Induction of apoptosis in HeLa cells by chloroform fraction of seed extracts of Nigella sativa

Abstract

Background: Cancer remains one of the most dreaded diseases causing an astonishingly high death rate, second only to cardiac arrest. The fact that conventional and newly emerging treatment procedures like chemotherapy, catalytic therapy, photodynamic therapy and radiotherapy have not succeeded in reverting the outcome of the disease to any drastic extent, has made researchers investigate alternative treatment options. The extensive repertoire of traditional medicinal knowledge systems from various parts of the world are being re-investigated for their healing properties. This study progresses in the direction of identifying component(s) from Nigella sativa with anti cancer activity. In the present study we investigated the efficacy of Organic extracts of Nigella sativa seed powder for its clonogenic inhibition and induction of apoptosis in HeLa cancer cell.

Results: Methanolic, n-Hexane and chloroform extracts of Nigella sativa seedz effectively killed HeLa cells. The IC50 values of methanolic, n-hexane, and chloroform extracts of Nigella sativa were 2.28 microg/ml, 2.20 microg/ml and 0.41 ng/ml, respectively. All three extracts induced apoptosis in HeLa cells. Apoptosis was confirmed by DNA fragmentation, western blot and terminal transferase-mediated dUTP-digoxigenin-end labeling (TUNEL) assay.

Conclusion: Western Blot and TUNEL results suggested that Nigella sativa seed extracts regulated the expression of pro- and anti- apoptotic genes, indicating its possible development as a potential therapeutic agent for cervical cancer upon further investigation.

Figure 1

A NS-Methanolic Extracts induced Clonogenic inhibition in HeLa cells. 1B. NS n-haxane Extracts induced Clonogenic inhibition in HeLa cells. 1C: NS-Chloroform Extract induced Clonogenic inhibition in HeLa cells

Figure 2

NS extracts induced apoptosis in HeLa cells. The bar graphs show the percentage of TUNEL-positive cells. Apoptosis in the treated cells was normalized to the baseline apoptosis in the untreated cells. The data shown are the means from two independent experiments.

Figure 3

DNA laddering: DNA fragmentation on 1% Agarose Gel Electrophoresis shows; Lane 1 100 bp marker, Lane 2 untreated cells, Lane 3 Chloroform extract treated cells, Lane 4 n-hexane extract treated cells and Lane 5 methanolic extract treated cells.

Figure 4

Expression of anti-apoptotic genes; bcl-2 and bcl-X_L in HeLa cells treated with methanolic, choloroform and n-haxane extract. The graph shows the intensity of bands (ImageJ sofware).

Figure 5

Expression of caspase -3, -8 and -9 in HeLa cells treated with methanolic, choloroform. and n-haxane extract. The graph shows the intensity of bands (ImageJ sofware).

Figure 6

Expression of pro-apoptotic gene, p53 in HeLa cells treated with methanolic, choloroform. and n-haxane extract. The graph shows the intensity of bands (ImageJ sofware).

Figure 7

Extraction and partitioning of bioactive compounds of N. sativa.