Apigenin shows synergistic anticancer activity with curcumin by binding at different sites of tubulin.

Apigenin, a natural flavone, present in many plants sources, induced apoptosis and cell death in lung epithelium cancer (A549) cells with an IC50 value of 93.7 ± 3.7 μM for 48 h treatment. Target identification investigations using A549 cells and also in cell-free system demonstrated that apigenin depolymerized microtubules and inhibited reassembly of cold depolymerized microtubules of A549 cells. Again apigenin inhibited polymerization of purified tubulin with an IC50 value of 79.8 ± 2.4 μM. It bounds to tubulin in cell-free system and quenched the intrinsic fluorescence of tubulin in a concentration- and time-dependent manner. The interaction was temperature-dependent and kinetics of binding was biphasic in nature with binding rate constants of 11.5 × 10(-7) M(-1) s(-1) and 4.0 × 10(-9) M(-1) s(-1) for fast and slow phases at 37 °C, respectively. The stoichiometry of tubulin-apigenin binding was 1:1 and binding the binding constant (Kd) was 6.08 ± 0.096 μM. Interestingly, apigenin showed synergistic anti-cancer effect with another natural anti-tubulin agent curcumin. Apigenin and curcumin synergistically induced cell death and apoptosis and also blocked cell cycle progression at G2/M phase of A549 cells. The synergistic activity of apigenin and curcumin was also apparent from their strong depolymerizing effects on interphase microtubules and inhibitory effect of reassembly of cold depolymerized microtubules when used in combinations, indicating that these ligands bind to tubulin at different sites. In silico modeling suggested apigenin bounds at the interphase of α-β-subunit of tubulin. The binding site is 19 Å in distance from the previously predicted curcumin binding site. Binding studies with purified protein also showed both apigenin and curcumin can simultaneously bind to purified tubulin. Understanding the mechanism of synergistic effect of apigenin and curcumin could be helped to develop anti-cancer combination drugs from cheap and readily available nutraceuticals.

[1]  George Huang,et al.  Antiproliferative activities of parthenolide and golden feverfew extract against three human cancer cell lines. , 2006, Journal of medicinal food.

[2]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[3]  Gopal Chakrabarti,et al.  Aqueous extract of ginger shows antiproliferative activity through disruption of microtubule network of cancer cells. , 2010, Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association.

[4]  Seema Singh.  From Exotic Spice to Modern Drug? , 2007, Cell.

[5]  S B Kaye,et al.  On the receiving end--patient perception of the side-effects of cancer chemotherapy. , 1983, European journal of cancer & clinical oncology.

[6]  G. Kuttan,et al.  Anti-tumour and antioxidant activity of natural curcuminoids. , 1995, Cancer letters.

[7]  Y. Liu,et al.  Curcumin upregulates transcription factor Nrf2, HO-1 expression and protects rat brains against focal ischemia , 2009, Brain Research.

[8]  K. Rathinasamy,et al.  Dietary antioxidant curcumin inhibits microtubule assembly through tubulin binding , 2006, The FEBS journal.

[9]  M. Kawai,et al.  Flavonoids and related compounds as anti-allergic substances. , 2007, Allergology international : official journal of the Japanese Society of Allergology.

[10]  P. Curmi,et al.  Benomyl and colchicine synergistically inhibit cell proliferation and mitosis: evidence of distinct binding sites for these agents in tubulin. , 2008, Biochemistry.

[11]  M. Mouret-Reynier,et al.  Phase I dose escalation trial of docetaxel plus curcumin in patients with advanced and metastatic breast cancer , 2010, Cancer biology & therapy.

[12]  Bipul R. Acharya,et al.  The microtubule depolymerizing agent naphthazarin induces both apoptosis and autophagy in A549 lung cancer cells , 2011, Apoptosis.

[13]  R. Agarwal,et al.  Silibinin strongly synergizes human prostate carcinoma DU145 cells to doxorubicin-induced growth Inhibition, G2-M arrest, and apoptosis. , 2002, Clinical Cancer Research.

[14]  Gopal Chakrabarti,et al.  Thermodynamics of Colchicinoid-Tubulin Interactions , 1996, The Journal of Biological Chemistry.

[15]  Paul Talalay,et al.  Analysis of combined drug effects: a new look at a very old problem , 1983 .

[16]  A. Doseff,et al.  Apigenin-induced-apoptosis is mediated by the activation of PKCdelta and caspases in leukemia cells. , 2006, Biochemical pharmacology.

[17]  Bipul R. Acharya,et al.  Vitamin K3 disrupts the microtubule networks by binding to tubulin: a novel mechanism of its antiproliferative activity. , 2009, Biochemistry.

[18]  P. Chakrabarti,et al.  Curcumin recognizes a unique binding site of tubulin. , 2011, Journal of medicinal chemistry.

[19]  Gopal Chakrabarti,et al.  1,4-Benzoquinone (PBQ) induced toxicity in lung epithelial cells is mediated by the disruption of the microtubule network and activation of caspase-3. , 2010, Chemical research in toxicology.

[20]  T. Chou,et al.  Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. , 1984, Advances in enzyme regulation.

[21]  S. Gupta,et al.  Selective growth-inhibitory, cell-cycle deregulatory and apoptotic response of apigenin in normal versus human prostate carcinoma cells. , 2001, Biochemical and biophysical research communications.

[22]  S. Sahoo,et al.  Coformulation of doxorubicin and curcumin in poly(D,L-lactide-co-glycolide) nanoparticles suppresses the development of multidrug resistance in K562 cells. , 2011, Molecular pharmaceutics.

[23]  A. Gescher,et al.  Dietary polyphenolic phytochemicals—promising cancer chemopreventive agents in humans? A review of their clinical properties , 2007, International journal of cancer.

[24]  E. Hamel,et al.  Glutamate-induced polymerization of tubulin: characteristics of the reaction and application to the large-scale purification of tubulin. , 1981, Archives of biochemistry and biophysics.

[25]  C. S. Yang,et al.  Inhibition of carcinogenesis by dietary polyphenolic compounds. , 2001, Annual review of nutrition.

[26]  J. Schwartz.  Dexamethasone premedication for prophylaxis of taxane toxicities: Can the doses be reduced when paclitaxel or docetaxel are given weekly? , 2012, Journal of oncology pharmacy practice : official publication of the International Society of Oncology Pharmacy Practitioners.

[27]  B. Bhattacharyya,et al.  -NH-dansyl isocolchicine exhibits a significantly improved tubulin-binding affinity and microtubule inhibition in comparison to isocolchicine by binding tubulin through its A and B rings. , 2005, Biochemistry.

[28]  H. Mukhtar,et al.  Involvement of nuclear factor-kappa B, Bax and Bcl-2 in induction of cell cycle arrest and apoptosis by apigenin in human prostate carcinoma cells , 2002, Oncogene.

[29]  A. Mobasheri,et al.  Curcumin modulates nuclear factor kappaB (NF-kappaB)-mediated inflammation in human tenocytes in vitro: role of the phosphatidylinositol 3-kinase/Akt pathway. , 2011, The Journal of biological chemistry.

[30]  P. V. Balaji,et al.  Griseofulvin stabilizes microtubule dynamics, activates p53 and inhibits the proliferation of MCF-7 cells synergistically with vinblastine , 2010, BMC Cancer.

[31]  T. Tseng,et al.  Apigenin inhibits HGF-promoted invasive growth and metastasis involving blocking PI3K/Akt pathway and beta 4 integrin function in MDA-MB-231 breast cancer cells. , 2008, Toxicology and applied pharmacology.

[32]  M. Klein,et al.  Flavonoid Biosynthesis in Barley Primary Leaves Requires the Presence of the Vacuole and Controls the Activity of Vacuolar Flavonoid Transport1 , 2007, Plant Physiology.

[33]  David S. Goodsell,et al.  Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function , 1998 .

[34]  Xianglin Shi,et al.  Benzo[a]pyrene-3,6-dione inhibited VEGF expression through inducing HIF-1alpha degradation. , 2007, Biochemical and biophysical research communications.

[35]  D. Panda,et al.  Curcumin suppresses the dynamic instability of microtubules, activates the mitotic checkpoint and induces apoptosis in MCF‐7 cells , 2010, The FEBS journal.

[36]  Jen-kun Lin,et al.  Stability of curcumin in buffer solutions and characterization of its degradation products. , 1997, Journal of pharmaceutical and biomedical analysis.

[37]  S. Hastie,et al.  Effect of the B ring and the C-7 substituent on the kinetics of colchicinoid-tubulin associations. , 1993, Biochemistry.

[38]  Kenneth H. Downing,et al.  Structure of the αβ tubulin dimer by electron crystallography , 1998, Nature.

[39]  Razelle Kurzrock,et al.  Phase II Trial of Curcumin in Patients with Advanced Pancreatic Cancer , 2008, Clinical Cancer Research.

[40]  D. Banji,et al.  Synergistic activity of curcumin with methotrexate in ameliorating Freund's Complete Adjuvant induced arthritis with reduced hepatotoxicity in experimental animals. , 2011, European journal of pharmacology.

[41]  Ricky A. Sharma,et al.  Curcumin: the story so far. , 2005, European journal of cancer.