RL66 a second-generation curcumin analog has potent in vivo and in vitro anticancer activity in ER-negative breast cancer models

Introduction

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 (ER)-negative breast cancer (1,2). In the search for effective drugs for ER-negative breast cancer, several lead compounds from natural products have emerged including curcumin (diferuloylmethane), the primary bioactive compound isolated from the rhizome of turmeric (Curcuma longa Linn.). Curcumin has numerous pharmacological, chemopreventative and chemotherapeutic actions, and in vitro studies have also demonstrated that curcumin exhibits potent cytoxicity toward numerous cell lines including ER-negative human breast cancer cells (3–9). Furthermore, in vivo studies have demonstrated decreased tumorigenesis of many organs, including the mammary gland (10–15). However, curcumin has shown limited clinical efficacy, due to its low bioavailability and low stability (11). Therefore, numerous groups have concentrated on improving drug efficacy, bioavailability and stability by synthesizing analogs of curcumin. Specifically, cyclohexanone analogs of curcumin have shown enhanced activity and stability compared to curcumin (16). Specifically, the cyclohexanone-containing curcumin derivative 2,6-bis((3-methoxy-4-hydroxyphenyl)methylene)-cyclohexanone (BMHPC) was cytotoxic towards ER-negative breast cancer cells (IC₅₀ of 5.0 μM) (17), although bioavailability and in vivo efficacy were still problematic. More recently the fluorinated cyclohexanone derivative EF24 has shown potent cytotoxicity toward MDA-MB-231 cells with an IC₅₀ value of 0.8 μM (18,19).

Our laboratory has been involved in the search for new drug treatments for ER-negative breast cancer and we have shown that 2nd generation heterocyclic cyclohexanone curcumin analogs exhibit potent cytotoxicity toward ER-negative breast cancer cells. This work demonstrated that 1-methyl-3,5-bis[(E)-4-pyri-dyl)methylidene]-4-piperidone (RL66) (Fig. 1) exhibited IC₅₀ values of 0.8, 0.5 and 0.6 μM for MDA-MB-231, MDA-MB-468 and SKBr3 breast cancer cells, respectively (20). It also induced apoptosis, as ∼18% of MDA-MB-231 cells underwent apoptosis after 12 h of RL66 treatment (2 μM) (20). Only one other compound synthesized (RL71) showed a more potent effect in vitro (21), but this compound did not suppress tumor growth in vivo. Therefore, this study was designed to comprehensively investigate the potency and mechanisms of action of RL66 in vitro and in vivo in order to determine its potential to be developed into a drug for ER-negative breast cancer.

Materials and methods

Materials

HUVEC, MDA-MB-231, MDA-MB-468 and SKBr3 cells were purchased from American...