Mechanisms for the activity of heterocyclic cyclohexanone curcumin derivatives in estrogen receptor negative human breast cancer cell lines.

Estrogen receptor (ER)-negative breast cancer is an aggressive form that currently requires more drug treatment options. Thus, we have further modified cyclohexanone derivatives of curcumin and examined them for cytotoxicity towards ER-negative human breast cancer cells. Two of the analogs screened elicited increased cytotoxic potency compared to curcumin and other previously studied derivatives. Specifically, 2,6-bis(pyridin-3-ylmethylene)-cyclohexanone (RL90) and 2,6-bis(pyridin-4-ylmethylene)-cyclohexanone (RL91) elicited EC(50) values of 1.54 and 1.10 µM, respectively, in MDA-MB-231 cells and EC(50) values of 0.51 and 0.23 in SKBr3 cells. All other new compounds examined were less potent than curcumin, which elicited EC(50) values of 7.6 and 2.4 µM in MDA-MB-231 and SKBr3 cells, respectively. Mechanistic analyses demonstrated that RL90 and RL91 significantly induced G(2)/M-phase cell cycle arrest and apoptosis. RL90 and RL91 also modulated the expression of key cell signaling proteins, specifically, in SKBr3 cells, protein levels of Her-2, Akt, and NFκB were decreased in a time-dependent manner, while activity of stress kinases JNK1/2 and P38 MAPK were increased. Signaling events in MDA-MB-231 cells were differently implicated, as EGFR protein levels were decreased and activity of GSK-3ß transiently decreased, while ß-catenin protein level and activity of P38 MAPK, Akt, and JNK1/2 were transiently increased. In conclusion replacement of the phenyl group of cyclohexanone derived curcumin derivatives with heterocyclic rings forms a class of second-generation analogs that are more potent than both curcumin and other derivatives. These new derivatives provide a platform for the further development of drugs for the treatment of ER-negative breast cancer.

Synthesis and cytotoxic potential of heterocyclic cyclohexanone analogues of curcumin.

A series of 18 heterocyclic cyclohexanone analogues of curcumin have been synthesised and screened for their activity in both adherent and non-adherent cancer cell models. Cytotoxicity towards MBA-MB-231 breast cancer cells, as well as ability to inhibit NF-kappaB transactivation in non-adherent K562 leukemia cells were investigated. Three of these analogues 3,5-bis(pyridine-4-yl)-1-methylpiperidin-4-one B1, 3,5-bis(3,4,5-trimethoxybenzylidene)-1-methylpiperidin-4-one B10, and 8-methyl-2,4-bis((pyridine-4-yl)methylene)-8-aza-bicyclo[3.2.1]octan-3-one C1 showed potent cytotoxicity towards MBA-MB-231, MDA-MB-468, and SkBr3 cell lines with EC50 values below 1 microM and inhibition of NF-kappaB activation below 7.5 microM. The lead drug candidate, B10, was also able to cause 43% of MDA-MB-231 cells to undergo apoptosis after 18 h. This level of activity warrants further investigation for the treatment of ER-negative breast cancer and/or chronic myelogenous leukemia as prototypical cellular models for solid and liquid tumors.

Coenzyme Q0 induces apoptosis and modulates the cell cycle in estrogen receptor negative breast cancer cells.

We postulated that methoxy-substituted cyclic compounds could inhibit estrogen receptor (ER) negative breast cancer growth in vitro. Therefore, this study assessed the cytotoxic potential of various methoxy-substituted cyclic compounds [7,8-dimethoxyflavone, 4-methoxyphenylacetic acid, 2-methoxyphenylacetic acid, 4-methoxybenzophenone, 5-methoxy-1-indanone, and coenzyme Q0 (CoQ0)] toward ER-negative human breast cancer cells (MDA-MB-231 and SKBr3). Cytotoxicity was assessed using the sulforhodamine B assay. CoQ0 demonstrated the strongest cytotoxicity toward MDA-MB-231 and SKBr3 cells with IC50 values of 1.7 micromol/l and 3.1 micromol/l, respectively, whereas the other compounds were either much less potent or completely lacked cytotoxicity toward both breast cancer cell lines. Therefore, only CoQ0 was examined for its ability to modulate cell cycle progression and induce apoptosis. Cell cycle experiments, using propidium iodide staining and flow cytometry, demonstrated that CoQ0 at 7.5 micromol/l increased the proportion of MDA-MB-231 cells in G1/G0-phase by 16.6+/-0.6% of control (P<0.05), and increased in the proportion of S-phase SKBr3 cells by 37.8+/-5.8% over control (P<0.05). Induction of apoptosis was determined using propidium iodide/Annexin-V-FLUOS staining followed by flow cytometry. The results demonstrated that treatment with CoQ0 (7.5 micromol/l) increased the proportion of apoptotic MDA-MB-231 and SKBr3 cells by 12-fold and 4-fold over control (P<0.05), respectively. Thus, CoQ0 is a potent cytotoxic drug that induces apoptosis and modulates cell cycle progression in ER-negative breast cancer cells. Therefore, CoQ0 is an appropriate candidate for further study and development as a potential drug for ER-negative breast cancer.

The combination of epigallocatechin gallate and curcumin suppresses ER alpha-breast cancer cell growth in vitro and in vivo.

Both epigallocatechin gallate (EGCG) and curcumin have shown efficacy in various in vivo and in vitro models of cancer. This study was designed to determine the efficacy of these naturally derived polyphenolic compounds in vitro and in vivo, when given in combination. Studies in MDA-MB-231 cells demonstrated that EGCG + curcumin was synergistically cytotoxic and that this correlated with G(2)/M-phase cell cycle arrest. After 12 hr, EGCG (25 microM) + curcumin (3 microM) increased the proportion of cells in G(2)/M-phase to 263 +/- 16% of control and this correlated with a 50 +/- 4% decrease in cell number compared to control. To determine if this in vitro result would translate in vivo, athymic nude female mice were implanted with MDA-MB-231 cells and treated with curcumin (200 mg/kg/day, po), EGCG (25 mg/kg/day, ip), EGCG + curcumin, or vehicle control (5 ml/kg/day, po) for 10 weeks. Tumor volume in the EGCG + curcumin treated mice decreased 49% compared to vehicle control mice (p < 0.05), which correlated with a 78 +/- 6% decrease in levels of VEGFR-1 protein expression in the tumors. Curcumin treatment significantly decreased tumor protein levels of EGFR and Akt, however the expression of these proteins was not further decreased following combination treatment. Therefore, these results demonstrate that the combination of EGCG and curcumin is efficacious in both in vitro and in vivo models of ER alpha-breast cancer and that regulation of VEGFR-1 may play a key role in this effect.

A novel plant toxin, persin, with in vivo activity in the mammary gland, induces Bim-dependent apoptosis in human breast cancer cells.

Phytochemicals have provided an abundant and effective source of therapeutics for the treatment of cancer. Here we describe the characterization of a novel plant toxin, persin, with in vivo activity in the mammary gland and a p53-, estrogen receptor-, and Bcl-2-independent mode of action. Persin was previously identified from avocado leaves as the toxic principle responsible for mammary gland-specific necrosis and apoptosis in lactating livestock. Here we used a lactating mouse model to confirm that persin has a similar cytotoxicity for the lactating mammary epithelium. Further in vitro studies in a panel of human breast cancer cell lines show that persin selectively induces a G2-M cell cycle arrest and caspase-dependent apoptosis in sensitive cells. The latter is dependent on expression of the BH3-only protein Bim. Bim is a sensor of cytoskeletal integrity, and there is evidence that persin acts as a microtubule-stabilizing agent. Due to the unique structure of the compound, persin could represent a novel class of microtubule-targeting agent with potential specificity for breast cancers.