Afzali M et al. (2017), Evaluation of the Cytotoxic Effect of the Britt...

ECB-ART-46365

Iran J Pharm Res 2017 Jan 01;16Suppl:216-226.

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Evaluation of the Cytotoxic Effect of the Brittle Star (Ophiocoma Erinaceus) Dichloromethane Extract and Doxorubicin on EL4 Cell Line.

Afzali M , Baharara J , Nezhad Shahrokhabadi K , Amini E .

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Leukemia is a blood disease that creates from inhibition of differentiation and increased proliferation rate. The nature has been known as a rich source of medically useful substances. High diversity of bioactive molecules, extracted from marine invertebrates, makes them as ideal candidates for cancer research. The study has been done to investigate cytotoxic effects of dichloromethane brittle star extract and doxorubicin on EL4 cancer cells. Blood cancer EL4 cells were cultured and treated at different concentrations of brittle star ( Ophiocoma erinaceus ) dichloromethane extract at 24, 48 and 72 h. Cell toxicity was studied using MTT assay. Cell morphology was examined using an invert microscope. Further, apoptosis was examined using Annexin V-FITC, propodium iodide, DAPI, and Acridine orange/propodium iodide staining. Eventually, the apoptosis pathways were analyzed using measurement of Caspase-3 and -9 activity. The statistical analysis was performed using SPSS, ANOVA software, and Tukey''s test. P<0.05 was considered to be significant. MTT assay and morphological observations showed that dichloromethane extract can inhibit cell growth in a dose dependent. The results considered 32 µg/mL of the extract as IC50. Also, doxorubicin suppressed EL4 proliferation as IC50=32 µg/mL. All experiments related to apoptosis analysis confirmed that dichloromethane brittle star extract and doxorubicin have a cytotoxic effect on EL4 cells inIC50 concentration. The study showed that dichloromethane brittle star extract is as an adjunct to doxorubicin in treatment of leukemia cells.

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Genes referenced: LOC115919910

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	Figure 1. Morphological effect control groups and groups treated with concentrations of 15, 31, 62, 125, 250 µg/ml brittle star dichloromethane extract O. erinaceus (a) and doxorubicin (b) 24 h after treatment observed by inverted microscope (magnification × 200) A: control, B: 15 µg/ml, C: 31 µg/ml, D: 62 µg/ml, E: 125 µg/ml, F: 250 µg/ml. c) Morphological changes under treatment with co-administration of brittle star dichloromethane extracts and doxorubicin after 24 h treatment. A: control group, B: 7.5 µg/ml DCM and 7.5 µg/ml doxorubicin (IC50) C: 31 µg/ml DCM and 7.5 µg/ml doxorubicin D: 7.5 µg/ml DCM and 31 µg/ml doxorubicin E: 15 µg/ml DCM and 31 µg/ml doxorubicin F: 31 µg/ml DCM and 15 µg/ml doxorubicin. (Magnification × 200).


	Figure 4. Apoptosis induced by brittle star dichloromethane fraction on EL4 cells. (A) Control (B) 31 and (C) 62 µg/ml of extract conducted by Annexin V/PI assay
	Figure 5. Flow cytometry assessed apoptosis induced by brittle star dichloromethane extract and synergic treatment brittle star dichloromethane extract and Doxorubicin on cells EL4 after staining with PI
	Figure 6. a,b.c. The fluorescence images shows AO/PI staining for induction of apoptosis on EL4 cells whit brittle star dichloromethane extract, Doxorubicin and synergic treatment different concentration Doxorubicin and brittle star dichloromethane extract (31, 62 and 7.5 DOX+ 7.5 DCM and 7.5 DOX+31 µg/mL DCM). (A)Control cells and (B) 31 µg/mL DCM (C) 62 µg/mL DCM (D), 31 µg/mL DOX and (E) 62 µg/mL DOX, (F) 7.5 DOX+7.5 DCM , (G) 7.5 DOX+31 DCM µg/mL (Magnification ×200).d) Fluorescence microscopic image of DAPI staining. A (control) B and C (treated EL4 cells with different concentration brittle star dichloromethane extract). (Magnification ×200).
	Figure 7. Effect of brittle star dichloromethane extract on Caspase-3 and caspase-9 activities in treated EL4 cells. (A) Histogram represents caspase-3 activity in untreated control and brittle star dichloromethane extract (31, 62 µg/mL). (B) Histogram represents caspase-9 activity in untreated control and brittle star dichloromethane extract (31, 62 µg/mL).
	Figure 8. Synergic effect of brittle star dichloromethane extract and Doxorubicin on Caspase-3 and 9 activity in EL4 cells. (A) Histogram represents caspase-3 activity in untreated and synergic treated EL4 cells whit brittle star dichloromethane extract and Doxorubicin (31+7.5 µg/mL) treated EL4 cells. (B) Histogram represents caspase-9 activity in untreated control and brittle star dichloromethane extract (31+7.5 µg/mL).

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