Comparative analysis of effects of conditioned mediums obtained from 2D or 3D cultured mesenchymal stem cells on kidney functions of diabetic rats: Early intervention could potentiate transdifferentiation of parietal epithelial cell into podocyte precursors.

AIM: The secretome of mesenchymal stem cells (MSCs) could be a potential therapeutic intervention for diabetes and associated complications like nephropathy. This study aims to evaluate the effects of conditioned mediums (CMs) collected from umbilical cord-derived MSCs incubated under 2-dimensional (2D) or 3D culture conditions on kidney functions of rats with type-I diabetes (T1D). MAIN METHODS: Sprague-Dawley rats were treated with 20 mg/kg streptozocin for 5 consecutive days to induce T1D, and 12 doses of CMs were applied intraperitoneally for 4 weeks. The therapeutic effects of CMs were comparatively investigated by biochemical, physical, histopathological, and immunohistochemical analysis. KEY FINDINGS: 3D-CM had significantly higher total protein concentration than the 2D-CM Albumin/creatinine ratios of both treatment groups were significantly improved in comparison to diabetes. Light microscopic evaluations showed that glomerular and cortical tubular damages were significantly ameliorated in only the 3D-CM applied group compared to the diabetes group, which were correlated with transmission electron microscopic observations. The nephrin and synaptopodin expressions increased in both treatment groups compared to diabetes. The WT1, Ki-67, and active caspase-3 expressions in glomeruli and parietal layers of the treatment groups suggest that both types of CMs suppress apoptosis and promote possible parietal epithelial cells' (PECs') transdifferentiation towards podocyte precursor cells by switching on WT1 expression in parietal layer rather than inducing new cell proliferation. SIGNIFICANCE: 3D-CM was found to be more effective in improving kidney functions than 2D-CM by ameliorating glomerular damage through the possible mechanism of transdifferentiation of PECs into podocyte precursors and suppressing glomerular apoptosis.

Preconditioning of human umbilical cord mesenchymal stem cells with deferoxamine potentiates the capacity of the secretome released from the cells and promotes immunomodulation and beta cell regeneration in a rat model of type 1 diabetes.

This study aimed to examine the effects of the secretome released by human umbilical cord-mesenchymal stem cells (MSC) as a result of preconditioning with deferoxamine (DFX), a hypoxia mimetic agent, on type 1 diabetes (T1D), by comparing it with the secretome produced by untreated MSCs. Initially, the levels of total protein, IL4, IL10, IL17, and IFNγ in the conditioned medium (CM) obtained from MSCs subjected to preconditioning with 150 µM DFX (DFX-CM) were analyzed in comparison to CM derived from untreated MSCs (N-CM). Subsequently, the CMs were administered to rats with T1D within a specific treatment plan. Following the sacrification, immunomodulation was evaluated by measuring serum cytokine levels and assessing the regulatory T cell (Treg) ratio in spleen mononuclear cells. Additionally, ß-cell mass was determined in the islets by immunohistochemical labeling of NK6 Homeobox 1 (Nkx6.1), Pancreatic duodenal homeobox-1 (Pdx1), and insulin antibodies in pancreatic sections. In vitro findings indicated that the secretome levels of MSCs were enhanced by preconditioning with DFX. In vivo, the use of DFX-CM significantly increased the Treg population, and accordingly, the level of inflammatory cytokines decreased. In ß-cell marker labeling, D + DFX-CM showed significantly increased PDX1 and insulin immunoreactivity. In conclusion, while the factors released by MSCs without external stimulation had limited therapeutic effects, substantial improvements in immunomodulation and ß-cell regeneration were seen with DFX-preconditioned cell-derived CM.

Investigation of conditioned medium properties obtained from human umbilical cord mesenchymal stem/stromal cells preconditioned with dimethyloxalylglycine in a correlation with ultrastructural changes.

Mesenchymal stem/stromal cells (MSCs) hold significant therapeutic value due to their regeneration abilities, migration capacity, and immunosuppressive and immunomodulatory properties. These cells secrete soluble and insoluble factors, and this complex secretome contributes to their therapeutic effect. Furthermore, stimulation of cells by various external stimuli lead to secretome modifications that can increase the therapeutic efficacy. So, this study examined the effect of dimethyloxalylglycine (DMOG), a hypoxia-mimetic agent, on secretome profiles and exosome secretions of MSCs by evaluating conditioned medium (CM) and ultrastructural morphologies of the cells in comparison with unpreconditioned MSCs. The appropriate dose and duration of the use of DMOG were determined as 1000 µM and 24 h by evaluating the HIF-1α expression. DMOG-CM and N-CM were collected from MSCs incubated in serum-free medium with/without DMOG for 24 h, respectively. The content analysis of conditioned mediums (CMs) revealed that VEGF, NGF, and IL-4 levels were increased in DMOG-CM. Subsequently, exosomes were isolated from the CMs and were shown by transmission electron microscopy and Western blot analysis in both groups. The effects of CMs on proliferation and migration were determined by in vitro wound healing tests; both CMs increased the fibroblast's migratory and proliferative capacities. According to the ultrastructural evaluation, autophagosome, autolysosome, myelin figure, and microvesicular body structures were abundant in DMOG-preconditioned MSCs. Consistent with the high number of autophagic vacuoles, Beclin-1 expression was increased in those cells. These findings suggested that DMOG could alter MSCs' secretion profile, modify their ultrastructural morphology accordingly, and make the CM a more potent therapeutic tool. RESEARCH HIGHLIGHTS: Preconditioning mesenchymal stem/stromal cells with dimethyloxalylglycine, a hypoxia-mimetic agent, could modify cellular metabolism. Hypoxic mechanisms lead to alterations in the ultrastructural characteristics of mesenchymal stromal/stem cells. Preconditioning with dimethyloxalylglycine leads to ultrastructural and metabolic changes of mesenchymal stromal/stem cells along with modifications in their secretome profiles. Preconditioning of mesenchymal stromal/stem cells could render them a more potent therapeutic tool.

2D and 3D cultured human umbilical cord-derived mesenchymal stem cell-conditioned medium has a dual effect in type 1 diabetes model in rats: immunomodulation and beta-cell regeneration.

BACKGROUND: Type 1 diabetes (T1D) is a T-cell-mediated autoimmune disease characterized by the irreversible destruction of insulin-producing ß-cells in pancreatic islets. Helper and cytotoxic T-cells and cytokine production, which is impaired by this process, take a synergetic role in ß-cell destruction, and hyperglycemia develops due to insulin deficiency in the body. Mesenchymal stem cells (MSCs) appear like an excellent therapeutic tool for autoimmune diseases with pluripotent, regenerative, and immunosuppressive properties. Paracrine factors released from MSCs play a role in immunomodulation by increasing angiogenesis and proliferation and suppressing apoptosis. In this context, the study aims to investigate the therapeutic effects of MSC's secretomes by conditioned medium (CM) obtained from human umbilical cord-derived MSCs cultured in 2-dimensional (2D) and 3-dimensional (3D) environments in the T1D model. METHODS: First, MSCs were isolated from the human umbilical cord, and the cells were characterized. Then, two different CMs were prepared by culturing MSCs in 2D and 3D environments. The CM contents were analyzed in terms of total protein, IL-4, IL-10, IL-17, and IFN-λ. In vivo studies were performed in Sprague-Dawley-type rats with an autoimmune T1D model, and twelve doses of CM were administered intraperitoneally for 4 weeks within the framework of a particular treatment model. In order to evaluate immunomodulation, the Treg population was determined in lymphocytes isolated from the spleen after sacrification, and IL-4, IL-10, IL-17, and IFN-λ cytokines were analyzed in serum. Finally, ß-cell regeneration was evaluated immunohistochemically by labeling Pdx1, Nkx6.1, and insulin markers, which are critical for the formation of ß-cells. RESULTS: Total protein and IL-4 levels were higher in 3D-CM compared to 2D-CM. In vivo results showed that CMs induce the Treg population and regulate cytokine release. When the immunohistochemical results were evaluated together, it was determined that CM application significantly increased the rate of ß-cells in the islets. This increase was at the highest level in the 3D-CM applied group. CONCLUSION: The dual therapeutic effect of MSC-CM on immunomodulation and homeostasis/regeneration of ß-cells in the T1D model has been demonstrated. Furthermore, this effect could be improved by using 3D scaffolds for culturing MSCs while preparing CM.

Therapeutic potential of conditioned medium obtained from deferoxamine preconditioned umbilical cord mesenchymal stem cells on diabetic nephropathy model.

BACKGROUND: The therapeutic potential of mesenchymal stem cells (MSCs)-derived conditioned media (CM) can be increased after preconditioning with various chemical agents. The aim of this study is comparative evaluation of effects of N-CM and DFS-CM which are collected from normal (N) and deferoxamine (DFS) preconditioned umbilical cord-derived MSCs on rat diabetic nephropathy (DN) model. METHODS: After incubation of the MSCs in serum-free medium with/without 150 µM DFS for 48 h, the contents of N-CM and DFS-CM were analyzed by enzyme-linked immunosorbent assay. Diabetes (D) was induced by single dose of 55 mg/kg streptozotocin. Therapeutic effects of CMs were evaluated by biochemical, physical, histopathological and immunohistochemical analysis. RESULTS: The concentrations of vascular endothelial growth factor alpha, nerve growth factor and glial-derived neurotrophic factor in DFS-CM increased, while one of brain-derived neurotrophic factor decreased in comparison with N-CM. The creatinine clearance rate increased significantly in both treatment groups, while the improvement in albumin/creatinine ratio and renal mass index values were only significant for D + DFS-CM group. Light and electron microscopic deteriorations and loss of podocytes-specific nephrin and Wilms tumor-1 (WT-1) expressions were significantly restored in both treatment groups. Tubular beclin-1 expression was significantly increased for DN group, but it decreased in both treatment groups. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive apoptotic cell death increased in the tubules of D group, while it was only significantly decreased for D + DFS-CM group. CONCLUSIONS: DFS-CM can be more effective in the treatment of DN by reducing podocyte damage and tubular apoptotic cell death and regulating autophagic activity with its more concentrated secretome content than N-CM.

Aspartame induces cancer stem cell enrichment through p21, NICD and GLI1 in human PANC-1 pancreas adenocarcinoma cells.

This study aimed to investigate the molecular effects of the common natural sugar glucose and artificial sweetener aspartame on cancer stem cell (CSC) population and cancer aggressiveness of PANC-1 human pancreas adenocarcinoma cells. According to our findings while aspartame exposure significantly increased the CSC population, high glucose had no effect on it. The epithelial-mesenchymal transition marker N-cadherin increased only in the aspartame group. The findings indicate that a high level of glucose exposure does not effect the invasion and migration of PANC-1 cells, while aspartame increases both of these aggressiveness criteria. The findings also suggest that a high concentration of glucose maintains CSC population through induction of nuclear Oct3/4 and differentiation to parental cells via increasing cytoplasmic c-myc. Aspartame exposure to PANC-1 cells activated AKT and deactivated GSK3ß by increasing levels of ROS and cytoplasmic Ca+2, respectively, through T1R2/T1R3 stimulation. Then p-GSK3ß(Ser9) boosted the CSC population by increasing pluripotency factors Oct3/4 and c-myc via NICD, GLI1 and p21. In the aspartame group, T1R1 silencing further increased the CSC population but decreased cell viability and suppressed the p21, NICD and GLI activation. The presence and amount of T1R subunits in the membrane fraction of PANC-1 cells are demonstrated for the first time in this study, as is the regulatory effect of T1R1's on CSC population. In conclusion, the present study demonstrated that long-term aspartame exposure increases CSC population and tumor cell aggressiveness through p21, NICD, GLI1. Moreover, while aspartame had no tumorigenic effect, it could potentially advance an existing tumor.

Efficacy of resveratrol in the wound healing process by reducing oxidative stress and promoting fibroblast cell proliferation and migration.

Oxidative stress (OS) is implicated in the pathogenesis of diabetic, chronic, and inflamed wounds. In these cases, wound healing is difficult and remains a big clinical challenge. As fibroblasts are crucial in wound healing by producing extracellular matrix and wound contraction, the researchers investigated the resveratrol (RES) effects as a potential therapeutic alternative, on an in vitro OS and wound healing model established by 3T3 Swiss Albino fibroblasts. OS was induced by H2 O2 , and 1-100 µM RES doses were applied on cells for up to 48 hours. The cell proliferation was assessed by the population doubling time and bromodeoxyuridine immunocytochemistry. The OS levels were determined by fluorescence staining and all values were calculated by the ImageJ program. The collagen-I expression (COL1A2 mRNA) was evaluated by a real-time PCR. Cell migration was evaluated by wound closure assay and the ultrastructure was evaluated by electron microscopy. The cell proliferation index significantly decreased in H2 O2 incubation and increased with RES application. RES application reduced OS levels and stabilized cell proliferation, but no differences were found in COL1A2 mRNA expression. RES-treated cells showed better proliferation, migration rates, and ultrastructural preservation. RES administration decreases OS levels and improves the healing process by increasing cell proliferation and migration quality, suggesting it is a powerful candidate for the treatment of skin wounds.

In-vitro investigation of calcitonin associated effects on the trophoblastic cells.

Calcitonin is expressed in the epithelium of endometrium, and modulates zonula adherens junctions which are composed of cadherin-catenins complex during the implantation window. Trophoblastic cells which have complex interaction with the epithelial cells of endometrium during implantation were demonstrated to have calcitonin receptors. Mechanism of action of calcitonin on trophoblastic cells has not yet been elucidated. Therefore, it was aimed to determine the effects of calcitonin on the expressions of ß-catenin and phospho-ß-catenin in a dose depended manner under the influence of progesterone and estrogen hormones (P + E) by using JAR cell line through the immunocytochemical and Western blot analyses. Moreover, adherens junctions (AJs) were ultrastructurally investigated to assess the involvement of cadherin-catenin complex in accordance with the changes in the specified parameters. Immunocytochemical analysis showed that only 10 nM calcitonin treated group had increased expression of membranous ß-catenin compared to the control group, while there was decreased expression of ß-catenin in the nucleus of all the experimental groups. Cytoplasmic expressions of the phospho-ß-catenin decreased in all experimental groups compared to the control group while the decrease in the nuclear expression was remarkable in the groups treated with P + E, and P + E + 250 nM calcitonin. Western blot analysis showed that total ß-catenin and phospho-ß-catenin expressions were not significantly different. Ultrastructural analysis showed that increase in the number of AJs was noticeable in the group treated with 10 nM calcitonin. Overall, the localization and expression levels of ß-catenin and phospho-ß-catenin suggest that calcitonin could show its effects through the non-canonical pathway in the trophoblastic cells.

Comparison of different cryopreservation protocols for human umbilical cord tissue as source of mesenchymal stem cells.

The main purpose of this study is to establish an effective cryopreservation protocol for the umbilical cord tissue as a source of mesenchymal stem cells (MSCs). In this context, it was aimed to use a cryoprotectant that could be an alternative to dimethyl sulfoxide (DMSO) which is commonly used despite the toxic side effects. Therefore, two different cryopreservation solutions were prepared using 10% DMSO and 10% 1,2 propanediol (PrOH). The fresh tissue group that was not performed cryopreservation was used as the control group. Following the cryopreservation step, MSCs were isolated from all groups and compared with each other to assess the efficiency of the cryopreservation solutions. The comparison was performed in terms of followings: morphology, immunophenotypes, growth kinetics, differentiation, and ultrastructural features. Based on the results, there were no significant morphological and immunophenotypic differences between the MSCs isolated from cryopreserved tissue groups and the MSCs isolated from the fresh tissue group. According to the growth kinetic analysis, the cells isolated from the PrOH group had a lower proliferation rate than the cells isolated from the fresh tissue. However, there was no significant difference between the cryopreserved groups in this respect. Osteogenic and adipogenic differentiation was observed in all groups. Upon comparison of the cryopreserved groups, PrOH group was discovered to hold a minor superiority in terms of these modes of differentiation. These results suggest that PrOH, which is considered as a cryoprotectant with low toxicity, could be used as a preferred cryoprotectant instead of DMSO concerning the process of cryopreservation of the umbilical cord.

Ultrastructural analysis of human umbilical cord derived MSCs at undifferentiated stage and during osteogenic and adipogenic differentiation.

Mesenchymal stem cells (MSCs) are considered as an important tool for regenerative medicine and experimental treatments. Unveiling the ultrastructural changes during the differentiation of MSCs might help us to understand the nature of the process and to develop novel therapeutic approaches. For this purpose, human umbilical cord (hUC) was chosen as MSC source. In the first place, MSCs were isolated from sub-amniotic, intervascular and perivascular areas of hUC by enzymatic and tissue explant method to determine the most favorable region of hUC and technique for further processing. Therefore, microscopic and growth kinetics analyses showed that there was no clear difference in the morphologies and proliferation rates among the hUC-MSC groups. Flow cytometric analysis showed that CD44 and CD90 MSC markers were highly expressed, while CD34 and CD45 hematopoietic stem cells markers were expressed at low degree. Because our preliminary results showed that there was no conspicuous superiority among the hUC-MSCs groups, whole UC was utilized as a source, and tissue explant method was applied to isolate MSCs for further differentiation analysis. At the 1st and 3rd week of osteogenic and adipogenic differentiation, ultrastructural analysis showed an increase in the number of secondary lysosomes in comparison with the undifferentiated status. Increase in the mitochondrial content was also detected at the 1st week of adipogenic differentiation. Consequently, ultrastructural changes including increase in the number of mitochondria and secondary lysosomes during the adipogenic and osteogenic differentiation could be attributed to the switch in energy metabolism of the MSCs and increment in the lysosomal activity respectively.

CB-1R and GLP-1R gene expressions and oxidative stress in the liver of diabetic rats treated with sitagliptin.

BACKGROUND: Type 2 diabetes is a major health problem affecting millions of people. Controlled eating and regular physical activity are important for the management of type 2 diabetes. Dipeptidyl peptidase-4 enzyme (DPP-4) inhibitor sitagliptin is a potent agent for the treatment of type-2 diabetes. The aim of this study was to examine the effects of sitagliptin on the liver of rats with streptozotocin (STZ)-induced diabetes, in terms of (i) the expression levels of the cannabinoid 1 receptor (CB-1R) and glucagon-like peptide 1 receptor (GLP-1R), (ii) alterations in the number and localization of these peptides, and (iii) changes in histological and oxidative damage. METHODS: Thirty-two neonatal (two-day-old) rats, which were divided into four groups, were treated with saline (control), sitagliptin (control; 1.5mg/kg/day for 15 days starting from day 5 of the experimental period), STZ (diabetes; 100mg/kg single dose), STZ+sitagliptin (diabetes+sitagliptin). After 20 days, hepatic tissues were obtained from rats. RESULTS: The expressions of GLP-1R and CB-1R mRNA increased approximately 1.89- and 2.94-fold, respectively, in the diabetes+sitagliptin group as compared to the diabetic group. Additionally the number of GLP-1R immunopositive cells decreased and CB-1R immunopositive cells increased in comparison to the diabetic group; however, this was not statistically significant. Glutathione levels increased, but malondialdehyde and protein carbonyl levels decreased in the diabetes+sitagliptin group more than the diabetic group. CONCLUSION: Our findings indicate that sitagliptin treatment regulates GLP-1R and CB-1R gene expressions, which are associated with appetite regulation in diabetic rat, and may decrease oxidative stress and liver tissue damage.

The role of ghrelin on apoptosis, cell proliferation and oxidant-antioxidant system in the liver of neonatal diabetic rats.

Ghrelin is a multifunctional peptide hormone which stimulates appetite and regulates glucose metabolism and adipogenesis. The purpose of this study was to investigate whether ghrelin has protective effects in the liver of streptozocin (STZ) diabetic rats or not. Wistar-type neonatal rats were divided into four groups: I. Controls, II. Ghrelin administrated controls, III. STZ-diabetic rats, and IV. Ghrelin administrated diabetic rats. On the second day after birth, 100 mg/kg STZ was administered intraperitoneally in a single dose to induce diabetes in rats. 100 µg/kg/day ghrelin was administrated to rats subcutaneously for 4 weeks. Ghrelin administration improved histopathologic changes in STZ-diabetic liver. Obestatin immunoreactivity has been shown in livers of neonatal rats. The immunoreactivity of obestatin increased in diabetic rats and a decline was observed in ghrelin administrated diabetic rats. Caspase 8 and 3 immunoreactivities increased in diabetic rats; however, ghrelin administration differently affected caspases 8 and 3 immunoreactivities. Proliferating cell nuclear antigen immunoreactivities decreased in diabetic rats and in ghrelin administrated diabetic rats. Serum alanine (P < 0.05) and aspartate transaminase (P < 0.0001) and serum alkaline phosphatase (P < 0.0001) activities were decreased in ghrelin administrated diabetic rats compared to the diabetic rats. Gamma glutamyl transferase activity (P < 0.001) decreased in ghrelin administrated diabetic rats compared to the diabetic rats. The response of antioxidants including glutathione levels, catalase and superoxide dismutase activities were altered in ghrelin administrated diabetic rats. Our findings indicate that ghrelin administration affects hepatic functions in neonatal diabetic rats and might be considered as a therapeutic agent.

Estradiol induces JNK-dependent apoptosis in glioblastoma cells.

Estrogens exert multiple regulatory actions on cellular events in a variety of tissues including the brain. In the present study, the signaling mechanisms of the concentration-dependent effects of 17-ß-estradiol (estradiol) on glioblastoma cells were investigated. Cell viability was evaluated by the trypan blue exclusion assay. Cell growth and kinase activities were evaluated by immunocytochemistry and Western blotting. The results showed that high concentrations of estradiol inhibit growth and induce apoptosis in C6 rat glioma and T98G human glioblastoma cells. The blockade of the c-jun NH(2)-terminal kinase (JNK) signaling pathway prevented these effects of estradiol, indicating the critical role of the JNK/c-jun signaling cascade in glioblastoma cell growth inhibition and cell death in response to high concentrations of estradiol. Collectively, these findings highlight the potential of new discoveries in sensitizing estrogen-sensitive tumors to chemotherapeutic drugs, and may lead to the development of new JNK-based effective therapies.

Effect of early preoperative 5-fluorouracil on the integrity of colonic anastomoses in rats.

AIM: To determine the effect of chemotherapy on wound healing by giving early preoperative 5-fluorouracil (5-FU) to rats with colonic anastomoses. METHODS: Sixty Albino-Wistar male rats (median weight, 235 g) were used in this study. The rats were fed with standard laboratory food and given tap water ad libitum. The animals were divided into three groups: Group 1: Control group (chemotherapy was not administered), Group 2: Intraperitoneally (i.p.) administered 5-FU group (chemotherapy was administered i.p. to animals at a dose of 20 mg/kg daily during the 5 d preceding surgery), Group 3: Intravenously (i.v.) administered 5-FU group. Chemotherapy was administered via the penil vein, using the same dosing scheme and duration as the second group. After a 3-d rest to minimize the side effects of chemotherapy, both groups underwent surgery. One centimeter of colon was resected 2 cm proximally from the peritoneal reflection, then sutured intermittently and subsequently end-to-end anastomosed. In each group, half the animals were given anaesthesia on the 3rd postoperative (PO) day and the other half on the 7th PO day, for in vivo analytic procedures. The abdominal incisions in the rats were dissected, all the new and old anastomotic segments were clearly seen and bursting pressures of each anastomotic segment, tissue hydroxyproline levels and DNA content were determined to assess the histologic tissue repair process. RESULTS: When the i.v. group was compared with the i.p. group, bursting pressures of the anastomotic segments on the 3rd and 7th PO days, were found to be significantly decreased, hydroxyproline levels at the anastomotic segment on the 7th PO day were significantly decreased (P < 0.01). CONCLUSION: In this study, we conclude that early preoperative 5-FU, administered i.v., negatively affects wound healing. However, i.p. administered 5-FU does not negatively affect wound healing.

A plant oxylipin, 12-oxo-phytodienoic acid, inhibits proliferation of human breast cancer cells by targeting cyclin D1.

Cyclin D1 overexpression has been associated with poor prognosis and resistance to therapy in human breast cancer. Thus, the development of therapeutic agents that selectively target cyclin D1 activity is of clinical interest. This study demonstrates that 12-oxo-phytodienoic acid (OPDA), a phytohormone with critical functions in growth and development in plants, induces growth arrest in MDA-MB-231 and T47D breast cancer cells. In response to OPDA treatment, the human breast cancer cell lines exhibit a progressive decline in cyclin D1 expression, which is tightly associated with the accumulation of hypophosphorylated form of the retinoblastoma protein (Rb) and G1 arrest. The decrease in cyclin D1 protein expression accompanies a dramatic decline in nuclear but not membranous beta-catenin expression and activation of glycogen synthase kinase-3-beta (GSK3beta) caused by inhibition of its serine-9 phosphorylation. The proteasome inhibitor MG132 blocks OPDA-mediated decrease in cyclin D1. In addition, the overexpression of T286A, a cyclin D1 mutant which is refractory to phosphorylation by GSK3beta and proteosomal degradation, is resistant to OPDA-mediated Rb dephosphorylation as well as G(1) cell cycle arrest. Thus, our results demonstrate that degradation of cyclin D1 protein is a key event in OPDA induced growth inhibition in breast cancer cells. These data provide the basic foundation for future efforts to develop OPDA-based approaches in the prevention and treatment of breast cancer and other types of cancer.

The effects of combined alpha-tocopherol, ascorbic acid, and selenium against cadmium toxicity in rat intestine.

In this study, the effects of combined antioxidants treatment against cadmium toxicity were investigated microscopically, immunohistochemically, and biochemically in small intestine of Sprague Dawley rats. The rats were subdivided into four groups as intact control, cadmium was administrated, and both control and cadmium groups treated with ascorbic acid, alpha-tocopherol, and selenium. Metallothionein expression was localized in the base of intestinal glands in control rats and similar expression was observed with antioxidants treatment. In cadmium-administrated rats, metallothionein expression was detected in surface epithelium, longitudinal muscle layer, meissner, and myenteric plexuses, but not in the base of intestinal gland. On the other hand, in the rats treated with antioxidants and cadmium, immunreactivity increased in the surface epithelium and in the base of intestinal glands according to cadmium-administrated rats but not changed in the plexuses and longitudinal muscle layer. Biochemically, lipid peroxidation levels increased and glutathione levels decreased significantly in intestine of the cadmium group compared to the control. Treatment with antioxidants in cadmium-administrated rats led to a decrease in lipid peroxidation levels and a significant increase in glutathione levels. As a result, the combination of ascorbic acid, alpha-tocopherol, and selenium shows a protective effect against cadmium toxicity in small intestine.

Simvastatin induces apoptosis in human breast cancer cells: p53 and estrogen receptor independent pathway requiring signalling through JNK.

The effect of simvastatin, a widely used statin for the treatment of hypercholesterolemia, was investigated in the estrogen receptor (ER)-positive MCF-7, and the ER-negative MDA-MB 231 human breast cancer cell lines. Simvastatin induced cell cycle arrest and apoptosis in both cells. These effects of simvastatin were not altered by 17-beta-estradiol treatment. MCF-7 cells express wild-type tumor suppressor protein p53, whereas MDA-MB 231 cells carry a p53 mutation. However, no alteration in the level or localisation of p53 was observed with simvastatin treatment in either cell line. On the other hand, simvastatin strongly stimulated phosphorylation of c-jun which was completely abolished by the c-jun NH2-terminal kinase (JNK) inhibitor SP600125, which also significantly reduced the antiproliferative and apoptotic effects of simvastatin in these cells. In conclusion, we describe here that simvastatin induces apoptosis via involvement of JNK in breast cancer cells independent of their ER or p53 expression status. These findings indicate a great potential for statins for the treatment of cancers resistant to currently used drugs, and target the JNK signalling pathway for a novel approach of breast cancer treatment.

JNK pathway regulates estradiol-induced apoptosis in hormone-dependent human breast cancer cells.

Estrogen is known to stimulate breast cancer development in humans. Ironically, high doses of estrogen can induce regression of hormone-dependent breast cancer in postmenopausal women. The mechanism by which estrogen induces tumour regression in breast cancer is still unknown. We found that under low growth-stimulated conditions, high concentrations of 17-beta-estradiol (estradiol) induces apoptosis and concomitantly increases phosphorylation of c-jun in estrogen receptor (ER)-positive breast cancer cell line, MCF-7, but not in ER-negative breast cancer cell line MDA-MB 231 suggesting an ER-mediated event. Interestingly, when the c-jun NH2-terminal kinase (JNK) signalling pathway was disrupted by the JNK inhibitor SP600125, the ability of estradiol to inhibit the growth of MCF-7 cells and to induce apoptosis was completely blocked. These data suggest that JNK plays a central role in mediating the anticancer effect of high concentrations of estradiol in MCF-7 cells. Our data showing the apoptotic effect of estradiol in low growth-stimulated conditions suggest potential implications for the pharmacological control of breast cancer with high dose estrogen in postmenopausal women. Furthermore, our results indicate that augmenting JNK activity could be an efficient novel approach for treating breast cancer.

The potential role of combined anti-oxidants against cadmium toxicity on liver of rats.

Cadmium (Cd), a widely distributed toxic trace metal, has been shown to accumulate in liver after long- and short-term exposure. Cd (2 mg/kg/day CdCl2) was intraperitoneally given to rats for eight days. Vitamin C (250 mg/kg/day) + vitamin E (250 mg/kg/day) + sodium selenate (0.25 mg/kg/day) were given to rats by oral means. The animals were treated by anti-oxidants one hour prior to treatment with Cd every day. The degenerative changes were observed in the groups given only Cd and anti-oxidants + Cd. Metallothionein (MT) immunoreactivity increased in cytoplasm of hepatocytes of the rats given Cd when compared with controls. In a number of cells with Cd and anti-oxidants treatment, immunoreactivity increase was more than in the group given Cd only and nuclear MT expression was also detected. Cell proliferation was assessed with proliferating cell nuclear antigen (PCNA) immunohistochemistry. PCNA expressions increased in all groups more than in the controls. Anti-oxidants treatment increased cell proliferation. In the animals administered with Cd, an increase in serum aspartate (AST) and alanine (ALT) aminotransferases, liver glutathione (GSH) and lipid peroxidation (LPO) levels were observed. On the other hand, in the rats treated with anti-oxidants and Cd, serum AST and ALT, liver glutathione and LPO levels decreased. As a result, these results suggest that combined anti-oxidants treatment might be useful in protection of liver against Cd toxicity.

Influence of combined antioxidants against cadmium induced testicular damage.

Acute effects of cadmium (Cd) and combined antioxidants were evaluated in Sprague-Dawley rat testes. The rats were subdivided into four groups. Cadmium chloride (2mg/kgday) injected intraperitoneally during 8 days. Vitamin C (250mg/kgday), vitamin E (250mg/kgday) and sodium selenate (0.25mg/kgday) were pretreated by gavage in both of control and cadmium injected rats. Testis lipid peroxidation and glutathione levels were determined by spectrophotometrically. In Cd treated rats, lipid peroxidation levels were increased and glutathione levels were decreased and combined antioxidants treatment was effective in preventing of lipid peroxidation and normalizing glutathione. In Cd treated animals, the degenerative changes were observed, but not observed in the administrated rats with Cd and antioxidants under the light microscope. Proliferating cell nuclear antigen, metallothionein and caspase-3 activities were evaluated by immunohistochemically. Proliferation activity was not seen in the spermatogonial cells of cadmium treated testis. Treatment with antioxidants in cadmium administrated testis leads to pronounced increase in proliferation activity. Cytoplasmic caspase-3 activity was determined in the spermatogenic cells but not spermatogonia in treatment of antioxidants with Cd. In control and treated with antioxidants animals, metallothionein expressions were localized in the cells of seminiferous tubules, although the expression only was observed in the interstitial cells of cadmium treated rats. Results demonstrated beneficial effects of combined vitamin C, vitamin E and selenium treatment in Cd toxicity.