Inhibitory effects of Hedyotis diffusa Willd. on colorectal cancer stem cells

Abstract

Cancer stem cells (CSCs) are proposed to be closely correlated with the development and progression of tumors, as well as with chemo- and radioresistance. Targeting CSCs may therefore be a promising potential strategy for the treatment of cancer. Currently, natural products have received great interest due to their therapeutic efficacy and reduced adverse effects compared with modern chemotherapeutics. As a significant component of a number of traditional Chinese medicine formulas, the medicinal herb Hedyotis diffusa Willd. (HDW) has long been utilized in China to clinically treat a variety of malignancies, including colorectal cancer (CRC). Previously, the authors of the present study reported that HDW suppressed CRC growth through multiple mechanisms, including promoting apoptosis, and inhibiting cell proliferation and tumor angiogenesis. To additionally investigate its mode of action, the present study isolated a stem-like side population (SP) from colorectal cancer HT-29 cells to investigate the effect of ethanol extract of HDW on CSCs. It was observed that HDW was able to markedly downregulate the expression of CSC marker leucine-rich repeat-containing G-protein coupled receptor 5 and also significantly decrease the proportion of SP in HT-29 cells, in a dose-dependent manner. Furthermore, HDW treatment significantly and dose-dependently inhibited the viability and sphere formation, and induced cell morphological changes of isolated HT-29 SP cells. In addition, HDW greatly suppressed the messenger RNA expression of several critical genes that mediate CSC features, including ATP-binding cassette, sub-family B, member 1, β-catenin, c-Myc, proliferating cell nuclear antigen and survivin. In conclusion, the present study indicates that HDW may exert inhibitory effects on cancer stem cells.

Keywords: Hedyotis diffusa Willd.; cancer stem cells; side population; traditional Chinese medicine.

Figure 1.

EEHDW inhibits the percentage of SP in human colorectal cancer HT-29 cells. (A) Following treatment with various concentrations of EEHDW (0, 0.5, 1 and 2 mg/ml) for 24 h, HT-29 cells were stained with Hoechst 33342 and percentages of SP were analyzed by FACS. Verapamil was used as a positive control. (B) Quantification of FACS analysis. Images are representative and data are expressed as the mean ± standard deviation of 3 independent experiments. *P<0.05 vs. untreated control cells. EEHDW, ethanol extract of Hedyotis diffusa Willd.; SP, side population; FACS, fluorescence-activated cell sorting.

Figure 2.

EEHDW inhibits the protein expression of Lgr5 in HT-29 cells. (A) The protein expression of Lgr5 in HT-29 cells was determined by western blot analysis. GAPDH was used as the internal control. (B) Densitometric analysis. The data were normalized to the mean protein expression of untreated control cells (100%). Images are representative and data are presented as the mean ± standard deviation of 3 independent experiments. *P<0.05 vs. untreated control cells. EEHDW, ethanol extract of Hedyotis diffusa Willd.; Lgr5, Leucine-rich repeat-containing G-protein coupled receptor 5; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 3.

EEHDW inhibits the sphere formation capacity and viability of isolated HT-29 SP cells. (A) Following treatment with various concentrations (0, 0.5, 1 and 2 mg/ml) of EEHDW, SP cells were grown in serum-free stem cell culture medium for 15 days. Spheroids (>50 cells) were counted and photographed. (B) Quantification of sphere formation analysis. Images are representative and data are presented as the mean ± standard deviation of 3 independent experiments. *P<0.05 vs. untreated control cells. (C) SP cells were treated with the indicated concentrations (0, 0.5, 1 and 2 mg/ml) of EEHDW for 24 h. Cell viability was determined by the water-soluble tetrazolium salts-1 assay. Data are presented as the mean ± standard deviation. *P<0.05 vs. untreated control cells. EEHDW, ethanol extract of Hedyotis diffusa Willd.; SP, side population.

Figure 4.

EEHDW induces morphological changes in isolated HT-29 SP cells. Sorted SP cells were treated with the indicated concentrations (0, 0.5, 1 and 2 mg/ml) of EEHDW for 24 h and morphological changes were observed using phase-contrast microscopy. The images were captured at magnification, ×200. Images are representative of 3 independent experiments. EEHDW, ethanol extract of Hedyotis diffusa Willd.; SP, side population.

Figure 5.

EEHDW suppresses the mRNA expression of ABCB1, β-catenin, c-Myc, PCNA and survivin in isolated HT-29 SP cells. (A) Following treatment with the indicated concentrations (0, 0.5, 1 and 2 mg/ml) of EEHDW for 24 h, the mRNA expression of ABCB1, β-catenin, c-Myc, PCNA and survivin in sorted HT-29 SP cells was determined by reverse transcription-polymerase chain reaction. GAPDH was used as the internal control. (B) Densitometric analysis. The data were normalized to the mean mRNA expression of untreated controls (100%). Images are representative and data are presented as the mean ± standard deviation of 3 independent experiments. *P<0.05 vs. untreated control cells. EEHDW, ethanol extract of Hedyotis diffusa Willd.; mRNA, messenger RNA; ABCB1, ATP-binding cassette, sub-family B, member 1; PCNA, proliferating cell nuclear antigen; SP, side population; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.