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Chelerythrine Chloride Downregulates β-Catenin and Inhibits Stem Cell Properties of Non-Small Cell Lung Carcinoma

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Chelerythrine Chloride Downregulates β-Catenin and Inhibits Stem Cell Properties of Non-Small Cell Lung Carcinoma

Win Sen Heng et al. Molecules.

Abstract

Plant secondary metabolites have been seen as alternatives to seeking new medicines for treating various diseases. Phytochemical scientists remain hopeful that compounds isolated from natural sources could help alleviate the leading problem in oncology-the lung malignancy that kills an estimated two million people annually. In the present study, we characterized a medicinal compound benzophenanthridine alkaloid, called chelerythrine chloride for its anti-tumorigenic activities. Cell viability assays confirmed its cytotoxicity and anti-proliferative activity in non-small cell lung carcinoma (NSCLC) cell lines. Immunofluorescence staining of β-catenin revealed that there was a reduction of nuclear content as well as overall cellular content of β-catenin after treating NCI-H1703 with chelerythrine chloride. In functional characterizations, we observed favorable inhibitory activities of chelerythrine chloride in cancer stem cell (CSC) properties, which include soft agar colony-forming, migration, invasion, and spheroid forming abilities. Interesting observations in chelerythrine chloride treatment noted that its action abides to certain concentration-specific-targeting behavior in modulating β-catenin expression and apoptotic cell death. The downregulation of β-catenin implicates the downregulation of CSC transcription factors like SOX2 and MYC. In conclusion, chelerythrine chloride has the potential to mitigate cancer growth due to inhibitory actions toward the tumorigenic activity of CSC in lung cancer and it can be flexibly adjusted according to concentration to modulate specific targeting in different cell lines.

Keywords: Wnt inhibitor; alternative medicine; apoptosis; benzophenanthridine alkaloid; dosing; herbal compound; lung cancer; natural compound.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Dose–response curves of chelerythrine chloride treatments in NCI-H1703, SK-LU-1, and human lung cancer stem cells (HLCSC). Representative dose–response curves show the kinetic response of (A) NCI-H1703, (B) SK-LU-1, and (C) HLCSC toward chelerythrine chloride in a span of 72 h treatments. Curves show dose–response of NCI-H1703, SK-LU-1, and HLCSC toward chelerythrine chloride after (D) 24, (E) 48, and (F) 72 h treatment. Dose–response kinetic curves in (AC) were plotted from duplicate data obtained in a representative of two independent experiments. Dose–response curves in (DF) were derived from means of CI at specified time-points from two independent experiments.
Figure 2
Figure 2
Molecular implications of chelerythrine chloride treatment in lung cancer cell lines. NCI-H1703, SK-LU-1, and HLCSC were treated with GSK3i for 24 h and sequentially treated with various concentrations of chelerythrine chloride for 24 h. Subsequently, immunofluorescence stainings of β-catenin were compared among the cell lines. (A) Representative micrographs show immunofluorescence stainings for chelerythrine chloride treatment in NCI-H1703. (B) Representative micrographs show immunofluorescence stainings for chelerythrine chloride treatment in SK-LU-1. (C) Representative micrographs show immunofluorescence stainings for chelerythrine chloride treatment in HLCSC. Scale bars represent 100 μm. (D) Representative quantification shows cell number with positive nuclear β-catenin in an independent immunostaining experiment. Error bars are expressed as mean ± SD. Most responsive chelerythrine chloride-treated cell lines—NCI-H1703′s—protein lysates were resolved using Western blotting. (E) Representative Western blots show the effect of chelerythrine chloride treatment toward the expression of CSC-related transcription factors, namely β-catenin, MYC, and SOX2. Statistical significance was expressed as *** p < 0.001.
Figure 3
Figure 3
Chelerythrine chloride induces apoptosis and inhibits CSC functions. (A) Parallel estimations of cell viability, cytotoxicity and apoptosis were performed after 24 h treatment of chelerythrine chloride in NCI-H1703 at indicated concentrations using ApoTox-Glo triplex assay. (B) Representative micrographs show three weeks grown soft agar colonies of NCI-H1703 upon treatment of chelerythrine chloride at indicated concentrations. Scale bars represent 1000 μm. (C) Representative micrographs show spheroids’ morphological characteristics after 24 h treatment of chelerythrine chloride at indicated concentrations. Scale bars represent 200 μm. (D) Dose–response bar graph show comparison of cytotoxicity of various concentration of chelerythrine chloride between monolayer and spheroid models of NCI-H1703. (E) Estimations of migration and invasion of NCI-H1703 after 16 h of chelerythrine chloride treatment at indicated concentrations using real-time cell analyzer (RTCA). (F) Effect of corresponding treatment concentrations in (E) to cell viability is shown in bar graph. All data were obtained from mean of three independent experiments. Error bars are expressed as mean ± SD. Statistical significance was expressed as *** p < 0.001 ** p < 0.01; * p < 0.05.

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