Effect Of Curcumin On The Growth Rate Of Npc Cells

Abstract Curcumin is a phenolic compound extracted from the rhizosome of Curcuma longa. Previous studies suggested that curcumin possesses pro-apoptic and anti-cancer features. Our further study focused on the effect of curcumin on the molecular basis of nasopharyngeal carcinoma. NPC cells treated with 30µM curcumin were subjected to several analysis using hemocytometry, DNA fragmentation, flow cytometry, Western blot analysis, RT-PCR and luciferase assay. The results showed that 30µM curcumin promoted cell cycle arrest at sub-G1 phase. It also induced apoptosis through inhibiting the anti-apoptotic pathways (Akt, NFkB and Bcl-2) and stimulating the pro-apoptotic pathways (Bax). These findings allowed us to have a better understanding on the effect of curcumin in NPC cells and suggested the potential therapeutic use of curcumin as a drug against NPC. Key words: Curcumin, Nasopharyngeal carcinoma, apoptosis Intro: The mechanism of anti-cancer effect of curcumin has been under intensive research in recent years. Abundant evidence showed that curcumin could induce apoptosis and cell cycle arresting in the G2/M phase (PMID9422331). With the dose dependent curcumin-treatment in various cancers in vitro and in vivo, their expression levels of anti-apoptotic genes such as Bcl2 and Bcl-xL (PMID15142674) and pro-proliferative genes cyclin E, cyclin D1 (PMID18156803) and Akt (PMID17332930) were deactivated while those of pro-apoptotic caspases (PMID11756235) and anti-proliferative genes such as cyclin-dependent kinase inhibitors (p16, p21, and p27) (PMID18156803) and p53 (PMID17332930) activated. Recent researches, monitoring reactive oxygen species (ROS), mitochondrial membrane potential (PMID20043100) and the release of cytochrome c (PMID19235267), further regarded mitochondrial signaling pathway as one of the curcumin-related pathways, which includes NF?B pathway (PMID18829502) and signaling pathway stimulated by Toll-like receptor (TLR) ligands (PMID19303754). Studies demonstrated that curcumin takes part in multiple signaling pathways; nevertheless, the underlying mechanism remains largely unidentified.

Curcurmin effectively inhibits the growth and induce the programmed cell death in NPC cells (PMID19317462). Various pivotal signaling events which govern the occurrence of apoptosis and inhibition of the cell proliferation were also identified to accompany with the curcumin treatment, including the DNA fragmentation (PMID19317462, PMID15601554) and the suppression of NF?B-mediated transcri ptional activity (PMID19317462). In addition, the upregulated expressions of Fas protein and cytochrome c leading to the activation of the caspase cascade (PMID17039805) further hinted their possible involvements in the curcumin-stimulated responses. Yet, the regulatory role of the hallmark cellular event by the Bcl-2 family and the exact participation of these signaling cascades remain largely undefined. In this paper, we attempt to delineate its underlying molecular mechanism with the Human epithelial nasopharyngeal cancer cell line CNE-2, that in hope provide new insights in the development of novel alternative therapy for the nasopharyngeal cancer.

Material & method Luciferase assay: Materials DH5? cells, NF?B-luc reporter and luciferin were products of Clontech (Mountain View, CA). Cell culture consumable were products of Invitrogen (Carlsbad, CA). Other chemicals were purchased from Sigma-Aldrich (St. Louis, MO). Method In this experiment, 15,000 HONE-1 cells were transfected with pNF?B-TA-Luc vector and seeded per well in a 96-well microplate. After overnight incubation, the cells were treated with medium containing 0 or 30 ?M of curcumin. They were then incubated for 48h at 37°C, and then harvested with luciferase lysis buffer. Luciferase activity was measured with a luciferase assay kit by a luminometer (Promega, Madison, WI) according to the manufacturer's protocol. All the experiments were done in triplicates and repeated 3 times. Results Reason for experiment To examine the inhibitory effect of curcumin on NFkB signaling cascade, after treatment with different concentrations of curcumin, the average luminescence intensity of Luciferase-expressing cells, which monitors the NF?B-luc reporter, will allow us to find out effect of curcumin one the expression of NF?B.

. Results of experiment In this luciferase assay experiment, cells tasnfected with NFkB-luc reporter are exposed to different concentrations of curcumin. As curcumin concentration increases, there is a significant decrease in the intensity reading after the cells are exposed to 3 uM of curcumin when compared to the control with no curcumin. The dunnett test clearly shows that the probability/chance is less that 0.001 and so it is significant.

. How to experiment? To do the luciferase assay experiment; firstly seed cells trasnfected with NF?B-luc reporter. The next day, replenish medium with 50 ?l serum free medium with 0 or 30 uM curcumin and incubate for two days. On the fourth day remove medium and replenish with 25?l lysis buffer, then monitor luminescence of the cells after Freeze-thraw. Finally wash the tublings to get rid of any remaining luciferin.

. Message NF?B-mediated luciferase expression was reduced in the presence of curcumin. Reason is that curcumin inhibited the anti-apoptotic NF?B-mediated gene transcri ption. So the average intensity will decrease as curcumin concentration increases. - Curcumin suppressed NF?B mediated transcri ptional activity To examine the inhibitory effect of curcumin on NFkB signaling cascade, HONE-1 cells transfected with NF?B-luc reporter were seeded and then treated with 0 or 30 ?M of curcumin. After four days of incubation, the cell lysates were subjected to the luciferase assay experiment to evaluate the transcri ptional activity of NF?B. The luminescence intensity of the luciferase produced by the cells exposed to 30 ?M curcumin decreased significantly (Paired-T test, p?0.001) when compared to the cells that were exposed to 0 ?M curcumin. The results show that NF?B-mediated transcri ptional activity was reduced in the presence of curcumin.

Discussion Discussion Bcl-2 cascade also participated in curcumin-induced apoptosis. The results of the western analysis showed that the increase in the curcumin concentration suppressed the phosphorylation of Akt and the expression of anti-apoptotic Bcl-2, similar to the previous study on apoptosis in a way that certain exogenous factors can induce apoptosis through inhibiting the Akt pathway (PMID8929531). The Akt and Bcl-2 are linked through an agonist of Bcl-2, the Bad protein. Bad is an apoptosis promoter. The heterodimerization with Bcl-2 removes the anti-apoptotic effect of Bcl-2. Upon the activation of Akt, Bad is phosphorylated (PMID8929531). The phosphorylated Bad dissociates from Bcl-2 to compete with the Bax protein for the rupture of the outer mitochondrial membrane (PMID11413467) and the subsequent apoptosis (PMID11048727, PMID8090205). Indeed, the ratio of Bad/Bcl-2 heterodimers is also a critical checkpoint for apoptosis (PMID9670005). When the level of phosphorylated Akt was suppressed by curcumin, the expression of Bax was up-regulated while the expression of Bcl-2 was down-regulated, leading to a higher ratio of Bad/Bcl-2 heterodimers. As a result, the anti-apoptotic functionality of Bcl-2 fades.