Human Gyrovirus Apoptin as a Potential Selective Anticancer Agent: An In Vitro Study

Background: Selective therapy has always been the main challenge in cancer treatments. Recently, it has been shown that Human Gyrovirus-derived protein apoptin (HGV-Apoptin) has selective cytotoxic effects on cancer cells similar to its homologue, Chicken Anemia Virus-derived Apoptin (CAV-Apoptin). However, apoptotic effects of Human Gyrovirus apoptin have been only evaluated on a few cancerous cell lines and need to be further investigated. In this study, we have evaluated the apoptotic effects of HGV-Apoptin and CAV-Apoptin expression on lung cancer (A549) and normal (HEK-293) cell lines, in order to provide more information about the specificity of these proteins on cancerous cells. Methods: Target cells were transfected by the calcium-phosphate precipitation method with constructed plasmids expressing HGV-Apoptin and CAV-Apoptin proteins as well as the control plasmid. Transfection efficiency was followed and imaged by fluorescence microscopy. Quantification of apoptosis was performed by flow cytometry. Measurements were compared by paired Student t-test. Results: Cells were successfully transfected with control and constructed plasmids. Flowcytometry analysis showed that A549 cells transfected with HGV-Apoptin and CAV-Apoptin expressing plasmids, undergone the apoptosis compared to A549 cells transfected with control plasmid (P<0.001). None of the plasmids could induce apoptosis in HEK-293 cells. Conclusion: Human Gyrovirus-derived apoptin (HGV-Apoptin) similar to its homologue, chicken anemia virus derived Apoptin (CAV-Apoptin) can induce apoptosis in Non-small-cell lung carcinoma cell line A549, but not in normal human embryonic kidney cell line HEK-293, which can be introduced as a promising novel specific antitumor agent

Optimal Electroporation Condition for Small Interfering RNA Transfection into MDA-MB-468 Cell Line

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 µF 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

New techniques and methods in the study of the invasion, cell migration and MMPs activity in vitro and in animal models

Background & Objective: Cancer metastasis is the primary cause of cancer morbidity and mortality, it accounts for about 90% of cancer deaths. Cancer treatment has improved significantly, due to early detection and inhibition of cancer growth. The ability to invade and migrate is important in malignant tumor cells. The study of cell migration is valuable in cancer diagnosis, prognosis, drug development and treatment. New methods are available to investigate the invasion, migration and the activity of enzymes involved in the invasion process in the laboratory. The effectiveness and efficacy of various anti-cancer drugs and compounds can be studied using these methods in laboratory and animal models. Conclusion: In this paper, we offer a summary of in-vitro migration assays, including the transwell migration assay, scratch wound assay, microfluidic chamber assay, exclusion zone assay, fence assay, micro carrier bead assay, spheroid migration assay and in-vivo approach, Chick chorioallantoic membrane (CAM) assay. This review also provides an overview of methods, In situ Zymography, ELISA, and FRET based measurement of MMP activity and Substrate Zymography for measuring the level of metalloproteinase enzyme as the major enzyme in the degradation of extracellular matrix