Induction of apoptosis in human lung cancer cells by curcumin
Introduction
Cancer causes significant morbidity and mortality and is a major public health problem worldwide. An effective cancer prevention program, diet, and exercise may decrease the incidence of cancer. Plant-derived compounds are known to have curative potential. Curcumin (diferuloylmethane) is a phenolic compound from the plant Curcuma longa (Linn). It is widely used as a coloring and flavoring agent in food [1]. Its anti-inflammatory activity is well documented. Curcumin is not toxic to mammals at very high doses (5–10% by weight of diet) [2].
Curcumin is found to have inhibitory function towards a broad range of tumors such as mammary adenocarcinoma, fore stomach, duodenal and colon cancer as well as 12-O-tetradecanoyl-13-phorbol ester (TPA) induced skin tumors in mice [2], [3]. Curcumin is a potent anti-cancer agent and affects cells in a cell type dependent manner. Its ability to induce apoptosis in different cancer cells indicates the possibility of developing curcumin as a universal cancer prophylactic agent.
The protein p53 has an important role in pathogenesis of neoplasia [4]. The mechanism involved entails a rapid increase in p53 protein levels and the mediation of several cellular responses including G1 arrest, DNA damage repair and induction of apoptosis [5]. One of the major signaling pathways involved in apoptotic cell death includes the intracellular caspases, a family of structurally related cysteine proteases [6]. Caspase activity is responsible, either directly or indirectly, for the cleavage of cellular proteins, which are characteristically proteolyzed during apoptosis. For example, caspases -2, -3, -6, -7 and -9 can cleave poly (ADP ribose) polymerase (PARP) [7]. Bcl-2 family proteins are one of the already identified regulators of apoptosis. Bcl-2 family of homologous proteins represents a critical checkpoint within most apoptotic pathways, acting upstream of such irreversible damage to cellular constituents [8]. At least 15 bcl-2 family members have been identified so far in mammalian cells. They function either as proapoptotic (Bax, Bak, Bad) or anti apoptotic (Bcl-2, Bcl-XL) regulators. These proteins form heterodimers of anti and pro-apoptotic members, thereby titrating one another's function [8]. The ratio of anti-apoptotic and proapoptotic proteins determines in part how cells respond to apoptotic or survival signals [9]. Despite extensive analysis of anti-tumor activities of curcumin, its ability to modulate lung cancer growth has not yet been well characterized. We used lung cancer cell lines to study the effect of different concentrations of curcumin on cell viability and genes related to apoptosis. Our results demonstrated that curcumin causes growth arrest and apoptosis in lung cancer cell lines and the growth inhibitory effects of curcumin appeared to be mediated by the regulation of bcl-2, bcl-xL, bax, caspase-1, caspase-3, c-myc and p53 genes. Curcumin caused induction of apoptosis and the induction was independent of p53 status of the cell lines.
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Cell lines
The human lung adenocarcinoma cell line A549 (ATCC, Manassas, VA, USA) and the large cell lung carcinoma cell line H1299 (ATCC, Manassas, VA, USA) were used for this study. Cell line A549 is p53 wild type and H1299 is devoid of endogenous p53. The cells were grown in RPMI1640 culture medium supplemented with 2 mM l-glutamine and 10% FCS, penicillin (50 IU/ml) and streptomycin (50 (g/ml) at a temperature of 37 °C in a humidified incubator with a 5% CO2 atmosphere.
Curcumin treatment of cells
Curcumin (Sigma Chemical Co, St
Effect of curcumin on the growth of A549 and H1299 cells
A549 and H1299 lung cancer cell lines were used as a model system to examine the effect of curcumin on their growth. The growth inhibitory effect of curcumin was concentration dependent in both the cell lines (Fig. 1, Fig. 2). H1299 cells also showed time dependent effect of curcumin and were more sensitive to curcumin treatment than A549 cells. The IC50 s at 24 exposure of curcumin were 50 and 40 μM for A549 and H1299 cells, respectively.
Induction of apoptosis
We analyzed PARP cleavage in H1299 cells by flow
Discussion
Our results, for the first time, demonstrate that curcumin induces apoptosis in human lung cancer cells. Curcumin is a hydrophobic molecule and passes easily through the plasma membrane into the cytosol [14]. The results of this study demonstrate that this phenolic substance has the ability to reduce the viability of lung cancer cells through induction of apoptosis. Forty to fifty micromoles of curcumin decreased the cell viability to 50%, whereas 160 μM Curcumin reduced the viability by 95%.
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Both authors contributed equally to this work.