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.

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“…Samples were then subjected to centrifugation at 8000 rpm for 20 min at 4°C, the supernatant was discarded and the pellet was air-dried and then resuspended in 30 µl of DEPC water. RNA concentration was determined by using a Nanodrop (Thermo Scientific, USA) [21].…”

“…Each cDNA reaction was made up to 20 µl containing 1 x first strand reaction Buffer (Invitrogen), 5 mM DTT, 200 units SuperScript III RT (Invitrogen) and 40 units of RNAse (Invitrogen). The PCR cycles used were: 25°C for 10 min followed by 37°C for 2 h, 85°C for 10 min and finally 4°C hold [21].…”

“…In a StepOnePlus Real-Time PCR machine (Applied Biosystems), the PCR cycles used were: 1 cycle of 95°C for 3 min to activate the enzyme, followed by 40 cycles of 95°C for 1 second (denaturation) and 95°C for 20 seconds (annealing and extension). Fold change in gene expression was calculated using the ∆∆CT method [21]. The sequences of the human genome based primers, probes and details of TaqMan assays used for RT-PCR are tabulated in supplementary file 1.…”

“…The luminescence was measured with a luminescence plate reader (BMG Labtech PHERAstar FS, Germany). Luminescence values were normalized to cell viability (measured using MTT assay) [21].…”

“…Samples were again centrifuged (1000 rpm for 5 min), the supernatant was removed and the pellets were resuspended in 50 µl RNAseA (Roche) (50 µg/ml) for 15 min at 37°C or 30 min at room temperature. 200 µl of PI Staining solution (0.1% Triton X-100, 2 mM MgCl 2 , 100 mM NaCl, 10 mM PIPES ph 6.8 and 10 µg/ml PI) was added per sample and incubated for 10 -15 min prior to analyzing on HTFC Screening System (IntelliCyt Corporation, Albuquerque, NM) [21].…”