Demethoxycurcumin induces Bcl-2 mediated G2/M arrest and apoptosis in human glioma U87 cells
Introduction
Curcumin inhibits growth of tumor cells and induces apoptosis in vitro[1], [2] and in vivo[3], [4] in a variety of tumor cells, and acts as anti-proliferative agent, interrupts the cell cycle (most often in G2/M phase) and induces cell death by inhibition of signal cascades involving cell survival [5], [6], [7]. The curcumin-induced apoptotic cell death and cell arrest in the G2/M phase has also been linked to down-regulation of Bcl-2 [8].
Bcl-2 proteins play critical roles in regulating programmed cell death (i.e., apoptosis) and share at least one of the four homologous regions known as Bcl homology (BH) domains (BH1–BH4). Some members of the Bcl-2 family, such as Bcl-2, Bcl-xL, Bcl-w and Mcl-1, block apoptosis, whereas others such as Bax, Bak, Bad and Bid promote apoptosis [9]. Bcl-2 upregulation inhibited the cell killing by blocking the apoptotic pathway [10]. Antagonizing the function of Bcl-2, would be a useful strategy for restoring normal apoptotic processes in cancer cells.
Curcumin, isolated from turmeric (Curcuma longa) contains curcumin I as major component but also contains curcumin II and curcumin III [11], which are identified as curcumin (C1), demethoxycurcumin (C2) and bisdemethoxycurcumin (C3), respectively [12]. In the present paper, docking of curcuminoids C1, C2 and C3 was carried out to envisage their binding to the modeled 3D structure of Bcl-2 protein. The effect of purified curcuminoids (C1, C2, and C3) was studied on human glioma U87 cell line and alteration in cellular and nuclear morphology, cytotoxicity, DNA fragmentation, cell cycle and Bcl-2 protein was studied to elucidate the molecular mechanism of their anticancer activity.
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Materials
MTT (Sigma–Aldrich), DMEM (Himedia, India), Fetal bovine serum (Hyclone), Antibiotic and antimycotic solution (Himedia, India), Hoechst 33342 (Sigma–Aldrich), PI (Invitrogen, USA), Phase lock gel heavy 2.0 ml tubes (Hysel India Pvt. Ltd.), anti-human Bcl-2 monoclonal antibody (Cell signaling Technology, USA), ECL Kit (Pierce, USA), Bcl-2 Protein–Maltose Binding Protein (MBP) fusion protein, C-terminal truncated (Sigma–Aldrich), Bak-BH3 domain GQVGRQLAIIGDDINR (R&D systems) were procured from
Docking analysis and validation of binding site of Bcl-2
The stereochemical quality of the modeled structure of Bcl-2 protein exhibited that around 91.1% of the residues were plotted in the most favored regions (A, B, L) of the Ramachandran plot and none of the residues were found to be in disallowed regions. The coordinates of the optimized human Bcl-2 protein 3D structure (PMDB ID: PM0075561) could be accessed from the Protein Model database (http://mi.caspur.it/PMDB). Seven cavities were obtained as apparent binding sites on Bcl-2. The multiple
Discussion
Computational methods and diversity of existing compound databases for identification of novel protein binding molecules has become a powerful tool for the discovery of organic ligands [22], [23]. In this approach 3D structure of human Bcl-2 protein was predicted by homology modeling, curcumin types (six compounds) were docked with 3D structure of Bcl-2 protein. The three compounds C1, C2, and C3 showed strongest in silico binding (Ki) as compared to other inhibitors (Table 1). Out of the three
Acknowledgments
The authors wish to thank Dr. B.S. Dwarakanath, Head, Department of Biocybernetics, Institute of Medicine and Allied Sciences (DRDO), New Delhi, India, for providing human glioma U87 cells and flow cytometery facility. Rakesh Kumar and Amresh Prakash are thankful to University Grant Commission and Department of Science and Technology, New Delhi, India, respectively, for financial support.
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