activator, SKA-121, without changes in those of VEGF and TGF-ß1. Furthermore, under in vitro experimental conditions that mimic extracellular K+ levels in the TME, IL-8 and IL-10 levels were both significantly elevated, and these increases were reversed by combined treatment with SKA-121. Among several signaling pathways potentially involved in the transcriptional regulation of IL-8 and IL-10, respective treatments with ERK and JNK inhibitors significantly repressed their transcriptions, and treatment with SKA-121 significantly reduced the phosphorylated ERK, JNK, c-Jun, and CREB levels. These results strongly suggest that the KCa3.1 activator may suppress IL-10-induced tumor immune surveillance escape and IL-8-induced tumorigenicity and metastasis by inhibiting their production from TAMs through ERK-CREB and JNK-c-Jun cascades.

KCa3.1 inhibition-induced activation of the JNK/c-Jun signaling pathway enhances IL-10 expression in peripherally-induced regulatory T cells.

The KCa3.1 inhibition up-regulates IL-10 expression in regulatory T (Treg) cells in the recovery phase of inflammatory bowel disease (IBD) model mice; however, the underlying signaling pathway remains unclear. We investigated the involvement of AP-1 (Fos/Jun) and NF-κB in the expression of IL-10 and its transcription factors (TFs) in in vitro-induced mouse splenic Treg cells. The pharmacological inhibition of JNK reversed KCa3.1 inhibition-induced increases in the expression of IL-10 and its TFs. The inhibition of KCa3.1 increased phosphorylated JNK and c-Jun levels. Therefore, the JNK/c-Jun signaling pathway may contribute to the KCa3.1 inhibition-induced up-regulation of IL-10 in peripherally-induced Treg cells.

KCa1.1 K+ Channel Inhibition Overcomes Resistance to Antiandrogens and Doxorubicin in a Human Prostate Cancer LNCaP Spheroid Model.

Several types of K+ channels play crucial roles in tumorigenicity, stemness, invasiveness, and drug resistance in cancer. Spheroid formation of human prostate cancer (PC) LNCaP cells with ultra-low attachment surface cultureware induced the up-regulation of cancer stem cell markers, such as NANOG, and decreased the protein degradation of the Ca2+-activated K+ channel KCa1.1 by down-regulating the E3 ubiquitin ligase, FBXW7, compared with LNCaP monolayers. Accordingly, KCa1.1 activator-induced hyperpolarizing responses were larger in isolated cells from LNCaP spheroids. The pharmacological inhibition of KCa1.1 overcame the resistance of LNCaP spheroids to antiandrogens and doxorubicin (DOX). The protein expression of androgen receptors (AR) was significantly decreased by LNCaP spheroid formation and reversed by KCa1.1 inhibition. The pharmacological and genetic inhibition of MDM2, which may be related to AR protein degradation in PC stem cells, revealed that MDM2 was responsible for the acquisition of antiandrogen resistance in LNCaP spheroids, which was overcome by KCa1.1 inhibition. Furthermore, a member of the multidrug resistance-associated protein subfamily of ABC transporters, MRP5 was responsible for the acquisition of DOX resistance in LNCaP spheroids, which was also overcome by KCa1.1 inhibition. Collectively, the present results suggest the potential of KCa1.1 in LNCaP spheroids, which mimic PC stem cells, as a therapeutic target for overcoming antiandrogen- and DOX-resistance in PC cells.

Ca2+ -activated K+ channel KCa 1.1 as a therapeutic target to overcome chemoresistance in three-dimensional sarcoma spheroid models.

The large-conductance Ca2+ -activated K+ channel KCa 1.1 plays a pivotal role in tumor development and progression in several solid cancers. The three-dimensional (3D) in vitro cell culture system is a powerful tool for cancer spheroid formation, and mimics in vivo solid tumor resistance to chemotherapy in the tumor microenvironment (TME). KCa 1.1 is functionally expressed in osteosarcoma and chondrosarcoma cell lines. KCa 1.1 activator-induced hyperpolarizing responses were significantly larger in human osteosarcoma MG-63 cells isolated from 3D spheroid models compared with in those from adherent 2D monolayer cells. The present study investigated the mechanisms underlying the upregulation of KCa 1.1 and its role in chemoresistance using a 3D spheroid model. KCa 1.1 protein expression levels were significantly elevated in the lipid-raft-enriched compartments of MG-63 spheroids without changes in its transcriptional level. 3D spheroid formation downregulated the expression of the ubiquitin E3 ligase FBXW7, which is an essential contributor to KCa 1.1 protein degradation in breast cancer. The siRNA-mediated inhibition of FBXW7 in MG-63 cells from 2D monolayers upregulated KCa 1.1 protein expression. Furthermore, a treatment with a potent and selective KCa 1.1 inhibitor overcame the chemoresistance of the MG-63 and human chondrosarcoma SW-1353 spheroid models to paclitaxel, doxorubicin, and cisplatin. Among several multidrug resistance ATP-binding cassette transporters, the expression of the multidrug resistance-associated protein MRP1 was upregulated in both spheroids and restored by the inhibition of KCa 1.1. Therefore, the pharmacological inhibition of KCa 1.1 may be an attractive new strategy for acquiring resistance to chemotherapeutic drugs in the TME of KCa 1.1-positive sarcomas.

Increased Interleukin-10 Expression by the Inhibition of Ca2+-Activated K+ Channel KCa3.1 in CD4+CD25+ Regulatory T Cells in the Recovery Phase in an Inflammatory Bowel Disease Mouse Model.

Inflammatory bowel diseases (IBD) are chronic inflammatory diseases of the gastrointestinal tract arising from abnormal responses of the innate and adaptative immune systems. Interleukin (IL)-10-producing CD4+CD25+ regulatory T (Treg) cells play a protective role in the recovery phase of IBD. In the present study, the effects of the administration of the selective Ca2+-activated K+ channel KCa3.1 inhibitor TRAM-34 on disease activities were examined in chemically induced IBD model mice. IBD disease severity, as assessed by diarrhea, visible fecal blood, inflammation, and crypt damage in the colon, was significantly lower in mice administered 1 mg/kg TRAM-34 than in vehicle-administered mice. Quantitative real-time polymerase chain reaction examinations showed that IL-10 expression levels in the recovery phase were markedly increased by the inhibition of KCa3.1 in mesenteric lymph node (mLN) Treg cells of IBD model mice compared with vehicle-administered mice. Among several positive and negative transcriptional regulators (TRs) for IL-10, three positive TRs-E4BP4, KLF4, and Blimp1-were upregulated by the inhibition of KCa3.1 in the mLN Treg cells of IBD model mice. In mouse peripheral CD4+CD25+ Treg cells induced by lectin stimulation, IL-10 expression and secretion were enhanced by the treatment with TRAM-34, together with the upregulation of E4BP4, KLF4, and Blimp1. Collectively, the present results demonstrated that the pharmacological inhibition of KCa3.1 decreased IBD symptoms in the IBD model by increasing IL-10 production in peripheral Treg cells and that IL-10high Treg cells produced by the treatment with KCa3.1 inhibitor may contribute to efficient Treg therapy for chronic inflammatory disorders, including IBD. SIGNIFICANCE STATEMENT: Pharmacological inhibition of Ca2+-activated K+ channel KCa3.1 increased IL-10 expression in peripheral Treg cells, together with the upregulation of the transcriptional regulators of IL-10: Krüppel-like factor 4, E4 promoter-binding protein 4, and/or B lymphocyte-induced maturation protein 1. The manipulation of IL-10high-producing Treg cells by the pharmacological inhibition of KCa3.1 may be beneficial in the treatment of chronic inflammatory diseases such as inflammatory bowel disease.

Downregulation of the Ca2+-activated K+ channel KCa3.1 in mouse preosteoblast cells treated with vitamin D receptor agonist.

The maturity of osteoblasts by proliferation and differentiation in preosteoblasts is essential for maintaining bone homeostasis. The beneficial effects of vitamin D on bone homeostasis in mammals have been demonstrated experimentally and clinically. However, the direct actions of vitamin D on preosteoblasts remain to be fully elucidated. In this study, we found that the functional activity of intermediate-conductance Ca2+-activated K+ channels (KCa3.1) positively regulated cell proliferation in MC3T3-E1 cells derived from mouse preosteoblasts by enhancing intracellular Ca2+ signaling. We examined the effects of treatment with vitamin D receptor (VDR) agonist on the expression and activity of KCa3.1 by real-time PCR examination, Western blotting, Ca2+ imaging, and patch clamp analyses in mouse MC3T3-E1 cells. Following the downregulation of KCa3.1 transcriptional modulators such as Fra-1 and HDAC2, KCa3.1 activity was suppressed in MC3T3-E1 cells treated with VDR agonists. Furthermore, application of the KCa3.1 activator DCEBIO attenuated the VDR agonist-evoked suppression of cell proliferation rate. These findings suggest that a decrease in KCa3.1 activity is involved in the suppression of cell proliferation rate in VDR agonist-treated preosteoblasts. Therefore, KCa3.1 plays an important role in bone formation by promoting osteoblastic proliferation under physiological conditions.

Possible Contribution of Inflammation-Associated Hypoxia to Increased K2P5.1 K+ Channel Expression in CD4+ T cells of the Mouse Model for Inflammatory Bowel Disease.

Previous studies have reported the up-regulation of the two-pore domain K+ channel K2P5.1 in the CD4+ T cells of patients with multiple sclerosis (MS) and rheumatoid arthritis (RA), as well as in a mouse model of inflammatory bowel disease (IBD). However, the mechanisms underlying this up-regulation remain unclear. Inflammation-associated hypoxia is involved in the pathogenesis of autoimmune diseases, such as IBD, MS, and RA, and T cells are exposed to a hypoxic environment during their recruitment from inflamed tissues to secondary lymphoid tissues. We herein investigated whether inflammation-associated hypoxia is attributable to the increased expression and activity of K2P5.1 in the splenic CD4+ T cells of chemically-induced IBD model mice. Significant increases in hypoxia-inducible factor (HIF)-1α transcripts and proteins were found in the splenic CD4+ T cells of the IBD model. In the activated splenic CD4+ T cells, hypoxia (1.5% O2) increased K2P5.1 expression and activity, whereas a treatment with the HIF inhibitor FM19G11 but not the selective HIF-2 inhibitor exerted the opposite effect. Hypoxia-exposed K2P5.1 up-regulation was also detected in stimulated thymocytes and the mouse T-cell line. The class III histone deacetylase sirtuin-1 (SIRT1) is a downstream molecule of HIF-1α signaling. We examined the effects of the SIRT1 inhibitor NCO-01 on K2P5.1 transcription in activated CD4+ T cells, and we found no significant effects on the K2P5.1 transcription. No acute compensatory responses of K2P3.1-K2P5.1 up-regulation were found in the CD4+ T cells of the IBD model and the hypoxia-exposed T cells. Collectively, these results suggest a mechanism for K2P5.1 up-regulation via HIF-1 in the CD4+ T cells of the IBD model.

[Ca2+-activated K+ channels as cancer therapeutic targets].

Similar to calcium (Ca2+) and chloride (Cl-) ion channels/transporters, potassium (K+) channels have been recognized as a crucial cancer treatment target. Recent studies have provided convincing evidences of positive correlation between elevated expression levels of Ca2+-activated K+ (KCa) channels and cancer proliferation, metastasis, and poor patient prognosis. In cancer cells, KCa1.1 and KCa3.1 KCa channels are co-localized with Ca2+-permeable Orai/TRP channels to provide a positive-feedback loop for Ca2+ entry. They are responsible for the promotion of cell growth and metastasis in the different types of cancer, and are therefore potential therapeutic targets and biomarkers for cancer. We determined the epigenetic and post-transcriptional dysregulation of KCa3.1 by class I histone deacetylase inhibitors in breast and prostate cancer cells. We further determined the transcriptional repression and protein degradation of KCa1.1 by vitamin D receptor agonists and androgen receptor antagonists, which are expected as potential therapeutic drugs for triple-negative breast cancer. The anti-inflammatory cytokine, interleukin-10 (IL-10) is an immunosuppressive factor involved in tumorigenesis, and plays a crucial role in escape from tumor immune surveillance. We determined KCa3.1 activators are a possible therapeutic option to suppress the tumor-promoting activities of IL-10. These results may provide new insights into cancer treatment focused on Ca2+-activated K+ channels.

Inhibition of Interleukin 10 Transcription through the SMAD2/3 Signaling Pathway by Ca2+-Activated K+ Channel KCa3.1 Activation in Human T-Cell Lymphoma HuT-78 Cells.

The hyperpolarization induced by intermediate-conductance Ca2+-activated K+ channel (KCa3.1) activation increases the driving force for Ca2+ influx, which generally promotes cell proliferation, migration, and cytokine production in immunocompetent cells. Interleukin-10 (IL-10) from tumor-infiltrating lymphocytes and macrophages, lymphoma, and carcinoma cells facilitates escape from cancer immune surveillance; however, the role of KCa3.1 in IL-10 production remains unclear. The objective of the present study was to elucidate the involvement of KCa3.1 in IL-10 expression and production using the human T-cell lymphoma HuT-78 cells. In HuT-78 cells, IL-10 gene expression and production were reduced by treatment with the KCa3.1 activator, as 6-hour Western blotting showed that the protein expression ratio of phosphorylated Smad2 (P-Smad2)/Smad2, but not P-Smad3/Smad3, was decreased by the treatment with KCa3.1 activator in HuT-78 cells. Concomitant with this, the nuclear translocation of P-Smad2 was inhibited by KCa3.1 activator. Furthermore, the KCa3.1 activator-induced transcriptional repression of IL-10 disappeared with pretreatment with the calmodulin kinase II (CaMKII) inhibitor KN-62 for 1 hour, and KCa3.1 activator-induced decreases in the nuclear translocation of P-Smad2 were also prevented by pretreatment with KN-62. Taken together, the KCa3.1 activator-induced transcriptional repression of IL-10 is due to the inhibition of the nuclear translocation of P-Smad2 in HuT-78 cells, resulting in the prevention of P-Smad2/3 complex formation in nuclei, and the activation of CaMKII induced by KCa3.1 activators suppresses the constitutive activation of P-Smad2/3 in HuT-78 cells. Therefore, KCa3.1 activators have potential as a therapeutic option to suppress the tumor-promoting activities of IL-10.

Histone Deacetylases Enhance Ca2+-Activated K⁺ Channel KCa3.1 Expression in Murine Inflammatory CD4⁺ T Cells.

The up-regulated expression of the Ca2+-activated K⁺ channel KCa3.1 in inflammatory CD4⁺ T cells has been implicated in the pathogenesis of inflammatory bowel disease (IBD) through the enhanced production of inflammatory cytokines, such as interferon-γ (IFN-γ). However, the underlying mechanisms have not yet been elucidated. The objective of the present study is to clarify the involvement of histone deacetylases (HDACs) in the up-regulation of KCa3.1 in the CD4⁺ T cells of IBD model mice. The expression levels of KCa3.1 and its regulators, such as function-modifying molecules and transcription factors, were quantitated using a real-time polymerase chain reaction (PCR) assay, Western blotting, and depolarization responses, which were induced by the selective KCa3.1 blocker TRAM-34 (1 µM) and were measured using a voltage-sensitive fluorescent dye imaging system. The treatment with 1 µM vorinostat, a pan-HDAC inhibitor, for 24 h repressed the transcriptional expression of KCa3.1 in the splenic CD4⁺ T cells of IBD model mice. Accordingly, TRAM-34-induced depolarization responses were significantly reduced. HDAC2 and HDAC3 were significantly up-regulated in the CD4⁺ T cells of IBD model mice. The down-regulated expression of KCa3.1 was observed following treatments with the selective inhibitors of HDAC2 and HDAC3. The KCa3.1 K⁺ channel regulates inflammatory cytokine production in CD4⁺ T cells, mediating epigenetic modifications by HDAC2 and HDAC3.

Transcriptional repression of human epidermal growth factor receptor 2 by ClC-3 Cl- /H+ transporter inhibition in human breast cancer cells.

Recent studies have indicated that the intracellular concentration of chloride ions (Cl- ) regulates gene expression in several types of cells and that Cl- modulators positively or negatively regulate the PI3K/AKT/mammalian target of rapamycin (mTOR) and signal transducer and activator of transcription (STAT)3 signaling pathways. We previously reported that the Ca2+ -activated Cl- channel anoctamine (ANO)1 regulated human epidermal growth factor receptor 2 (HER2) transcription in breast cancer YMB-1 cells. However, the mechanisms underlying ANO1-regulated HER2 gene expression have not yet been elucidated. In the present study, we showed the involvement of intracellular organelle ClC-3 Cl- /H+ transporter in HER2 transcription in breast cancer MDA-MB-453 cells. The siRNA-mediated inhibition of ClC-3, but not ANO1, markedly repressed HER2 transcription in MDA-MB-453 cells. Subsequently, treatments with the AKT inhibitor AZD 5363 and mTOR inhibitor everolimus significantly enhanced HER2 transcription in MDA-MB-453 cells, whereas that with the STAT3 inhibitor 5,15-diphenylporphyrin (5,15-DPP) inhibited it. AKT and mTOR inhibitors also significantly enhanced HER2 transcription in YMB-1 cells. The siRNA-mediated inhibition of ClC-3 and ANO1 resulted in increased AKT phosphorylation and decreased STAT3 phosphorylation in MDA-MB-453 and YMB-1 cells, respectively. The intracellular Cl- channel protein CLIC1 was expressed in both cells; however, its siRNA-mediated inhibition did not elicit the transcriptional repression of HER2. Collectively, our results demonstrate that intracellular Cl- regulation by ANO1/ClC-3 participates in HER2 transcription, mediating the PI3K/AKT/mTOR and/or STAT3 signaling pathway(s) in HER2-positive breast cancer cells, and support the potential of ANO1/ClC-3 blockers as therapeutic options for patients with resistance to anti-HER2 therapies.

Transcriptional Repression and Protein Degradation of the Ca2+-Activated K+ Channel KCa1.1 by Androgen Receptor Inhibition in Human Breast Cancer Cells.

The large-conductance Ca2+-activated K+ channel KCa1.1 plays an important role in the promotion of breast cancer cell proliferation and metastasis. The androgen receptor (AR) is proposed as a therapeutic target for AR-positive advanced triple-negative breast cancer. We herein investigated the effects of a treatment with antiandrogens on the functional activity, activation kinetics, transcriptional expression, and protein degradation of KCa1.1 in human breast cancer MDA-MB-453 cells using real-time PCR, Western blotting, voltage-sensitive dye imaging, and whole-cell patch clamp recording. A treatment with the antiandrogen bicalutamide or enzalutamide for 48 h significantly suppressed (1) depolarization responses induced by paxilline (PAX), a specific KCa1.1 blocker and (2) PAX-sensitive outward currents induced by the depolarizing voltage step. The expression levels of KCa1.1 transcripts and proteins were significantly decreased in MDA-MB-453 cells, and the protein degradation of KCa1.1 mainly contributed to reductions in KCa1.1 activity. Among the eight regulatory ß and γ subunits, LRRC26 alone was expressed at high levels in MDA-MB-453 cells and primary and metastatic breast cancer tissues, whereas no significant changes were observed in the expression levels of LRRC26 and activation kinetics of PAX-sensitive outward currents in MDA-MB-453 cells by the treatment with antiandrogens. The treatment with antiandrogens up-regulated the expression of the ubiquitin E3 ligases, FBW7, MDM2, and MDM4 in MDA-MB-453 cells, and the protein degradation of KCa1.1 was significantly inhibited by the respective siRNA-mediated blockade of FBW7 and MDM2. Based on these results, we concluded that KCa1.1 is an androgen-responsive gene in AR-positive breast cancer cells, and its down-regulation through enhancements in its protein degradation by FBW7 and/or MDM2 may contribute, at least in part, to the antiproliferative and antimetastatic effects of antiandrogens in breast cancer cells.

Role of Glyceraldehyde-Derived AGEs and Mitochondria in Superoxide Production in Femoral Artery of OLETF Rat and Effects of Pravastatin.

A complication of diabetes mellitus is the over-production of vascular superoxides, which contribute to the development of arteriosclerosis and peripheral arterial disease (PAD). Hyperglycemia induces the formation and accumulation of advanced glycation end-products (AGEs), which in turn stimulate vascular superoxide production. The mechanism underlying AGE-mediated vascular superoxide production remains to be clarified in lower limb complications associated with diabetes. In the present study, we investigated the role of AGEs and the mitochondrial respiratory complex in superoxide production in femoral arteries using the type 2 diabetes model Otsuka Long-Evans Tokushima Fatty (OLETF) rats [vs. non-diabetic Long-Evans Tokushima Otsuka (LETO) rats]. The effects of in vivo administration of pravastatin on superoxide production in femoral arteries were also examined. Using chemiluminescent assays, luminescence microscopy, and competitive enzyme-linked immunosorbent assay (ELISA), we determined that vascular superoxide production and serum glyceraldehyde-derived AGEs (Glycer-AGEs) increased in OLETF rats. Pravastatin inhibited these responses without changing serum total cholesterol concentrations. The mitochondrial complex II inhibitor thenoyltrifluoroacetone (TTFA) also inhibited vascular superoxide production. Application of Glycer-AGEs in situ increased superoxide production in the vascular wall of femoral arteries from pravastatin-treated OLETF rats, which was then inhibited by TTFA. These results suggest that hyperglycemia increases serum Glycer-AGEs, which subsequently induce superoxide production in the femoral artery of OLETF rats in a mitochondrial complex II-dependent manner. Collectively, our results have partially elucidated the pathological mechanisms leading to diabetes-related PAD, and indicate dual beneficial actions of pravastatin for the prevention of oxidative damage to the vascular wall.

Enhancement of Nitric Oxide Production Is Responsible for Minimal Intimal Hyperplasia of Autogenous Rabbit Arterial Grafts.

BACKGROUND: Vascular endothelium induces smooth muscle cell (SMC) relaxation mainly mediated by endothelium-derived nitric oxide (EDNO) and endothelium-derived hyperpolarizing factor (EDHF). It has previously been reported that functions of these endothelium factors have been greatly impaired in vein grafts. The present study was undertaken to determine whether the functions of EDNO and EDHF might be altered in artery graft.Methods and Results:In rabbits, the right carotid artery was excised and implanted in its original position as an autogenous graft ("artery graft") and the non-operated left carotid artery served as the "control artery". Histochemical changes, acetylcholine (ACh)-induced effects on the intracellular concentration of Ca2+([Ca2+]i) in endothelial cells, endothelium-dependent SMC hyperpolarization and relaxation, and tissue cGMP content were examined on post-operative day 28. "Artery graft" displayed a minimal amount of intimal hyperplasia. When compared with the "control artery", it exhibited greater ACh-induced, endothelium-dependent relaxation, but the reverse was true when EDNO production was blocked. In the "artery graft" (vs. the "control artery"), basal cGMP content was greater, whereas the [Ca2+]iincrease in endothelial cells and the endothelium-dependent SMC-hyperpolarization induced by ACh were less. CONCLUSIONS: It is suggested that the [Ca2+]i-independent EDNO production covers the loss of function of endothelium-dependent SMC hyperpolarization and minimizes intimal hyperplasia caused by surgical operation in autogenous carotid artery graft.

Dipeptidyl peptidase 4 inhibitor reduces intimal hyperplasia in rabbit autologous jugular vein graft under poor distal runoff.

BACKGROUND: Dipeptidyl peptidase 4 inhibitors are widely used in patients with type 2 diabetes mellitus to accomplish glycemic control through an increase in the blood glucagon-like peptide 1 (GLP-1) concentration. These agents also inhibit vascular inflammation (eg, in atherosclerosis). This study was undertaken to determine whether and how vildagliptin (a potent dipeptidyl peptidase 4 inhibitor) might reduce intimal hyperplasia in vein grafts. METHODS: Twelve rabbits were randomly divided into two groups; one group received vildagliptin orally (10 mg/kg/d; n = 6), whereas the control group (n = 6) did not. Vildagliptin administration was started 7 days before rabbits underwent interposition reversed autologous jugular vein grafting and ended at graft harvesting (28 days after the operation). Histochemical changes in the vascular wall were examined, as were changes in the acetylcholine-induced effects on the endothelial Ca(2+) concentration ([Ca(2+)]i) and endothelium-dependent relaxation. RESULTS: Under fasting conditions, vildagliptin increased the plasma GLP-1 concentration, without affecting plasma glucose or insulin. Acetylcholine induced endothelium-dependent relaxation only in the vildagliptin group, and this was blocked by the nitric oxide synthase inhibitor N(ω)-nitro-l-arginine. Acetylcholine did not modify the endothelial [Ca(2+)]i in either the control or vildagliptin group. Intimal hyperplasia was significantly less in the vildagliptin group (0.11 ± 0.02 mm, n = 5) than in the controls (0.31 ± 0.06 mm, n = 4; P < .01). CONCLUSIONS: Vildagliptin increased the plasma GLP-1 concentration. It also enhanced acetylcholine-induced [Ca(2+)]i-independent endothelial nitric oxide release and reduced vein graft intimal hyperplasia, independently of any glycemic control action.

Aortic superoxide production at the early hyperglycemic stage in a rat type 2 diabetes model and the effects of pravastatin.

Endothelium-derived superoxide induces vascular dysfunctions. The aim of this study was to examine the activity of protein kinase C (PKC) isoforms and endothelial nitric oxide synthase (eNOS), which leads to vascular superoxide production in type 2 diabetes, in addition to the effects of pravastatin. We studied these mechanisms in Otsuka Long-Evans Tokushima Fatty (OLETF) rats (type 2 diabetes model) at the early hyperglycemic stage (vs. non-diabetic Long-Evans Tokushima Otsuka [LETO] rats). Superoxide production and catalase activity were measured in aortas, as were the protein expressions of PKCδ and phospho-Ser(1177) eNOS. Superoxide production was increased in OLETF rats, and this increase was inhibited by the selective conventional PKC (cPKC) inhibitor Gö6976 and by the non-selective cPKC and novel PKC inhibitor GF109203X. Phospho-Ser(1177) eNOS was significantly increased in OLETF rats, whereas the protein expressions of PKCδ and phosopho-Thr(505) PKCδ and catalase activity were all greatly reduced. Pravastatin administration to OLETF rats in vivo had normalizing effects on all of these variables. The increment in superoxide production seen in OLETF rats (but not the production in pravastatin-treated OLETF rats) was abolished by high concentration of N(ω)-nitro-L-arginine methyl ester (electron transport inhibitor of eNOS), by sepiapterin (precursor of tetrahydrobiopterin), and by LY294002 (phosphatidylinositol 3-kinase [PI3-kinase] inhibitor). In OLETF rats at the early hyperglycemic stage, aortic superoxide production is increased owing to activation of uncoupled eNOS through phosphorylation by PI3-kinase/Akt. This may be related to the observed reduction in PKCδ/catalase activities. Pravastatin inhibited endothelial superoxide production via normalization of PKCδ/catalase activities.