Cytotoxicity and apoptosis induced by a plumbagin derivative in estrogen positive MCF-7 breast cancer cells

Abstract

Plumbagin [5-hydroxy- 2-methyl-1, 4-naphthaquinone] is a well-known plant derived anticancer lead compound. Several efforts have been made to synthesize its analogs and derivatives in order to increase its anticancer potential. In the present study, plumbagin and its five derivatives have been evaluated for their antiproliferative potential in one normal and four human cancer cell lines. Treatment with derivatives resulted in dose- and time-dependent inhibition of growth of various cancer cell lines. Prescreening of compounds led us to focus our further investigations on acetyl plumbagin, which showed remarkably low toxicity towards normal BJ cells and HepG2 cells. The mechanisms of apoptosis induction were determined by APOPercentage staining, caspase-3/7 activation, reactive oxygen species production and cell cycle analysis. The modulation of apoptotic genes (p53, Mdm2, NF-kB, Bad, Bax, Bcl-2 and Casp-7) was also measured using real time PCR. The positive staining using APOPercentage dye, increased caspase-3/7 activity, increased ROS production and enhanced mRNA expression of proapoptotic genes suggested that acetyl plumbagin exhibits anticancer effects on MCF-7 cells through its apoptosis-inducing property. A key highlighting point of the study is low toxicity of acetyl plumbagin towards normal BJ cells and negligible hepatotoxicity (data based on HepG2 cell line). Overall results showed that acetyl plumbagin with reduced toxicity might have the potential to be a new lead molecule for testing against estrogen positive breast cancer.

Fig. (1)

Structures of PL and its derivatives (AP, BP, CP, IP, and PP).

Fig. (2)

PL and derivatives (AP, BP, CP, IP, and PP) induced growth inhibition in cancer and normal cells within 24 h of treatment (read 0 µM as untreated). Growth inhibition was assessed using MTT assay. Data are mean ± standard deviation S.D. (n=3). Growth inhibition at 24 h for (a) BJ cells, (b) MCF-7 cells, (c) BT20 cells, (d) HepG2 cells, and (e) DU145 cells.

Fig. (3)

PL-and AP- dependent induction of apoptosis in BJ and MCF-7 cells. Cells were incubated with 5 and 10 µM of PL and AP for 12 and 24 h. (a) A representative image of at least two independent experiments each having quadruplicates is shown. H2O2 is used as a positive control to show that assay is working. Percentages of apoptotic cells (based on dye uptake) at 12 and 24 h in: (b) BJ cells, and (c) MCF-7 cells. Data are mean±S.D. (n=4), *p<0.05 significant difference to untreated control.

Fig. (4)

PL- and AP- dependent induction of caspase-3/7 activity in BJ, MCF-7 and BT20 cells. Cells were incubated with 5 and 10 µM of PL and AP for 1, 2, 4, 8 and 16 h. Fold change in caspase-3/7 activity after treatment with PL and AP in a time-course experiment in (a) BJ cells, (b) MCF-7 cells, and (c) BT20 cells. Docetaxel (200nM) is used as positive control at all time points to show that assay is working and representative activity is shown for 16 h only. Data are mean±S.D. (n=4), *p<0.05 significant difference to untreated (Untx) control.

Fig. (5)

Effect of 10 and 20 µM PL and derivatives on ROS production in MCF-7 and BT20 cells. ROS activity in cells was determined by staining with the DCFDA dye and cells were using flow cytometry. The percentages of stained cells are shown on the histograms. Data are mean ± standard deviation S.D. (n=3). Cells were treated with 10mM H₂O₂ for 1 h as a positive control to show that assay was working.

Fig. (6)

Molecular profiling by measuring mRNA levels of key apoptosis-associated genes using RT-PCR. The MCF-7 cells were treated with 5 and 10 µM of PL and AP for 2 and 6 h in duplicate experiments. Bar graph representing the expression profiles of p53, NF-Kb, Bax, Bcl2, Mdm2, Casp7, and Bad genes after treating the MCF-7 cells for (a) 2 h, and (b) 6 h. Data are mean±S.D. (n=3), *p<0.05 significant difference to untreated (Untx) control. (c) Pathway diagram showing the interplay of the tested genes during the apoptotic cell death.

Fig. (7)

An overview of the cell death mechanisms induced by PL in various cancer cells. Black arrows represent the activation, black bars represent the inactivation, blue ovals represent the genes/mechanisms targeted by PL and pink arrows represent the biological processes or pathways affected by PL in various types of cancers [6, 13-14, 16-17, 22, 26, 50-53].