Targeting apoptosis Pathways on Malignant Human Glioma Cell Line by Cytochalasin H

Targeting apoptosis Pathways on Malignant Human Glioma Cell Line by Cytochalasin H
samaneh heidarzadeh,^1,*
1. Department of Biology, Faculty of Science, University of Zabol, Zabol, Iran
Introduction: Glioblastoma is one of the most malignant central nervous system tumors located in the brain . This is characterized by the code 3/9440 in the International Classification of Diseases for Oncology. Despite the efforts carried out to improve the glioma tumor’s treatment, these tumors are not curable. Although chemotherapy is effective in the tumor treatment but the drugs used in the treatment have side effects for the patients and sometimes, drug resistance causes limitations in the treatment of the patients. Therefore, the investigation of new therapeutic approaches for this disease is very important. we are needed to explore molecular mechanism for treatment. Cytochalasins connect to their sub-units, leading to some alterations in the cytoskeleton structure and preventing polymerization. Thus, formation of microfilaments is significantly inhibited .Moreover, cytochalasins prevent cell transfer and create enucleated cells by penetrating the cell membrane. Apoptosis is the consequence of a planned intracellular cascade of genetically controlled stages. On this basis, the aim of current study was to provide a proof-of-concept on the mechanism of caspases-activities in the apoptotic pathway after treatment of malignant human glioma cell line (U87MG) by cytochalasin H and stud ied the morphology of the cells treated with cytochalasin H by inverted microscope and Fluorescence Microscope.
Methods: we examined the cytochalasin H cytotoxicity activities as a new agent therapeutic on the human malignant glioma cell line (U87MG) in vitro. The cells were cultured and treated with 10 -5 - 10-9 M of cytochalasin H for 24, 48 and 72 h. MTT assay was used to assess Cytochalasin H cytotoxic effect on human malignant glioma cell line (U87MG). Different concentrations of cytochalasin H (10-5 and 10-9 M) for 24, 48 and 72 h and each concentration has 8 replicate wells. Fluorometric of caspase 3, 8, and 9 activities were carried out using the caspase assay kit (Novex)and Morphology changes in the U87MG cells were examined by fluorescence microscope.
Results: MTT assay showed that cytochalasin H at concentrations of 10-5 M inhibited the U87MG cancer cells proliferation for 48 h (p<0.05). but there was no cytochalasin H toxic effects on the cell line U87MG for 24 and 72 h (p>0.05) and as well as was not observed cytochalasin H toxicity effects on normal cell line HEK Compared with the U87MG cancer cells (p>0.05). After treatment U87MG with 10-5 M CH for 48 h, Using a caspase assay kit (Novex). Activity of caspase-3, caspase-8 and caspase-9 were increased following CH treatment (17% , 12% and 7% respectively) but this was not a significant difference (p > 0.05). The structural changes of the cells treated with CH compared with the control was investigated.Moreover ,the fluorescence microscopy indicated morphological changes due to apoptosis in U87MG cancer cells after treatment with cytochalasin H. The results obtained from this study show that the enzyme activity of the caspases is not sufficient to start the caspase-dependent apoptosis process. These results were inconsistent with the findings obtained from testing the cells by fluorescent microscope. The cell nuclei were observed, condensed, and fragmented under fluorescent microscope, and this is how the cells were led towards apoptosis. So, the effect of cytochalasin H on the induction of apoptosis in the U87MG cells could probably be attributed to the caspase-independent apoptosis pathway.
Conclusion: This study shows the effect of caspase-independent pathways of the programmed cell death on the U87MG cancer cell line under cytochalasin H treatment. Further studies are needed to explore the another mechanism.
Keywords: Caspases, Cytochalasin H, Glioblastoma, apoptosis