Combined Treatment with Cryptocaryone and Ultraviolet C Promotes Antiproliferation and Apoptosis of Oral Cancer Cells.

Cryptocaryone (CPC) was previously reported as preferential for killing natural products in oral cancer cells. However, its radiosensitizing potential combined with ultraviolet C (UVC) cell killing of oral cancer cells remains unclear. This study evaluates the combined anti-proliferation effect and clarifies the mechanism of combined UVC/CPC effects on oral cancer cells. UVC/CPC shows higher anti-proliferation than individual and control treatments in a low cytotoxic environment on normal oral cells. Mechanistically, combined UVC/CPC generates high levels of reactive oxygen species and induces mitochondrial dysfunction by generating mitochondrial superoxide, increasing mitochondrial mass and causing the potential destruction of the mitochondrial membrane compared to individual treatments. Moreover, combined UVC/CPC causes higher G2/M arrest and triggers apoptosis, with greater evidence of cell cycle disturbance, annexin V, pancaspase, caspases 3/7 expression or activity in oral cancer cells than individual treatments. Western blotting further indicates that UVC/CPC induces overexpression for cleaved types of poly (ADP-ribose) polymerase and caspase 3 more than individual treatments. Additionally, UVC/CPC highly induces γH2AX and 8-hydroxy-2'-deoxyguanosine adducts as DNA damage in oral cancer cells. Taken together, CPC shows a radiosensitizing anti-proliferation effect on UVC irradiated oral cancer cells with combined effects through oxidative stress, apoptosis and DNA damage.

Withholding of M-CSF Supplement Reprograms Macrophages to M2-Like via Endogenous CSF-1 Activation.

Macrophage colony-stimulating factor (M-CSF or CSF-1) is known to have a broad range of actions on myeloid cells maturation, including the regulation of macrophage differentiation, proliferation and survival. Macrophages generated by M-CSF stimulus have been proposed to be alternatively activated or M2 phenotype. M-CSF is commonly overexpressed by tumors and is also known to enhance tumor growth and aggressiveness via stimulating pro-tumor activities of tumor-associated macrophages (TAMs). Currently, inhibition of CSF-1/CSF-1R interaction by therapeutic antibody to deplete TAMs and their pro-tumor functions is becoming a prevalent strategy in cancer therapy. However, its antitumor activity shows a limited single-agent effect. Therefore, macrophages in response to M-CSF interruption are pending for further investigation. To achieve this study, bone marrow derived macrophages were generated in vitro by M-CSF stimulation for 7 days and then continuously grown until day 21 in M-CSF absence. A selective pressure for cell survival was initiated after withdrawal of M-CSF. The surviving cells were more prone to M2-like phenotype, even after receiving interleukin-4 (IL-4) stimulation. The transcriptome analysis unveiled that endogenous CSF-1 level was dramatically up-regulated and numerous genes downstream to CSF-1 covering tumor necrosis factor (TNF), ras-related protein 1 (Rap1) and phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT) signaling pathway were significantly modulated, especially for proliferation, migration and adhesion. Moreover, the phenomenal increase of miR-21-5p and genes related to pro-tumor activity were observed in parallel. In summary, withholding of CSF-1/CSF-1R interaction would rather augment than suspend the M-CSF-driven pro-tumor activities of M2 macrophages in a long run.

Apoptotic effects of dillapiole on maturation of mouse oocytes, fertilization and fetal development.

Previously, we reported that dillapiole, a phenylpropanoid with antileishmanial, anti-inflammatory, antifungal and acaricidal activities, is a risk factor for normal embryonic development that triggers apoptotic processes in the inner cell mass of mouse blastocysts, leading to impaired embryonic development and cell viability. In the current study, we investigated the deleterious effects of dillapiole on mouse oocyte maturation, in vitro fertilization (IVF) and subsequent pre- and post-implantation development, both in vitro and in vivo. Notably, dillapiole induced significant impairment of mouse oocyte maturation, decrease in the IVF rate and inhibition of subsequent embryonic development in vitro. Pre-incubation of oocytes with dillapiole during in vitro maturation led to an increase in post-implantation embryo resorption and decrease in mouse fetal weight. In an in vivo animal model, 2.5, 5 or 10 µM dillapiole provided in drinking water caused a decrease in oocyte maturation and IVF, and led to deleterious effects on early embryonic development. Importantly, pre-incubation of oocytes with a caspase-3-specific inhibitor effectively blocked dillapiole-triggered deleterious effects, clearly implying that embryonic injury induced by dillapiole is mediated via a caspase-dependent apoptotic mechanism. To the best of our knowledge, this is the first study to establish the impact of dillapiole on maturation of mouse oocytes, fertilization and sequential embryonic development.

Characterization of apoptosis induced by emodin and related regulatory mechanisms in human neuroblastoma cells.

Emodin (1,3,8-trihydroxy-6-methylanthraquinone), a major constituent of rhubarb, has a wide range of therapeutic applications. Recent studies have shown that emodin can induce or prevent cell apoptosis, although the precise molecular mechanisms underlying these effects are unknown. Experiments from the current study revealed that emodin (10-20 µM) induces apoptotic processes in the human neuroblastoma cell line, IMR-32, but exerts no injury effects at treatment doses below 10 µM. Treatment with emodin at concentrations of 10-20 µM led to a direct increase in the reactive oxygen species (ROS) content in IMR-32 cells, along with significant elevation of cytoplasmic free calcium and nitric oxide (NO) levels, loss of mitochondrial membrane potential (MMP), activation of caspases-9 and -3, and cell death. Pretreatment with nitric oxide (NO) scavengers suppressed the apoptotic biochemical changes induced by 20 µM emodin, and attenuated emodin-induced p53 and p21 expression involved in apoptotic signaling. Our results collectively indicate that emodin at concentrations of 10-20 µM triggers apoptosis of IMR-32 cells via a mechanism involving both ROS and NO. Based on the collective results, we propose a model for an emodin-triggered apoptotic signaling cascade that sequentially involves ROS, Ca²âº, NO, p53, caspase-9 and caspase-3.

Ochratoxin a inhibits mouse embryonic development by activating a mitochondrion-dependent apoptotic signaling pathway.

Ochratoxin A (OTA), a mycotoxin found in many foods worldwide, causes nephrotoxicity, hepatotoxicity, and immunotoxicity, both in vitro and in vivo. In the present study, we explored the cytotoxic effects exerted by OTA on the blastocyst stage of mouse embryos, on subsequent embryonic attachment, on outgrowth in vitro, and following in vivo implantation via embryo transfer. Mouse blastocysts were incubated with or without OTA (1, 5, or 10 µM) for 24 h. Cell proliferation and growth were investigated using dual differential staining; apoptosis was measured using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay; and embryo implantation and post-implantation development were assessed by examination of in vitro growth and the outcome of in vivo embryo transfer, respectively. Blastocysts treated with 10 µM OTA displayed a significantly increased level of apoptosis and a reduction in total cell number. Interestingly, we observed no marked difference in implantation success rate between OTA-pretreated and control blastocysts either during in vitro embryonic development (following implantation in a fibronectin-coated culture dish) or after in vivo embryo transfer. However, in vitro treatment with 10 µM OTA was associated with increased resorption of post-implantation embryos by the mouse uterus, and decreased fetal weight upon embryo transfer. Our results collectively indicate that in vitro exposure to OTA triggers apoptosis and retards early post-implantation development after transfer of embryos to host mice. In addition, OTA induces apoptosis-mediated injury of mouse blastocysts, via reactive oxygen species (ROS) generation, and promotes mitochondrion-dependent apoptotic signaling processes that impair subsequent embryonic development.

Regenerative Effect of Mesenchymal Stem Cell on Cartilage Damage in a Porcine Model.

Osteoarthritis (OA) is a major public and animal health challenge with significant economic consequences. Cartilage degradation plays a critical role in the initiation and progression of degenerative joint diseases, such as OA. Mesenchymal stem cells (MSCs) have become increasingly popular in the field of cartilage regeneration due to their promising results. The objective of this preclinical study was to evaluate the regenerative effects of mesenchymal stem cells (MSCs) in the repair of knee cartilage defects using a porcine model. Seven healthy LYD breed white pigs, aged 9-10 weeks and weighing approximately 20 ± 3 kg, were used in the experimental protocol. Full-thickness defects measuring 8 mm in diameter and 5 mm in depth were induced in the lateral femoral condyle of the posterior limbs in both knee joints using a sterile puncture technique while the knee was maximally flexed. Following a 1-week induction phase, the pig treatment groups received a 0.3 million/kg MSC transplant into the damaged knee region, while the placebo group received a control solution as a treatment. Magnetic resonance imaging (MRI), computerized tomography (CT), visual macroscopic examination, histological analysis, and cytokine concentration analysis were used to assess cartilage regeneration. The findings revealed that human adipose-derived mesenchymal stem cells (hADSCs) were more effective in repairing cartilage than pig umbilical cord-derived mesenchymal stem cells (pUCMSCs). These results suggest that MSC-based treatments hold promise as a treatment option for cartilage repair, which aid in the treatment of OA. However, further studies with larger sample sizes and longer follow-up periods are required to fully demonstrate the safety and efficacy of these therapies in both animals and humans.

High levels estradiol affect blastocyst implantation and post-implantation development directly in mice.

BACKGROUND: Previous studies have demonstrated that high levels of estradiol (E2) impair blastocyst implantation through effects on the endometrium; however, whether high E2 directly affects blastocysts is not well established. The present study sought to clarify the direct impacts of high E2 levels on blastocysts in vitro. METHODS: ICR virgin albino mice were used. Using an in-vitro 8-day blastocyst culture model, immunofluorescence staining for the estrogen receptor (ER), blastocyst outgrowth assays, differential staining and TUNEL assays of blastocysts, and embryo transfer, we investigated the main outcomes of exposure to different E2 concentrations (10-7 to 10-4 M) in vitro and in vivo. RESULTS: ERα and ERß expression were detected in pre-implantation stage embryos. In vitro exposure of blastocysts to 10-4 M E2 for 24 h followed by 7 days culture in the absence of E2 caused severe inhibition of implantation and post-implantation development. The late adverse effects of E2 on post-implantation development still occurred at concentrations of 10-7 to 10-5 M. In addition, blastocyst proliferation was reduced and apoptotic cells were increased following exposure to 10-4 M E2. Using an in vivo embryo-transfer model, we also showed that treatment with high E2 resulted in fewer implantation sites (38% vs. 72% in control) and greater resorption of implanted blastocysts (81% vs. 38% in control). CONCLUSION: Exposure to high E2 concentrations in vitro is deleterious to blastocyst implantation and early post-implantation development, mainly owing to direct impacts of E2 on implanting blastocysts. In clinical assisted reproductive technique (ART), high serum E2 concentrations not only affects the endometrium, but also affects blastocysts directly at the period of implantation.

Dietary Supplementation of Two-Stage Fermented Feather-Soybean Meal Product on Growth Performance and Immunity in Finishing Pigs.

This study investigates the effects of two-stage fermented feather meal-soybean meal product (TSFP) on growth performance, blood characteristics, and immunity of finishing pigs. Firstly, feather meal-soybean meal is subjected to aerobic fermentation with Bacillus subtilis var. natto N21, B. subtilis Da2 and Da15, B. amyloliquefaciens Da6, Da16 for two days, and anaerobic fermentation with B. coagulans L12 for three days. Then, the fermented product is air-dried into an end product-TSFP. Eighty hybrid pigs (Duroc x KHAPS) with equal numbers of both sexes are randomly assigned into 3% fish meal, 0%, 2.5%, or 5.0% TSFP groups with five replicates per group. Our results show that the average daily feed intake and feed conversion rate of TSFP groups are significantly better than the other groups at 0-3 weeks (p < 0.05). The 5% TSFP group significantly increased HDL-C in the blood (p < 0.05), and decreased LDL-C and blood urea nitrogen content (p < 0.05). The lipopolysaccharide (LPS) and concanavalin A (ConA) in 5% TSFP group and interferon-γ (IFN-γ) content in 2.5% and 5% TSFP groups are significantly higher than the other groups (p < 0.05). The phagocytic oxygen burst capacity and serum IgA content of the 5% TSFP group are significantly higher than those of the fishmeal group (p < 0.05). The CD3, CD4, and CD4 + CD8 + T cells subsets in 2.5% and 5% TSFP groups are significantly higher than the control group (p < 0.05). In conclusion, TSFP has a positive effect on the growth performance and immunity of finishing pigs with the best performance on 5% TSFP.

Non-embryotoxic dosage of alternariol aggravates ochratoxin A-triggered deleterious effects on embryonic development through ROS-dependent apoptotic processes.

Alternariol (AOH) and ochratoxin A (OTA), two mycotoxins found in many foods worldwide, exhibit cytotoxicity and embryotoxicity, triggering apoptosis and cell cycle arrest in several mammalian cells and mouse embryos. The absorption rate of AOH from dietary foodstuff is low, meaning that the amount of AOH obtained from the diet rarely approaches the cytotoxic threshold. Thus, the potential harm of dietary consumption of AOH is generally neglected. However, previous findings from our group and others led us to question whether a low dosage of AOH could aggravate the cytotoxicity of other mycotoxins. In the present study, we examined how low dosages of AOH affected OTA-triggered apoptosis and embryotoxicity and investigated the underlying regulatory mechanism in mouse blastocysts. Our results revealed that non-cytotoxic concentrations of AOH (1 and 2 µM) could enhance OTA (8 µM)-triggered apoptotic processes and embryotoxicity in mouse blastocysts. We also found that AOH can enhance OTA-evoked intracellular reactive oxygen species (ROS) generation and that this could be prevented by pretreatment with the potent ROS scavenger, N-acetylcysteine. Finally, we observed that this ROS generation acts as a key inducer of caspase-dependent apoptotic processes and subsequent impairments of embryo implantation and pre- and post-implantation embryonic development. In sum, our results show that non-cytotoxic dosages of AOH can aggravate OTA-triggered apoptosis and embryotoxicity through ROS- and caspase-dependent signaling pathways.

Hazardous effects of curcumin on mouse embryonic development through a mitochondria-dependent apoptotic signaling pathway.

In this study, we examined the cytotoxic effects of curcumin, the yellow pigment of Curcuma longa, on the blastocyst stage of mouse embryos, subsequent embryonic attachment, and outgrowth in vitro and in vivo implantation by embryo transfer. Mouse blastocysts were incubated in medium with or without curcumin (6, 12 or 24 µM) for 24 h. Cell proliferation and growth were investigated using dual differential staining, apoptosis was analyzed with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), and implantation and post-implantation development of embryos were measured by in vitro development analysis and in vivo embryo transfer, respectively. Blastocysts treated with 24 µM curcumin displayed significantly increased apoptosis and decreased total cell number. Interestingly, we observed no marked differences in the implantation success rates between curcumin-pretreated and control blastocysts during in vitro embryonic development through implantation with a fibronectin-coated culture dish. However, in vitro treatment with 24 µM curcumin was associated with decreased implantation rate and increased resorption of postimplantation embryos in mouse uterus, as well as decreased fetal weight in the embryo transfer assay. Our results collectively indicate that in vitro exposure to curcumin triggers apoptosis and retards early postimplantation development after transfer to host mice. In addition, curcumin induces apoptotic injury effects on mouse blastocysts through ROS generation, and further promotes mitochondria-dependent apoptotic signaling processes to impair sequent embryonic development.

Dosage effects of ginkgolide B on ethanol-induced cell death in human hepatoma G2 cells.

Ginkgolide B is a major active component of Ginkgo biloba extracts, which has been shown to confer anticancer effects by inducing apoptosis or inhibiting oxidative stress generation. Ethanol induces a wide range of cellular toxicities, many of which have been linked to free radical generation. To further elucidate the cellular effects of ginkgolide B, we examined the dose-response effect of ginkgolide B on ethanol-induced toxicity in human Hep G2 cells. TUNEL and MTT assays revealed that ethanol (50-400 mM) induced apoptotic cell death in human Hep G2 cells, and that this effect was inhibited by low (5-25 microM) doses of ginkgolide B, but enhanced by high (50-100 microM) doses of ginkgolide B. Additional experiments revealed that ethanol treatment directly increased intracellular oxidative stress; this effect was enhanced by high doses of ginkgolide B but decreased following treatment with low concentrations of ginkgolide B. The dose-response effects of ginkgolide B on reactive oxygen species (ROS) generation were directly correlated with cell apoptotic biochemical changes including c-Jun N-terminal kinase (JNK) activation, caspase-3 activation, and DNA fragmentation. These results indicate that treatment dosage may determine the effect of ginkgolide B on ethanol-induced ROS generation and cell apoptosis, and support the notion that an appropriate dosage of ginkgolide B may aid in decreasing the toxic effects of ethanol.

Epigallocatechin gallate dose-dependently induces apoptosis or necrosis in human MCF-7 cells.

The catechins, a family of polyphenols found in tea, can evoke various responses, including cell death. However, the precise molecular mechanisms of these effects are unknown. Here, we demonstrate that treatment of human MCF-7 cells with 50 microM (-)-Epigallocatechin-3-gallate (EGCG), a catechin that is highly abundant in green tea, can induce apoptotic changes, including mitochondrial membrane potential changes and activation of c-Jun N-terminal kinase (JNK), caspase-9, and caspase-3. In contrast, higher concentrations of EGCG (100-400 microM) do not induce apoptosis, but rather trigger necrotic cell death in MCF-7 cells. Investigations of the possible mechanisms underlying these differences revealed that treatment with lower concentrations of EGCG (10-50 microM) directly increased intracellular oxidative stress, while higher concentrations (100-400 microM) did not. Immunoblotting revealed that treatment of MCF-7 cells with 10-50 microM EGCG caused increases in Bax protein levels and decreases in Bcl-2 protein levels, shifting the Bax-Bcl-2 ratio to favor apoptosis, while treatment with 100-400 microM EGCG had no such effect. Moreover, we observed a dose-dependent decrease in intracellular ATP levels in cells treated with high-dose EGCG. Blockade of reactive oxygen species (ROS) generation and ATP synthesis using antioxidants and ATP synthesis inhibitors revealed that ROS and ATP play important roles to switch cell death types with apoptosis or necrosis. Collectively, these results indicate for the first time that EGCG treatment has a dose-dependent effect on ROS generation and intracellular ATP levels in MCF-7 cells, leading to either apoptosis or necrosis, and that the apoptotic cascade involves JNK activation, Bax expression, mitochondrial membrane potential changes, and activation of caspase-9 and caspase-3.

Interaction of vitamin E and exercise training on oxidative stress and antioxidant enzyme activities in rat skeletal muscles.

It has been shown that free radicals are increased during intensive exercise. We hypothesized that vitamin E (vit E) deficiency, which will increase oxidative stress, would augment the training-induced adaptation of antioxidant enzymes. This study investigated the interaction effect of vit E and exercise training on oxidative stress markers and activities of antioxidant enzymes in red quadriceps and white gastrocnemius of rats in a 2x2 design. Thirty-two male rats were divided into trained vit E-adequate, trained vit E-deficient, untrained vit E-adequate, and untrained vit E-deficient groups. The two trained groups swam 6 h/day, 6 days/week for 8 weeks. The two vit E-deficient groups consumed vit E-free diet for 8 weeks. Vitamin E-training interaction effect was significant on thiobarbituric acid reactive substances (TBARSs), glutathione peroxidase (GPX), and superoxide dismutase (SOD) in both muscles. The trained vit E-deficient group showed the highest TBARS and GPX activity and the lowest SOD activity in both muscles. A significant vit E effect on glutathione reductase and catalase was present in both muscles. Glutathione reductase and catalase activities were significantly lower in the two vit E-adequate groups combined than in the two vit E-deficient groups combined in both muscles. This study shows that vit E status and exercise training have interactive effect on oxidative stress and GPX and SOD activities in rat skeletal muscles. Vitamin E deprivation augmented the exercise-induced elevation in GPX activity while inhibiting exercise-induced SOD activity, possibly through elevated oxidative stress.

Protective effects of resveratrol on ethanol-induced apoptosis in embryonic stem cells and disruption of embryonic development in mouse blastocysts.

Previous studies have established that ethanol induces apoptosis, but the precise molecular mechanisms are currently unclear. Here, we show that 0.3-1.0% (w/v) ethanol induces apoptosis in mouse blastocysts and that resveratrol, a grape-derived phytoalexin with known antioxidant and anti-inflammatory properties, prevents ethanol-induced apoptosis and inhibition of cell proliferation. Moreover, ethanol-treated blastocysts show normal levels of implantation on culture dishes in vitro but a reduced ability to reach the later stages of embryonic development. Pretreatment with resveratrol prevented ethanol-induced disruption of embryonic development in vitro and in vivo. In an in vitro cell-based assay, we further found that ethanol increases the production of reactive oxygen species in ESC-B5 embryonic stem cells, leading to an increase in the intracellular concentrations of cytoplasmic free Ca(2+) and NO, loss of mitochondrial membrane potential, mitochondrial release of cytochrome c, activation of caspase-9 and -3, and apoptosis. These changes were blocked by pretreatment with resveratrol. Based on these results, we propose a model for the protective effect of resveratrol on ethanol-induced cell injury in blastocysts and ESC-B5 cells.

Curcumin inhibits ROS formation and apoptosis in methylglyoxal-treated human hepatoma G2 cells.

Methylglyoxal (MG) is a reactive dicarbonyl compound endogenously produced mainly from glycolytic intermediates. Elevated MG levels in diabetes patients are believed to contribute to diabetic complications. MG is cytotoxic through induction of apoptosis. Curcumin, the yellow pigment of Curcuma longa, is known to have antioxidant and anti-inflammatory properties. In the present study, we investigated the effect of curcumin on MG-induced apoptotic events in human hepatoma G2 cells. We report that curcumin prevented MG-induced cell death and apoptotic biochemical changes such as mitochondrial release of cytochrome c, caspase-3 activation, and cleavage of PARP (poly [ADP-ribose] polymerase). Using the cell permeable dye 2',7'-dichlorofluorescein diacetate (DCF-DA) as an indicator of reactive oxygen species (ROS) generation, we found that curcumin abolished MG-stimulated intracellular oxidative stress. The results demonstrate that curcumin significantly attenuates MG-induced ROS formation, and suggest that ROS triggers cytochrome c release, caspase activation, and subsequent apoptotic biochemical changes.

Effects of retinoic acid on the inner cell mass in mouse blastocysts.

This is the first evidence that excess retinoic acid has a direct cellular response from proliferation to cell death (apoptosis) and affects in vitro development in mouse inner cell mass.

Effect of curcumin on in vitro early post-implantation stages of mouse embryo development.

OBJECTIVES: This study was designed to examine the embryotoxic potential of the curcumin at the blastocyst stage and during early post-implantation development of mouse embryos in vitro. STUDY DESIGN: Curcumin was administered to ICR mice embryos at a dose of 0, 6, 12, 24 µM throughout in vitro culture. A total of 1015 embryos were randomly assigned to the different dosage groups. The embryotoxic effects were studied by the exposure of curcumin at the blastocyst, implanted blastocyst and early egg cylinder stages, respectively. For assessment of implantation in vitro and further embryonic differentiation, blastocysts were cultured for 8 days. The cell proliferation of outgrowth blastocysts was analysed by Giemsa staining. RESULTS: Exposure to 24 µM of curcumin at the implanted blastocyst stage or early egg stage cause adverse effects on development. The percentage of embryos in the later stages of development was changed depending upon the dose of curcumin used. Furthermore, exposure to 24 µM of curcumin at the blastocyst stage was lethal to all embryos. The number of nuclei per outgrowth of the blastocyst decreased significantly after curcumin pre-treatment. The percentage of trophoblastic giant cells per outgrowth increased significantly after curcumin pre-treatment. CONCLUSIONS: These findings demonstrate that curcumin exerts an adverse effect on mouse embryos during the early post-implantation stages of development, equivalent to day 3-day 8 of gestation in vivo. Curcumin treatment or administration should be used carefully at the early post-implantation stage of gestation.

Ginkgolide B induces apoptosis via activation of JNK and p21-activated protein kinase 2 in mouse embryonic stem cells.

Ginkgolide B (GKB), the major active component of Ginkgo biloba extracts, can both stimulate and inhibit apoptotic signaling. We previously showed that ginkgolide treatment of mouse blastocysts induces apoptosis, decreases cell numbers, retards the proliferation and development of mouse embryonic stem cells and blastocysts in vitro, and causes developmental injury in vivo. However, the precise molecular mechanisms underlying its actions are currently unknown. Here, our study further revealed that GKB induced apoptotic biochemical changes, including activation of JNK, caspase-3, and p21-activated protein kinase 2 (PAK2), in ESC-B5 mouse embryonic stem cells. Treatment of ESC-B5 cells with a JNK-specific inhibitor (SP600125) reduced GKB-induced activation of both JNK and caspase-3, indicating that JNK activity is required for GKB-induced caspase activation. Experiments using caspase-3 inhibitors and antisense oligonucleotides against PAK2 showed that caspase-3 activation is required for PAK2 activation and both of these activations are required for GKB-induced apoptosis in ESC-B5 cells.

Androgenic and antiandrogenic effects and expression of androgen receptor in mouse embryonic stem cells.

OBJECTIVE: To investigate the effects of androgen and antiandrogen and the expression of androgen receptor on mouse embryonic stem cells (ESCs) and the inner cell mass. DESIGN: Controlled laboratory study. SETTING: Academic university hospital. ANIMAL(S): Blastocysts from mice developed at the Institute for Cancer Research and 129/Sv mice embryonic stem cell line. INTERVENTION(S): Cultured mouse ESCs were exposed to testosterone (T), dihydrotestosterone (DHT), or the antiandrogen nilutamide. MAIN OUTCOME MEASURE(S): Immunohistochemistry for androgen receptor (AR), quantitative real-time polymerase chain reaction analysis, cell colorimetric assays, and Western blot analysis. RESULT(S): Androgen receptor messenger RNA (mRNA) was first detected both in the inner cell mass from blastocysts and in undifferentiated ESCs. It increased stage-dependently during ESC differentiation. Although both T and DHT had marginal effects on AR mRNA expression level and cell growth in vitro, the nonsteroidal antiandrogen nilutamide significantly stimulated ESC growth and induced Akt expression. The enhancing effects of nilutamide on mouse ESCs indicated that the Akt pathway may be involved in nilutamide-promoted ESC growth. CONCLUSION(S): These findings provide the first evidence of the existence of AR in ESCs. During differentiation, the expression level of AR was increased in a stage-dependent but not a ligand-dependent manner. Nilutamide promoted cell growth and increased Akt expression in ESCs.

Adverse effects of retinoic acid on embryo development and the selective expression of retinoic acid receptors in mouse blastocysts.

BACKGROUND: All-trans retinoic acid (RA), the oxidative metabolite of vitamin A, is essential for normal development. In addition, high levels of RA are teratogenic in many species. We have previously shown that excess RA results in immediate effects on the preimplantation embryo and on blastocyst development. This study was conducted to clarify the long-term survival of mouse blastocyst and the effect of RA on gene expression. METHODS AND RESULTS: Using an in vitro model, we identified the immediate adverse impact of RA on mouse blastocyst development. This involved an inhibition of cell proliferation and growth retardation. Using an in vivo model, we also identified the resorption of postimplanted blastocysts that had been treated with excess RA. Analysis of RA-mediated gene induction was also included. The retinoic acid receptors RARalpha and RARgamma were constitutively expressed in the blastocyst and the inner cell mass, whereas RARbeta was induced upon RA treatment. CONCLUSIONS: This is the first evidence to show the impacts of RA on mouse blastocysts in vitro and any carry-over effects in the uterus. There is a retardation of early postimplantation blastocyst development and then subsequent blastocyst death. Our findings also show that there is some degree of selective induction of retinoic acid receptors when excess RA is administered to the blastocysts.