Objective: Since cancer stem cells (CSCs) hypothesis has emerged, cancer has been considered as a stem cell disease. Due to some problems in isolation of CSCs throughout methods like FACS and MACS, establishment culture combined with anti cancer drugs has recently been used to do so. In current study, vincristine as an antimitotic drug has been used in order to isolation and purification of CSCs from human breast cancer cell line MDA-MB231.Materials and Methods: There were two sets of experiments: At first, Determination of optimal dose of vincristine in order to isolate cancer stem cells from human breast cancer cell line MDA-MB231. Mentioned, conducted by cells treatment with 2, 4, 6 and 8 ng/ml of vincristine (72 hours) followed by MTT assay and trypan blue dye exclusion. Then, identification, purification and characterization of isolated cells which was performed by RT-PCR and immunofluorescence staining upon exposure to vincristine, culture of cells in non adherent culture condition in CSC medium and formation of mamosphere forming unit (MFU); respectively.Results: Both MTT and trypan blue dye exclusion assay demonstrated significant decrease in viability of cells treated with high doses of vincristine (6 and 8 ng/ml) in comparison with low ones (2 and 4 ng/ml) (p<0.05). So, the later (4 ng/ml) was selected as an optimal dose of vincristine in order to combine with establishment culture condition. RT-PCR showed isolated cells upon exposure to vincristine expressed Oct3/4, Nanog, Sox2 and nocleostemin. Also, immunofluorescence staining demonstrated that isolated cells were CD44 positive. Furthermore, results of MFU assay displayed remarkable increase in percentage of MFU among cells pretreated with vincristine (3.3%) in comparison with those were not treated (0.4%) (p<0.05).Conclusion: Non adherent culture condition in CSC medium (establishment culture) combined with vincristine can be considered as an appropriate method for cancer stem cells isolation from human breast cancer cell line MDA-MB231

It has been reported that different species of Euphorbia (Euphorbiaceae) have antitumor activity. Some reports also show that these plants have potential cytotoxic effect against different cell lines. In a program to screen the cytotoxicity of Iranian native plants, Euphorbia macroclada Boiss. was collected, identified, and the cytotoxic activity of dichloromethane, ethylacetate, methanol extracts, and the plant latex were determined against MDA-MB-468 cell line. Different concentration of extracts and latex were added to 24 h cultured cells and then incubated for 72 h under specific condition (37oC, 5% CO2). Cell survival was evaluated using MTT assay. The results of this study indicated that, dichloromethane and ethylacetate extracts had cytotoxic effects on cell line, while the methanol extract and latex were not cytotoxic at the tested concentrations. The data from this investigation suggest that the nonpolar extracts of E. macroclada possess higher cytotoxic activity.

Background: Breast cancer is the major cause of death from cancer among women around the world. Given the drug resistance in the treatment of this disease, it is very important to identify new therapies and anticancer drugs. Many studies demonstrated that hypericin could induce apoptosis in different cancer cell lines; however, the underlying mechanism is not well understood yet. Therefore, this study aimed to evaluate the anticancer effect of hypericin in two breast cancer cell lines, one with wild type P53 and the other with mutant P53. Methods: In this study, the MDA-MB-231 and MDA-MB-175-VII cell lines were treated with different concentrations of hypericin for 24 and 48 hours. The measurement of cell death was performed by MTT assay. The cell apoptosis rate was measured using annexin V/propidium iodide assay through flow cytometry. The level of expression in P21 and P53 genes was evaluated by real time PCR. Immunocytochemistry (ICC) analysis was performed for P21 (direct target for P53 protein) to confirm the results. Results: The results showed that hypericin could have dose-dependent cytotoxic effects on the MDA-MB-231 and MDA-MB-175-VII cell lines, and its cytotoxicity is much higher in the latter cells. According to flow cytometry results, 86% of MDA-MB-175-VII cells underwent apoptosis with IC50 dose of hypericin for MDA-MB-231 cells after 24 hours. Moreover, after 24 hours of exposure to hypericin with MDA-MB-231 IC50 concentration, the expression of P53 and P21 genes upregulated in MDA-MB-175-VII much more than MDA-MB-231 when both cell lines were treated with 24 hours IC50 dose of MDA-MB-231. The ICC analysis on P21 confirmed that by treating both cell lines with MDA-MB-231 IC50 dose of hypericin for 24 hours, this protein is overexpressed much more in MDA-MB-175-VII cells. Conclusion: The results of this study demonstrated that hypericin’ s apoptotic and cytotoxic effects on cancer cells may be mediated via P53 overexpression, cell cycle arrest and the subsequent apoptosis. Therefore, it is of great importance to consider that hypericin would have better impact on cells or tumors with wild type P53.

Objective: Epithelial-mesenchymal transition (EMT) is an important mechanism in tumor progression and metastasis. In addition to the uncontrolled epithelial proliferation and angiogenesis, the role of this mechanism in solid tumors such as breast cancer is also important for cancer progression and metastasis. The role of cytokines such as TGF-b and SDF-1 in triggering the EMT mechanism is previously demonstrated. In contrast to the SDF-1 with angiogenic roles, the chemokine IP-10 has been documented with anti-angiogenic activities but there is no research which shows the effect of IP-10 on EMT. Thus, in this study we investigated the effect of IP-10 overexpression on EMT mechanism in tumor cells. Besides, because of the key role of tumor microenvironment on tumor progression and the importance of ASCs, we also investigated the effect of these cells on the EMT mechanism.Materials and Methods: ASCs were isolated from 6 breast cancer patients and 6 normal individuals and then cultured. MDA-MB-468 breast cancer cell line was transfected by IP-10 plasmid through electroporation. Transfected cell line was co-cultured with ASCs using transwell system. After 4 days, proteins were extracted and the expressions of MMP9 and MMP13 were defined by western blotting.Results: Results of western blot for MMP9 and MMP13 protein expressions showed that the expression of both proteins were downregulated in IP-10 transfected MDAMB- 468 cells compared to the untransfected cells. Presence of ASCs causes the upregulation of MMP9 in MDA-MB468 cell line.Conclusion: Based on the results of this study, IP-10 may downregulate the EMT markers in MDA-MB-468 cancer cell line. No different was found between the ASCs from different sources but the presence of ASCs in tumor microenvironment can lead to the up-regulation of EMT markers. Accordingly, IP-10 may use as an antitumor therapeutic agent to suppress tumor progression and metastasis.

Interaction between urokinase-type plasminogen activator (uPA) and its receptor (uPAR) plays an important role in the progression of numerous cancer types including breast cancer by promoting tumor initiating, proliferation, invasion and metastasis. Hence, disruption of this interaction inhibits their downstream cascades and subsequently tumor growth. For this, we created two series of 8 and 10 amino acids linear peptides, derived from uPA binding region to target uPAR and studied the inhibition of proliferation in MDA-MB-231 cell line. Results revealed that all of the 10-mer peptides inhibited breast cancer cell proliferation significantly with maximum 40% inhibition of 103 peptides. Meanwhile, none of the 8-mer peptides showed significant toxicity. Current results indicate that the linear 10-mer peptides which mimic a small part of a sequence of a binding domain of uPA to uPAR could be exploited to design a novel class of anti-cancer agents.

Background: Electroporation is a valuable tool for small interfering RNA (siRNA) delivery into cells because it efficiently transforms a wide variety of cell types. Since electroporation condition for each cell type must be determined experimentally, this study presents an optimal electroporation strategy to reproducibly and efficiently transfect MDA-MB 468 human breast cancer cell with siRNA.Methods: To identify the best condition, the cells were firstly electroporated without siRNA and cell viability was determined by trypan blue and MTT assays. Then siRNA transfection in the best condition was performed. Western blot analysis was used for monitoring successful siRNA transfection.Results: The best condition for electroporation of this cell line was 220 volt and 975 mF in exponential decay using the Gene Pulser X cell electroporation system. Our data demonstrated that by using proper electroporation condition, DNA methyl transferase mRNA was silenced by 10 nmol DNMT1 siRNA in MDA-MB 468 cells when compared with negative control siRNA electroporation. Analysis of cell viability demonstrated that optimal electroporation condition resulted in 74% and 78% cell viability by trypan blue staining and MTT assay, respectively.Conclusion: Transfection of the MDA-MB-468 breast cancer cell line with siRNA in the obtained electroporation condition was successful and resulted in effective gene silencing and high cellular viability.