Upregulation of C-X-C chemokine receptor type 4 (CXCR4) in the breast cancer stem like cells.

BACKGROUND AND OBJECTIVES: Breast cancer stem like cells (CSCs) as a subset of cancer cells exhibit similar properties with normal stem cells. These cells are responsible for cancer metastasis and recurrence. Pivotal roles of CXCR4 in metastasis, chemoresistance and stemness of tumor cells have been showed previously. Here, we aim to explore the relationship between CXCR4 and CSCs in primary and metastatic breast tumor cells. METHODS AND RESULTS: Primary and highly metastatic breast tumor cells were isolated in our laboratory. Spheroid formation was used to confirm the presence of CSCs and their self-renewal capability. CXCR4 expression was evaluated using real-time polymerase chain reaction in monolayer culture and multicellular spheroids. Our data showed that in all tested cells, CXCR4 expression was significantly increased in CSCs. In parallel, compared with primary tumor cells, downregulation of CXCR4 in metastatic tumor cells was confirmed. CONCLUSION: These results provided new insights related to significant alteration of CXCR4 expression in multicellular spheroids. Analysis of molecular properties of spheroids could be used to detect molecular and genetic aspects of CSCs and also created a targeted therapeutic strategy against breast CSCs.

Evaluation of real-time NASBA assay for the detection of SARS-CoV-2 compared with real-time PCR.

PURPOSE: In January 2020, the COVID-19 pandemic started and has severely affected all countries around the world. The clinical symptoms alone are not sufficient for a proper diagnosis. Thus, molecular tests are required. Various institutes and researchers developed real-time PCR-based methods for the detection of the virus. However, the method needs expensive equipment. In the present study, we developed a real-time NASBA assay for the detection of SARS-CoV-2. METHODS: Primers and molecular beacon probes for RdRp and N genes were designed. In silico analysis showed that primers and the probes were specific for SARS-CoV-2. The standard samples with known copy numbers of the virus were tested using the NASBA assay and an FDA-approved real-time PCR kit. A series of standard samples were prepared and tested. Clinical sensitivity, precision analysis, and clinical assessment of the assay were performed. RESULTS: The limit of detection of the assay was 200 copies/mL. The clinical sensitivity of the assay was 97.64%. The intra-assay and inter-assay for both N and RdRp genes were less than 5% and 10%, respectively. Clinical assessment of the assay showed that the positive agreement rate and negative agreement rate of the assays were determined to be 97.64% and 100%, respectively. CONCLUSIONS: The results of the present study show that the developed real-time NASBA is a sensitive and specific method for the detection of SARS-CoV-2 and is comparable with real-time PCR. NASBA is an isothermal signal amplification method, and if stand-alone fluorescent readers are available, the real-time NASBA can be used without the need for expensive thermocyclers. In addition compared to other isothermal methods like LAMP, the primer design is straightforward. Thus, real-time NASBA could be a suitable method for inexpensive SARS-CoV-2 detection.

Mesenchymal Stromal Cells and their EVs as Potential Leads for SARSCoV2 Treatment.

In December 2019, a betacoronavirus was isolated from pneumonia cases in China and rapidly turned into a pandemic of COVID-19. The virus is an enveloped positive-sense ssRNA and causes a severe respiratory syndrome along with a cytokine storm, which is the main cause of most complications. Therefore, treatments that can effectively control the inflammatory reactions are necessary. Mesenchymal Stromal Cells and their EVs are well-known for their immunomodulatory effects, inflammation reduction, and regenerative potentials. These effects are exerted through paracrine secretion of various factors. Their EVs also transport various molecules such as microRNAs to other cells and affect recipient cells' behavior. Scores of research and clinical trials have indicated the therapeutic potential of EVs in various diseases. EVs also seem to be a promising approach for severe COVID-19 treatment. EVs have also been used to develop vaccines since EVs are biocompatible nanoparticles that can be easily isolated and engineered. In this review, we have focused on the use of Mesenchymal Stromal Cells and their EVs for the treatment of COVID-19, their therapeutic capabilities, and vaccine development.

New design and optimization of an in-house quantitative TaqMan Real-Time PCR-based assay for the detection and monitoring of occult hepatitis B virus (genotype A-J) infection.

PURPOSE: HBV DNA quantification is used for individuals with uninterpretable serological tests, occult HBV infections, decreasing the window period of the disease, and treatment follow-up. Although there are commercial qPCR assays, they are expensive. In this study, we developed a highly sensitive quantitative TaqMan Real-Time PCR with an exogenous internal control to quantify HBV DNA in serum/plasma. METHODS: A specific primer/probe set was designed for the S conserved region of various HBV genotypes. The primer/probe set was evaluated experimentally and in-silico. An exogenous internal control was included to monitor the effects of inhibitors. The standard plasmid was titrated using three different methods to prepare the seven standards for the assay. The functional characteristics of the in-house assay were evaluated using the standards. Two hundred clinical specimens were also tested. RESULTS: The LOD of the in-house assay was 40 IU/mL, and the assay was linear from 3.26Log10 to 9.26Log10 IU/mL. The analytical and clinical sensitivity of the assay was 100% and 92.15%, respectively. The analytical and clinical specificity of the assay was 100% and 98.97%, respectively. The positive and negative predictive values of the assay were determined to be 98.94% and 92.38%, respectively. The highest coefficient of variation of the inter/intra-assay was 5.1%. The accuracy was close to 100% for all standards, and the correlation between the in-house assay and commercial kit AltoStar® PCR Kits 1.5 was remarkable. The results of the clinical samples using the standards titrated using AcroMetrix™ HBV Panel, Artus® HBV RG PCR Kit, and AltoStar® PCR Kits 1.5 were comparable (r â€‹= â€‹0.942, 0.951, 0.951). CONCLUSIONS: The results indicate that the in-house assay is highly sensitive and specific, reproducible, and cost-benefit. Thus, it can be used to detect and quantify HBV DNA in research and clinical settings.

Effects of Sambucus ebulus Extract on Cell Proliferation and Viability of Triple- Negative Breast Cancer: An In Vitro and In Vivo Study.

BACKGROUND: Triple-negative breast cancer (TNBC) accounts for 15% of all breast cancer (BC) cases and is a severe type of BC. Since medicinal herbs containing biocompatible substances that are accepted by patient more than chemical therapeutics, they can be considered a safe option for treating BC. OBJECTIVE: This study evaluated the effect of Sambucus Ebulus (S. ebulus) extract on a model of TNBC. METHODS: S. ebulus extract was prepared using petroleum ether, ethyl acetate, and methanol. The petroleum ether extract was fractionated and analyzed using vacuum liquid chromatography and GC-MS, respectively. MDAMB- 231 and MCF-10A were used as TNBC and normal breast cells, respectively. Flowcytometry and MTT assays were performed to evaluate cell cycle, apoptosis, and viability of the cells. Gene expression analysis was performed using RT-qPCR. Nude mouse allograft tumor models were used, and pathological sections were evaluated. RESULTS: The findings indicated that S. ebulus extract remarkably decreased cell proliferation and viability. The extract had no toxicity to the normal breast cells but efficiently killed the cancer cells. Cell cycle- and apoptosisrelated gene expression showed that fraction 4 of S. ebulus extract significantly increased the expression of Bax, Bak, P53, and c-MYC. CONCLUSION: This study showed satisfactory results of the effect of S. ebulus extract on clearing BC cells both in vitro and in vivo. Thus, S. ebulus extract may be a safe herbal compound for eliminating BC cells without toxicity to host cells.

Design and development of a simple method for the detection and quantification of residual host cell DNA in recombinant rotavirus vaccine.

Rotavirus recombinant vaccine is usually produced in Vero cells. Residual host DNA may reside in the final product and is considered a source of contamination. WHO protocols indicate that biological products should be free of any type of impurity such as nucleic acids, endotoxins, and host cell intermediate materials. Therefore, all recombinant biological therapeutics should be assessed for residual host DNA. In the present study, a sensitive and specific real-time PCR method was developed to detect residual host cell DNA in the final product. The Beta-actin gene of Vero cells was selected to detect residual host cell DNA. One set of primers and a TaqMan probe were designed for the gene using AlleleID 6 software. Real-time PCR reactions were set up, and efficiency of 84% was obtained. The sensitivity and limit of detection of the assay were determined to be 0.176 Fg/µl and 0.044 Fg/µl, respectively. The intra-assay and inter-assay variations were 4.4% and 1.04%, respectively. Furthermore, the specificity and sensitivity of the assay were high enough, and the detection limit was lower than that of the FDA and WHO standards. This indicates that our assay is highly specific and sensitive to detect residual host DNA of Vero cells in the recombinant rotavirus vaccine.

High percentage of cancer stem cells in metastatic locations; a comment on a claim "variation in cancer risk among tissues can be explained by the number of stem cell divisions".

Metastasis is the major cause of cancer-related deaths. Cancer relapse and metastasis are associated with a part of cancer cells with stem cell properties. These cancer stem cells (CSCs) are resistant to treatments. In a recent survey, we observed that the population of cancer stem-like cells among metastatic tumor cells was significantly higher than that among the primary tumor cells. This high percentage can partly explain the reasons for chemoresistance and relapse in metastatic cancers. Analysis of the role of CSCs in metastasis has been mainly conceptual and speculative, and the reasons for a higher number of CSCs in the metastatic loci are questionable. Tomasetti and Vogelstein's claim can partly answer the question. They postulated that the proliferation rate of normal stem cells in some tissue is greater than that of other tissues, and accordingly, the incidence of cancer in these tissues is high. In compliance with CSCs paradigm, resident normal stem cells of tissues are the most probable source of CSCs. After homing of metastatic cancer cells in a tissue with high rate of normal stem cell proliferation, there is a big opportunity for cancer cells to convert normal stem cells to cancer stem cells. This is the powerful effect of cancerous microenvironment on resident stem cells of tissue. Therefore, in metastatic cancers, the number of CSCs in primary tumor or in each metastatic location is relevant to the proliferation rate of resident normal stem cells of the location. This concept is a confirmation of Tomasetti and Vogelstein's claim and can answer some fundamental questions about metastasis process.

Exosomal miRNAs from highly metastatic cells can induce metastasis in non-metastatic cells.

AIMS: Breast cancer is a high prevalence cancer among women worldwide. 15-20% of breast cancer cases are triple-negative with a poor prognosis. miRNA aberrant expression is one of the reasons of cancer development and metastasis. Exosomes are vesicles that carry cargos such as miRNAs to other cells. Therefore, we hypothesized that miRNAs transported by exosomes to other cells can induce malignant transformation. MATERIALS AND METHODS: We extracted exosomes from highly metastatic MDA-MB-231 cells and characterized them using Dynamic light scattering, scanning and transmitting electron microscopy as well as western blot. Then, we treated non-metastatic MCF-7 cells with the exosomes. Afterwards, we evaluated exosome uptake by MCF-7 cells using PHK67 staining. Finally, we used soft agar colony formation, migration, and invasion assays to explore any increase in/induction of metastatic behavior of exosome-treated MCF-7 cells. KEY FINDINGS: Our result indicated that the particles extracted from MDA-MB-231 cells' supernatant were actually exosomes. PKH67 staining and confocal microscopy showed that the exosomes were actively taken up by MCF-7 cells. Treatment of MCF-7 cells with the exosomes resulted in increased ability of MCF-7 cells to grow independent of anchorage. In addition, migration and invasion capacity of exosome-treated MCF-7 cells increased in a dose-dependent manner. SIGNIFICANCE: Along with our previous study, we here indicate that highly metastatic MDA-MB-231 cells' exosomes and exosomal miRNAs may induce malignant transformation in non-metastatic MCF-7 cells, thus introducing a novel route of cancer development and metastasis.

Evaluation of exosomal miR-9 and miR-155 targeting PTEN and DUSP14 in highly metastatic breast cancer and their effect on low metastatic cells.

Breast cancer is one of the most prevalent cancers in women. Triple-negative breast cancer consists 15% to 20% of breast cancer cases and has a poor prognosis. Cancerous transformation has several causes one of which is dysregulation of microRNAs (miRNAs) expression. Exosomes can transfer miRNAs to neighboring and distant cells. Thus, exosomal miRNAs can transfer cancerous phenotype to distant cells. We used gene expression omnibus (GEO) datasets and miRNA target prediction tools to find overexpressed miRNA in breast cancer cells and their target genes, respectively. Exosomes were extracted from MDA-MB-231 and MCF-7 cells and characterized. Overexpression of the miRNAs of MDA-MB-231 cells and their exosomes were analyzed using quantitative Real-time PCR. The target genes expression was also evaluated in the cell lines. Luciferase assay was performed to confirm the miRNAs: mRNAs interactions. Finally, MCF-7 cells were treated with MDA-MB-231 cells' exosomes. The target genes expression was evaluated in the recipient cells. GSE60714 results indicated that miR-9 and miR-155 were among the overexpressed miRNAs in highly metastatic triple negative breast cancer cells and their exosomes. Bioinformatic studies showed that these two miRNAs target PTEN and DUSP14 tumor suppressor genes. Quantitative Real-time PCR confirmed the overexpression of the miRNAs and downregulation of their targets. Luciferase assay confirmed that the miRNAs target PTEN and DUSP14. Treatment of MCF-7 cells with MDA-MB-231 cells' exosomes resulted in target genes downregulation in MCF-7 cells. We found that miR-9 and miR-155 were enriched in metastatic breast cancer exosomes. Therefore, exosomal miRNAs can transfer from cancer cells to other cells and can suppress their target genes in the recipient cells.

Is miR-144 an effective inhibitor of PTEN mRNA: a controversy in breast cancer.

Breast cancer is the first common cancer among women worldwide. One of the major signaling pathways playing a role in the onset and progression of this disease is PI3K/Akt/mTOR, which can be inhibited by PTEN. miRNAs are small non-coding molecules that regulate the expression of their targets by inhibition or suppression, and thus, their dysregulated expression results in the development of cancer. Using various software applications predicting miRNAs and evaluating GEO microarray data, miR-144 was selected as an inhibitor of PTEN. The expression of miR-144 and PTEN was evaluated in 18 triple negative breast cancer (TNBC) clinical samples and cell lines including 4T1, MDA-MB-231, MDA-MB-468, SK-BR-3, and MCF-7 in comparison with normal cells. PTEN and miR-144 expression analysis revealed their elevated expression in MCF-7 cells. MDA-MB-468, SK-BR-3, and MDA-MB-231 cells showed decreased levels of PTEN and increased levels of miR-144. In contrast, 4T1 cells had an increased expression of PTEN and decreased expression of miR-144. In clinical samples, miR-144 was up-regulated in 22% of the cases and PTEN was down-regulated in 78% of the cases. The results showed that the expression of PTEN and miR-144 was inversely correlated in metastatic breast cancer cell lines. However, in TNBC clinical samples, there was no correlation between the expression of miR-144 and PTEN. Literature shows that there are other influencing factors affecting the expression of miRNAs. Therefore, care should be taken in interpreting the results of gene expression studies and its relation with cancer diagnosis/prognosis.

A lentiviral vaccine expressing KMP11-HASPB fusion protein increases immune response to Leishmania major in BALB/C.

Hydrophilic acylated surface protein B (HASPB) is an immunogenic Leishmania-specific protein that antibodies are produced against it in the sera of Leishmania-infected individuals. Kinetoplastid membrane protein 11 (KMP11) is another Leishmania antigen and considered as the suitable candidate for vaccine development Leishmaniasis. It is a highly conserved surface protein expressed in both promastigotes and amastigotes. In this study, KMP11 and HASPB coding sequences were cloned into a pCDH-cGFP lentiviral vector as a fusion protein to be used as a DNA vaccine against L. major. The KMP11-HASPB fusion protein was successfully expressed as evidenced by RT-PCR and Western blot assays. The effect of the vaccine was determined by evaluating the level of IFN-γ, IL-10, IgG1, and IgG2a performed using ELISA as well as determining the parasite load after challenge with L. major in vaccinated mice. The results revealed that IFN-γ, IL-10, IgG1, and IgG2a significantly increased after vaccination using KMP11-HASPB-expressing lentiviruses in BALB/c mice. It is noteworthy that the level of IFN-γ and IgG2a was higher than that of IL-10 and IgG1, respectively, which indicates the activation Th1 cells, macrophages, and cellular immunity. Moreover, the parasite load in the spleen and lymph node of vaccinated mice after challenge was significantly lower than that of controls.

Quantitative real-time PCR technique for the identification of E. coli residual DNA in streptokinase recombinant product.

Recombinant streptokinase is a biopharmaceutical which is usually produced in E. coli. Residual DNA as a contamination and risk factor may remain in the product. It is necessary to control the production procedure to exclude any possible contamination. The aim of the present study was to develop a highly specific and sensitive quantitative real-time PCR-based method to determine the amount of E. coli DNA in recombinant streptokinase. A specific primers and a probe was designed to detect all strains of E. coli. To determine the specificity, in addition to using NCBI BLASTn, 28 samples including human, bacterial, and viral genomes were used. The results confirmed that the assay detects no genomic DNA but E. coli's and the specificity was determined to be 100%. To determine the sensitivity and limit of detection of the assay, a 10-fold serial dilution (101 to 107 copies/µL) was tested in triplicate. The sensitivity of the test was determined to be 101 copies/µL or 35 fg/µL. Inter-assay and intra-assay were determined to be 0.86 and 1.69%, respectively. Based on the results, this assay can be used as an accurate method to evaluate the contamination of recombinant streptokinase in E. coli.

Design and validation of a new method to detect and quantify residual host cell DNA in human recombinant erythropoietin (rEPO).

During the purification of human recombinant erythropoietin (rEPO) from host cells, residual DNA may remain in final products. This contamination is a risk factor for patients and may result in the inactivation of some tumor suppressor genes or activation of oncogenes if its concentration is more than the standard defined by WHO. Based on WHO's criteria, acceptable level of residual DNA in biopharmaceuticals is less than 10-100 pg/dose. In this study, we have designed a sensitive and specific quantitative real-time polymerase chain reaction (PCR) assay for the detection of residual DNA in human rEPO products. All reported sequences of CHO's GAPDH gene were retrieved from GenBank, and a multiple alignment was performed using Mega 6 software to find conserved regions of the gene. Primers and probe were designed by AlleleID7 software for the highly conserved region. Quantitative real-time PCR showed an R2 value more than 0.99 and the efficiency equal to 101% indicating a highly accurate and efficiency of the reaction, respectively. Based on the standard curve, the limit of detection of the assay was determined to be 10 copies/µL (0.00967 fg/µL). In addition, the inter- and intra-assay of the test were determined to be 1.14% and 0.65%, respectively, which are in acceptable range according to the WHO's guidelines.

Simultaneous Detection and Genotype Determination of HSV 1 and 2 by Real-time PCR Using Melting Curve Analysis and a Unique Pair of Primers.

Herpes simplex virus (HSV) is a human pathogen that causes different pathologic manifestations. Rapid and feasible detection and discrimination methods for HSV genotyping is a challenge in clinical laboratories, especially in children suffering from herpetic encephalitis. A quantitative real-time polymerase chain reaction (PCR)-based genotyping assay using SYBR Green I was established. We designed only 1 pair of primer for HSV 1 and 2, targeting thymidine kinase gene conserved region. HSV genotypes were determined by PCR using melting curve analysis with LightCycler. Different HSV genotypes were successfully detected in all clinical samples. The melting temperature for HSV 1 and 2 was 85.5±0.78°C and 89±0.53°C, respectively. These 2 genotypes were completely distinguished by means of the accurate melting assay. Importantly, detection was reliably performed within only 1 hour. The assay had no cross-reactivity across species, an excellent dynamic range from 10 to 10 copies per reaction, a good intra-assay and interassay reproducibility, and a detection limit of a single copy per reaction. Our homebrew designed and validated quantitative real-time PCR followed by a melting curve analysis provided a rapid and convenient screening test for differential identification of HSV genotypes 1 and 2. We recommend the large-scale application of this method for HSV 1 and 2 detection.

WNT1 Gene from WNT Signaling Pathway Is a Direct Target of miR-122 in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is an invasive form of hepatic cancer arising from the accumulation of multiple genetic alterations. In this study, the causal role of disturbed canonical Wnt/ß-catenin pathway was approved, and some of HCC-driven important gene candidates were determined. MicroRNAs (miRNAs), small non-coding RNAs, are the key regulators of important cancer genes, and their participation in tumorigenesis has been shown. By reviewing literature, WNT1 gene with functional significance was selected to approve miRNAs as new subjects for targeted therapy.For proper and fast miRNA detection and also confirmation of the role of bioinformatics in obtaining practical data, we benefited from different bioinformatics tools such as TargetScan, miRanda, and DIANA. In order to use an HCC model, we used HepG2 cell line. Luciferase assay was applied to assess the ability of the selected miRNAs in targeting WNT1 3'-UTR. To overexpress the selected miRNA in HepG2 cell line, viral construct was prepared. Quantitative real-time PCR was performed to evaluate selected miRNA and target gene expression levels. miR-122 was selected according to data concerning various bioinformatics tools.miR-122 was downregulated and WNT1 gene expression was upregulated in HepG2 cell line. After viral construct transduction, miR-122 expression was elevated and WNT1 expression was notably declined. Finally, we introduced WNT1 gene as one of the important genes in HCC, and also, we showed that miR-122 can regulate WNT1 gene expression.Moreover, our study determines the potential of bioinformatics analyses in providing accurate and reliable data for miRNA: messenger RNA (mRNA) prediction.

Development of an in-House TaqMan Real-Time PCR-Based Method to Detect Residual Host Cell DNA in HBV Vaccine.

Biological therapeutic products such as recombinant hepatitis B virus (HBV) vaccine, produced by microbial fermentation in complex media, should be evaluated for host cell DNA contamination in purification steps. Eliminating these contaminations increases the efficacy of the vaccine and decreases its side effects. The objective of the present study is to trace the residual host cell DNA (HCD) in recombinant HBV vaccine by developing a TaqMan Real-Time PCR method which is more sensitive, specific, and reproducible than traditional methods such as Picogreen analysis and Threshold DNA assay. Primers and a probe were designed for the most highly conserved regions of Pichia pastoris genome. To determine the specificity of the assay, in addition to performing a BLAST for the primers and the probe in NCBI nucleotide database, 20 different human genomes and 8 bacterial and viral genomes were used. Moreover, serial dilutions of plasmids, from 10(2) to 10(7) copies/µL (from 0.00064 to 6.4 pg/µL), were prepared to find the sensitivity and the limit of detection (LOD) of the assay. Using 28 different genome samples, the specificity of the assay was determined to be 100 %. In addition, the sensitivity and LOD of the method was 0.39 × 10(-5) pg/µL. Moreover, the reproducibility of the assay based on intra- and inter-assay was 1.03 and 1.06 %, respectively. Considering the suitable specificity and sensitivity, ease of use, relatively low cost, and rapidity of the assay, it can be a reproducible and sensitive method to examine recombinant vaccines for P. pastoris residual DNA.

Bioinformatics Prediction and In Vitro Analysis Revealed That miR-17 Targets Cyclin D1 mRNA in Triple Negative Breast Cancer Cells.

Breast cancer is one of the most prevalent malignancies among women worldwide. Triple negative breast cancer (TNBC) is a type of breast cancer in which estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (HER-2) are not expressed. There is no targeted therapy for this type of cancer, and available therapies have poor therapeutic effects. Performing a preliminary research, we selected cyclin D1 (CCND1) gene of Wnt signaling pathway which is a target of miRNAs, a promising set of biomolecules in diagnosis and treatment of breast cancer. In this study using bioinformatic analyses, miR-17 was selected as it targets the 3'UTR of CCND1 gene with the highest score. Luciferase assay results also confirmed the bioinformatic prediction. Decreased expression of miR-17 in MDA-MB-231 cell line was observed using qRT-PCR method. After lentiviral transduction of miR-17 to the target cells, gene expression analysis showed decreased expression of CCND1 gene. We found miR-17 as an attractive molecule that after intensive research can probably be used as a biomarker in TNBC.

Inhibition of silibinin on migration and adhesion capacity of human highly metastatic breast cancer cell line, MDA-MB-231, by evaluation of ß1-integrin and downstream molecules, Cdc42, Raf-1 and D4GDI.

Metastasis is a property of malignant cancer cells that requires integrins which with their downstream molecules participate in a number of signaling events in cells with pivotal roles in malignancy, migration and invasion of tumor cells. Silibinin, a flavonoid antioxidant from milk thistle (Silybum marianum L.), has attracted attention in the last decades for chemoprevention and chemotherapy of tumor cells. In the present study, the effect of silibinin on migration and adhesion capacity of MDA-MB-231 cells, a highly metastatic human breast cancer cell line, was investigated by evaluation of ß1-integrin and its important downstream molecules. MTT, migration and adhesion assays were performed to evaluate the silibinin effects on proliferation, migration and adhesion of MDA-MB-231 cells. In addition, the influence of the silibinin on the expression of ß1-integrin, Raf-1, Cdc42 and D4-GDI mRNAs was assessed by RT-PCR. Results showed significant dose-dependent inhibitory effect of silibinin on proliferation, migration and adhesion of MDA-MB-231 cells. It significantly inhibited the expression of Cdc42 and D4-GDI mRNAs but had no statistically significant effect on the expression of ß1-integrin and Raf-1 mRNAs although it indirectly but effectively modulated ß1-integrin signaling pathway and RAF1 function. In conclusion, the results showed the silibinin effectson reducing the rate of metastasis, migration and adhesion of MDA-MB-231 to distant organs.