Razieh Amini

Resveratrol Inhibits Proliferation, Invasion, and Epithelial–Mesenchymal Transition by Increasing miR‐200c Expression in HCT‐116 Colorectal Cancer Cells

Objective: colorectal cancer (CRC) is one of the most common malignancies, associated with high rates of relapse. A notable challenge in treatment is low response rate to current therapies for advanced CRC. The miR‐200c plays an essential role in tumor suppression by inhibiting epithelial–mesenchymal transition (EMT). Resveratrol, a natural compound found in red wine, reveals anti‐cancer properties in several types of cancers such as CRC. The aim of current study was to evaluate the effects of resveratrol on proliferation, apoptosis, and invasion of HCT‐116 cells and also expression of EMT‐related genes in presences or absence of miR‐200c. Methods: the effect of resveratrol on viability was examined by MTT assay. LNA‐anti‐miR‐200c transfection of HCT‐116 cells was carried out in a time dependent manner. Then, the expression of miR‐200c and EMT‐related genes were quantified by qRT‐PCR. Further, expression of EMT‐related proteins, apoptosis, and invasion were analyzed by Western blot, Annexin V/PI staining and scratch test, respectively. Results: resveratrol could significantly inhibit viability of HCT‐116 cells. LNA‐anti‐miR‐200c suppressed the endogenous miR‐200c in transfected cells compared with the control. qRT‐PCR and Western blot analysis of LNA‐anti‐miR‐200c transfected cells revealed a considerable increase in vimentin and ZEB‐1 expression, with a concomitant reduction in E‐cadherin expression level. Migration of HCT‐116 cells increased, and apoptosis significantly reduced in transfected cells. While, resveratrol could entirely reverse these changes by modulation of miR‐200c expression. Conclusion: our findings revealed a major role of resveratrol in apoptosis, invasion, and switching of EMT to MET phenotype through upregulation of miR‐200c in CRC. J. Cell. Biochem. 118: 1547–1555, 2017.

Involvement of miR-155/FOXO3a and miR-222/PTEN in acquired radioresistance of colorectal cancer cell line

PurposeFinding a novel biomarker for determining the radiosensitivity of colorectal cancer (CRC) is critical. The aim of this study is to evaluate the role of two main miRNAs including miR-222 and miR-155 in radiation response of CRC.Materials and methodsThe radioresistant CRC cell lines were established by exposing the HCT 116 cell line to fractional X-ray radiation. SubG1 fraction analysis, MTT and clonogenic assays were applied to evaluate acquired radioresistant cell line radiosensitivity. miR-222/PTEN and miR-155/FOXO3a expressions were detected by RT PCR.ResultsThe clonogenic assay and sub-G1fraction analysis indicated that the RR2 sub-line was significantly more resistant than the parental cell line. MiR-222 and miR-155 were significantly upregulated in the radioresistant cell lines compared with the parental cell lines. The PTEN and FOXO3a expressions in the radioresistant cell lines were significantly higher than in the parental line.ConclusionThese observations indicate that miR-222 and miR-155 could induce radiation resistance in colorectal cancer by targeting PTEN and FOXO3a genes, respectively. Therefore, miR-222 and miR-155 can be suggested as good biomarkers of CRC radiation response.

Binding site identification of anticancer drug gefitinib to HSA and DNA in the presence of five different probes

This study was carried out to evaluate the binding interaction of gefitinib (GEF) with human serum albumin (HSA) and calf thymus DNA (ct-DNA) using fluorescence, UV–Visible, zeta potential measurements and molecular docking methods in order to understand its pharmacokinetic mechanism. By increasing the temperature, a steady decrease in Stern–Volmer quenching constants was observed for HSA binding properties; this indicates a static type of fluorescence quenching. Negative values were calculated for Gibbs free energy (ΔG), enthalpy (ΔH), and entropy (ΔS) changes, indicating that the reaction is spontaneous and enthalpy-driven. Probe competitive experimental results showed that GEF contains the same binding site as warfarin and are consistent with modeling results. The zeta potential of the HSA increased with increasing GEF, which represents the presence of electrostatic interactions in the system. DNA binding properties were investigated in the presence of three probes. The experimental results showed that by increasing GEF to DNA-AO (acridine-orange) and DNA-MB (methylene-blue) system, the fluorescence intensity and absorbance spectra had no considerable change. Furthermore, with the addition of GEF to DNA, the zeta potential decreased gradually, indicating that the hydrophobic interaction between the GEF and the bases of DNA is the major factor. Thus, GEF can bind to DNA via a groove binding mode. It was also found that GEF entered into the minor groove in the A–T rich region of DNA fragment and bind via van der-Waals forces and three H-bond with double strands of DNA. This is in good agreement with experimental results.

The effect of mesenchymal stem cells combined with platelet-rich plasma on skin wound healing

Mesenchymal stem cells (MSCs) are multipotent stem cells that have the potential of proliferation, high self‐renewal, and the potential of multilineage differentiation. The differentiation potential of the MSCs in vivo and in vitro has caused these cells to be regarded as potentially appropriate tools for wound healing. After the burn, trauma or removal of the tumor of wide wounds is developed. Although standard treatment for skin wounds is primary healing or skin grafting, they are not always practical mainly because of limited autologous skin grafting.

Zerumbone inhibits epithelial-mesenchymal transition and cancer stem cells properties by inhibiting the β-catenin pathway through miR-200c: DERMANI et al.

Colorectal cancer (CRC) is one of the most lethal and rampant human malignancies in the world. Zerumbone, a sesquiterpene isolated from subtropical ginger, has been found to exhibit an antitumor effect in various cancer types. However, the effect of Zerumbone on the biological properties of CRC, including epithelial‐mesenchymal transition (EMT) and cancer stem cells (CSCs) has not been fully elucidated. Here, we investigated the inhibitory action of Zerumbone on the EMT process, CSC markers, and the β‐catenin signaling pathway in the presence or absence of miR‐200c. The effect of Zerumbone on HCT‐116 and SW‐48 cells viability was examined by 3‐(4, 5‐dimethylthiazol‐2‐yl)‐2, 5‐diphenyltetrazolium bromide assay. The effects of Zerumbone on EMT‐related genes, CSCs markers, cell migration, invasion, sphere‐forming, and β‐catenin signaling pathway were explored. To evaluate the role of miR‐200c in anticancer effects by Zerumbone, miR‐200c was downregulated by LNA‐anti‐miR‐200c. Zerumbone significantly inhibited cell viability, migration, invasion, and sphere‐forming potential in HCT‐116 and SW‐48 cell lines. Zerumbone significantly suppressed the EMT and CSC properties as well as downregulated the β‐catenin. Silencing of miR200c reduced the inhibitory effects of Zerumbone on EMT and CSCs in CRC cells. These data indicated that Zerumbone may be a promising candidate for reducing the risk of CRC progression by suppressing the β‐catenin pathway via miR‐200c.

miR-200c, a tumor suppressor that modulate the expression of cancer stem cells markers and epithelial-mesenchymal transition in colorectal cancer

Colorectal cancer (CRC) is the most frequently diagnosed cancer and the most common gastrointestinal cancer worldwide. Due to the presence of populations of cancer stem cells (CSCs) that cause recurrence, the possibility of cancer treatment is very low. The aim of current study was to evaluate the inhibitory role of miR‐200c on EMT, CSCs markers and β‐catenin in HCT‐116 and SW48 cell lines. The expression of miR‐200c, EMT‐related genes, CSCs markers, and β‐catenin were quantified by qRT‐PCR. Further, expression of β‐catenin and EMT‐related proteins, and migration were analyzed by Western blot, and migration assay kit, respectively. Spheroid formation assay was used to enrich colorectal CSCs from colorectal cancer cell lines. LNA‐anti‐miR‐200c suppressed the endogenous miR‐200c in transfected cells compared with the control. qRT PCR and Western blot analysis of LNA‐anti‐miR‐200c transfected cells revealed a considerable increase in CSCs markers, vimentin, ZEB‐1, N‐cadherin, and β‐catenin expression, with a concomitant reduction in E‐cadherin expression level. Migration and sphere forming ability of HCT‐116 and SW48 cells increased in transfected cells. The results of current study revealed that downregulation of miR‐200c may be an important factor for the overexpression of CSCs markers and EMT related genes via β‐catenin upregulation in CRC.

Anticancer effects of miR-200c in colorectal cancer through BMI1: KARIMI MAZRAEHSHAH et al.

Colorectal cancer (CRC) is one of the most leading cancer deaths throughout the world. MiR‐200c has been shown to have a critical role in cancer initiation and progression. In this study, we investigated the miR‐200c expression in CRC tissues and its effects on CRC cell lines which were mediated by polycomb complex protein (BMI1). Quantitative reverse transcription polymerase chain reaction (QRT‐PCR) and immunohistochemistry were used to detect miR‐200c and BMI1 expression in tumor tissues from 38 patients with CRC and 38 normal colon tissues. HCT‐116 and SW‐48 cells were transfected by locked nucleic acid (LNA)‐anti‐miR‐200c. Western blot analysis and real‐time PCR were applied to determine the BMI1 protein and microRNA (miRNA) levels. The apoptosis was analyzed via annexin/propidium iodide staining, and cell invasion was evaluated by transwell assay. MiR‐200c was markedly downregulated in CRC tissues, whereas the protein expression of BMI1 in CRC tissues was upregulated compared with normal colon tissues. In the colon cancer cell lines, transfection of LNA‐anti‐miR‐200c increased BMI1 gene and protein expression as well as the cell invasion. Downregulation of miR‐200c by LNA decreased the apoptotic cells. The results from this study revealed that miR‐200c may have antitumor effects through inhibition of BMI1 expression.

MiR-185 enhances radiosensitivity of colorectal cancer cells by targeting IGF1R and IGF2

OBJECTIVEnRadioresistance is a significant obstacle for effective treatment of colorectal cancer (CRC). Recent studies have indicated that miR-185 inhibits proliferation, survival, and invasion of CRC; however, the role of this miRNA in radioresistance of CRC has not been identified yet. The aim of this study is to investigate the role of miR-185 in radiosensitivity of CRC.nnnMETHODSnAfter transfecting the cells with mimic miR-185, expressions of IGF1R and IGF2 were evaluated by real-time PCR and western blot. The radiation response of transfected cells was also examined by colony forming assay. Sub-G1 fraction analysis through flow cytometry and caspase 3 activity was used to evaluate apoptosis.nnnRESULTSnThe results of real-time PCR and western blot indicated that IGF1R and IGF2 are downregulated in the transfected cells. Colony forming assay revealed that transfected cells were more radiosensitive than other cells. On the other hand,following irradiation the rate of apoptosis was significantly higher in the transfected cells than in the other cells.nnnCONCLUSIONnIn summary, our study is the first to show that upregulation of miR-185 enhances the sensitivity of CRC cells to ionizing radiation. miR-185 may act as a novel biomarker of radioresistance and may clinically enhance the radiation response of CRC.

DCLK1 plays an important role in colorectal cancer tumorgenesis through the regulation of miR-200c

Doublecortin-like kinase 1 (DCLK1) is a protein kinase that is known as a specific cancer stem cell (CSC) marker in colorectal cancer (CRC). Deregulation of DCLK1 expression has been reported in various cancers. We measured the protein expression of DCLK1 in 38 CRC and normal colon samples by immunohistochemistry (IHC). HCT-116 and SW-48 cells were transfected with DCLK1 siRNA and analyzed for expression of DCLK1 and miR-200c. The effects of DCLK1 knockdown on cell migration, invasion, sphere-forming, and apoptosis were explored. It was found that DCLK1 protein expression levels were significantly higher in CRC tissue than in normal colon specimens. Silencing of DCLK1 significantly inhibited cell migration, invasion, and sphere-forming potential; it also induced apoptosis as well as increased expression of miR-200c. Furthermore, silencing of miR-200c significantly up-regulated DCLK1 expression. Overall, our data demonstrated that DCLK1 plays an important role in cancer progression and is involved in the regulation of miR-200c expression.

Ferula pseudalliacea induces apoptosis in human colorectal cancer HCT-116 cells via mitochondria-dependent pathway

Abstract Ferula species have diverse biological functions. This study set out to investigate the anti-proliferative effects of methanolic extract of F. pseudalliacea against human colon cancer HCT-116 cell line. Cytotoxic effects of F. pseudalliacea on HCT-116 cells was estimated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay. Real-time polymerase chain reaction (PCR) and Western blot were employed to analyze BAX and Bcl2 expression. Cell cycle analysis and apoptosis were conducted using flowcytometry and Annexin V/ propidium iodide (PI) staining. Rhodamine 123 staining and enzyme-linked immunosorbent assay (ELISA) assay were employed to quantify the mitochondria membrane potential (MMP) and caspase 3 activity, respectively. F. pseudalliacea markedly decreased HCT-116 cells viability. The gene and protein expression of BAX were increased, whereas Bcl-2 was decreased in F. pseudalliacea treated cells. F. pseudalliacea induced apoptosis via promotion of cell cycle arrest, caspase 3 activation, and destruction of MMP. These results demonstrate that F. pseudalliacea extract is able to induce apoptosis in HCT-116 cells mainly by activation of the mitochondrial pathway.