title: +TIP EB1 downregulates paclitaxel‑induced proliferation inhibition and apoptosis in breast cancer cells through inhibition of paclitaxel binding on microtubules.
abstract: Microtubule plus‑end‑binding protein (+TIP) EB1 has been shown to be upregulated in breast cancer cells and promote breast tumor growth in vivo. However, its effect on the cellular actions of microtubule‑targeted drugs in breast cancer cells has remained poorly understood. By using cellular and biochemical assays, we demonstrate that EB1 plays a critical role in regulating the sensitivity of breast cancer cells to anti‑microtubule drug, paclitaxel (PTX). Cell viability assays revealed that EB1 expression in the breast cancer cell lines correlated with the reduction of their sensitivity to PTX. Knockdown of EB1 by enzymatically‑prepared siRNA pools (esiRNAs) increased PTX‑induced cytotoxicity and sensitized cells to PTX‑induced apoptosis in three breast cancer cell lines, MCF‑7, MDA MB‑231 and T47D. Apoptosis was associated with activation of caspase‑9 and an increase in the cleavage of poly(ADP‑ribose) polymerase (PARP). p53 and Bax were upregulated and Bcl2 was downregulated in the EB1‑depleted PTX‑treated MCF‑7 cells, indicating that the apoptosis occurs via a p53‑dependent pathway. Following its upregulation, the nuclear accumulation of p53 and its association with cellular microtubules were increased. EB1 depletion increased PTX‑induced microtubule bundling in the interphase cells and induced formation of multiple spindle foci with abnormally elongated spindles in the mitotic MCF‑7 cells, indicating that loss of EB1 promotes PTX‑induced stabilization of microtubules. EB1 inhibited PTX‑induced microtubule polymerization and diminished PTX binding to microtubules in vitro, suggesting that it modulates the binding sites of PTX at the growing microtubule ends. Results demonstrate that EB1 downregulates inhibition of PTX‑induced proliferation and apoptosis in breast cancer cells through a mechanism in which it impairs PTX‑mediated stabilization of microtubule polymerization and inhibits PTX binding on microtubules.
authors.affiliation: School of Biology, Indian Institute of Science Education and Research, CET Campus, Thiruvananthapuram 695016, Kerala, India.
authors.forename: Geethu Emily
authors.initials: GE
authors.lastname: Thomas
authors.affiliation: School of Biology, Indian Institute of Science Education and Research, CET Campus, Thiruvananthapuram 695016, Kerala, India.
authors.forename: Jamuna S
authors.initials: JS
authors.lastname: Sreeja
authors.affiliation: School of Biology, Indian Institute of Science Education and Research, CET Campus, Thiruvananthapuram 695016, Kerala, India.
authors.forename: K K
authors.initials: KK
authors.lastname: Gireesh
authors.affiliation: School of Biology, Indian Institute of Science Education and Research, CET Campus, Thiruvananthapuram 695016, Kerala, India.
authors.forename: Hindol
authors.initials: H
authors.lastname: Gupta
authors.affiliation: School of Biology, Indian Institute of Science Education and Research, CET Campus, Thiruvananthapuram 695016, Kerala, India.
authors.forename: Tapas K
authors.initials: TK
authors.lastname: Manna
mesh_terms: D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D001943:Breast Neoplasms / Q000235:genetics / Q000473:pathology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects / Q000235:genetics; D015536:Down-Regulation; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D005260:Female; D006801:Humans; D061986:MCF-7 Cells; D008869:Microtubule-Associated Proteins / Q000037:antagonists & inhibitors / Q000502:physiology*; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism; D017239:Paclitaxel / Q000378:metabolism / Q000494:pharmacology*; D034741:RNA, Small Interfering / Q000494:pharmacology
chemical_list: C500985:MAPRE1 protein, human; D008869:Microtubule-Associated Proteins; D034741:RNA, Small Interfering; D017239:Paclitaxel