Increased chemosensitivity and radiosensitivity of human breast cancer cell lines treated with novel functionalized single-walled carbon nanotubes

Abstract

Hypoxia is a major cause of treatment resistance in breast cancer. Single-walled carbon nanotubes (SWCNTs) exhibit unique properties that make them promising candidates for breast cancer treatment. In the present study, a new functionalized single-walled carbon nanotube carrying oxygen was synthesized; it was determined whether this material could increase chemosensitivity and radiosensitivity of human breast cancer cell lines, and the underlying mechanisms were investigated. MDA-MB-231 cells growing in folic acid (FA) free medium, MDA-MB-231 cells growing in medium containing FA and ZR-75-1 cells were treated with chemotherapy drugs or radiotherapy with or without tombarthite-modified-FA-chitosan (R-O₂-FA-CHI)-SWCNTs under hypoxic conditions, and the cell viability was determined by water-soluble tetrazolium salts-1 assay. The cell surviving fractions were determined by colony forming assay. Cell apoptosis induction was monitored by flow cytometry. Expression of B-cell lymphoma 2 (Bcl-2), survivin, hypoxia-inducible factor 1-α (HIF-1α), multidrug resistance-associated protein 1 (MRP-1), P-glycoprotein (P-gp), RAD51 and Ku80 was monitored by western blotting. The novel synthesized R-O₂-FA-CHI-SWCNTs were able to significantly enhance the chemosensitivity and radiosensitivity of human breast cancer cell lines and the material exhibited its expected function by downregulating the expression of Bcl-2, survivin, HIF-1α, P-gp, MRP-1, RAD51 and Ku80.

Keywords: breast cancer; chemotherapy sensitivity; hypoxia; radiotherapy sensitivity; single-walled carbon nanotubes.

Figure 1.

(A) X-ray photoelectron spectroscopy analysis of solutions of R-O₂-SWCNTs. (B) Ultraviolet-visible spectra of solutions of R-O₂-SWCNTs, free FA, and R-O₂-FA-CHI-SWCNTs. R-O₂-SWCNTs, tombarthite-modified-single-walled carbon nanotubes; FA, folic acid; CHI, chitosan.

Figure 2.

Effects of R-O₂-FA-CHI-SWCNTs at various doses on the viability of MDA-MB-231 and ZR-75-1 cells under normoxic and hypoxic conditions. (A) MDA-MB-231 cells were incubated with R-O₂-FA-CHI-SWCNTs at concentrations from 5–500 µg/ml for 48 h. The number of viable cells was measured by WST-1 assay. *P<0.05 vs. control group in normoxic conditions. ^#P<0.05 vs. control group in hypoxic conditions. (B) ZR-75-1 cells were incubated with treatment R-O₂-FA-CHI-SWCNTs at concentrations from 5–500 µg/ml for 48 h. The number of viable cells was measured by WST-1 assay. *P<0.05 vs. control group in normoxic conditions. ^#P<0.05 vs. control group in hypoxic conditions. Data are presented as the mean ± standard deviation. R-O₂-SWCNTs, tombarthite-modified-single-walled carbon nanotubes; FA, folic acid; CHI, chitosan; WST-1, water-soluble tetrazolium salts-1.

Figure 3.

Cell survival rates of MDA-MB-231 and ZR-75-1 cells cultured with or without FA. The cells were divided into four groups: Control, M, chemotherapeutic drug (5-Fu, pirarubicin, epirubicin or paclitaxel) and chemotherapeutic drug + M groups. Cell survival was determined using water-soluble tetrazolium salts-1 assays. (A) FR-positive MDA-MB-231 cells cultured in FA-free medium. (B) MDA-MB-231 cells cultured in FA-containing medium. (C) FR-negative ZR-75-1 cells. Each value represents the mean ± standard deviation (n=3). *P<0.05 vs. control group. **P<0.05 vs. chemotherapy group. R-O₂-FA-CHI-SWCNTs is denoted as M in the figure. R-O₂-SWCNTs, tombarthite-modified-single-walled carbon nanotubes; FA, folic acid; FR, FA receptor; CHI, chitosan; 5-Fu, fluorouracil.

Figure 4.

Cell apoptosis and death detected by flow cytometry. Untreated MDA-MB-231 cells under hypoxic conditions served as a control group. MDA-MB-231 cells were incubated with 100 µg/ml R-O₂-FA-CHI-SWCNTs under hypoxic conditions. MDA-MB-231 cells were incubated with carboplatin under hypoxic conditions. MDA-MB-231 cells were incubated with carboplatin plus 100 µg/ml R-O₂-FA-CHI-SWCNTs under hypoxic conditions. The populations in quadrants 2 and 4 indicate the percentage of necrotic and early-apoptotic cells, respectively. R-O₂-FA-CHI-SWCNTs are denoted as M in the figure. Data in the graph are presented as the mean ± standard deviation. R-O₂-FA-CHI-SWCNTs, tombarthite-modified-folic acid-chitosan-single-walled carbon nanotubes. *P<0.05, **P<0.01.

Figure 5.

(A) MDA-MB-231 cells in FA-free medium were treated with R-O₂-FA-CHI-SWCNTs, irradiation (4 Gy) or a combination of R-O₂-FA-CHI-SWCNTs and irradiation (4 Gy), respectively. Control cells were untreated. Colony forming assays for these four groups are shown. (B) Survival curves with SF normalized to the plating efficiency were fitted according to the linear quadratic equation: SF=exp[-(αD+βD2)]. Data are presented as the mean ± standard deviation from three independent experiments. *P<0.05, vs. irradiation treated cells. R-O₂-FA-CHI-SWCNTs are denoted as M in the figure. FA, folic acid; R-O₂-SWCNTs, tombarthite-modified-single-walled carbon nanotubes; CHI, chitosan; SF, surviving fractions; D, dose; IR, irradiation.

Figure 6.

Cell apoptosis and death detected by flow cytometry. Untreated MDA-MB-231 cells under hypoxic conditions served as a control group. MDA-MB-231 cells were incubated with 100 µg/ml R-O₂-FA-CHI-SWCNTs under hypoxic conditions. MDA-MB-231 cells were irridiated with 4 Gy under hypoxic conditions. MDA-MB-231 cells were irradiated with 4 Gy plus 100 µg/ml R-O₂-FA-CHI-SWCNTs under hypoxic conditions. The populations in quadrants 2 and 4 indicate the percent of necrotic and early-apoptotic cells. R-O₂-FA-CHI-SWCNTs are denoted as M in the figure. R-O₂-FA-CHI-SWCNTs, tombarthite-modified-folic acid-chitosan-single-walled carbon nanotubes; IR, irradiation. *P<0.05, **P<0.01.

Figure 7.

(A) Epirubicin group, western blot analysis was performed against Bcl-2, survivin, P-gp, MRP-1 and HIF-1α. β-actin was used as a loading control. (B) Radiotherapy group, western blot analysis was performed against Bcl-2, survivin, RAD51, Ku80 and HIF-1α. β-actin was used as a loading control. M, tombarthite-modified-folic acid-chitosan-single-walled carbon nanotubes; IR, irradiation; Bcl-2, B-cell lymphoma 2; P-gp, P-glycoprotein; MRP-1, multidrug resistance-associated protein 1; Hif-1α, Hypoxia-inducible factor 1-α.