¹Department of Molecular Genetics, College of Medicine, University of Illinois at Chicago, Chicago, Illinois 60607, USA

²Department of Pharmaceutics and Pharmacodynamics, Center for Pharmaceutical Biotechnology, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois 60607, USA

³Department of Surgery, Cook County Hospital, Chicago, Illinois 60612, USA

⁴Department of Microbiology and Immunology, Baylor College of Medicine, Houston, Texas 77030, USA

^aAuthor for correspondence

^bAA Shtil and S Mandlekar contributed equally to this work

Drug design targeted at microtubules has led to the advent of some potent anti-cancer drugs. In the present study, we demonstrated that microtubule-binding agents (MBAs) taxol and colchicine induced immediate early gene (c-jun and ATF3) expression, cell cycle arrest, and apoptosis in the human breast cancer cell line MCF-7. To elucidate the signal transduction pathways that mediate such biological activities of MBAs, we studied the involvement of mitogen-activated protein (MAP) kinases. Treatment with taxol, colchicine, or other MBAs (vincristine, podophyllotoxin, nocodazole) stimulated the activity of c-jun N-terminal kinase 1 (JNK1) in MCF-7 cells. In contrast, p38 was activated only by taxol and none of the MBAs changed the activity of extracellular signal-regulated protein kinase 2 (ERK2). Activation of JNK1 or p38 by MBAs occurred subsequent to the morphological changes in the microtubule cytoskeleton induced by these compounds. Furthermore, baccatine III and -lumicolchicine, inactive analogs of taxol and colchicine, respectively, did not activate JNK1 or p38. These results suggest that interactions between microtubules and MBAs are essential for the activation of these kinases. Pretreatment with the antioxidants N-acetyl-L-cysteine (NAC), ascorbic acid or vitamin E, blocked H₂O₂- or doxorubicin-induced JNK1 activity, but had no effect on JNK1 activation by MBAs, excluding a role for oxidative stress. However, BAPTA/AM, a specific intracellular Ca²⁺ chelator, attenuated JNK1 activation by taxol but not by colchicine, and had no effect on microtubule changes induced by taxol. Thus, stabilization or depolymerization of microtubules may regulate JNK1 activity via distinct downstream signaling pathways. The differential activation of MAP kinases opens up a new avenue for addressing the mechanism of action of anti-microtubule drugs.

taxol; colchicine; MAP kinases; micro-tubules; breast cancer

ATF, activation transcription factor; BAPTA/AM, 1,2-bis-(o-Aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetra-(acetoxymethyl)-ester; ERK, extracellular signal-regulated protein kinase; GST, glutathione S-transferase; JNK, c-jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MBA, microtubule binding agent; NAC, N-acetyl-L-cysteine; ROI, reactive oxygen intermediates