Heat shock protein 90 inhibitor RGRN-305 potently attenuates skin inflammation.

Introduction: Chronic inflammatory skin diseases may have a profound negative impact on the quality of life. Current treatment options may be inadequate, offering an unsatisfactory response or side effects. Therefore, ongoing efforts exist to identify novel effective and safe treatments. Heat shock protein (HSP) 90 is a chaperone that promotes the activity of a wide range of client proteins including key proinflammatory molecules involved in aberrant inflammation. Recently, a proof-of-concept clinical trial of 13 patients suggested that RGRN-305 (an HSP90 inhibitor) may be an oral treatment for psoriasis. However, HSP90 inhibition may be a novel therapeutic approach extending beyond psoriasis to include multiple immune-mediated inflammatory skin diseases. Methods: This study aimed to investigate (i) the anti-inflammatory effects and mechanisms of HSP90 inhibition and (ii) the feasibility of topical RGRN-305 administration (new route of administration) in models of inflammation elicited by 12-O-tetradecanoylphorbol-13-acetate (TPA) in primary human keratinocytes and mice (irritative dermatitis murine model). Results/Discussion: In primary human keratinocytes stimulated with TPA, a Nanostring® nCounter gene expression assay demonstrated that HSP90 inhibition with RGRN-305 suppressed many proinflammatory genes. Furthermore, when measured by quantitative real-time polymerase chain reaction (RT-qPCR), RGRN-305 significantly reduced the gene expression of TNF, IL1B, IL6 and CXCL8. We next demonstrated that topical RGRN-305 application significantly ameliorated TPA-induced skin inflammation in mice. The increase in ear thickness (a marker of inflammation) was significantly reduced (up to 89% inhibition). In accordance, RT-qPCR of the ear tissue demonstrated that RGRN-305 robustly reduced the gene expression of proinflammatory markers (Tnf, Il1b, Il6, Il17A and Defb4). Moreover, RNA sequencing revealed that RGRN-305 mitigated TPA-induced alterations in gene expression and suppressed genes implicated in inflammation. Lastly, we discovered that the anti-inflammatory effects were mediated, at least partly, by suppressing the activity of NF-κB, ERK1/2, p38 MAPK and c-Jun signaling pathways, which are consistent with previous findings in other experimental models beyond skin inflammation. In summary, HSP90 inhibition robustly suppressed TPA-induced inflammation by targeting key proinflammatory cytokines and signaling pathways. Our findings suggest that HSP90 inhibition may be a novel mechanism of action for treating immune-mediated skin disease beyond psoriasis, and it may be a topical treatment option.

Heat shock protein 90 inhibition attenuates inflammation in models of atopic dermatitis: a novel mechanism of action.

Background: Heat shock protein 90 (HSP90) is an important chaperone supporting the function of many proinflammatory client proteins. Recent studies indicate HSP90 inhibition may be a novel mechanism of action for inflammatory skin diseases; however, this has not been explored in atopic dermatitis (AD). Objectives: Our study aimed to investigate HSP90 as a novel target to treat AD. Methods: Experimental models of AD were used including primary human keratinocytes stimulated with cytokines (TNF/IFNγ or TNF/IL-4) and a mouse model established by MC903 applications. Results: In primary human keratinocytes using RT-qPCR, the HSP90 inhibitor RGRN-305 strongly suppressed the gene expression of Th1- (TNF, IL1B, IL6) and Th2-associated (CCL17, CCL22, TSLP) cytokines and chemokines related to AD. We next demonstrated that topical and oral RGRN-305 robustly suppressed MC903-induced AD-like inflammation in mice by reducing clinical signs of dermatitis (oedema and erythema) and immune cell infiltration into the skin (T cells, neutrophils, mast cells). Interestingly, topical RGRN-305 exhibited similar or slightly inferior efficacy but less weight loss compared with topical dexamethasone. Furthermore, RNA sequencing of skin biopsies revealed that RGRN-305 attenuated MC903-induced transcriptome alterations, suppressing genes implicated in inflammation including AD-associated cytokines (Il1b, Il4, Il6, Il13), which was confirmed by RT-qPCR. Lastly, we discovered using Western blot that RGRN-305 disrupted JAK-STAT signaling by suppressing the activity of STAT3 and STAT6 in primary human keratinocytes, which was consistent with enrichment analyses from the mouse model. Conclusion: HSP90 inhibition by RGRN-305 robustly suppressed inflammation in experimental models mimicking AD, proving that HSP90 inhibition may be a novel mechanism of action in treating AD.

Connexin 43 confers chemoresistance through activating PI3K.

Circumventing chemoresistance is crucial for effectively treating cancer including glioblastoma, a lethal brain cancer. The gap junction protein connexin 43 (Cx43) renders glioblastoma resistant to chemotherapy; however, targeting Cx43 is difficult because mechanisms underlying Cx43-mediated chemoresistance remain elusive. Here we report that Cx43, but not other connexins, is highly expressed in a subpopulation of glioblastoma and Cx43 mRNA levels strongly correlate with poor prognosis and chemoresistance in this population, making Cx43 the prime therapeutic target among all connexins. Depleting Cx43 or treating cells with αCT1-a Cx43 peptide inhibitor that sensitizes glioblastoma to the chemotherapy temozolomide-inactivates phosphatidylinositol-3 kinase (PI3K), whereas overexpression of Cx43 activates this signaling. Moreover, αCT1-induced chemo-sensitization is counteracted by a PI3K active mutant. Further research reveals that αCT1 inactivates PI3K without blocking the release of PI3K-activating molecules from membrane channels and that Cx43 selectively binds to the PI3K catalytic subunit ß (PIK3CB, also called PI3Kß or p110ß), suggesting that Cx43 activates PIK3CB/p110ß independent of its channel functions. To explore the therapeutic potential of simultaneously targeting Cx43 and PIK3CB/p110ß, αCT1 is combined with TGX-221 or GSK2636771, two PIK3CB/p110ß-selective inhibitors. These two different treatments synergistically inactivate PI3K and sensitize glioblastoma cells to temozolomide in vitro and in vivo. Our study has revealed novel mechanistic insights into Cx43/PI3K-mediated temozolomide resistance in glioblastoma and demonstrated that targeting Cx43 and PIK3CB/p110ß together is an effective therapeutic approach for overcoming chemoresistance.

The connexin 43 carboxyl terminal mimetic peptide αCT1 prompts differentiation of a collagen scar matrix in humans resembling unwounded skin.

Phase II clinical trials have reported that acute treatment of surgical skin wounds with the therapeutic peptide alpha Connexin Carboxy-Terminus 1 (αCT1) improves cutaneous scar appearance by 47% 9-month postsurgery. While Cx43 and ZO-1 have been identified as molecular targets of αCT1, the mode-of-action of the peptide in scar mitigation at cellular and tissue levels remains to be further characterized. Scar histoarchitecture in αCT1 and vehicle-control treated skin wounds within the same patient were compared using biopsies from a Phase I clinical trial at 29-day postwounding. The sole effect on scar structure of a range of epidermal and dermal variables examined was that αCT1-treated scars had less alignment of collagen fibers relative to control wounds-a characteristic that resembles unwounded skin. The with-in subject effect of αCT1 on scar collagen order observed in Phase I testing in humans was recapitulated in Sprague-Dawley rats and the IAF hairless guinea pig. Transient increase in histologic collagen density in response to αCT1 was also observed in both animal models. Mouse NIH 3T3 fibroblasts and primary human dermal fibroblasts treated with αCT1 in vitro showed more rapid closure in scratch wound assays, with individual cells showing decreased directionality in movement. An agent-based computational model parameterized with fibroblast motility data predicted collagen alignments in simulated scars consistent with that observed experimentally in human and the animal models. In conclusion, αCT1 prompts decreased directionality of fibroblast movement and the generation of a 3D collagen matrix postwounding that is similar to unwounded skin-changes that correlate with long-term improvement in scar appearance.

Development of PLGA nanoparticles for sustained release of a connexin43 mimetic peptide to target glioblastoma cells.

Effective therapeutic delivery of peptide and protein drugs is challenged by short in vivo half-lives due to rapid degradation. Sustained release formulations of αCT1, a 25 amino acid peptide drug, would afford lower dosing frequency in indications that require long term treatment, such as chronic wounds and cancers. In this study, rhodamine B (RhB) was used as a model drug to develop and optimize a double emulsion-solvent evaporation method of poly(lactic-co-glycolic acid) (PLGA) nanoparticle synthesis. Encapsulation of αCT1 in these nanoparticles (NPs) resulted in a sustained in vitro release profile over three weeks, characterized by an initial burst release of approximately 50% of total encapsulated drug over the first three days followed by sustained release over the remaining two and a half weeks. NP uptake by glioblastoma stem cells was through endocytosis and RhB and αCT1 were observed in cells after at least 4 days.

Novel approach to temozolomide resistance in malignant glioma: connexin43-directed therapeutics.

Resistance of malignant glioma, including glioblastoma (GBM), to the chemotherapeutic temozolomide (TMZ) remains a key obstacle in treatment strategies. The gap junction protein connexin43 (Cx43) has complex roles in the establishment, progression, and persistence of malignant glioma. Recent findings demonstrate that connexins play an important role in the microenvironment of malignant glioma and that Cx43 is capable of conferring chemotherapeutic resistance to GBM cells. Carboxyl-terminal Cx43 peptidomimetics show therapeutic promise in overcoming TMZ resistance via mechanisms that may include modulating junctional activity between tumor cells and peritumoral cells and/or downstream molecular signaling events mediated by Cx43 protein binding. High levels of intra-tumor and inter-tumor heterogeneity make it difficult to clearly define specific populations for Cx43-targeted therapy; hence, development of in vitro models that better mimic the microenvironment of malignant glioma, and the incorporation of patient-derived stem cells, could provide opportunities for patient-specific drug screening. This review summarizes recent advances in understanding the roles of Cx43 in malignant glioma, with a special focus on tumor microenvironment, TMZ resistance, and therapeutic opportunity offered by Cx43 peptidomimetics.

Targeting the tight junction protein, zonula occludens-1, with the connexin43 mimetic peptide, αCT1, reduces VEGF-dependent RPE pathophysiology.

A critical target tissue in age-related macular degeneration (AMD) is the retinal pigment epithelium (RPE), which forms the outer blood-retina barrier (BRB). RPE-barrier dysfunction might result from attenuation/disruption of intercellular tight junctions. Zonula occludens-1 (ZO-1) is a major structural protein of intercellular junctions. A connexin43-based peptide mimetic, αCT1, was developed to competitively block interactions at the PDZ2 domain of ZO-1, thereby inhibiting ligands that selectively bind to this domain. We hypothesized that targeting ZO-1 signaling using αCT1 would maintain BRB integrity and reduce RPE pathophysiology by stabilizing gap- and/or tight-junctions. RPE-cell barrier dysfunction was generated in mice using laser photocoagulation triggering choroidal neovascularization (CNV) or bright light exposure leading to morphological damage. αCT1 was delivered via eye drops. αCT1 treatment reduced CNV development and fluid leakage as determined by optical coherence tomography, and damage was correlated with disruption in cellular integrity of surrounding RPE cells. Light damage significantly disrupted RPE cell morphology as determined by ZO-1 and occludin staining and tiling pattern analysis, which was prevented by αCT1 pre-treatment. In vitro experiments using RPE and MDCK monolayers indicated that αCT1 stabilizes tight junctions, independent of its effects on Cx43. Taken together, stabilization of intercellular junctions by αCT1 was effective in ameliorating RPE dysfunction in models of AMD-like pathology. KEY MESSAGE: The connexin43 mimetic αCT1 accumulates in the mouse retinal pigment epithelium following topical delivery via eye drops. αCT1 eye drops prevented RPE-cell barrier dysfunction in two mouse models. αCT1 stabilizes intercellular tight junctions. Stabilization of cellular junctions via αCT1 may serve as a novel therapeutic approach for both wet and dry age-related macular degeneration.

A Multicenter Randomized Controlled Trial Evaluating a Cx43-Mimetic Peptide in Cutaneous Scarring.

The transmembrane protein Cx43 has key roles in fibrogenic processes including inflammatory signaling and extracellular matrix composition. aCT1 is a Cx43 mimetic peptide that in preclinical studies accelerated wound closure, decreased inflammation and granulation tissue area, and normalized mechanical properties after cutaneous injury. We evaluated the efficacy and safety of aCT1 in the reduction of scar formation in human incisional wounds. In a prospective, multicenter, within-participant controlled trial, patients with bilateral incisional wounds (≥10 mm) after laparoscopic surgery were randomized to receive acute treatment (immediately after wounding and 24 hours later) with an aCT1 gel formulation plus conventional standard of care protocols, involving moisture-retentive occlusive dressing, or standard of care alone. The primary efficacy endpoint was average scarring score using visual analog scales evaluating incision appearance and healing progress over 9 months. There was no significant difference in scar appearance between aCT1- or control-treated incisions after 1 month. At month 9, aCT1-treated incisions showed a 47% improvement in scar scores over controls (Vancouver Scar Scale; P = 0.0045), a significantly higher Global Assessment Scale score (P = 0.0009), and improvements in scar pigmentation, thickness, surface roughness, and mechanical suppleness. Adverse events were similar in both groups. aCT1 has potential to improve scarring outcome after surgery.

Connexin 43, breast cancer tumor suppressor: Missed connections?

Connexins are a family of transmembrane proteins that are characterized by their capacity to form intercellular channels called gap junctions that directly link the cytoplasm of adjacent cells. The formation of gap junctions by connexin proteins facilitates intercellular communication between neighboring cells by allowing for the transfer of ions and small signaling molecules. Communication through gap junctions is key to cellular equilibrium, where connexins, and the gap junction intercellular communication that connexins propagate, have roles in cellular processes such as cell growth, differentiation, and tissue homeostasis. Due to their importance in maintaining cellular functions, the disruption of connexin expression and function underlies the etiology and progression of numerous pathologies, including cancer. Over the past half a century, the role of connexins and gap junction intercellular communication have been highlighted as critical areas of research in cellular malignancies, and much research effort has been geared toward understanding their dysfunction in human cancers. Although ample evidence supports the role of connexins in a variety of human cancers, detailed examination in specific cancers, such as breast cancer, is still lacking. This review highlights the most abundant gap junction connexin isoform in higher vertebrate organisms, Connexin 43, and its role in breast cancer.

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR.

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death. Common sites of metastatic spread include lung, lymph node, brain, and bone. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

Targeting connexin 43 with α-connexin carboxyl-terminal (ACT1) peptide enhances the activity of the targeted inhibitors, tamoxifen and lapatinib, in breast cancer: clinical implication for ACT1.

BACKGROUND: Treatment failure is a critical issue in breast cancer and identifying useful interventions that optimize current cancer therapies remains a critical unmet need. Expression and functional studies have identified connexins (Cxs), a family of gap junction proteins, as potential tumor suppressors. Studies suggest that Cx43 has a role in breast cancer cell proliferation, differentiation, and migration. Although pan-gap junction drugs are available, the lack of specificity of these agents increases the opportunity for off target effects. Consequently, a therapeutic agent that specifically modulates Cx43 would be beneficial and has not been tested in breast cancer. In this study, we now test an agent that specifically targets Cx43, called ACT1, in breast cancer. METHODS: We evaluated whether direct modulation of Cx43 using a Cx43-directed therapeutic peptide, called ACT1, enhances Cx43 gap junctional activity in breast cancer cells, impairs breast cancer cell proliferation or survival, and enhances the activity of the targeted inhibitors tamoxifen and lapatinib. RESULTS: Our results show that therapeutic modulation of Cx43 by ACT1 maintains Cx43 at gap junction sites between cell-cell membrane borders of breast cancer cells and augments gap junction activity in functional assays. The increase in Cx43 gap junctional activity achieved by ACT1 treatment impairs proliferation or survival of breast cancer cells but ACT1 has no effect on non-transformed MCF10A cells. Furthermore, treating ER+ breast cancer cells with a combination of ACT1 and tamoxifen or HER2+ breast cancer cells with ACT1 and lapatinib augments the activity of these targeted inhibitors. CONCLUSIONS: Based on our findings, we conclude that modulation of Cx43 activity in breast cancer can be effectively achieved with the agent ACT1 to sustain Cx43-mediated gap junctional activity resulting in impaired malignant progression and enhanced activity of lapatinib and tamoxifen, implicating ACT1 as part of a combination regimen in breast cancer.

Topical administration of a connexin43-based peptide augments healing of chronic neuropathic diabetic foot ulcers: A multicenter, randomized trial.

Nonhealing neuropathic foot ulcers remain a significant problem in individuals with diabetes. The gap-junctional protein connexin43 (Cx43) has roles in dermal wound healing and targeting Cx43 signalling accelerates wound reepithelialization. In a prospective, randomized, multicenter clinical trial we evaluated the efficacy and safety of a peptide mimetic of the C-terminus of Cx43, alpha connexin carboxy-terminal (ACT1), in accelerating the healing of chronic diabetic foot ulcers (DFUs) when incorporated into standard of care (SOC) protocols. Adults with DFUs of at least four weeks duration were randomized to receive SOC with or without topical application of ACT1. Primary outcome was mean percent ulcer reepithelialization and safety variables included incidence of treatment related adverse events (AEs) and detection of ACT1 immunogenicity. ACT1 treatment was associated with a significantly greater reduction in mean percent ulcer area from baseline to 12 weeks (72.1% vs. 57.1%; p = 0.03). Analysis of incidence and median time-to-complete-ulcer closure revealed that ACT1 treatment was associated with a greater percentage of participants that reached 100% ulcer reepitheliazation and a reduced median time-to-complete-ulcer closure. No AEs reported were treatment related, and ACT1 was not immunogenic. Treatment protocols that incorporate ACT1 may present a therapeutic strategy that safely augments the reepithelialization of chronic DFUs.

The effect of a connexin43-based Peptide on the healing of chronic venous leg ulcers: a multicenter, randomized trial.

The gap junction protein, connexin43 (Cx43), has critical roles in the inflammatory, edematous, and fibrotic processes following dermal injury and during wound healing, and is abnormally upregulated at the epidermal wound margins of venous leg ulcers (VLUs). Targeting Cx43 with ACT1, a peptide mimetic of the carboxyl-terminus of Cx43, accelerates fibroblast migration and proliferation, and wound reepithelialization. In a prospective, multicenter clinical trial conducted in India, adults with chronic VLUs were randomized to treatment with an ACT1 gel formulation plus conventional standard-of-care (SOC) protocols, involving maintaining wound moisture and four-layer compression bandage therapy, or SOC protocols alone. The primary end point was mean percent ulcer reepithelialization from baseline to 12 weeks. A significantly greater reduction in mean percent ulcer area from baseline to 12 weeks was associated with the incorporation of ACT1 therapy (79% (SD 50.4)) as compared with compression bandage therapy alone (36% (SD 179.8); P=0.02). Evaluation of secondary efficacy end points indicated a reduced median time to 50 and 100% ulcer reepithelialization for ACT1-treated ulcers. Incorporation of ACT1 in SOC protocols may represent a well-tolerated, highly effective therapeutic strategy that expedites chronic venous ulcer healing by treating the underlying ulcer pathophysiology through Cx43-mediated pathways.

Cardiac to cancer: connecting connexins to clinical opportunity.

Gap junctions and their connexin components are indispensable in mediating the cellular coordination required for tissue and organ homeostasis. The critical nature of their existence mandates a connection to disease while at the same time offering therapeutic potential. Therapeutic intervention may be offered through the pharmacological and molecular disruption of the pathways involved in connexin biosynthesis, gap junction assembly, stabilization, or degradation. Chemical inhibitors aimed at closing connexin channels, peptide mimetics corresponding to short connexin sequences, and gene therapy approaches have been incredibly useful molecular tools in deciphering the complexities associated with connexin biology. Recently, therapeutic potential in targeting connexins has evolved from basic research in cell-based models to clinical opportunity in the form of human trials. Clinical promise is particularly evident with regards to targeting connexin43 in the context of wound healing. The following review is aimed at highlighting novel advances where the pharmacological manipulation of connexin biology has proven beneficial in animals or humans.

Impact of the controlled release of a connexin 43 peptide on corneal wound closure in an STZ model of type I diabetes.

The alpha-carboxy terminus 1 (αCT1) peptide is a synthetically produced mimetic modified from the DDLEI C-terminus sequence of connexin 43 (Cx43). Previous research using various wound healing models have found promising therapeutic effects when applying the drug, resulting in increased wound healing rates and reduced scarring. Previous data suggested a rapid metabolism rate in vitro, creating an interest in long term release. Using a streptozotocin (STZ) type I diabetic rat model with a surgically induced corneal injury, we delivered αCT1 both directly, in a pluronic gel solution, and in a sustained system, using polymeric alginate-poly-l-ornithine (A-PLO) microcapsules (MC). Fluorescent staining of wound area over a 5 day period indicated a significant increase in wound closure rates for both αCT1 and αCT1 MC treated groups, withαCT1 MC groups showing the most rapid wound closure overall. Analysis of inflammatory reaction to the treatment groups indicated significantly lower levels of both Interferon Inducible T-Cell Alpha Chemoattractant (ITAC) and Tumor Necrosis Factor Alpha (TNFα) markers using confocal quantification and ELISA assays. Additional analysis examining genes selected from the EMT pathway using RT-PCR and Western blotting suggested αCT1 modification of Transforming Growth Factor Beta 2 (TGFß2), Keratin 8 (Krt8), Estrogen Receptor 1 (Esr1), and Glucose Transporter 4 (Glut4) over a 14 day period. Combined, this data indicated a possible suppression of the inflammatory response by αCT1, leading to increased wound healing rates.

A Connexin43 Mimetic Peptide Promotes Regenerative Healing and Improves Mechanical Properties in Skin and Heart.

SIGNIFICANCE: Evidence is building that the gap junction protein connexin43 (Cx43) is an important molecule in regenerative healing of skin and heart. Excess scarring from skin wound healing is a continuing clinical problem. Humans generally lack the ability to regenerate tissue following injury, and some degree of fibrotic repair occurs. In the skin, this results in unsightly scars with inferior mechanical properties. In the heart, scarring causes disruption in the contractility of cardiac muscle and increases the risk of deadly arrhythmia. Therapies that tip the balance of wound healing away from scar tissue and toward regeneration would thus represent a significant medical advance. RECENT ADVANCES: A cell-permeant peptide, αCT1 (alpha connexin carboxyl-terminal peptide), based on the carboxyl-terminus of connexin43, has been shown to elicit changes in gap junction organization and intracellular communication. In the skin, αCT1 applied at acute time points results in decreased inflammatory response, reduced area of scar progenitor tissue, and restoration of more normal dermal structure and mechanical strength. αCT1 application to infarcted hearts improved cardiac contractility, reduced the propensity for arrhythmia, and increased conduction velocity through the injured heart. CRITICAL ISSUES: Application of therapies like αCT1 could reduce cutaneous scarring and improve mechanical properties of healed skin and the contractile function and electrical stability of the heart following injury or surgery. FUTURE DIRECTIONS: αCT1 is a potential therapy for cutaneous wounds that could lead to reduced scarring and improvements in the mechanical properties of healed skin. For injured myocardial tissues, this Cx43 mimetic peptide may also provide a therapeutic approach for targeting pathological fibrosis and reducing the likelihood of sudden death from cardiac arrhythmias.

2-Methoxyestradiol induced Bax phosphorylation and apoptosis in human retinoblastoma cells via p38 MAPK activation.

Retinoblastoma (Rb) is a common childhood intraocular cancer that affects approximately 300 children each year in the United States alone. 2-Methoxyestradiol (2ME), an endogenous metabolite of 17-ß-estradiol that dose not bind to nuclear estrogen receptor, exhibits potent apoptotic activity against rapidly growing tumor cells. Here, we report that 2ME induction of apoptosis was demonstrated by early fragmented DNA after 48 h of incubation with 10 µM 2ME in Rb cell lines. Subsequently, a decrease of proliferation was observed in a time- and dose-dependent manner. Further analysis of the mechanism indicates that p38 kinase plays a critical role in 2ME-induced apoptosis in Y79 cells, even though ERK was also activated by 2ME under the same conditions. Activation of p38 kinase also mediates 2ME induced Bax phosphorylated at Thr(167) after a 6 h treatment of 2ME, which in turn prevents formation of the Bcl-2-Bax heterodimer. Both p38 specific inhibitor, SB 203580, or p38 knockdown by specific siRNA, blocked 2ME induction of Bax phosphorylation. Furthermore, only transiently transfected mutant BaxT167A, but not Bax S163A, inhibited 2ME-induced apoptosis. In summary, our data suggest that 2ME induces apoptosis in human Rb cells by causing phosphorylation of p38 Mitogen-activated protein kinase (MAPK), which appears to be correlated with phosphorlation of Bax. This understanding of 2ME's ability may help develop it as a promising therapeutic candidate by inducing apoptosis in a Rb.

Diazoxide prevents diabetes through inhibiting pancreatic beta-cells from apoptosis via Bcl-2/Bax rate and p38-beta mitogen-activated protein kinase.

Increased apoptosis of pancreatic beta-cells plays an important role in the occurrence and development of type 2 diabetes. We examined the effect of diazoxide on pancreatic beta-cell apoptosis and its potential mechanism in Otsuka Long Evans Tokushima Fatty (OLETF) rats, an established animal model of human type 2 diabetes, at the prediabetic and diabetic stages. We found a significant increase with age in the frequency of apoptosis, the sequential enlargement of islets, and the proliferation of the connective tissue surrounding islets, accompanied with defective insulin secretory capacity and increased blood glucose in untreated OLETF rats. In contrast, diazoxide treatment (25 mg.kg(-1).d(-1), administered ip) inhibited beta-cell apoptosis, ameliorated changes of islet morphology and insulin secretory function, and increased insulin stores significantly in islet beta-cells whether diazoxide was used at the prediabetic or diabetic stage. Linear regression showed the close correlation between the frequency of apoptosis and hyperglycemia (r = 0.913; P < 0.0001). Further study demonstrated that diazoxide up-regulated Bcl-2 expression and p38beta MAPK, which expressed at very low levels due to the high glucose, but not c-jun N-terminal kinase and ERK. Hence, diazoxide may play a critical role in protection from apoptosis. In this study, we demonstrate that diazoxide prevents the onset and development of diabetes in OLETF rats by inhibiting beta-cell apoptosis via increasing p38beta MAPK, elevating Bcl-2/Bax ratio, and ameliorating insulin secretory capacity and action.

Hypoglycemia induced changes in cell death and cell proliferation in the organogenesis stage embryonic mouse heart.

BACKGROUND: Hypoglycemia is a side effect of diabetes therapy and causes abnormal heart development. Embryonic heart cells are largely resistant to teratogen-induced apoptosis. METHODS: Hypoglycemia was tested for effects on cell death and cell proliferation in embryonic heart cells by exposing mouse embryos on embryonic day (E) 9.5 (plug = E0.5) to hypoglycemia (30-50 mg/dl glucose) in vivo or in vitro for 24 hr. Long-term effects of in vivo exposure on conceptus viability were evaluated at E18.5. Cell death was evaluated on E10.5 by: 1) two TUNEL assays in sectioned embryos to demonstrate DNA fragmentation; 2) confocal microscopy in whole embryos stained with Lysotracker; 3) flow cytometry in dispersed heart cells stained for TUNEL and myosin heavy chain (MHC) to quantify and characterize cell type susceptibility; and 4) immunohistochemistry (IHC) and Western analysis in sectioned embryos to evaluate potential involvement of caspase-3 active subunit and p53. Effects on cell proliferation were evaluated by IHC and Western analysis of proliferating cell nuclear antigen (PCNA). RESULTS: In vivo hypoglycemic exposure on E9.5 reduced viability in conceptuses examined on E18.5. Hearts examined on E10.5 demonstrated increased TUNEL and Lysotracker staining. In hearts of embryos exposed to hypoglycemia, flow cytometry demonstrated increased TUNEL-positive cells and cells dual-labeled for TUNEL and MHC. Protein expression of caspase-3 active subunit and p53 was increased and PCNA was markedly reduced in hearts of embryos exposed to hypoglycemia. CONCLUSIONS: Hypoglycemia reduces embryonic viability, induces significant cell death, and reduces cell proliferation in the E9.5 mouse heart, and these processes may involve active caspase-3 and p53.