Apoptosis Inducing Effect of Plumbagin on Colonic Cancer Cells Depends on Expression of COX-2

Plumbagin, a quinonoid found in the plants of the Plumbaginaceae, possesses medicinal properties. In this study we investigated the anti-proliferative and apoptotic activity of plumbagin by using two human colonic cancer cell lines, HT29 and HCT15. IC50 of Plumbagin for HCT15 and HT29 cells (22.5 µM and 62.5 µM, respectively) were significantly different. To study the response of cancer cells during treatment strategies, cells were treated with two different concentrations, 15 µM, 30 µM for HCT15 and 50 µM, 75 µM for HT29 cells. Though activation of NFκB, Caspases-3, elevated levels of TNF-α, cytosolic Cytochrome C were seen in both HCT15 cells HT29 treated with plumbagin, aberrant apoptosis with decreased level of pEGFR, pAkt, pGsk-3β, PCNA and Cyclin D1was observed only in 15 µM and 30 µM plumbagin treated HCT15 and 75 µM plumbagin treated HT29 cells. This suggests that plumbagin induces apoptosis in both HCT15 cells and HT29 treated, whereas, proliferation was inhibited only in 15 µM and 30 µM plumbagin treated HCT15 and 75 µM plumbagin treated HT29 cells, but not in 50 µM plumbagin treated HT29 cells. Expression of COX-2 was decreased in 75 µM plumbagin treated HT29 cells when compared to 50 µM plumbagin treated HT29 cells, whereas HCT15 cells lack COX. Hence the observed resistance to induction of apoptosis in 50 µM plumbagin treated HT29 cells are attributed to the expression of COX-2. In conclusion, plumbagin induces apoptosis in colonic cancer cells through TNF-α mediated pathway depending on expression of COX-2 expression

Plumbagin induces apoptotic in colonic cancer epithelial cells.

<p><i>a. Immunoblotting analysis of NFκB activation, Caspase-3 activation and cytochrome C release</i>. Lane 1- Control HCT15; Lane 2- 15 µM plumbagin treated HCT15; Lane 3- 30 µM plumbagin treated HCT15; Lane 4- Control HT29; Lane 5- 50 µM plumbagin treated HT29; Lane 6- 75 µM plumbagin treated HT29. <i>b.i. Cell cycle analysis of control and plumbagin treatment HCT15 and HT29 cells by Flow Cytometry</i>. Compared with control HCT15, HCT15 treated with plumbagin shows marked rise in sub-G1 fraction suggesting that these cells are undergoing apoptosis. HT29 cells exposed to 75 µM of Plumbagin alone exhibited increase in sub-G1 fraction whereas HT29 cells exposed to 50 µM of Plumbagin sub-G1 fraction was much lesser. <i>b.ii. Quantitative data of cell cycle analysis</i>. All the experiments were done in triplicates and expressed as the mean ± SD. Significance is indicated as *<i>p<0.001</i>.</p

Analysis of apoptotic inducing effect of plumbagin on HCT15 and HT29 cells assessed by comet assay.

<p>HCT15 cells treated with 30 µM plumbagin and HT29 cells treated with 75 µM plumbagin showed increased extant of DNA damage. The length of the comet tail was ten and six times that of the control and 15 µM plumbagin treated HCT15, respectively, while, 50 µM and 75 µM plumbagin - treated HT29 cells shows four and two time of span of tail, respectively, compared to control HT29 cells. DNA damage was not observed, as the halo surrounding cell nuclei was clearly visible in control cells.</p

Effect of Plumbagin on Colonic cancer cells and normal PBMCs.

<p><i>a. Cytotoxicity effect of various concentrations of Plumbagin on HCT15 cells</i>. Dose dependent cytotoxicity effects of Plumbagin on HCT15 cells were represented in the above graph. HCT15 cells were more sensitive to plumbagin as IC₅₀ at 24 hours was 22.5 µM. All the experiments were done in triplicates and expressed as the mean ± SD. Significance is indicated as *<i>p<0.001</i>. <i>b. Cytotoxicity effect of various concentrations of Plumbagin on HT29 cells</i>. Dose dependent cytotoxicity effects of Plumbagin on HT29 cells were represented in the above graph. HT29 cells were more sensitive to plumbagin as IC₅₀ at 24 hours was 62.5 µM. All the experiments were done in triplicates and expressed as the mean ± SD. Significance is indicated as *<i>p<0.001</i>. <i>c. Cytotoxicity effect of various concentrations of Plumbagin on PBMC cells</i>. Dose dependent cytotoxicity effects of Plumbagin on PBMC cells were represented in the above graph. PBMC cells were resistance to plumbagin induced cytotoxicity even at 100 µM concentration. All the experiments were done in triplicates and expressed as the mean ± SD. Significance is indicated as *<i>p<0.001</i>.</p

Expression analysis of TNF-α, COX-2 and GAPDH by RT-PCR.

<p>Lane 1- Control HCT15; Lane 2- 15 µM plumbagin treated HCT15; Lane 3- 30 µM plumbagin treated HCT15; Lane 4- Control HT29; Lane 5- 50 µM plumbagin treated HT29; Lane 6- 75 µM plumbagin treated HT29. a. Plumbagin - treated HCT15 and HT29 cells showed increased amounts of RNA transcripts of the <i>TNF-α</i> gene compared to the untreated HCT15 and HT29 cells. b. Levels of RNA transcript of <i>cox-2</i> was decreased in 75 µM plumbagin treated HT29 cells, when compared to 50 µM plumbagin treated HT29 cells, while plumbagin treated HCT15 cells as well as untreated HCT15 did not show any expression of <i>cox-2</i> RNA transcript. c. Showing level of GAPDH RNA transcripts in control and Plumbagin - treated HCT15 and HT29 cells. d. Represents densitometric analysis showing relative expression of <i>TNF-α</i> and <i>cox-2</i> to GAPDH. Represented data values were obtained from triplicate analysis and expressed as the mean ± SD. Significance is indicated as *<i>p<0.05</i>; **<i>p<0.001</i>.</p

Western blotting analysis of phosphorylated Akt, phosphorylated EGFR, PCNA and cyclin D1 in control and plumbagin treated HCT15 and HT29 cells.

<p>Lane 1- Control HCT15; Lane 2- 15 µM plumbagin treated HCT15; Lane 3- 30 µM plumbagin treated HCT15; Lane 4- Control HT29; Lane 5- 50 µM plumbagin treated HT29; Lane 6- 75 µM plumbagin treated HT29. Expression of PCNA, cyclin D1 along with phosphorylation of Akt and EGFR were significantly decreased in 15 µM and 30 µM plumbagin treated HCT15 and in 75 µM plumbagin when compared to Control HCT15, Control HT29 and HT29 cells treated with 75 µM plumbagin. β-Actin served as internal control.</p

Primer sequence of GAPDH, COX-2 and TNF-α.

<p>Primer sequence of GAPDH, COX-2 and TNF-α.</p

Effect of different concentrations of Plumbagin on HCT15 and HT29 cell proliferation.

<p><i>a. HCT15 cells. b. HT29 cells</i>. Proliferation of HCT15 cells treated with 15 µM and 30 µM of plumbagin and HT29 cells treated with 75 µM plumbagin increased significantly at 48 h and 72 h, whereas , 50 µM plumbagin treated HT29 cells tend to proliferate. All the experiments were done in triplicates and expressed as the mean ± SD. Significance is indicated as *<i>p<0.001</i>.</p