Downregulation of Stemness Genes and Induction of Necrosis in Rat LA7 Cancer Stem Cells Induced Tumors Treated with Starved Fibroblasts Culture Supernatant.

BACKGROUND: Stem cell differentiation therapy is a promising strategy in cancer treatment. we show that protein cocktail prepared from serum starved fibroblasts has therapeutic potential based on this strategy. METHODS: The condition medium was prepared from foreskin isolated fibroblasts and analyzed by Liquid chromatography electrospray ionization mass spectrometry-mass spectrometry (LC-ESI-MS/MS). LA7 mammary gland cancer stem cells originated tumors were induced in Sprague Dawley rats. The rats treated subcutaneously with DMEM (group A), condition medium (group B), or normal saline (group C) once daily for 7 days. Then the tumors were removed and divided into the two parts, one part was used to quantify gene expression by stem-loop RT-qPCR assay and the other part was used for Hematoxylin & Eosin (H & E), Giemsa, and immunohistochemistry (IHC) staining. RESULTS: All induced tumors appeared as sarcomatoid carcinoma (SC). Immunohistochemistry staining confirmed this conclusion by recognizing the tumor as Ki67+, cytokeratin+, vimentine+, and estrogen receptor negative SC. RT-qPCR analysis revealed that Oct4-, Sox-2, Nanog- gene expression was much reduced in the condition medium treated tumors versus proper controls (p< 0.05). Tissue necrosis was more prevalent in this group while tumors volume was diminished almost by 40%. The LC-ESI-MS/MS analysis unrevealed the stemness reducing and the cell death inducing proteins such as, pigment epithelium-derived factor (PEDF), insulin like growth factor binding protein-5 (IGFBP-5) and -7 (IGFBP-7) in the condition medium. CONCLUSION: This study showed that the substances released from starved human fibroblasts were able to down-regulate the stemness-related genes and induce necrosis in LA7 derived tumors.

SCF Improves In Vitro Differentiation of SSCs Through Transcriptionally Up-regulating PRTM1, STRA8, c-KIT, PIWIL2, and OCT4 Genes.

Several lines of evidence strongly suggest that retinoic acid (RA) and stem cell factor (SCF)/c-Kit signal transduction pathways are involved in the differentiation of spermatogonial stem cells (SSCs). This study was aimed to investigate the effect of RA and SCF on in vitro differentiation of SSCs via evaluation of the mRNA expression of meiosis-specific genes in cultured testicular tissues. Testicular tissue samples were obtained from bilaterally vasectomized rats and also healthy adult rats and then were cultured for 25, 30, and 35 days on different conditions. The cultured testicular pieces were sectioned and stained with PAS to histological analysis. The total RNA was extracted from cultured testicular samples, and the expression of ACR, PRTM1, SYCP3, STRA8, c-KIT, PIWIL2, and OCT4 genes at mRNA level was quantified using real-time polymerase chain reaction (qPCR) procedure. After 1-month surgery, bilateral testicular weight showed a significant decrease in vasectomized adult rats compared with healthy adult rats (P < 0.05). Reduction in the diameter of the seminiferous tubules and depletion of advanced germinal elements in vasectomized rats compared with healthy adult rats were also observed. Our findings also demonstrated that the mRNA expression level of PRTM1, STRA8, c-KIT, PIWIL2, and OCT4 genes in cultured testicular tissues significantly up-regulated in experimental group II compared with the control group (P < 0.001). Our findings lead us to conclude that SCF improves in vitro differentiation of SSCs in the OA rats, at least partially, by transcriptionally upregulating PRTM1, STRA8, c-KIT, PIWIL2, and OCT4 genes.

Targeting LA7 breast cancer stem cells of rat through repressing the genes of stemness-related transcription factors using three different biological fluids.

Down-regulation of stemness genes expression is important in differentiation therapy against cancer stem cells (CSCs). The aim of this study was to evaluate the Oct4 , Sox2, Nanog, and C-myc expression in rat breast cancer stem cells (LA7) which treated with human ovarian follicular fluid (FF), replicative senescent fibroblast culture supernatant (P14), and 16 h serum starved fibroblast supernatant (16 h-SFS). The cells were exposed to these biological fluids for 24 h, 72 h, and 7 days. Stem-loop RT-qPCR assay was used to quantify the expression of above mentioned genes. Results showed that FF had the least cytotoxic effect on the LA7 cells. Except for Nanog gene, exposure of LA7 cell line to 16 h-SFS and P14 decreased significantly expression of the three other genes after 24 h (P < 0.05). Nanog and Sox2 genes expression was also decreased in LA7 cells which have been already treated with FF for 24 h. Moreover, compared to the control solution, the expression of Oct4 increased significantly after 7 days exposure to FF (P < 0.05). Annexin V-PE /7-AAD-, acridine orange/ethidium bromide staining and doubling time assays revealed apoptosis and necrosis induction by these biological fluids in LA7 cells. Moreover, in an in vitro model of metastasis assay, i.e., scratch test, these fluids exhibited anti-LA7 migration activity which culminated in 16 h-SFS treated cells. Generally, this study showed that FF, 16 h-SFS, and P14 have positive effects on down-regulation of Nanog, Oct4, Sox2 and C-myc expression, and consequently can increase the differentiation of breast cancer stem cells. For the first time, this study provided some evidence indicating that some biological fluids have potential to differentiate the CSCs, show anti- survival, growth-, and cell migration activity.

Protective Effect of N-Acetyl Cysteine on Chlorpyrifos-Induced Testicular Toxicity in Mice.

BACKGROUND: Chlorpyrifos (CPF), an organophosphate pesticide, is widely used in farms in order to preserve crops and fruits. Previous studies have shown that CPF exposure might cause chronic toxicity in male genital system. The present study investigated the protective effect of N-Acetyl Cysteine (NAC), a potent antioxidant against testicular toxicity of CPF in male mice. MATERIALS AND METHODS: In this experimental study, 42 adult male mice were divided into seven groups, CPF low (0.5 mg/kg.b.w) and high (5 mg/kg.b.w) doses groups, NAC group (35 mg/kg.b.w), NAC+CPF 0/5 mg/kg.b.w, NAC+CPF 5 mg/kg.b.w, dimethyl sulfoxide (DMSO, 0.75% solution mg/kg.b.w) and control group. All treatment were done intraperitoneally. Treatment was conducted for four consecutive weeks (five days each week). However NAC was injected to NAC+CPF groups five days before initiation of the treatment procedure. One week after the last injection, mice were sacrificed using anesthetic gas to evaluate alterations in testicular histology and sperm parameters. RESULTS: Seminiferous tubules area and diameter were significantly diminished in the group treated with 5 mg/kg CPF (P<0.05). CPF also statistically reduced sperm parameters (count and motility) and damaged sperm morphology) at both doses (P<0.05). However, NAC significantly improved spermatogonia, spermatocytes, spermatid cell counts as well as sperm parameters in mice treated with both CPF concentrations (P<0.05). CONCLUSION: According to our results, NAC may significantly ameliorate CPF-induced damages to spermatogonia, spermatocytes, spermatids cell counts and sperm parameters.

Evaluation of Novel Mouse-Specific Germ Cell Gene Expression in Embryonic Stem Cell-Derived Germ Cell-Like Cells In Vitro with Retinoic Acid Treatment.

We designed a study to induce differentiation of Oct4-GFP (expression of Green Fluorescent Protein of oct4) embryonic stem cells (ESCs) by embryoid body (EB) culture system into germ cells (GCs) using retinoic acid (RA) and evaluated the expression level of (Fkbp6, Mov10l1, 4930432K21Rik, and Tex13) in differentiated cells. The expression levels of four GC-related genes, Oct4, Mvh, Scp3, and Stra8, was determined by quantitative real-time polymerase chain reaction (q-RT-PCR). Immunostaining and flow cytometry were used as additional tests to confirm q-RT-PCR findings. A significant increase occurred in the expression of meiotic markers and specific genes, Fkbp6 (p = 0.00), Mov10l1 (p = 0.01), and Tex13 (p = 0.00) in ESCs treated with RA (+RA) compared with the controls (-RA). Oct4 expression was decreased in all studied groups. The expression levels of 4930432K21Rik, Mvh, Stra8, and Scp3 in the +RA group was higher than that of the -RA group. Flow cytometry analysis showed that mean number of Mvh-positive cells in the +RA group was greater as compared with ESCs, -RA and EB7 groups (p = 0.00). Downregulation of Oct4 as a pluripotency factor as well as the expression of meiosis markers, this hypothesis is raised that ESCs are differentiated by RA, and have been introduced into the zygote/pachytene of first meiosis as GC-like cells.

Impact of pre-incubation time of silk fibroin scaffolds in culture medium on cell proliferation and attachment.

Cell behaviours such as proliferation and attachment can be affected by the length of pre-incubation period of the scaffolds in the culture medium for long term. The aim of this study was to investigate the long term pre-incubation of 3D silk fibroin scaffolds in complete culture medium on cell attachment and proliferation. After the preparation of silk fibroin scaffolds by the technique of freeze drying, the scaffolds were pre-incubated in complete culture medium for 2 d, 6 d or 10 d before apical papilla stem cells (SCAP) seeding. Modifications of the scaffold surface and wettability were examined by FE-SEM and water contact angle, respectively. Results showed a decrease both in roughness and water contact angle as pre-incubation time increases. DNA measurement after 18h and 10 d cell seeding showed a significant increase of DNA concentration which represents better attachment and proliferation with pre-incubation time increase. Qualitative examination, live&dead assay or H&E staining method after 30h and 10 d cell seeding respectively, indicated that pre-incubation of scaffolds has time dependent effect on cell proliferation and attachment. This suggests that improvement of cell attachment and proliferation may be mediated by differences in the amount of wettability (decreased water contact angle) after exposure of scaffold to culture medium for long term which, in turn, causes more protein adsorption in the surface of silk fibroin scaffold (decreased roughness).

Cytochrome P-450 1B1 Leu432Val Polymorphism Does Not Show Association With Breast Cancer in Northern Iranian Women With a History of Infertility.

The Cytochrome P-4501B1 (CYP1B1) Leu432Val polymorphism has been previously shown to be associated with some types of cancer and affects CYP1B1-mediated metabolism of various infertility drugs. To establish the frequency of CYP1B1 Leu432Val polymorphism among women with a history of infertility drug use, we studied the genotypes of 147 patients with breast cancer with a history of infertility and 150 cancer-free, infertile women (control group) in Northern Iran. A polymerase chain reaction-based restriction fragment length polymorphism assay was used to detect GG (Val/Val), CG (Leu/Val), and CC (Leu/Leu) genotype frequencies, which did not vary significantly between the 2 patient groups (P = .847). We established for the first time that the incidence of CYP1B1 Leu432Val polymorphism is 46.6% among women with infertility history and breast cancer in Northern Iran. Finally, our results do not show any significant association between CYP1B1 Leu432Val polymorphism and breast cancer in infertile women in this region, who have also received infertility treatment.

Histopathological, histomorphometrical, and radiographical evaluation of injectable glass-ceramic-chitosan nanocomposite in bone reconstruction of rat.

Background. Bone defects following tumor resection and osteolysis due to bone lesions, periodontal tissue disorders, and bone reconstruction are challenges that surgeons face. Gass-ceramic-chitosan nanocomposite contains chitosan, a derivative of crustaceans' exoskeleton. Methods. Thirty-two 6-8-week-old male Wistar rats were chosen. One hole on each right and left tibia was made. The right tibia holes were filled with injectable glass-ceramic-chitosan nanocomposite, and the left tibia holes were left empty. After 7, 14, 28, and 60 days, histopathological, histomorphometrical, and radiographical assessments were performed. Results. Radiographic density on days 7 and 14 was significantly higher in the right tibias than in the left tibias. Trabecular bone thickness, which was higher in the right tibias, increased from day 7 to day 60 in both right and left tibias, although not significantly. Conclusions. Glass-ceramic-chitosan nanocomposite is suggested for use in bone repair in cases of bone loss. More histopathological, histomorphometrical, and radiographical assessments are also recommended.

Immunotoxicity effects of carbaryl in vivo and in vitro.

Carbaryl is a pesticide for controlling pests in agricultural industry. To determine of immunotoxicity effects of carbaryl, rats were exposure with carbaryl for 28 days. The lymphoid organ weight, lymphocyte proliferation, IL-2, IFN-γ, IL-4, IL-10, IL-1ß and TNF-α cytokines level were measured, respectively. Exposure with carbaryl significantly reduced both thymus and spleen weight and also suppressed lymphocyte proliferation. In addition, carbaryl significantly decreased IL-2, IFN-γ, IL-1ß and TNF-α and also increased IL-4, IL-10 cytokines. These findings suggest that exposure to carbaryl can induce immunotoxicity effects on lymphoid organ weight, suppresses the functions of lymphocyte and macrophage, Th2 polarization in Th1/Th2 balance by reducing of IFN-γ and increasing of IL-4 and IL-10 cytokines. Therefore, carbaryl can contribute to the development of allergic, autoimmune, cancer or infection diseases through immunotoxicity effects and unbalancing of Th1/Th2 immune response however, further study is necessary.

Evaluation of preincubation time interval in testicular biopsy to obtain optimum sperm parameters.

OBJECTIVE: In sever oligospermia; one of the paths used for surgical sperm retrieval (SSR) is to extract sperm via a testicular biopsy. The aim of our study is to determine the reliable time interval between testicular biopsy and intracytoplasmic sperm injection (ICSI) procedure in order to obtain optimumsperm parameters (count, motility and normal morphology). MATERIALS AND METHODS: This cohort study was carried out on 30 patients which were candidates for ICSI. After collection and keeping the samples obtained from the testicular biopsy in Ham's F10 environment, the concentration, motility and morphology of the sperm in each sample was evaluated immediately as well as 2 and 4 hours after processing. The Data were then compared with each other. For the statistical analysis, Friedman, Willcoxon and Cochran tests were used. RESULTS: The mean of sperm concentration was 5.69 ± 6.14 million and the motility was10.83 ± 12.63% at 2 hours following biopsy which was significantly higher than those obtained after 0 and 4 hours of the biopsy (p <0.05). CONCLUSION: The reliable preincubation time which resulted in the highest rate of spermatozoa parameters after testicular biopsy and before incubation was 2 hours.