Haematococcus pluvialis Microalgae Extract Inhibits Proliferation, Invasion, and Induces Apoptosis in Breast Cancer Cells

Abstract

Breast cancer (BC) is the most common malignant cancer in females worldwide. Drug resistance, toxicity, and the failure of current therapies to completely cure BC has challenged conventional medicine. Consequently, complementary alternative medicine has become popular due to its safety and efficacy. Haematococcus pluvialis (H. pulvialis) is a green microalga living in fresh water, and its crude extract is rich of bioactives, including carotenoids, known to inhibit cancer cell growth. In the present study, we investigated the effects of a methanol crude extract called "T1" of H. pulvialis on cell growth and migration/invasion of the BC cell line MDA-MB-231 in comparison to the fibroblast control cells. TI significantly suppressed BC cell growth, inhibited migration and invasion and induced apoptosis. Interestingly, apoptosis was mediated by a significant loss of mutant p53 protein, and increased Bax/Bcl2 ratio. Our findings support our hypothesis that T1 exerts its anti-cancer effects by inhibiting BC invasion and inducing apoptosis mediated, at least, via the p53/Bax/Bcl2 pathway. Ongoing experiments aim to identify the molecular mechanisms underpinning T1-inhibited BC cell invasion using pre-designed metastasis gene-based array method.

Keywords: Haematococcus pluvialis microalgae; apoptosis; breast cancer; cell proliferation; invasion.

FIGURE 1

(A) Image of magnified H. pluvialis culture (magnification, 40×). (B) HPLC-chromatograph of the pigments extracted from H. pluvialis biomass.

FIGURE 2

(A) Effect of different concentrations of T1 extract on cell viability of MDA-MB-231 cells after 48 h of treatment. Data indicate an inverse relationship between concentrations of T1-extract and cell proliferation in MDA-MB-231 cells as compared to the vehicle control (methanol). (B) Effect of T1 on cell viability of fibroblasts after 48 h of treatment. Data are presented as a percentage of treatment relative to the control (Mean ± SEM; n = 3). ***p < 0.0001. VC, Vehicle control.

FIGURE 3

Effect of T1-extract on the morphology of MDA-MB-231. T1 induced cell death and the formation of a monolayer of cells in the MDA-MB-231 cells (black arrows indicate loss of cell-cell adhesion), in comparison with untreated (control) and fibroblast cells, which show no cytotoxic effect, displaying a round phenotype and form multilayers; Black arrows indicate epithelial morphology with clear cell-cell adhesion.

FIGURE 4

Effect of T1 on migration of MDA-MB-231 using wound healing assay. T1-extract sup-pressed cell motility of MDA-MB-231 cells in comparison to methanol treated (VC: vehicle control) and non-treated cells. Representative images are shown from three independent experiments and dark lines define the areas lacking cells (wound area, ImageJ). Values of percentage wound closure ± SEM (n = 3). *p < 0.01, **p < 0.001.

FIGURE 5

Effect of T1-extract on cell invasion of MDA-MB-231 breast cancer cell line using Boyden chamber assay. T1-extract significantly decreased cell invasion ability of MDA-MB-231 by ∼60% in comparison to control cells (VC: vehicle control) (***p < 0.0001).

FIGURE 6

Expression patterns of proteins associated with T1-induced apoptosis. Western blot analysis of T1 extract- inhibited mutant p53 expression, while upregulating Bax/Bcl ratio, in comparison with their vehicle control (VC). β-actin was used as a control for the proteins amount in this assay. Cells were treated with 5% of T1-extract for 48 h, as explained in the materials and methods and the results sections. ***p < 0.0001.