Benzene-poly-carboxylic acid complex, a novel anti-cancer agent induces apoptosis in human breast cancer cells.

Some cases of breast cancer are composed of clones of hormonal-independent growing cells, which do not respond to therapy. In the present study, the effect of Benzene-Poly-Carboxylic Acid Complex (BP-C1) on growth of human breast-cancer cells was tested. BP-C1 is a novel anti-cancer complex of benzene-poly-carboxylic acids with a very low concentration of cis-diammineplatinum (II) dichloride. Human breast cancer cells, MCF-7 and T47D, were used. Cell viability was detected by XTT assay and apoptosis was detected by Flow Cytometry and by annexin V/FITC/PI assay. Caspases were detected by western blot analysis and gene expression was measured by using the Applied Biosystems® TaqMan® Array Plates. The results showed that exposure of the cells to BP-C1 for 48 h, significantly (P<0.001) reduced cell viability, induced apoptosis and activated caspase 8 and caspace 9. Moreover, gene expression experiments indicated that BP-C1 increased the expression of pro-apoptotic genes (CASP8AP1, TNFRSF21, NFkB2, FADD, BCL10 and CASP8) and lowered the level of mRNA transcripts of inhibitory apoptotic genes (BCL2L11, BCL2L2 and XIAP. These findings may lead to the development of new therapeutic strategies for treatment of human cancer using BP-C1 analog

Effects of <i>BP-C1</i> on annexin V level on MCF-7 CELLS.

<p>Cells were treated with <i>BP-C1</i> (750 µg/ml) for 48 h and Flow cytometric analysis of annexin V-FITC/PI double-stained was performed. In each plot, the lower left quadrant (Q3) represents viable cells, the upper left quadrant (Q1) indicates necrotic cells, the lower right quadrant (Q4) denotes early apoptotic cells, and the upper right quadrant (Q2) represents necrotic or late apoptotic cells.</p

The effect of <i>BP-C1</i> on cell cycle progression.

<p>MCF-7 cells were treated with 750 µg/ml for 48 h. followed by treatment with RNase (100 µg/ml) and DNA staining with propidium iodide (PI). Cell cycle was analyzed using the FACSCalibur Cell Sorter.</p

Effects of <i>BP-C1</i> on annexin V level on T47D CELLS.

<p>Cells were treated with <i>BP-C1</i> (750 µg/ml) for 48 h and Flow cytometric analysis of annexin V-FITC/PI double-stained was performed. In each plot, the lower left quadrant (Q3) represents viable cells, the upper left quadrant (Q1) indicates necrotic cells, the lower right quadrant (Q4) denotes early apoptotic cells, and the upper right quadrant (Q2) represents necrotic or late apoptotic cells.</p

The effect of <i>BP-C1</i> on gene expression.

<p>MCF-7 cells were treated with 750 µg/ml for 48 h. After treatment, total RNA was isolated and reverse transcribed.Gene expression of pro-apoptotic genes was detected using the Applied Biosystems® TaqMan® Array Plates. HPRT1 and GAPDH genes were used as housekeeping genes for internal control to correct the potential variation in RNA loading.</p

Activation of caspase 8 (A) and caspase 9 (C) by <i>BP-C1</i>.

<p>Cells were treated with or without 750/ml of BP-C1 for 48 h. Cells were harvested and lysed. Activation of caspase-9 was determined by Western blotting as described under “Materials of Methods”. TA represents paclitaxel and 32c refers to Lx2-32c. Columns represent the densitometry analysis of the indicated proteins. The level of actin in the cells is represented in B and D.</p