Nicotinic receptors modulate antitumor therapy response in triple negative breast cancer cells

Abstract

Background: Triple negative breast cancer is more aggressive than other breast cancer subtypes and constitutes a public health problem worldwide since it has high morbidity and mortality due to the lack of defined therapeutic targets. Resistance to chemotherapy complicates the course of patients' treatment. Several authors have highlighted the participation of nicotinic acetylcholine receptors (nAChR) in the modulation of conventional chemotherapy treatment in cancers of the airways. However, in breast cancer, less is known about the effect of nAChR activation by nicotine on chemotherapy treatment in smoking patients.

Aim: To investigate the effect of nicotine on paclitaxel treatment and the signaling pathways involved in human breast MDA-MB-231 tumor cells.

Methods: Cells were treated with paclitaxel alone or in combination with nicotine, administered for one or three 48-h cycles. The effect of the addition of nicotine (at a concentration similar to that found in passive smokers' blood) on the treatment with paclitaxel (at a therapeutic concentration) was determined using the 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The signaling mediators involved in this effect were determined using selective inhibitors. We also investigated nAChR expression, and ATP "binding cassette" G2 drug transporter (ABCG2) expression and its modulation by the different treatments with Western blot. The effect of the treatments on apoptosis induction was determined by flow cytometry using annexin-V and 7AAD markers.

Results: Our results confirmed that treatment with paclitaxel reduced MDA-MB-231 cell viability in a concentration-dependent manner and that the presence of nicotine reversed the cytotoxic effect induced by paclitaxel by involving the expression of functional α7 and α9 nAChRs in these cells. The action of nicotine on paclitaxel treatment was linked to modulation of the protein kinase C, mitogen-activated protein kinase, extracellular signal-regulated kinase, and NF-κB signaling pathways, and to an up-regulation of ABCG2 protein expression. We also detected that nicotine significantly reduced the increase in cell apoptosis induced by paclitaxel treatment. Moreover, the presence of nicotine reduced the efficacy of paclitaxel treatment administered in three cycles to MDA-MB-231 tumor cells.

Conclusion: Our findings point to nAChRs as responsible for the decrease in the chemotherapeutic effect of paclitaxel in triple negative tumors. Thus, nAChRs should be considered as targets in smoking patients.

Keywords: Breast cancer; Drug therapy; Drug transporter; Nicotinic acetylcholine receptors; Paclitaxel; Signal transduction.

Figure 1

Diagram of the administration schedule for the determination of cell viability or cell sensitivity to chemotherapy. PX: Paclitaxel; NIC: Nicotine.

Figure 2

MDA-MB-231 cell viability. A: Concentration-response curves of nicotine on cell viability in the absence or presence of nicotinic antagonists: mecamylamine [non-selective for nicotinic acetylcholine receptors (nAChRs)], methyllycaconitine (selective for α7 nAChRs), or luteolin (selective for α9 nAChRs) at a concentration of 10^-6 mol/L. Values are the mean ± SD of five experiments performed in duplicate. ^cP < 0.001 vs control; ^dP < 0.001 vs nicotine; ^eP < 0.001 vs control or nicotine; B: Western blot assay to detect α7 and α9 nAChR expression. Molecular weights are indicated on the right. The expression of glyceraldehyde 3-phosphate dehydrogenase was used as the loading control. One representative experiment of three is shown. MM: Mecamylamine; MLA: Methyllycaconitine; Lut: Luteolin; nAChRs: Nicotinic acetylcholine receptors; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase.

Figure 3

MDA-MB-231 cell viability. Concentration-response curves of paclitaxel on cell viability in the absence or presence of nicotine (NIC) (10^-10 mol/L). Values are the mean ± SD of six experiments performed in duplicate. ^aP < 0.05; ^cP < 0.001 vs Control; ^dP < 0.001 vs +NIC. NIC: Nicotine.

Figure 4

Sensitivity of MDA-MB-231 cells to chemotherapy. Concentration-response curves of paclitaxel (PX) on the viability of surviving cells after three cycles of PX treatment (10^-7 mol/L) in the absence or presence of nicotine (10^-10 mol/L). Values are the mean ± SD of three experiments performed in duplicate. ^bP < 0.01; ^cP < 0.001 vs surviving cells after three cycles of PX treatment. PX: Paclitaxel; NIC: Nicotine.

Figure 5

Effect of nicotine on paclitaxel treatment. A: Viability determination of MDA-MB-231 cells treated with nicotine (10^-10 mol/L) and paclitaxel (10^-7 mol/L) alone or in combination, in the absence or presence of nicotinic antagonists: mecamylamine [non-selective for nicotinic acetylcholine receptors (nAChRs)], methyllycaconitine (selective for α7 nAChRs), or luteolin (selective for α9 nAChRs) at a concentration of 10^-6 mol/L; B: Determination of percentage of living MDA-MB-231 cells treated with the same drug combinations as those shown in Figure 5A. Values are the mean ± SD of four experiments performed in duplicate. ^bP < 0.01; ^cP < 0.001 vs control, considered as 100%. MM: Mecamylamine; MLA: Methyllycaconitine; Lut: Luteolin; PX: Paclitaxel; NIC: Nicotine.

Figure 6

Effect of paclitaxel and nicotine on MDA-MB-231 cell viability. A: Cells were treated with paclitaxel (PX) (10^-7 mol/L) and the mediators were evaluated in the absence or presence of the kinase inhibitors for: PKC (staurosporine, 10^-8 mol/L), MEK (PD098059 PD, 10^-5 mol/L), Ras (S-trans, trans-farnesylthiosalicylic acid, 10^-6 mol/L), ERK1/2 (U126, 10^-5 mol/L), p38MAPK (SB203580, 10^-5 mol/L) or IKKβ (IMD354, 5 × 10^-8 mol/L); B: Cells were treated with the combination of PX and nicotine (NIC) (10^-10 mol/L) as well as with the same inhibitors as those shown in Figure 6A. Values are the mean ± SD of four experiments performed in duplicate. ^aP < 0.05; ^bP < 0.01 vs PX. ^cP < 0.05; ^dP < 0.01; ^eP < 0.001 vs PX+NIC. FTS: S-trans, trans-farnesylthiosalicylic acid; SB: SB203580; Stau: Staurosporine; PD: PD098059; PX: Paclitaxel; NIC: Nicotine.

Figure 7

Effect of nicotine on paclitaxel-induced apoptosis in MDA-MB-231 cells. Tumor cells were treated with paclitaxel (10^-7 mol/L) in the absence or presence of the following nicotinic antagonists: mecamylamine [non-selective for nicotinic acetylcholine receptors (nAChRs)], methyllycaconitine (selective for α7 nAChRs), or luteolin (selective for α9 nAChRs) at a concentration of 10^-6 mol/L. The percentage of apoptotic cells was determined by flow cytometry. Values are the mean ± SD of four experiments performed in duplicate. ^aP < 0.05; ^bP < 0.01; ^cP < 0.001 vs control; ^dP < 0.001. MM: Mecamylamine; MLA: Methyllycaconitine; Lut: Luteolin; PX: Paclitaxel; NIC: Nicotine.

Figure 8

Effect of nicotine on paclitaxel-induced expression of ATP binding cassette transporter G2 protein in MDA-MB-231 cells. A: ATP binding cassette transporter G2 expression was determined by Western blot assays in cells treated with paclitaxel (10^-7 mol/L), nicotine (10^-10 mol/L) or both, in the absence or presence of the nicotinic antagonists mecamylamine [non-selective for nicotinic acetylcholine receptors (nAChRs)], methyllycaconitine (selective for α7 nAChRs) or luteolin (selective for α9 nAChRs) at a concentration of 10^-6 mol/L. Molecular weights are indicated on the right; B: The densitometric analysis of the bands is expressed as optical density units relative to the expression of glyceraldehyde 3-phosphate dehydrogenase protein used as the loading control. One representative experiment of three is shown. Values are the mean ± SD of three experiments. ^aP < 0.05; ^cP < 0.001 vs Control; ^dP < 0.001 vs PX+NIC. ABCG2: ATP “binding cassette” G2 drug transporter; O.D.: Optical density; MM: Mecamylamine; MLA: Methyllycaconitine; Lut: Luteolin; nAChRs: Nicotinic acetylcholine receptors; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; PX: Paclitaxel; NIC: Nicotine.

Figure 9

Possible signal transduction pathways in MDA-MB-231 cells activated by paclitaxel in the absence or presence of nicotine. NIC: Nicotine; PX: Paclitaxel; nAChR: Nicotinic acetylcholine receptors; ABCG2: ATP “binding cassette” G2 drug transporter; PKC: Protein kinase C; MEK: Mitogen-activated protein kinase kinase; ERK: Extracellular signal-regulated kinases; p38MAPK: p38 Mitogen-activated protein kinases; IKKβ: IκB kinase; IκBα: κB inhibitors.