Study of New Curcumin Analogs for the Treatment of Erα Negative Breast Cancers

Abstract

Breast cancer is the most prevalent form of cancer diagnosed in women, and there continues to be limited drug treatment options for the ~30% of patients with estrogen receptor (ERα)-negative breast cancers. In the search for effective drugs for ERα negative breast cancer, several lead compounds from natural products such as curcumin (diferuloylmethane), the primary bioactive compound isolated from the rhizome of turmeric (Curcuma longa Linn.), have emerged. Though curcumin showed promising anticancer activity in various in vitro and in vivo models of breast cancer, its clinical application was limited due to its low bioavailability and low stability in physiological media. Therefore, research groups have concentrated on the synthesis and characterization of curcumin analogs. Our lab previous developed cyclohexanone curcumin analogs and showed enhanced anticancer activity towards ERα negative breast cancer cells compared to curcumin. To search for more potent compounds, this study was designed to develop second generation heterocyclic cyclohexanone curcumin analogs and also examine their anticancer activity in various in vitro and in vivo models of ERα-negative breast cancer. This work demonstrated that among 20 heterocyclic curcumin analogs screened, 3,5-bis(3,4,5- trimethoxybenzylidene)-1-methylpiperidine-4-one (RL71) and 1-methyl-3,5-bis[(E)-(4-pyridyl) methylidene]-4-piperidone (RL66) showed the most potent anticancer activity with IC50 values in submicromolar range (<1μM) in MDA-MB-231, MDA-MB-468 and SKBr3 breast cancer cells. Further in vitro mechanism of action studies of RL71 and RL66 demonstrated a cell cycle arrest in G2/M phase or S/G2/M phase and induction of apoptosis in all the three ERα negative breast cancer cells in a concentration-, cell line- and time-dependent manner. Moreover, the effect on various cell signaling proteins involved in cell proliferation and cell death was also examined by Western blotting. Both compounds showed a similar type of result. RL71 and RL66 significantly increased the phosphorylation of stress activated protein kinases, p38 and JNK1/2 in a concentration and time-dependent manner. Moreover, both the compounds modulated the PI3K/Akt/mTOR pathway and showed activation of p27kip1 and cleaved caspase-3 in a time- and cell-line dependent manner. In HER2 overexpressing SKBr3 cells, RL71 and RL66 significantly down regulated phosphorylation of HER2.

The study also involved assessment of anti-angiogenic activity of both the compounds in vitro. The results showed that at 1 μM, RL71 and RL66 inhibited HUVEC cell invasion by 46% and 48% respectively compared to control, along with the ability of these cells to form tube like networks and thus showed anti-angiogenic potential in vitro. In addition, RL71 (1 μM) and RL66 (2 μM) inhibited migration of MDA-MB-231 cells in vitro. Both the drugs were further tested for their bioavailability in mice. The results showed that RL71 and RL66 were bioavailable after an oral dose of 8.5 mg/kg dose. The next study was designed to study the anticancer potential of both the compounds in vivo. For this, female athymic nude mice were subcutaneously inoculated with MDA-MB-468 (8 x 106) breast cancer cells and were treated with either vehicle (water) or RL71 or RL66 at 0.85 mg/kg or 8.5 mg/kg dose for 10 weeks. The results showed that RL71 did not significantly reduce the tumor volume compared to vehicle treated mice. However, RL66, at a dose of 8.5 mg/kg significantly reduced the tumor volume by 48% compared to the vehicle group. Both the compounds did not produce any toxicity after 10 weeks treatment as the total body weight remained unchanged. Moreover, RL66 treatment showed no significant change in major organ weight and gave normal plasma ALT values. The mechanism of tumor reduction by RL66 was further studied by examining its effect on various cell signaling proteins by Western blotting. The results showed that the tumors treated with 8.5 mg/kg of RL66 had a marginally reduced expression of EGFR whereas the expression of NF-κB and the phosphorylation of Akt were considerably increased compared to vehicle treatment. Moreover, the expression of p27kip1 was up regulated and the phosphorylation of mTOR was down regulated compared to vehicle treatment. However, none of the effects were significantly different compared to control. Further mechanistic studies by immunohistochemistry showed a significant reduction in the microvessel density of tumors as seen by a 59% reduction in CD105 protein in tumors from mice treated with 8.5 mg/kg of RL66 compared to vehicle treatment. In conclusion we have shown that RL71 and RL66 have potent anticancer and anti-angiogenic properties in vitro. However, RL71 needs further modification to enhance its anticancer effect in vivo, whereas RL66 has potential for development into novel treatments for ERα negative breast cancer.