Synergistic Roles of Curcumin in Sensitising the Cisplatin Effect on a Cancer Stem Cell-Like Population Derived from Non-Small Cell Lung Cancer Cell Lines

Abstract

Cancer stem cells (CSCs) represent a small subpopulation within a tumour. These cells possess stem cell-like properties but also initiate resistance to cytotoxic agents, which contributes to cancer relapse. Natural compounds such as curcumin that contain high amounts of polyphenols can have a chemosensitivity effect that sensitises CSCs to cytotoxic agents such as cisplatin. This study was designed to investigate the efficacy of curcumin as a chemo-sensitiser in CSCs subpopulation of non-small cell lung cancer (NSCLC) using the lung cancer adenocarcinoma human alveolar basal epithelial cells A549 and H2170. The ability of curcumin to sensitise lung CSCs to cisplatin was determined by evaluating stemness characteristics, including proliferation activity, colony formation, and spheroid formation of cells treated with curcumin alone, cisplatin alone, or the combination of both at 24, 48, and 72 h. The mRNA level of genes involved in stemness was analysed using quantitative real-time polymerase chain reaction. Liquid chromatography-mass spectrometry was used to evaluate the effect of curcumin on the CSC niche. A combined treatment of A549 subpopulations with curcumin reduced cellular proliferation activity at all time points. Curcumin significantly (p < 0.001) suppressed colonies formation by 50% and shrank the spheroids in CSC subpopulations, indicating inhibition of their self-renewal capability. This effect also was manifested by the down-regulation of SOX2, NANOG, and KLF4. Curcumin also regulated the niche of CSCs by inhibiting chemoresistance proteins, aldehyde dehydrogenase, metastasis, angiogenesis, and proliferation of cancer-related proteins. These results show the potential of using curcumin as a therapeutic approach for targeting CSC subpopulations in non-small cell lung cancer.

Keywords: cisplatin; curcumin; lung cancer stem cells; non-small cell lung cancer; preventive; sensitisation.

Figure 1

Cell proliferation activity of NSCLC CSCs of A549 and H2170 cells before single and combination treatment after 24 h, 48 h, and 72 h. Each bar represents the average mean ± SD of triplicate samples. Statistical significance was measured with the two-way ANOVA. *** p < 0.001 compared with untreated cells. ## p < 0.01, ### p < 0.001 compared with curcumin alone.

Figure 2

Effect of curcumin on cell cycle distribution on CD166 + EpCAM + CSCs subpopulation and CD166-EpCAM- non-CSCs subpopulation of A549 and H2170 cell lines in both rescue (synergistic) and preventive (sensitisation) treatment groups. In rescue (synergistic) treatment group, the cells were treated with single and combined treatment on day 7 after the formation of colonies or spheroids. In the preventive (sensitisation) treatment groups the cells were treated with single and combined treatment, 48 h post-treatment. Cell cycle analysis was performed by flow cytometry. Each bar represents the average mean ± SD of triplicate samples. Statistical significance was measured with the two-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with untreated cells. # p < 0.05, ## p < 0.01, ### p < 0.001 compared with curcumin alone.

Figure 3

The effect of curcumin on colonies formation as the rescue and the preventive agents. (A): Curcumin suppresses colonies formation using both synergistic (i,ii) and sensitisation (iii,iv) assays in both CD166 + EpCAM + CSCs subpopulation and CD166EpCAM- non-CSCS subpopulations of A549 and H2170 cells. In this experiment, the colonies were allowed to develop before the treatment with curcumin was given. (B): Curcumin acts as preventive agent where the cells were treated with curcumin before colonies were developed. Each bar represents the average mean ± SD of triplicate samples. Statistical significance was measured with the two-way ANOVA. ** p < 0.01, *** p < 0.001 compared with untreated cells. # p < 0.05, ### p < 0.001 compared with curcumin alone.

Figure 3

The effect of curcumin on colonies formation as the rescue and the preventive agents. (A): Curcumin suppresses colonies formation using both synergistic (i,ii) and sensitisation (iii,iv) assays in both CD166 + EpCAM + CSCs subpopulation and CD166EpCAM- non-CSCS subpopulations of A549 and H2170 cells. In this experiment, the colonies were allowed to develop before the treatment with curcumin was given. (B): Curcumin acts as preventive agent where the cells were treated with curcumin before colonies were developed. Each bar represents the average mean ± SD of triplicate samples. Statistical significance was measured with the two-way ANOVA. ** p < 0.01, *** p < 0.001 compared with untreated cells. # p < 0.05, ### p < 0.001 compared with curcumin alone.

Figure 4

(A,B): Curcumin inhibits the spheroid formation of the CD166 + EpCAM + CSCs subpopulation and A549 CD166-EpCAM- non-CSCs subpopulation of A549 and H2170 cells in rescue (A) and preventive (B) treatment groups. (i) Boxplot illustrated the sphere size analysis. Upper and lower boxplot margins represent the interquartile range, and the middle bar indicated the median. The whisker defines the range of values. (ii) Representative photomicrographs of spheroid formation are presented for both CD166 + EpCAM + CSCs subpopulation and A549 CD166-EpCAM- non-CSCs subpopulation of A549 and H2170 cells at ×20 magnification. In the rescue treatment group, spheroid cells were treated with single and combined treatment and were evaluated after day-21. While in the preventive treatment group, cells were treated with either single or combined treatment after 72 h of cultured and were continued growing the colonies until 14 days before staining. Each bar represents the average mean ±SD of triplicate samples. Statistical significance was measured with the two-way ANOVA. ** p < 0.01, *** p < 0.001 compared with untreated cells. # p < 0.05 compared with curcumin alone.

Figure 5

The mRNA expression of stemness genes (SOX2, NANOG, KLF4, and POU51F) after treated with either single (curcumin or cisplatin) or combined treatment, 48 h post-treatment in CD166 + EpCAM + and CD166-EpCAM- non-CSCs subpopulations of A549 (A) and H2170 (B) in both synergistic effect (rescue) and sensitisation (preventive) treatments. Detectable expression levels of the genes were found in all H2170 subpopulations. Each bar represents the average mean ±SD of triplicate samples. Statistical significance was measured with the two-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with untreated cells. # p < 0.05, ## p < 0.01, ### p < 0.001 compared with curcumin alone.

Figure 6

Expression of proteins in CD166 + EpCAM + CSCs (A) and CD166-EpCAM-non-CSCs (B) subpopulations of A549 cell following treatments with curcumin and in combination with cisplatin as compared to the untreated group. The sample profile in all 12 samples are colour-coded and range from −4 to 4 log.