Crude ethyl acetate extract of marine microalga, Chaetoceros calcitrans, induces Apoptosis in MDA-MB-231 breast cancer cells

Abstract

Background: Marine brown diatom Chaetoceros calcitrans and green microalga Nannochloropsis oculata are beneficial materials for various applications in the food, nutraceutical, pharmaceutical and cosmeceutical industries.

Objective: This study investigated cytotoxicity of different crude solvent extracts from C. calcitrans and N. oculata against various cancer cell lines.

Materials and methods: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was carried out to screen the cytotoxic effects of hexane (Hex), dichloromethane (DCM), ethyl acetate, and methanol extract from C. calcitrans and N. oculata toward various cancer cell lines. Flow cytometry cell cycle was used to determine the cell cycle arrest while the mode of cell death was investigated through acridine orange/propidium iodide (AOPI) staining, Annexin V-Fluorescein Isothiocyanate (FITC) and Terminal deoxynucleotidyl transferase-mediated d-UTP Nick End Labeling (TUNEL) assays. Expression profile of apoptotic and proliferative-related genes was then determined using the multiplex gene expression profiler (GeXP).

Results: Crude ethyl acetate (CEA) extract of C. calcitrans inhibited growth of MDA-MB-231 cells, with IC50 of 60 μg/mL after 72 h of treatment. Further studies were conducted to determine the mode of cell death at various concentrations of this extract: 30, 60 and 120 μg/mL. The mode of cell death was mainly apoptosis as shown through apoptosis determination test. The expression data from GeXP showed that caspase-4 was upregulated while B-cell leukemia/lymphoma 2(Bcl-2) was down regulated. Thus, caspase-4 induction endoplasmic reticulum death pathway is believed to be one of the mechanisms underlying the induction of apoptosis while Bcl-2 induced S and G2/M cell cycle phase arrest in MDA-MB-231 cells.

Conclusion: CEA extract of C. calcitrans showed the highest cytotoxicity on MDA-MB-231 via apoptosis.

Keywords: Apoptosis; Chaetoceros calcitrans; MDA-MB-231; crude ethyl acetate extract; gene expression profiler.

Figure 1

Percentage of cell death on MDA-MB-231 which treated with different concentrations of crude EA extract at 24, 48 and 72 hours

Figure 2

Flow cytometric analysis of apoptosis (sub G1) and cell cycle distribution on the MDA-MB-231 cells treated 72 h with different concentration of Crude ethyl acetate extract from Chaetoceros calcitrans. Data represent percentage mean±standard deviation, n = 3. Data marked with alphabet are significantly different at P < 0.05, in comparison with control culture

Figure 3

MDA-MB-231 cells with different features characteristic of apoptosis and secondary necrosis as detected by dual staining with AO and PI after 72 hours treatment with EA extract of C. calcitrans. Apoptotic cells showed membrane blebbing, DNA fragmentation and chromatin condensation. Necrotic cells showed diffused chromatin with apparent nucleus membrane disruption. (a) Untreated control; (b) Low concentration (30 ug/mL); (c) IC50 (60 ug/mL); (d) High concentration (120 ug/mL) (magnification: ×400)

Figure 4

Flow cytometric analysis of phosphatidylserine-annexin V labeled on the MD Anderson-metastatic breast-231MDA-MB-231 cells treated 72 h with different concentration of Crude ethyl acetate extract from Chaetoceros calcitrans. Data represent percentage mean±standard deviation, n = 3. Data from the same group marked with different alphabet are significantly different at P < 0.05

Figure 5

Apoptosis induced by ethyl acetate extract of Chaetoceros calcitrans was determined by the presence of apoptotic deoxyribonucleic acid (DNA) strand breaks detected via Terminal deoxynucleotidyl transferase-mediated d-UTP Nick End Labeling TUNEL staining flow cytometry is shown. DNA fragmentation is obviously increased from low concentration treatment (b) to high concentration of treatment (d), compared with the control (a). Data represent percentage mean±standard deviation (n = 3), *P < 0.05