Anti-metastatic effect of taraxasterol on prostate cancer cell lines.

Background and purpose: Prostate cancer is the second cause of death among men. Nowadays, treating various cancers with medicinal plants is more common than other therapeutic agents due to their minor side effects. This study aimed to evaluate the effect of taraxasterol on the prostate cancer cell line. Experimental approach: The prostate cancer cell line (PC3) was cultured in a nutrient medium. MTT method and trypan blue staining were used to evaluate the viability of cells in the presence of different concentrations of taraxasterol, and IC50 was calculated. Real-time PCR was used to measure the expression of MMP-9, MMP-2, uPA, uPAR, TIMP-2, and TIMP-1 genes. Gelatin zymography was used to determine MMP-9 and MMP-2 enzyme activity levels. Finally, the effect of taraxasterol on cell invasion, migration, and adhesion was investigated. Findings/Results: Taraxasterol decreased the survival rate of PC3 cells at IC50 time-dependently (24, 48, and 72 h). Taraxasterol reduced the percentage of PC3 cell adhesion, invasion, and migration by 74, 56, and 76 percent, respectively. Real-time PCR results revealed that uPA, uPAR, MMP-9, and MMP-2 gene expressions decreased in the taraxasterol-treated groups, but TIMP-2 and TIMP-1 gene expressions increased significantly. Also, a significant decrease in the level of MMP-9 and MMP-2 enzymes was observed in the PC3 cell line treated with taraxasterol. Conclusion and implications: The present study confirmed the therapeutic role of taraxasterol in preventing prostate cancer cell metastasis in the in-vitro study.

Aflatoxin G1 exposure altered the expression of BDNF and GFAP, histopathological of brain tissue, and oxidative stress factors in male rats.

Background and purpose: Aflatoxins are highly toxic compounds that can cause acute and chronic toxicity in humans and animals. This study aimed to evaluate the expression of BDNF and GFAP, histopathological changes, and oxidative stress factors in brain tissue exposed to aflatoxin G1 (AFG1) in male rats. Experimental approach: Twenty-eight male Wistar rats were used. Animals were randomly divided into 4 groups of 7 each. The control group received 0.2 mL of corn oil and the treatment groups were exposed to AFG1 (2 mg/kg) intra-peritoneally for 15, 28, and 45 days. The tissue was used for histopathological studies, and the level of TAC, SOD, and MDA, and the expression of BDNF and GFAP genes were evaluated. Findings/Results: Real-time PCR results showed that AFG1 increased GFAP expression and decreased BDNF expression in AFG1-treated groups compared to the control group. The tissue level of TAC and SOD over time in the groups receiving AFG1 significantly decreased and the tissue level of MDA increased compared to the control group. Histopathological results showed that AFG1 can cause cell necrosis, a reduction of the normal cells number in the hippocampal region of CA1, cerebral edema, shrinkage of nerve cells, formation of space around neuroglia, and diffusion of gliosis in the cerebral cortex after 45 days. Conclusion and implication: AFG1, by causing pathological complications in cortical tissue, was able to affect the exacerbation of nerve tissue damage and thus pave the way for future neurological diseases.

Protective effect of gallic acid on nicotine-induced testicular toxicity in mice.

BACKGROUND AND PURPOSE: Nicotine is an alkaloid found in many nutrients and tobacco that can cause infertility in men. Gallic acid is a powerful antioxidant that possesses antimutagenic and anticancer activities. This study aimed to determine the potential protective effect of gallic acid against nicotine-induced testicular toxicity in male mice. EXPERIMENTAL APPROACH: In this in vivo study, forty-eight mice were equally divided into eight groups intraperitoneally receiving normal saline (control), nicotine (0.6 mg/kg), gallic acid (5, 10, and 15 mg/kg), and gallic acid (5, 10, and 15 mg/kg) plus nicotine. Nicotine was injected intraperitoneally for 14 days and gallic acid was administered concomitantly with nicotine and continued for 7 days later. Then, body and testicular weights, the sperm parameters (viability, number, motility, and morphology of sperm), and testicular histology were evaluated. Also, serum levels of nitric oxide, total antioxidant, superoxide dismutase, malondialdehyde, and testosterone were measured. FINDINGS/RESULTS: The results showed that the administration of nicotine significantly reduced testis and body weight, sperm count, viability, normal morphology and motility, seminiferous tubules diameter, testosterone levels, serum levels of total antioxidants, and superoxide dismutase compared to the control group (P < 0.05). It also significantly increased the level of nitric oxide and malondialdehyde (P < 0.05). Increasing the dose of gallic acid along with nicotine significantly increased body weight, sperm count, viability, normal morphology and motility, the diameter of seminiferous, testosterone concentration, total antioxidant levels (P < 0.05). This combination also significantly decreased malondialdehyde and nitric oxide levels compared to the nicotine-receiving group (P < 0.05). CONCLUSION AND IMPLICATIONS: Gallic acid had a protective effect on nicotine-induced testicular toxicity in mice. It can neutralize the harmful effect of nicotine on male fertility in smokers.

Expression of cyclin D1, p21, and estrogen receptor alpha in aflatoxin G1-induced disturbance in testicular tissue of albino mice.

BACKGROUND AND PURPOSE: Aflatoxin (AF) is a mycotoxin produced by various strains of the Aspergillus family. AFG1 as one of the most important types is highly found in cereals and grains. AF affects sperm production or even its quality. This study was designed to test the effects of AFG1 on mice testicular tissue. EXPERIMENTAL APPROACH: Twenty-four Albino mice were divided into four groups of 6 each; a control group (0.2 mL corn oil and ethanol), three treatment groups with different periods (20 µg/kg AFG1 for 7, 15, and 35 consecutive days). All treatments were applied intraperitoneally. Biosynthesis of cyclin D1, p21, and estrogen receptor alpha (ERα) proteins was evaluated by immunohistochemistry (IHC) staining. Levels of cyclin D1, p21, and ERα mRNA were evaluated by the real-time polymerase chain reaction (RT-PCR) technique. Tubular differentiation index (TDI), reproductive index (RI), and spermiogenesis indices were also analyzed. FINDINGS/RESULTS: AFG1 increased the percentage of seminiferous tubules with negative TDI, RI, and SPI compared to the control group (P < 0.05). RT-PCR and IHC analyses illustrated time-dependent enhancement in p21 expression and cyclin D1 biosynthesis in AFG1-treated groups significantly (P < 0.05). While the protein and mRNA levels of ERα were significantly (P < 0.05) decreased in a time-dependent manner. CONCLUSION AND IMPLICATIONS: The chronic exposure to AFG1 reduced the expression and synthesis of ERα, increased the expression and synthesis of p21 and cyclin D1, impaired apoptosis, which in turn could impair spermatogenesis.

Anti-proliferative and cytotoxic effect of Iranian snake (Vipera raddei kurdistanica) venom on human breast cancer cells via reactive oxygen species-mediated apoptosis.

BACKGROUND AND PURPOSE: Breast cancer is the most commonly occurring cancer in women around the world. Despite new advances in cancer therapy, breast cancer remains a disease with high morbidity and mortality. Snake venom is a poisonous mixture of different molecules, such as carbohydrates, nucleosides, amino acids, lipids, proteins, and peptides. Previous studies demonstrated that some snake venoms showed in vitro anti-cancer effects. In this study, the effects of the Iranian snake (Vipera raddei kurdistanica) venom on breast cancer cells were investigated. EXPERIMENTAL APPROACH: The effect of increasing concentrations of snake venom on breast cell viability was assessed by trypan blue, MTT, and lactate dehydrogenase measurements. Apoptosis was detected and quantified by fluorescent staining and DNA fragmentation assay. The expression level of some apoptotic-related genes was investigated using real-time polymerase chain reaction (RT-PCR). The Western blotting method was also used to detect the protein expression profiles in the cells. FINDINGS / RESULTS: After treatment for 24, 48, 72, and 96 h, the cell viability was significantly reduced in a time- and dose-dependent manner (P < 0.05). The venom effect on normal breast cells was significantly smaller than cancer cells (P > 0.05). Apoptosis was significantly increased (P < 0.05). The RT-PCR and western blot data confirmed the increase of apoptosis in cells treated with venom. CONCLUSION AND IMPLICATIONS: These data suggested that the vipera raddei kurdistanica venom had a cytotoxic property via activation of apoptosis in breast cancer cells.

Resveratrol Effect on Adipose-Derived Stem Cells Differentiation to Chondrocyte in Three-Dimensional Culture.

Purpose: Adipose stem cells (ASCs) are pluripotent cells with the ability of self-renewal and differentiation into different types of mesenchymal cells. As cartilage repair is difficult due to lack of blood capillary, resveratrol (Res) is a polyphenolic compound with diverse biological properties to be possibly used in this case. The aim of the present study was to investigate the effect of Res on differentiation of ASCs into chondrocyte in a three-dimensional (3D) culture model. Methods: Subcutaneous adipose tissues were prepared and digested enzymatically, and passed through cell strainer. ASCs were harvested in the fourth passage, and divided into five groups. The control group received chondrogenic differentiation medium (CDM) while the experimental groups received CDM plus different doses of Res (1, 10, 20, and 50 µM) for 21 days. Expression of cartilage specific genes and Sirtuin1 (SIRT 1), cell viability, apoptosis and ferric reducing antioxidant power (FRAP) were detected using reverse transcription polymerase chain reaction (RT-PCR), MTT assay, TUNEL and acridine orange/ethidium bromide (AO/EB) staining. One-way ANOVA and non-parametric Mann-Whitney U test were used for data analyses. Results: ASCs were differentiated to chondrocyte by CDM in a three-dimensional culture. 10 and 20 µM doses of Res showed the most proliferating effect on ADSCs. The SIRT 1 genes expression and FRAP level also increased significantly compared to the control group (P<0.05). Also, OD of cell increased whereas apoptosis decreased. Conclusion: 3D culture was a suitable condition for ASCs differentiation to chondrocyte, and lower doses of Res exert proliferation effect on ASCs.

Temozolomide and tranilast synergistic antiproliferative effect on human glioblastoma multiforme cell line (U87MG).

Background: Glioblastoma multiforme (GBM) is the most malignant primary brain tumor. Temozolomide (TMZ) is a chemotherapeutic agent that has been used in GBM treatment. Resistance to TMZ is a major obstacle to successful GBM treatment. The aim of the present study was to investigate the effect of TMZ and tranilast on human GBM cell line (U87MG). Methods: In this in vitro experimental study, the effect of TMZ and tranilast on cell proliferation was measured using the MTT assay. Median effect analysis was performed to determine the TMZ and tranilast interaction. Lactate dehydrogenase assay was used to determine TMZ and tranilast cytotoxicity. Cell fluorescent staining and real-time PCR were used for apoptosis evaluation. The effect of TMZ and tranilast on U87MG nitric oxide (NO) production was evaluated by Griess assay. Results: TMZ and tranilast had a significant dose- and time-dependent inhibitory effect on cell proliferation. The mean combination index values represented a synergistic effect, and dose reduction index values suggested the advantages of reducing the toxicity, adverse effects, and drug resistance in combination of TMZ and tranilast. Apoptosis cell death was induced by TMZ and/or tranilast in cells. TMZ and tranilast reduced NO. production in cells. Conclusion: TMZ and tranilast combination inhibited the GBM cells growth effectively.

Antiproliferative Effect of Trifolium Pratens L. Extract in Human Breast Cancer Cells.

Breast cancer is currently the most common cancer in women worldwide. Tamoxifen is used for the treatment of both early and advanced estrogen receptor positive breast cancer. Trifolium pratense L. has been suggested for cancer treatment in traditional medicine. The effect of T. pratense extract on breast cell lines (MCF-7 and MDA-MB-231) was investigated here. The cells were treated with T. pratense extract and cell viability was evaluated using trypan blue staining, MTT assay and lactate dehydrogenase activity measurement. Apoptosis, autophagy cell death was detected using fluorescent dye staining. Griess assay was performed to nitric oxide (No) measurement. Change in expression level of some apoptotic and autophagic- related genes was detected using real-time PCR. The combination effect of exteract and tamoxifen was evaluated by calculating the combination index and dose reduction index (DRI) values. T. pratense extract reduced the cell viability in dose and time- dependent manner. Apoptosis and autophagy was increased after treatment with T. pratense extract. Nitric oxide production significantly reduced in cells and combination of tamoxifen and T. pratense extract had a synergistic cytotoxic effect on breast cancer cell lines. T. pratense as a dietary supplement can be beneficial for some patients with breast cancer who receive conventional chemotherapy.

Synergistic effect of temozolomide and thymoquinone on human glioblastoma multiforme cell line (U87MG).

AIMS: Temozolomide (TMZ) is an alkylating agent used for glioblastoma multiforme (GBM) treatment. Nevertheless, resistance to TMZ is a major obstacle to successful treatment of this cancer. The aim of the present study was to investigate the effects of TMZ and thymoquinone (TQ) on U87MG cell line. MATERIALS AND METHODS: The effect of TMZ and/or TQ on viability and invasion potential of U87MG cells was evaluated using various techniques including 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, lactate dehydrogenase activity, cell invasion, migration, and adhesion assays. Enzyme-linked immunosorbent assay and polymerase chain reaction were used to study the expression and secretion of matrix metalloproteinases (MMPs). RESULTS: Combination of TMZ and TQ had a synergistic cytotoxic effect on U87MG cells. TMZ and/or TQ significantly reduced the potential of U87MG cells invasion. In addition, after treating with TMZ and/or TQ, there was a decrease in the levels of matrix matrix metalloproteinase 2 nad 9 (MMP 2 and 9) expression and secretion in U87MG cells. CONCLUSIONS: The combination of TMZ and TQ may emerge as a promising strategy for the successful treatment of GBM.

Protective effect of hydroalcoholic extracts of Trifolium pratense L. on pancreatic ß cell line (RIN-5F) against cytotoxicty of streptozotocin.

Natural plants have traditionally been used throughout the world for their anti-diabetic effects. The aim of the present study was to investigate the protective effect of hydroalcoholic extract of Trifolium pratens L. (T. pratense) on streptozotocin (STZ) cytotoxicity and insulin concentration from RIN-5F pancreatic ß cell line. In this study, possible cytoprotective action of T. pratense extract (using pre-treatment, simultaneous, and post-treatment schedules) against STZ (30 mM) was evaluated using MTT assay. Apoptosis was quantified by fluorescent dye staining. Also, the effect of extract on insulin secretion in low and high glucose media was examined. Data were analyzed by one-way ANOVA test and P < 0.05 was considered significant. The viability of RIN-5F cells in 10, 20, 30, 40, and 60 µg/mL doses of T. pratense extract showed significant increases compared to control group (P < 0.001). STZ significantly reduced cell viability in a dose-dependent manner (P < 0.05). T. pratense extract in 20, 30, and 40 µg/mL doses had significant cytoprotective effect (P < 0.05) on cytotoxic action of STZ and this effect is greater in simultaneous treatment. STZ-mediated apoptotic death is reduced by extract. T. pratense extract treatment also, increased insulin concentration in cell culture medium. T. pratense had potent cytoprotective action, prevented apoptosis and increased insulin concentration in cell culture medium via the increase in pancreatic ß cell number and/or insulin secretion. In addition, T. pratense enhanced viability of RIN-5F. Thus, T. pratense not only has anti-diabetic actions on ß cells but also enhances their viability.

Evaluation of Hydro-Alcoholic Extract of Trifolium Pratens L. for Its Anti-Cancer Potential on U87MG Cell Line.

OBJECTIVE: Glioblastoma multiforme is the most malignant form of brain tumors. Trifolium pratense L. has been suggested for cancer treatment in traditional medicine. Here we have investigated the effects of T. pratense extract on glioblastoma multiforme cell line (U87MG). MATERIALS AND METHODS: In this experimental study, the effect of T. pratense extract on cell viability was investigated using trypan blue staining, MTT assay, and lactate dehydrogenase activity measurement. Apoptosis and autophagy cell death were detected by fluorescent staining. Nitric oxide (No) production was measured using Griess reaction. Expression levels of some apoptotic and autophagic-related genes were detected using real-time polymerase chain reaction (PCR). The combination effects of T. pratense extract and temozolomide (TMZ) were evaluated by calculating the combination index and dose reduction index values. RESULTS: After treatment with T. pratense extract, the cell viability was significantly reduced in a time- and dosedependent manner (P<0.05). Apoptosis and autophagy of U87MG cells were significantly increased (P<0.05). Also, T. pratense extract significantly decreased NO production (P<0.05) by U87MG cells. Combination of TMZ and T. pratense extract had a synergistic cytotoxic effect. CONCLUSION: T. pratense showed anti-cancer properties via induction of apoptosis and autophagy cell death.

Temozolomide-Mediated Apoptotic Death Is Improved by Thymoquinone in U87MG Cell Line.

Apoptosis induction of cancer cells can be an appropriate strategy by which chemotherapeutic agents kill tumor cells. The aim of the present study was to investigate the effect of temozolomide and thymoquinone combination on apoptotic pathway of human glioblastoma multiforme cell line (U87MG). U87MG cells were cultured, treated with temozolomide and thymoquinone, and cell proliferation was measured. Apoptosis cell death and its possible mechanism were investigated by various methods. Combination of temozolomide and thymoquinone had a synergistic effect on cells viability. Thymoquinone intensified the temozolomide-induced apoptosis. Combination of temozolomide and thymoquinone can be a good strategy for treatment of glioblastoma.

Thymoquinone synergistically potentiates temozolomide cytotoxicity through the inhibition of autophagy in U87MG cell line.

OBJECTIVES: Glioblastoma multiforme (GBM) is one of the most lethal forms of human cancer and temozolomide (TMZ) is currently part of the standard treatment for this disease. Combination therapy using natural substances can enhance the anti-cancer activity of TMZ. The purpose of this study was to evaluate the effect of TMZ in combination with thymoquinone (TQ) on human GBM cell line (U87MG). MATERIALS AND METHODS: The cell line was treated with TMZ and/or TQ. Cell viability was assessed using trypan blue and MTT assay. The effect of TMZ and/or TQ on colony-forming ability of the cells was investigated. Apoptosis and autophagy were quantified by fluorescent dye staining. The expression level of two autophagy related genes (ATG) were assessed using RT-PCR. Furthermore, nitric oxide (NO) production was detected by Griess reaction. RESULTS: After treatment with TMZ and/or TQ, the cell viability was reduced in a time- and dose-dependent manner, and the cell survival fraction (SF) was significantly decreased (P=0.000). Apoptosis index of U87MG cells was also significantly increased (P=0.000). Autophagy was significantly increased by TMZ (P=0.000) and decreased by TQ (P=0.018). Also TMZ and/or TQ significantly decreased NO production by U87MG cell (P=0.000). CONCLUSION: TQ enhanced the anti-cancer activity of TMZ by inhibition of autophagy at the transcriptional level and decreased the colony-forming ability and NO production of U87MG cell line.