Genistein abrogates G2 arrest induced by curcumin in p53 deficient T47D cells

Abstract

Background: The high cost and low level of cancer survival urge the finding of new drugs having better mechanisms. There is a high trend of patients to be "back to nature" and use natural products as an alternative way to cure cancer. The fact is that some of available anticancer drugs are originated from plants, such as taxane, vincristine, vinblastine, pacitaxel. Curcumin (diferuloylmethane), a dietary pigment present in Curcuma longa rizhome is reported to induce cell cycle arrest in some cell lines. Other study reported that genistein isolated from Glycine max seed inhibited phosphorylation of cdk1, gene involved during G2/M transition and thus could function as G2 checkpoint abrogator. The inhibition of cdk1 phosphorylation is one of alternative strategy which could selectively kill cancer cells and potentially be combined with DNA damaging agent such as curcumin.

Methods: T47D cell line was treated with different concentrations of curcumin and genistein, alone or in combination; added together or with interval time. Flow Cytometry and MTT assay were used to evaluate cell cycle distribution and viability, respectively. The presence of apoptotic cells was determined using acridine orange-ethidium bromide staining.

Results: In this study curcumin induced G2 arrest on p53 deficient T47D cells at the concentration of 10 μM. Increasing concentration up to 30 μM increased the number of cell death. Whilst genistein alone at low concentration (≤10 μM) induced cell proliferation, addition of genistein (20 μM) 16 h after curcumin resulted in more cell death (89%), 34% higher than that administered at the same time (56%). The combination treatment resulted in apoptotic cell death. Combining curcumin with high dose of genistein (50 μM) induced necrotic cells.

Conclusions: Genistein increased the death of curcumin treated T47D cells. Appropriate timing of administration and concentration of genistein determine the outcome of treatment and this method could potentially be developed as an alternative strategy for treatment of p53 defective cancer cells.

Figure 1

Cell cycle analysis of T47D cell using flowcytometry after treatment with different concentration of curcumin. T47D cells were plated onto 6 well plate and treated overnight with (A) control untreated cells, (B) 10 μM curcumin, (C), 20 μM curcumin and (D) 30 μM curcumin. Arrow indicated the presence of sub G1 population.

Figure 2

Cell cycle analysis of T47D cell using flowcytometry after treatment with curcumin and genistein. T47D cells were plated onto 6 well plate and treated with (A) control untreated cells, (B) 10 μM curcumin, (C), 10 μM curcumin overnight followed by 20 μM genistein and (D) 10 μM curcumin overnight followed by 50 μM genistein. Arrow indicated the presence of subG1 population.

Figure 3

Morphology of cells treated with curcumin, genistein or in combination. T47D cells were plated onto 6 well plate and treated with (A) control cells, (B) 10 μM curcumine only, (C) curcumin (10 μM) was added overnight followed by treatment with genistein (20 μM), (D) curcumin (10 μM) was added overnight followed by treatment with genistein (50 μM).