Autophagy inhibition of cancer stem cells promotes the efficacy of cisplatin against non-small cell lung carcinoma

Abstract

Background: Clinical treatment of non-small cell lung carcinoma (NSCLC) by cisplatin eventually results in drug resistance, which cancer stem cells and autophagy are believed to be involved in. In the present study, we aimed to explore the effect of autophagy-inhibited cancer stem cells in NSCLC.

Methods: Cancer stem cells were identified by CD133 expression levels detected by immunochemistry, real-time polymerase chain reaction, western blot, and flow cytometry. Stemness was detected by sphere-forming assays of tumor cells. Autophagy was determined by LC3-II expression at mRNA and protein levels. The effect of chloroquine (CQ) on autophagy was detected by real-time polymerase chain reaction, western blot and sphere-forming assay in vitro, and tumor growth in male NOD/SCID mice.

Results: Cisplatin (CDDP) treatment enhanced CD133⁺ cell ratios in clinical NSCLC specimens and NSCLC cell line A549. The CD133⁺ cells enriched by CDDP exhibited higher autophagy levels. Autophagy inhibition by CQ inhibited CD133⁺ stemness and promoted CDDP efficiency in A549 cells. In addition, the combination of CDDP and CQ treatment significantly inhibited autophagy levels and cancer stem cell proportions in vitro, and dramatically suppressed tumor growth compared with individual agents.

Conclusion: Autophagy inhibition of cancer stem cells could promote the efficacy of cisplatin against NSCLC.

Keywords: autophagy; cancer stem cell; cisplatin; non-small cell lung carcinoma.

Figure 1.

CDDP treatment elevates the ratio of CD133⁺ cells within specimens and cell line of NSCLC. (a) Representative images showed the immunohistochemistry of CD133 staining in the clinical specimens before and post CDDP treatment from patient with NSCLC. Scale bar, 20 μm. (b) mRNA and protein level of CD133 in the clinical specimens before and post CDDP treatment from patients with NSCLC. Data represent means ± SD. ^**p < 0.005, n = 6. (c) A549 cells were treated with different concentrations of CDDP for 48 h. The cell viability was determined by an MTT assay. (d) Percentages of CD133⁺ CSCs in A549 cells with or without the treatment of low dose of cisplatin for 48 h, respectively. The CDDP concentration was IC₂₀, about 2.5 μM. CSCs were stained with anti-CD133-PE antibody and analyzed by flow cytometer. Data are shown as mean ± SD (n = 6). (e) mRNA levels of ‘stemness’-associated genes in A549 cells after CDDP treatment. A549 cells were treated with 2.5 μM CDDP for 48 h. The mRNA levels of ‘stemness’-associated genes were analyzed by qPCR, the mRNA levels of genes were normalized against the expression level of housekeeping gene GAPDH, ^*p < 0.05, ^**p < 0.005, ^***p < 0.001, n = 6. (f) CDDP-treated A549 cells were subjected to the tumor sphere-forming assay. Scale bar, 100 μm. CDDP, cisplatin; CSC, cancer stem cell; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NSCLC, non-small cell lung cancer; qPCR, quantitative polymerase chain reaction; SD, standard deviation.

Figure 2.

CDDP-enriched CD133⁺ cancer stem cells exhibit enhanced levels of autophagy. (a) CD133 and CD133⁺ cells were sorted from A549 cells were treated with 2.5 μM CDDP for 48 h. The purities of CD133^– and CD133⁺ cells were evaluated by flow cytometry and western blotting. (b) Western blotting and densitometric analysis of LC3-II levels in CD133 and CD133⁺ A549 cells. GAPDH was used as a loading control. The signal intensities were quantified using software ImageJ (version 1.47) (n = 6). (c) Fluorescent microscopy images of CD133 and CD133⁺ EGFP-LC3/A549 cells. Bright punctate dots indicated the induction of autophagy. Scale bar, 10 µm. CDDP, cisplatin; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 3.

CQ-mediated inhibition of autophagy reduces the ‘stemness’ of CD133⁺ A549 cells and promotes efficiency of CDDP in A549 cells. (a) Protein expression of LC3 and p62 in CD133⁺ A549 cells after incubation with different concentration of CQ for 48 h and densitometric analysis, GAPDH was used as a loading control. The signal intensities were quantified using software ImageJ (version 1.47) (n = 6). (b) The proportion of CD133⁺ A549 cells in sorted cells after treatment with CQ (10 μM) for 48 h. Data represent means ± SD. ^**p < 0.005, n = 6. (c) CQ-treated sorted CD133⁺ A549 cells were subjected to the colony-forming assay. (d) CQ-treated sorted CD133⁺ A549 cells were subjected to sphere-forming assay. Scale bar, 100 μm. (e) Percentage of CD133⁺ cells in A549 cells after co-incubation with CDDP and CQ for 48 h, the concentrations of CDDP and CQ were 2.5 μM and 10 μM, respectively. Data represent means ± SD. ^**p < 0.005, n = 6. (f) The ability of A549 cells to regenerate spheres. A549 cells were treated with CDDP and CQ for 48 h, and then cells were seeded onto a six-well ultra-low attachment plate with complete spheres medium (100 cells per well). After 10 days, the number of newly formed spheres per well was determined under the microscope. Data represent means ± SD. ^**p < 0.005, (CDDP + CQ versus CDDP), n = 6. (g) MTT analyses of the viability of A549 cell after treatment with CDDP, CQ or a combination for 48 h. Cell viability was normalized to that of PBS-treated cells which served as the indicator of 100% cell viability. Data represent means ± SD. ^**p < 0.005 (CDDP + CQ versus CDDP), n = 6. The concentrations of CDDP and CQ were 2.5 μM and 10 μM, respectively. CDDP, cisplatin; CQ, chloroquine; PBS, phosphate-buffered saline; SD, standard deviation.

Figure 4.

In vivo anti-tumor effect of CDDP, CQ and a combination of two agents. (a) Inhibition of tumor growth by various treatments in A549 tumor-bearing NOD/SCID mice (n = 6). Male NOD/SCID mice bearing A549 tumors of ~50 mm³ received PBS, CDDP, CQ or combination of CDDP and CQ every 3 days five times, the administration dosage of CDDP and CQ were 2.5 mg/kg (i.v.) and 10.0 mg/kg (i.v.), respectively. ^**p < 0.005, ^***p < 0.001 (versus CDDP group). (b) Tumor weights of different groups at the end of treatments. ^***p < 0.001 (versus CDDP group); (c) PCNA analysis (immunohistochemistry) and TUNEL analysis (immunofluorescence) of tumor tissues after treatment with various therapeutic agents. The PCNA-positive proliferating cells are stained brown, TUNEL-positive cells are green. Scale bar was 20 μm. CDDP, cisplatin; CQ, chloroquine; i.v., intravenously; PBS, phosphate-buffered saline; PCNA, Proliferating cell nuclear antigen; SD, standard deviation; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

Figure 5.

Combination therapy inhibits autophagy and suppresses CSC subpopulation in vivo. (a) Protein expressions of LC3-I and LC3-II in tumor tissues after treatment with CDDP, CQ or a combination of two agents. GAPDH was used as a loading control. (b) The immunofluorescence of p62 protein in tumor cells after treatment with various agents. The nuclei were stained with DAPI (blue), p62 protein was stained with anti-p62 antibody labeled with Alexa Fluor 647. (c) Xenografted tumor at the end of therapy were dissociated into single cells and stained with anti-CD133 labeled with PE for flow cytometry analysis. (d) Quantitative analysis of CD133⁺ cells in tumor tissues. CDDP treatment significantly increase CD133⁺ cells and cotreatment of autophagy inhibitor CQ blocked the induction effect of CDDP on the percentage of CD133⁺ cells. Data represent means ± SD. n = 6, ^**p < 0.005. (e) mRNA expression levels of stemness-associated genes (including Sox2, Oct4, Nanog, ABCG2 and CD133) in A549 tumor tissue at the end-point of treatment. CDDP, Cisplatin; DAPI, 4′,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PE, phycoerythrin.