Down-Regulation of Mcl-1 by Small Interference RNA Induces Apoptosis and Sensitizes HL-60 Leukemia Cells to Etoposide

Karami, Hadi,Baradaran, Behzad,Esfehani, Ali,Sakhinia, Masoud,Sakhinia, Ebrahim Asian Pacific Journal of Cancer Prevention 2014 Asian Pacific journal of cancer prevention Vol.15 No.2

Background: Acute myeloid leukemia (AML) is a fatal hematological malignancy which is resistant to a variety of chemotherapy drugs. Myeloid cell leukemia-1 (Mcl-1), a death-inhibiting protein that regulates apoptosis, has been shown to be overexpressed in numerous malignancies. In addition, it has been demonstrated that the expression level of the Mcl-1 gene increases at the time of leukemic relapse following chemotherapy. The aim of this study was to target Mcl-1 by small interference RNA (siRNA) and analyze its effects on survival and chemosensitivity of acute myeloid leukemia cell line HL-60. Materials and Methods: siRNA transfection was performed with a liposome approach. The expression levels of mRNA and protein were measured by real-time quantitative PCR and Western blot analysis, respectively. Trypan blue assays were performed to evaluate tumor cell growth after siRNA transfection. The cytotoxic effects of Mcl-1 siRNA (siMcl-1) and etoposide were determined using MTT assay on their own and in combination. Apoptosis was quantified using a DNA-histone ELISA assay. Results: Transfection with siMcl-1 significantly suppressed the expression of Mcl-1 mRNA and protein in a time-dependent manner, resulting in strong growth inhibition and spontaneous apoptosis. Surprisingly, pretreatment with siMcl-1 synergistically enhanced the cytotoxic effect of etoposide. Furthermore, Mcl-1 down-regulation significantly increased apoptosis sensitivity to etoposide. No significant biological effects were observed with negative control siRNA treatment. Conclusions: Our results suggest that specific suppression of Mcl-1 by siRNA can effectively induce apoptosis and overcome chemoresistance of leukemic cells. Therefore, siMcl-1 may be a potent adjuvant in leukemia chemotherapy.

siRNA-mediated Silencing of Survivin Inhibits Proliferation and Enhances Etoposide Chemosensitivity in Acute Myeloid Leukemia Cells

Karami, Hadi,Baradaran, Behzad,Esfahani, Ali,Estiar, Mehrdad Asghari,Naghavi-Behzad, Mohammad,Sakhinia, Masoud,Sakhinia, Ebrahim Asian Pacific Journal of Cancer Prevention 2013 Asian Pacific journal of cancer prevention Vol.14 No.12

Background: Overexpression of survivin, a known inhibitor of apoptosis, is associated with tumor progression and drug resistance in numerous malignancies, including leukemias. The aim of this study was to investigate the effect of a specific survivin small interference RNA (siRNA) on proliferation and the sensitivity of HL-60 acute myeloid leukemia (AML) cells to the chemotherapeutic drug etoposide. Materials and Methods: The cells were transfected with siRNAs using Lipofectamine $^{TM}2000$ transfection reagent. Relative survivin mRNA and protein levels were measured by quantitative real-time PCR and Western blotting, respectively. Trypan blue exclusion assays were performed to monitor tumor cell proliferation after siRNA transfection. The cytotoxic effects of etoposide and survivin siRNA, alone and in combination, on leukemic cells were determined using MTT assay. Apoptosis was assessed by ELISA cell death assay. Results: Survivin siRNA markedly reduced both mRNA and protein expression levels in a time-dependent manner, leading to distinct inhibition of cell proliferation and increased spontaneous apoptosis. Surprisingly, survivin siRNA synergistically increased the cell toxic effects of etoposide. Moreover, survivin down-regulation significantly enhanced its induction of apoptosis. Conclusions: Our study suggests that down-regulation of survivin by siRNA can trigger apoptosis and overcome drug resistance of leukemia cells. Therefore, survivin siRNA may be an effective adjuvant in AML chemotherapy.

Bcl-2 Gene Expression in Human Breast Cancers in Iran

Rostamizadeh, Leila,Fakhrjou, Ashraf,Montazeri, Vahid,Estiar, Mehrdad Asghari,Naghavi-Behzad, Mohammad,Hosseini, Somayyeh,Sakhinia, Masoud,Sakhinia, Ebrahim Asian Pacific Journal of Cancer Prevention 2013 Asian Pacific journal of cancer prevention Vol.14 No.7

Background: Breast cancer is among the five most common cancers and ranks first among cancers diagnosed in Iranian women. Screening and treatment of this disease with molecular methods, especially regarding high incidences at early age and advanced stage, is essential. Several genes with altered expression have been identified by cDNA microarray studies in breast cancer, with the Bcl-2 gene indicated as a likely candidate. In this study, we studied Bcl-2 gene expression levels in parallel tumor and non-tumor breast tissues. Materials and Methods: Forty samples including 21 tumor, 16 non tumor (marginal) and 3 benign breast tissues which were all pathologically diagnosed, were subjected to RNA extraction and polyA RT-PCR with the expression level of Bcl-2 quantified using real-time PCR. Results: There is higher expression levels of the Bcl-2 gene in tumor samples compared with marginal samples, but not attaining significance(p>0.05). Bcl-2 expression in 14 (66.7%) of the cases of tumor samples and 9 (56.3%) cases of the marginal samples were positive. Comparison of the expression of the Bcl-2 gene in histological grade showed that a high expression of Bcl-2 was associated with a high histological grade (p<0.41). Conclusions: Our data suggests that dysregulated Bcl-2 gene expression is potentially involved in the pathogenesis of breast cancer. Using gene expression analysis may significantly improve our ability for screening cancer patients and will prove a powerful tool in the diagnosis and prognostic evaluation of the disease whilst aiding the cooperative group trials in the Bcl-2 based therapy project.

BMP3 promoter hypermethylation in plasma-derived cell-free DNA in colorectal cancer patients

Parisa Rokni,Afsaneh Mojtabanezhad Shariatpanahi,Ebrahim Sakhinia,Mohammad Amin Kerachian 한국유전학회 2018 Genes & Genomics Vol.40 No.4

Detecting cfDNA in plasma or serum could serve as a ‘liquid biopsy’, for circulating tumor DNA with aberrant methylation patterns offer a possible method for early detection of several cancers which could avoid the need for tumor tissue biopsies. Bone Morphogenetic Protein 3 (BMP3) was identified as a candidate tumor suppressor gene putatively down-regulated in colorectal cancer (CRC). In this study, we aimed to assess the potential role of BMP3 promoter methylation changes in plasma DNA for detection of colorectal cancerous and precancerous lesions. Plasma DNA samples were extracted from 50 patients with histologically diagnosed polyps or tumor and 50 patients reported negative for polyps or tumors. The procedure consists of bisulfite conversion of the extracted DNA, purification of bis-DNA, and BMP3 methylation status analysis by using the bisulfite specific high resolution melting analysis. This study demonstrated that there was a significantly higher frequency of BMP3 methylated DNA in plasma in patients with polyps versus healthy controls with a sensitivity and specificity of 40 and 94%, respectively. In conclusion, our results demonstrated that BMP3 DNA methylation in plasma had not have sufficient sensitivity and it should be used in combination with other biomarkers for the detection of CRC.

The effect of GGCX and CYP4F2 gene polymorphisms in genotype-guided dosing of warfarin in patients with a history of cardiac surgery

Maryam Azarara,Abbas Afrasibirad,Negin Farzamikia,Aylar Alijani,Ebrahim Sakhinia 한국약제학회 2017 Journal of Pharmaceutical Investigation Vol.47 No.4

Cytochrome P450 4F2 (CYP4F2) and γ-glutamyl carboxylase (GGCX) have small but significant roles in the maintenance dose of warfarin, an oral anticoagulant. CYP4F2 1347 C > T and GGCX 12970 C > G polymorphisms have been used in the pharmacogenetic dosing algorithms of warfarin for Caucasians, Chinese, Turkish and Indian populations. There are no reports about the genotype frequencies of these polymorphisms in Iranian population. In the present study, we aimed to find out the genotype frequencies of CYP4F2 1347 C > T and GGCX 12970 C > G polymorphisms and the impression on warfarin dosage in an Iranian-Azari population. CYP4F2 1347 C > T and GGCX 12970 C > G polymorphisms were genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in 100 cases receiving warfarin after cardiac surgery. No significant differences were found between prescribed and protocol doses of warfarin based on variants of CYP4F2 1347 C > T and GGCX 12970 C > G polymorphisms. Patients with combination genotypes 1347CC/12970CG and 1347TT/12970CG demonstrated significantly different prescribed and protocol doses of warfarin. Age of patients was negatively correlated with prescribed dose of warfarin. Variations of CYP4F2 1347 C > T and GGCX 12970 C > G polymorphisms play a role in determining the required dose of warfarin in patients with cardiac surgery of Iranian-Azari population.