N-Methyl-D-Aspartate Receptors Antagonist Prevents Secondary Ischemic Brain Injury Associated With Lipopolysaccharide-Induced Sepsis-Like State Presumably via Immunomodulatory Actions.

Infection is a major reason for poor stroke outcomes, and sepsis is a major cause of stroke-elated deaths. We herein examined whether NMDA receptor blockade, which was reported to exert anti-inflammatory actions, protects against the deleterious consequences of lipopolysaccharide (LPS)-induced sepsis-like state in adult male NMRI mice exposed to transient intraluminal middle cerebral artery occlusion (MCAO). At 24 h post-ischemia, vehicle or Escherichia coli LPS (2 or 4 mg/kg) was intraperitoneally administered, whereas 30 min later vehicle or ketamine (10 mg/kg), which is a non-competitive NMDA receptor antagonist, was intraperitoneally applied. Delivery of LPS at a dosage of 4 mg/kg induced a sepsis-like state characterized by a rectal temperature reduction by ∼4.0°C, increased neurological deficits in Clark score, cylinder and open-field tests, increased brain infarct volume and reduced neuronal survival in the previously ischemic tissue. Notably, additional treatment with ketamine (10 mg/kg) significantly attenuated the sepsis-associated rectal temperature reduction by ∼1.5°C, reduced neurological deficits, reduced infarct volume, and promoted neuronal survival. Ketamine alone did not influence infarct volume or neurological deficits. Real-time PCR data analysis showed that GFAP, CD86, CD206, IL-1ß, and IL-10 mRNA levels were significantly increased in ischemic brains of LPS-treated compared with vehicle-treated mice. Additional treatment with ketamine significantly decreased IL-1ß and IL-10, but not GFAP, CD86, and CD206 mRNA levels. Our data show that ketamine at a dose that on its own does not confer neuroprotection reverses the adverse effects of LPS-induced sepsis-like state post-ischemia, presumably via immunomodulatory actions.

Evaluation of apoptotic effects of mPEG-b-PLGA coated iron oxide nanoparticles as a eupatorin carrier on DU-145 and LNCaP human prostate cancer cell lines.

Many studies have so far confirmed the efficiency of phytochemicals in the treatment of prostate cancer. Eupatorin, a flavonoid with a wide range of phytomedical activities, suppresses proliferation of and induces apoptosis of multiple cancer cell lines. However, low solubility, poor bioavailability, and rapid degradation limit its efficacy. The aim of our study was to evaluate whether the use of mPEG-b-poly (lactic-co-glycolic) acid (PLGA) coated iron oxide nanoparticles as a carrier could enhance the therapeutic efficacy of eupatorin in DU-145 and LNcaP human prostate cancer cell lines. Nanoparticles were prepared by the co-precipitation method and were fully characterized for morphology, surface charge, particle size, drug loading, encapsulation efficiency and in vitro drug-release profile. The inhibitory effect of nanoparticles on cell viability was evaluated by MTT test. Apoptosis was then determined by Hoechest staining, cell cycle analysis, NO production, annexin/propidium iodide (PI) assay, and Western blotting. The results indicated that eupatorin was successfully entrapped in Fe3O4@mPEG-b-PLGA nanoparticles with an efficacy of (90.99 ± 2.1)%. The nanoparticle's size was around (58.5 ± 4) nm with a negative surface charge [(-34.16 ± 1.3) mV]. In vitro release investigation showed a 30% initial burst release of eupatorin in 24 h, followed by sustained release over 200 h. The MTT assay indicated that eupatorin-loaded Fe3O4@mPEG-b-PLGA nanoparticles exhibited a significant decrease in the growth rate of DU-145 and LNcaP cells and their IC50 concentrations were 100 µM and 75 µM, respectively. Next, apoptosis was confirmed by nuclear condensation, enhancement of cell population in the sub-G1 phase and increased NO level. Annexin/PI analysis demonstrated that eupatorin-loaded Fe3O4@mPEG-b-PLGA nanoparticles could increase apoptosis and decrease necrosis frequency. Finally, Western blotting analysis confirmed these results and showed that Bax/Bcl-2 ratio and the cleaved caspase-3 level were up-regulated by the designing nanoparticles. Encapsulation of eupatorin in Fe3O4@mPEG-b-PLGA nanoparticles increased its anticancer effects in prostate cancer cell lines as compared to free eupatorin. Based on these results, this formulation can provide a sustained eupatorin-delivery system for cancer treatment with the drug remaining active at a significantly lower dose, making it a suitable candidate for pharmacological uses.

Inhibitory effect of flavonoid xanthomicrol on triple-negative breast tumor via regulation of cancer-associated microRNAs.

Breast cancer is the leading cause of cancer death in women worldwide. Due to the side effects of current chemo-reagents on healthy tissues, it is essential to search for alternative compounds with less toxicity and better efficacy. In the present study, we have investigated the anticancer effects of flavonoid xanthomicrol on the mice breast cancer model using MTT assay, cell cycle and Annexin/PI analysis, colony formation assay, H&E staining, immunohistochemistry, and miRNA analysis. Our results demonstrated that xanthomicrol decreased the cell viability and clonogenic capability, induced G1-arrest and apoptosis in the breast cancer cells in vitro, and caused a significant reduction in the volume and weight of mice tumors in vivo. In addition, xanthomicrol reduced the expression of TNFα, VEGF, MMP9, and Ki67, while upregulating the expression of apoptotic markers such as Bax, caspase3, and caspase9. Finally, the expression of miR21, miR27, and miR125, known as oncomirs, decreased significantly after xanthomicrol administration, while the expression of miR29 and miR34, functioning as tumor suppressors, increased significantly (p < .001). Our data demonstrated that xanthomicrol can induce apoptosis and suppress angiogenesis in breast cancer cells due to its inhibitory effect on oncomirs and its stimulatory effect on tumor suppressor miRNAs.

Evaluation of apoptotic effects of mPEG-b-PLGA coated iron oxide nanoparticles as a eupatorin carrier on DU-145 and LNCaP human prostate cancer cell lines / 药物分析学报

Many studies have so far confirmed the efficiency of phytochemicals in the treatment of prostate cancer.Eupatorin,a flavonoid with a wide range of phytomedical activities,suppresses proliferation of and in-duces apoptosis of multiple cancer cell lines.However,low solubility,poor bioavailability,and rapid degradation limit its efficacy.The aim of our study was to evaluate whether the use of mPEG-b-poly(lactic-co-glycolic)acid(PLGA)coated iron oxide nanoparticles as a carrier could enhance the therapeutic efficacy of eupatorin in DU-145 and LNcaP human prostate cancer cell lines.Nanoparticles were prepared by the co-precipitation method and were fully characterized for morphology,surface charge,particle size,drug loading,encapsulation efficiency and in vitro drug-release profile.The inhibitory effect of nanoparticles on cell viability was evaluated by MTT test.Apoptosis was then determined by Hoechest staining,cell cycle analysis,NO production,annexin/propidium iodide(PI)assay,and Western blotting.The results indicated that eupatorin was successfully entrapped in Fe3O4@mPEG-b-PLGA nanoparticles with an efficacy of(90.99 ± 2.1)％.The nanoparticle's size was around(58.5 ± 4)nm with a negative surface charge[(-34.16 ± 1.3)mV].In vitro release investigation showed a 30％initial burst release of eupatorin in 24 h,followed by sustained release over 200 h.The MTT assay indicated that eupatorin-loaded Fe3O4@mPEG-b-PLGA nanoparticles exhibited a significant decrease in the growth rate of DU-145 and LNcaP cells and their IC50 concentrations were 100 μM and 75 μM,respectively.Next,apoptosis was confirmed by nuclear condensation,enhancement of cell population in the sub-G1 phase and increased NO level.Annexin/PI analysis demonstrated that eupatorin-loaded Fe3O4@mPEG-b-PLGA nanoparticles could increase apoptosis and decrease necrosis frequency.Finally,Western blotting analysis confirmed these results and showed that Bax/Bcl-2 ratio and the cleaved caspase-3 level were up-regulated by the designing nanoparticles.Encapsulation of eupatorin in Fe3O4@mPEG-b-PLGA nanoparticles increased its anticancer effects in prostate cancer cell lines as compared to free eupatorin.Based on these results,this formulation can provide a sustained eupatorin-delivery system for cancer treatment with the drug remaining active at a significantly lower dose,making it a suitable candidate for pharmacological uses.

Flavonoid calycopterin triggers apoptosis in triple-negative and ER-positive human breast cancer cells through activating different patterns of gene expression.

Breast cancer is the most common cause of death related to cancer in women, and several studies proved that flavonoids could induce apoptosis in this cancer through different pathways. Calycopterin is a flavonoid which was shown to induce preferential antiproliferative effects on some cancers; however, no information is available on its effect on breast cancer. Therefore, in this paper, the apoptotic effect of calycopterin and its underlying mechanism in two different breast cancer cells, MDA-MB-231 and MCF7 cell lines were investigated. MTT assay showed that calycopterin reduced the proliferation of both cancer lines with no adverse effect on normal cells. The clonogenic assay showed that calycopterin treatments decreased the colony numbers and sizes, and wound healing assay demonstrated the inhibition of migration in both cancer cells. Cell cycle and annexin/PI analyses indicated that calycopterin augmented sub-G1 population and annexin/PI-positive cells. Gene expression revealed that Bax/Bcl2 increased in the MDA-MB-231 cell line, while no change was observed in that of the MCF7 line. Expression of gene caspase-8 was augmented in both lines, although increased expression of caspase-3 was found just in MDA-MB-231 cells. Our results validated the apoptotic effect of calycopterin on both breast cancer lines with more potency on triple-negative ones.

Efficient synergistic combination effect of Quercetin with Curcumin on breast cancer cell apoptosis through their loading into Apo ferritin cavity.

Combination of natural agents has received a great attention in cancer treatment because of synergistically increased apoptotic effect on cancer cell lines by triggering several apoptotic signaling pathways. However, the hydrophobic nature, poor bioavailability and low cellular uptake of most natural agents limit their therapeutic effectiveness. The purpose of this study was to design Apoferritin nanoparticles loaded with Quercetin and Curcumin (Que-Cur-HoS-Apo NPs) and to test their synergistic antitumor properties on a breast cancer cell line (MCF7). The physico-chemical characterization of the Que-Cur-HoS-Apo NPs by Size Exclusion Chromatography (FPLC) and Dynamic Light Scattering (DLS) confirmed the encapsulation of the compounds in the protein cage with narrow size distribution in the range 17.4 ±â€¯1.2 nm. Cell viability study indicated that Que-Cur-HoS-Apo NPs were able to exert a more pronounced effect at lower dose on the MCF7 cell line when compared to the free combination of the drugs. The Que-Cur-HoS-Apo system allowed cellular uptake of natural agents thus triggering enhanced apoptosis. These effects were confirmed by Annexin-V/7-AAD Staining Assay and intracellular Reactive Oxygen Species (ROS) quantitative detection. These results suggest the potential of Que-Cur-HoS-Apo NPs as a promising anti-cancer agent in breast cancer therapy and pave the way to examine Que-Cur-HoS-Apo NPs effect in vivo.

Flavonoid Calycopterin Induces Apoptosis in Human Prostate Cancer Cells In-vitro.

Prostate cancer is enumerated as one of the most prevalent cancers in men, with a mortality rate of 18%. Chemotherapy is considered as a common strategy for cancer treatment; however, toxic side effects and drug resistance associated with chemotherapy are major drawbacks with this approach. It is well known that a diet rich in flavonoids can reduce the incidence of many types of cancer in a significant manner, and it was proved that methoxy flavones have greater bioavailability compared to the nonmethylated ones. Calycopterin is a tetramethoxy flavone which was demonstrated to have anti-proliferative effects on colon, gastric, and osteosarcoma cancer cells. Therefore, in the current study, we have evaluated the apoptotic effects of this flavonoid on two prostate cancer cell lines in-vitro. The MTT assay revealed that after 48 h treatment with this flavonoid, cell viability reduced to 50% compared to the control group. However, calycopterin treatment of healthy HUVEC did not cause any significant reduction in cell viability. Moreover, the clonogenic assay demonstrated that after 14 days, colony size and numbers reduced significantly in calycopterin treated cells. In addition, the percentage of the sub-G1 population in calycopterin-treated cells augmented significantly compared to untreated group. Also, calycopterin-treated cells demonstrated shiny condensed nuclei with fragmented DNA indicative of apoptosis. Finally, a significant reduction in the migration ability was observed in both lines subjected to calycopterin after 48 h. To conclude, our results demonstrated the apoptotic and anti-metastatic effects of calycopterin in both hormone-dependent and independent prostate cancer cell lines.

Gallic acid and curcumin induce cytotoxicity and apoptosis in human breast cancer cell MDA-MB-231.

Introduction: Gallic acid (GA) and curcumin (Cur) are natural phenolic compounds that their anti-tumor effects on many types of cancers have been proved. In the current study, the effect of the combination of these agents on MDA-MB-231 breast cancer cells was investigated. Methods: Inhibition of cell proliferation (MTT assay), light microscopy, fluorescence microscopy, cell cycle analysis, nitrite detection, ROS levels, measurement of the mitochondrial membrane potential, GSH level, Annexin V assay, RT-PCR and Western blotting methods were applied. Results: The results revealed the combination of GA and Cur strongly decreased MDA-MB-231 cell growth. Moreover, this combination increased ROS level and cytotoxic activity along with the glutathione depletion in MDA-MB-231 cells. Flow cytometry analysis showed the combination of GA and Cur increased sub-G1 cell population. Furthermore, fluorescent staining and Annexin V/PI assay showed that apoptotic cells were significantly increased in the presence of GA and Cur. At last, protein expression evaluation showed that the combination of GA and Cur significantly decreased Bcl-2 level while increased Bax, cleaved-caspase3 and PARP levels in MDA-MB-231 cells. Conclusion: These results suggest that GA in combination with Cur could be a possible candidate for chemoprevention agent of triple negative breast cancer.

Eupatorin and Salvigenin Potentiate Doxorubicin-Induced Apoptosis and Cell Cycle Arrest in HT-29 and SW948 Human Colon Cancer Cells

Background: Cancer persists as one of the world's most pressing maladies. Notable points about chemotherapy are drug side effects which are almost universally encountered. Emerging knowledge focusing on mechanisms of toxicity due to chemotherapy has led to characterization of novel methods, including the exploitation of natural compounds, in combination therapies. Flavonoids are natural polyphenolic compounds that play protective roles against tumor cell development. The focus of this study was apoptotic effects of two flavonoids, eupatorin and salvigenin, in combination with doxorubicin on a cellular model of colon cancer. Method: Upon establishing a non-effective dose of doxorubicin, and effective doses of eupatorin (100µM) and salvigenin (150µM) via MTT, morphological features of apoptosis were distinguished using DAPI staining and cell cycle blockage in the sub-G1 phase. Apoptosis was determined by annexin/ PI and western blotting. ROS levels and MMP were measured to show any role of mitochondria in apoptosis. Results: Co-administration of flavonoids with doxorubicin induced apoptosis via the mitochondrial pathway as mitochondrial membrane potential and ROS production were changed. Annexin/PI analysis demonstrated that apoptosis frequency was increased with the combination treatments in colon cancer cells. Finally, the combination of these flavonoids with doxorubicin increased the Bax/Bcl-2 ratio, caspase-3 expression and PARP cleavage. Conclusion: Combination of flavonoids with doxorubicin induces apoptosis and enhances effect on cancer cells which might allow amelioration of side effects by dose lowering.

Combination of arabinogalactan and curcumin induces apoptosis in breast cancer cells in vitro and inhibits tumor growth via overexpression of p53 level in vivo.

PURPOSE: Increased mortality associated with breast cancer in women has spurred the studies to develop new drugs. Arabinogalactan (AG) and curcumin (Cur) are two natural products broadly explored in cancer therapy. Our major goal in the current study was to assess anticancer properties of combination these reagents in vitro on human breast cancer cells and in vivo utilizing animal model of breast cancer. EXPERIMENTAL DESIGN: We evaluated cell proliferation, apoptosis, cell cycle, and protein expression in vitro on MDA-MB-231 human breast cancer cells. For in vivo studies, murine breast cancer cells were implanted into BALB/c mice. Thereafter, volume of the developing tumor was calculated and expression of Ki67 and p53 proteins was evaluated to analyze cell proliferation and apoptosis. RESULTS: Combination of AG and Cur significantly decreased cell growth in human breast cancer cells without any significant effect on normal cell growth. This combination could increase cell population in sub-G1 phase, which was indicative of apoptosis. Western blotting showed that the combination of AG and Cur significantly increased Bax/Bcl2 ratio as well as cleaved-caspase3 level in MDA-MB-231 cells. Combination of AG and Cur promoted apoptosis by increasing ROS level, changing mitochondrial membrane and reduction of glutathione. In addition, in vivo studies in mouse showed that this combination could inhibit the progression of breast tumors through over-expression of p53 and reduction of Ki67 levels. CONCLUSION: Our findings suggest that the combination of AG and Cur is of great potential to induce apoptosis in breast cancer cells in vitro and in vivo.

Collagen Extracted from Persian Gulf Squid Exhibits Anti-Cytotoxic Properties on Apple Pectic Treated Cells: Assessment in an In Vitro Bioassay Model.

BACKGROUND: Collagen-based three-dimensional (3D) in vitro systems have been introduced to study the physiological states of cells. As a biomolecule, collagen is usually extracted from terrestrial animals whilst aquatic animals like squid contain large amounts of collagen. METHODS: In order to make effective use of marine organisms, we selected Persian Gulf squid in 2015 to extract the required collagen. Then, a 3D culture system based on the extracted collagen was applied to investigate cellular mechanisms in a native microenvironment. The formed collagen gel was used to investigate the growth of MDA-MB-231 breast cancer cells as well as responses to pectic acid. RESULTS: The results revealed that the extracted collagen contained α, ß and γ components with high water holding capacity. This collagen formed a gel-like structure, which could promote the proliferation of MDA-MB-231 breast cancer cells. The MDA-MB-231 cells' viability in presence of pectic acid, demonstrating the cells' behavior in a 3D culture system. CONCLUSION: It seems that the collagen extracted from squid skin has type I collagen properties. It might be used as a substrate in 3D cell culture systems.

Apple pectin: A natural source for cancer suppression in 4T1 breast cancer cells in vitro and express p53 in mouse bearing 4T1 cancer tumors, in vivo.

PURPOSE: Increase in the number of cancer related deaths has made the study on developing new drugs and treatments essential. One of the main aims in developing new therapies is to use natural resources which have the ability to induce apoptosis. Pectin is one of these natural compounds, a complex polysaccharide found in apples with anti-cancer properties. The aim of this study was to examine anti-cancer properties of pectic acid both in vitro in 4T1 breast cancer cells and in vivo using an animal model of breast cancer. EXPERIMENTAL DESIGN: MTT cell proliferation assays, double fluorescence staining (acridine orange/ethidium bromide) and cell cycle analysis were employed to measure apoptosis in vitro. 4T1 cells were implanted into female BALB/c mice for in vivo studies. Then tumor volumes, histological analysis and immunohistochemical staining of P53 and tunnel test were applied to evaluate apoptosis in tumors. RESULTS: The results of in vitro studies showed that concentration of 0.1% of pectic acid could induce apoptosis, inhibit cell growth (p<0.001) and reduce cell attachment, fragmented chromatin, and membrane blebbing as well as blocking the sub-G1 phase (p<0.001). In addition, in vivo studies showed that pectic acid could inhibit the progression of tumors through over-expression of P53 and increasing the number of apoptotic cells. CONCLUSION: Our results demonstrated that pectic acid, a natural component of apple, can prevent metastasis in both cancer cell lines and primary tumors. This potential effect is mainly due to its ability to induce apoptosis.

Antioxidant and Anticancer Activities of Walnut (Juglans regia L.) Protein Hydrolysates Using Different Proteases.

Walnut (Juglans regia L.) contains approximately 20-25 % protein with abundant essential amino acids. The enzymatic hydrolysate of Persian walnut (Chandler) seed proteins was prepared by incubation with three different proteases, including pancreatic chymotrypsin and trypsin, and a microbial enzyme proteinase K. The hydrolysates were found to possess excellent antioxidant capacities. The peptide fractions scavenged the 2, 2'-anizo-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) free radicals and inhibited the activity of reactive oxygen species. Walnut protein hydrolysates were also tested, for the first time, against the viability of human breast (MDA-MB231) and colon (HT-29) cancer cell lines. MTT, [3-(4, 5dimethylthiazolyl)-2,5-diphenyl-tetrazolium bromide], assay was used to assess in vitro cancer cell viability upon treatment with the peptide fractions. The peptide fractions showed cell growth inhibition of 63 ± 1.73 % for breast cancer and 51 ± 1.45 % for colon cancer cells. Thus, a direct correlation between antioxidant and anticancer activities of walnut peptide fractions exists and supports their potential therapeutic benefit.

Pectic-Oligoshaccharides from Apples Induce Apoptosis and Cell Cycle Arrest in MDA-MB-231 Cells, a Model of Human Breast Cancer.

BACKGROUND: The effects of plant products on cancer cells has become a field of major importance. Many substancesmay induce apoptosis in anti-cancer treatment. Pectins, a family of complex polysaccharides, and their degradation products may for exasmple exert apoptotic effects in cancer cells. Apples and citrus fruits are the main sources of pectin which can be applied for anti-cancer research. The present study concerned an intact form of pectic-oligoshaccharide named pectic acid (poly galactronic acid). MATERIALS AND METHODS: Inhibition of cell proliferation assays (MTT), light microscopy, fluorescence microscopy (acridin orange/ethidium bromide), DNA fragmentation tests, cell cycle analysis, annexin PI and Western blotting methods were applied to evaluate apoptosis. RESULTS: The results indicated that pectic acid inhibited cell growth and reduced cell attachment after 24h incubation. This did not appear to be due to necrosis, since morphological features of apoptosis were detected with AO/EB staining and cell cycling was blocked in the sub-G1 phase. Annexin/PI and DNA fragmentation findings indicated that apoptosis frequency increased after 24h incubation with pectic acid. In addition, the data showed pectic acid induced caspase-dependent apoptosis. CONCLUSIONS: These data indicate that apple pectic acid without any modification could trigger apoptosis in MDA-MB-231 human breast cancer cells and has potential to improve cancer treatment as a natural product.

Alterations in the hippocampal phosphorylated CREB expression in drug state-dependent learning.

The present study investigated the possible alterations of hippocampal CREB phosphorylation in drug state-dependent memory retrieval. One-trial step-down passive avoidance task was used to assess memory retrieval in adult male NMRI mice. Pre-training administration of ethanol (1g/kg, i.p.) induced amnesia. Pre-test administration of ethanol (1g/kg, i.p) or nicotine (0.7 mg/kg, s.c.) reversed ethanol-induced amnesia, indicating ethanol- or ethanol-nicotine induced state-dependent learning (STD). Using Western blot analysis, it was found that the p-CREB/CREB ratio in the hippocampus increased in the mice that showed successful memory retrieval as compared with untrained mice. In contrast, pre-training administration of ethanol (1g/kg, i.p.) decreased the hippocampal p-CREB/CREB ratio in comparison with the control group. The hippocampal p-CREB/CREB ratio enhanced in ethanol- and ethanol-nicotine induced STD. Moreover, memory impairment induced by pre-training administration of WIN (1 mg/kg, i.p.) improved in the animals that received pre-test administration of WIN (1 mg/kg, i.p.), ethanol (0.5 g/kg, i.p.) or nicotine (0.7 mg/kg, s.c.), suggesting a cross STD between the drugs. The p-CREB/CREB ratio in the hippocampus decreased in the of WIN-induced amnesia and STD groups in comparison with the control group. In addition, cross state-dependent learning between WIN and ethanol or nicotine was associated with the increase of the hippocampal p-CREB/CREB ratio. It can be concluded that phosphorylation of CREB in the hippocampus is a critical event underlying the interaction of co-administration of drugs on memory retrieval in passive avoidance learning.

Efficient induction of pluripotency in primordial germ cells by dual inhibition of TGF-ß and ERK signaling pathways.

Primordial germ cells (PGCs) have the ability to be reprogrammed into a pluripotent state and are defined as embryonic germ cells (EGCs) in vitro. EGC formation is more efficient, has a shorter culture period than somatic cell reprogramming, and does not require exogenous genetic manipulation. Therefore, EGCs are a good model to analyze mechanisms by which committed cells acquire a pluripotent state. In the present study we have attempted to elucidate a more defined and robust protocol that promotes EGC generation through the suppression of transforming growth factor-ß (TGF-ß) and extracellular signal-regulated kinase (ERK) signaling pathways by SB431542 (SB) and PD0325901 (PD), respectively. Under this condition the efficiency of transformation of PGCs into EGCs was more than the inhibition of glycogen synthase kinase 3 and ERK signaling pathways. Pluripotency of the resultant-derived EGC lines were further confirmed by gene expression, immunofluorescent staining, directed differentiation ability, teratoma formation, and their contribution to chimeric mice and germ-line transmission. These results showed that PGCs from different embryonic stages (E8.5 and E12.5) could be reprogrammed, maintained, and expanded efficiently under feeder- and serum-free chemically defined conditions by dual inhibition of TGF-ß and ERK signaling pathways, regardless of the animal's genetic background.

Inhibition of glycogen synthase kinase-3 promotes efficient derivation of pluripotent stem cells from neonatal mouse testis.

BACKGROUND: Several studies have demonstrated the derivation of multi- or pluripotent stem cells from testicular cells of both newborn and adult mice by a spontaneous conversion process, when these cells are cultured in vitro for an extended time. To obtain a better and robust derivation, we have attempted to identify small molecules (SMs) that induce reprogramming of testicular cells in culture into germline-derived pluripotent stem cells (gPSCs). METHODS: We tested several SMs based on previous reports that have shown enhancement of establishment of induced pluripotent stem cells or embryonic stem cells (ESCs) on mouse NMRI (outbred strain) and C57BL/6 (inbred strain) testicular cells. After appearance of ESC-like colonies at Day 6, they were passaged on mitotically arrested mouse embryonic fibroblasts in mouse ESC medium in the absence or presence of SMs up to Day 14. The generated cells were characterized using a variety of experimental approaches. RESULTS: The application of several SMs involved in pluripotent reprogramming led to the discovery that CHIR99021 (CHIR), a glycogen synthase kinase-3 (GSK-3) inhibitor, promotes efficient derivation of gPSCs from neonatal mouse NMRI and C57BL/6 testes. The pluripotency of the generated cell lines has been confirmed by in vitro spontaneous and direct differentiation toward cardiac and neural lineages, and formation of chimeras after injection of gPSCs into blastocysts. We have shown that the generated gPSCs could be maintained and expanded under chemically defined serum and feeder-free conditions by inhibition of both the extracellular signal-regulated kinases (Erk1/2) and GSK-3. CONCLUSIONS: To our knowledge, this is the first report of a simple and efficient protocol to reprogram gPSCs from testicular cells solely by inhibition of GSK-3 in two strains of mice with different genetic backgrounds. Additionally, this brings us closer to eliminating the need for genetic modification in pluripotent reprogramming. Future studies will determine whether the inhibition of GSK-3 could affect the generation of naïve gPSCs lines in other mammals.

Combination effect of PectaSol and Doxorubicin on viability, cell cycle arrest and apoptosis in DU-145 and LNCaP prostate cancer cell lines.

The effect of PectaSol on Dox (Doxorubicin) cytotoxicity in terms of apoptosis and cell cycle changes in PCa (prostate cancer) cell lines (DU-145 and LNCaP) has been investigated. Combination of PectaSol and Dox resulted in a viability of 29.4 and 32.6% (P<0.001) in DU-145 and LNCaP cells. The IC50 values decreased 1.5-fold and 1.3-fold in the DU-145 and LNCaP cells respectively. In the DU-145 cells, combination of PectaSol and Dox resulted in a reduction in p27 gene and protein expression (P<0.001). In LNCaP cells, this combination increased p53, p27 and Bcl-2 expression. Treatment with both drugs in DU-145 cells led to an increase in sub-G1 arrest (54.6% compared with 12.2% in Dox). In LNCaP cells, combination of the drugs led to an increased in G2/M arrest (61.7% compared with 53.6% in Dox). Based on these findings, progressive cytotoxicity effect of Dox and PectaSol together rapidly induce cell death in DU-145 through apoptosis and in LNCaP cells through cell cycle arrest (G2/M arrest).

Learning impairment caused by intra-CA1 microinjection of testosterone increases the number of astrocytes.

The hippocampus is essentially involved in learning and memory, and is known to be a target for androgen actions. As neuron-astrocyte interactions play a crucial role during development and in the adult brain, it is thought that astrocytes support learning and memory through specific mechanisms. In this study, the effect of testosterone-induced impairment of acquisition and retrieval of long-term memory, on the number of astrocytes was studied. Adult male albino Wistar rats were bilaterally cannulated into the CA1 region and received 80 microg/0.5 microl of testosterone enanthate dissolved in DMSO as vehicle. In trained groups injection was done 30 min before training. Our results showed that pre-training administration of testosterone increased escape latency and the traveled distance, as compared to intact or DMSO administered (sham-operated) counterparts. Testosterone administration also increased the number of astrocytes in rats, compared to sham-operated and intact animals in both trained and non-trained groups. We have also found that spatial learning could increase the number of astrocytes in the hippocampus as a center of long-term learning and memory and recall. It is concluded that, testosterone-induced impairment in learning and memory, causes an increase in the number of astrocytes in the CA1 region of rat hippocampus.

Apoptotic and necrotic effects of pectic acid on rat pituitary GH3/B6 tumor cells.

BACKGROUND: Pectin is composed of complex polysaccharides that can inhibit cancer metastasis and proliferation with no evidence of toxicity. In the present study, the apoptotic and necrotic effects of pectic acid (PA) on the rat pituitary GH3/B6 tumor cells has been investigated. METHODS: GH3/B6 cells were cultured in the Ham's F12 medium enriched with 15% horse serum and 2.5% fetal bovine serum for 3 days. Then, they were treated by various amounts of PA in different periods (6, 24 and 48 hours). Bromocriptine was used as positive control and the cell viability was detected by MTT test. The nuclear morphology of cells was explored by florescent stains including acridine orange (AO)/ethidium bromide (EB). In addition, percentages of apoptotic and necrotic cells were studied with triphosphate nick-end labeling (TUNEL) assay, cell cycle analysis and propidium iodide (PI) staining. RESULTS: Long-term incubation with PA results in increased cell death and DNA damage as detected by MTT assay and AO/EB staining. TUNEL assay showed that PA (100 microg/ml to 1 mg/ml) could induce apoptosis in a dose-dependent manner, while higher concentrations of PA (2.5 and 5 mg/ml) induced necrosis which was confirmed by PI staining. Furthermore, cell cycle analysis indicated that PA induced sub G1 events, and DNA fragmentation was also correlated with the number of the apoptotic cells. CONCLUSION: It can be concluded that PA is responsible for apoptosis in the rat pituitary tumor cells. Therefore, one may suggest that this group of polysaccharides can be used in treatment of pituitary tumors.