According to Roverti et al. (2003
), 11b is more cytotoxic than resveratrol for HL60 premyelocytic leukemia cells. The anti-proliferative activity (IC50
) and apoptotic-inducing activity (AC50
) of resveratrol towards HL60 cells was 5 ± 0.9 µM and 50 ± 8.2 µM, respectively. In contrast, the IC50
values of 11b were at least 10-fold lower, 0.3 ± 0.001 µM for IC50
and 0.04 ± 0.0045 µM for the AC50
value (Roverti et al., 2003
). Here, using cell lines derived from two other types of cancers, we found that resveratrol also had significantly less cytotoxic activity than 11b.
This study is the first to report that 11b is superior to resveratrol for killing and/or inhibiting the growth of breast and pancreatic cancer cell lines. These effects of 11b are likely to be related to the ability of 11b to disrupt microtubule polymerization in vitro. Simulations of the molecular docking between α,β-tubulin and either colchicine or 11b suggest that the dimethoxyphenyl group of 11b could occupy almost exactly the same space as the trimethoxyphenyl group of colchicine. In addition, since the hydroxyl group of 11b could potentially form an H-bond with Val-α181 of tubulin (), our simulations suggest a mechanistic explanation for the greater potency of 11b (i.e., resveratrol does not form H-bonds in our binding pocket simulations). In our simulation 11b is buried in the hydrophobic cavity created by three tubulin residues, Leuβ238, Leuβ242, and Valβ318 (). Ensembles of 11b revealed H-bond interaction with Val-α181 due to the induced fit effect upon ligand binding, which suggests that ligand-induced conformational changes can be critical to the protein active site side chains during ligand binding. Thus, the dimethoxyphenyl moiety buried deep into the hydrophobic pocket can make interactions with β-tubulin similar to the interactions identified for the trimethoxyphenyl moiety of DMA-colchicine.
Our results also suggest that the molecular level effects of 11b described here, binding to tubulin, inducing changes in the tubulin structure and increasing the expression and/or phosphorylation of mitosis checkpoint protein such as BubR1, p-HH3, Aurora B, and Cyclin B, may at least partially explain the biological effects of 11b on breast and pancreatic cancer cells. That is, it has been previously shown that defects in microtubule polymerization prevent microtubules from attaching to centromeres during metaphase (Weaver and Cleveland, 2005
) and that the absence of normal centromere-microtubule interactions activates mitotic checkpoint proteins, resulting in a block of cell cycle progression into anaphase and failure to form anaphase-promoting complex (Weaver and Cleveland, 2005
). Although our data does not allow us to decide which of the biochemical effects of 11b described here, blocking microtubule polymerization or inducing/activating cell cycle checkpoint proteins, is the primary effect, numerous studies suggest that blocking microtubule polymerization activates cell cycle checkpoints (reviewed in Cuschieri et al., 2007
The results of our study are also consistent published studies suggesting that apoptosis pathways can be activated by treatments with agents that block microtubule polymerization (Ikegami et al., 1997
; Gorman et al., 1999
). Our studies found that treatments with low concentrations of 11b caused 98% (MDA-MB-231) and 88% (PANC-1) of live cells (excluding sub-G0/G1 fraction) to accumulate at the G2/M transition. Long-term arrest of cells in the M phase is known to drive cells to apoptotic cell death mediated by Bax, PARP, and caspase-3 (Tao et al., 2005
). In this study, we found that 11b treatments caused 57% (MDA-MB-231) and 86% (PANC-1) of total cells to accumulate at a position of hypoploid fraction, indicative of apoptosis, in a standard FACS analysis. However, our study does not provide direct evidence that the increased cells in sub-G0/G1 fractions are apoptotic cells.
In these studies, we showed that 11b has greater activity than resveratrol for blocking microtubule polymerization in vitro, for blocking in vivo cell proliferation and for promoting cell death. Further study of the effects of and mechanism(s) involved in 11b-caused cytotoxicity, especially in appropriate animal models are warranted to determine if this resveratrol derivative may have potential in humans, either as a chemopreventive agent for pancreatic cancer or as an anti-tumor agent for breast cancer.