Int J Oncol. 2012 Nov;41(5):1723-32. doi: 10.3892/ijo.2012.1625.

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

Yadav B1, Taurin S, Larsen L, Rosengren RJ.

1Department of Pharmacology and Toxicology, University of Otago, Dunedin, New Zealand.



There is a need for the development of new safe and efficacious drug therapies for the treatment of estrogen receptor (ER) negative breast cancers. 1-methyl-3,5-bis[(E)-4-pyridyl)methylidene]-4-piperidone (RL66) is a second generation curcumin analog that exhibited potent cytotoxicity towards a variety of ER-negative breast cancer cells. Therefore, we have further examined the mechanism of this novel drug in in vitro and in vivo models of ER-negative breast cancer. The mechanistic studies demonstrated that RL66 (2 µM) induced cell cycle arrest in the G2/M phase of the cell cycle. Moreover, RL66 (2 µM) caused 30% of SKBr3 cells to undergo apoptosis after 48 h and this effect was time-dependent. This correlated with an increase in cleaved caspase-3 as shown by Western blotting. RL66 (2 µM) also decreased HER2/neu phosphorylation and increased p27 in SKBr3 cells. While in MDA-MB-231 and MDA-MB-468 cells RL66 (2 µM) significantly decreased Akt phosphorylation and transiently increased the stress kinases JNK1/2 and MAPK p38. In addition, RL66 exhibited anti-angiogenic potential in vitro as it inhibited HUVEC cell migration 46% and the ability of these cells to form tube like networks. RL66 (8.5 mg/kg) suppressed the growth of MDA-MB-468 xenograft tumors by 48% compared to vehicle control following 10 weeks of daily oral administration. Microvessel density in the tumors from treated mice was also decreased 57% compared to control. Thus our findings demonstrate that RL66 has potent proapoptotic and anti-angiogenic properties in vivo and in vitro and has the potential to be further developed as a drug for the treatment of ER negative breast cancer.

PMID: 22971638



Our laboratory has been involved in the search for new drug treatments for estrogen receptor (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 study was designed to comprehensively investigate the potency and mechanisms of action of 1-methyl-3,5-bis[(E)-4-pyridyl)methylidene]-4-piperidone (RL66) in vitro and in vivo in order to determine its potential to be developed into a drug for ER-negative breast cancer.

In vitro assays measuring cell migration (i.e., Boyden chamber assay and scratch assay) showed that RL66 significantly decreased the migration of both endothelial (HUVEC) and MDA-MB-231 breast cancer cells in culture (Figures 1 and 2). These in vitro indicators of a drug’s potential as an inhibitor of angiogenesis were also seen in vivo where RL66 at both 0.85 and 8.5 mg/kg decreased tumor microvessle density as shown by the number of CD105 positive cells in tumor sections from treated mice (Figure 9 in original article). Further evaluation of drug-mediated changes in the tumor following RL66 was conducted via Western blotting of tumor extracts. mTOR was decreased 41% following treatment of RL66 (8.5 mg/kg), while p27kipI was increased 145% (Figure 3). Similar effects were seen in MDA-MB-231 cells in culture where RL66 (2 µM) decreased mTOR by 49% after 24 h (Figure 6 in original article). p27/kipI was also increased 205% after 12 h incubation with RL66 (3 µM) in MDA-MB-468 and 248% in SKBr3 cells following 2 µM (Figures 5 and 7 in original article). Thus it appears that mTOR and p27/kipI both play an important mechanistic role in the ability of RL66 to suppress tumor growth and decrease microvessel density in tumors. Furthermore, since similar findings were produced in the cell culture studies as well as in tumors from treated mice, the in vitro assays were useful in predicting both the potency and mechanism of RL66 in vivo.

Rhonda Rosengren-fig1In the manuscript, we showed that RL66 causes cell cycle arrest, induces apoptosis as well as modulates a variety of signaling pathways that all culminate in potent cytotoxicity toward 3 different ER-negative breast cancer cells. In SkBr3 cells inhibi­tion of HER2/neu and induction of p27 are key mechanisms for this potent cytotoxicity. Importantly, RL66 suppressed tumor growth in an in vivo model of TNBC and this correlated with a decrease in tumor microvessel density as well as anti-angiogenic effects in vitro.

Rhonda Rosengren-fig2

While other 2nd generation heterocyclic cyclohexanone curcumin derivatives have shown potent in vitro actions, RL66 is the first drug in this class to elicit tumor suppression in a model of triple negative breast cancer in vivo. RL66 will continue to be examined for its ability to suppress metastasis in aggressive breast cancer in vivo. Future studies will also investigate the ability of this drug to be delivered using nanotechnology in order to increase efficacy and prolong drug half-life.



The study was supported by a grant from the Breast Cancer Cure Research Trust.

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