Effect Of Chetomin On The Growth And Stemness Phenotype Of Non-small Cell Lung Cancer Cells

Objective:The aim of this study was to investigate the effect of chetomin on the growth and stemness phenotype of non-small cell lung cancer（NSCLC）cells.The findings will be helpful in adding new information to therapeutics against NSCLC,and providing new ideas and strategies for clinical treatment of NSCLC.Methods:Firstly,the effect of chetomin on cell viability was detected by MTT assay in a number of NSCLC cell lines including H1299,H460,H226B,H1944,H460/R（paclitaxel-resistant）and H226B/R（paclitaxel-resistant）,and the IC₅₀values of chetomin for each cell line was calculated to find the appropriate drug concentration for subsequent experiments.Next,the effect of chetomin on the proliferation and apoptosis of NSCLC cells were detected by MTT assay,plate clone formation assay,flow cytometry and Western blot analysis.And then,immunohistochemical staining（IHC）was used to detect the expression of stem cell markers in tumor tissues before and after chemotherapy in two NSCLC patients with driver gene-negative,and the effect of chetomin on the expression of stem cell markers in H1299 cells was detected by Western blot analysis.In vitro,lung cancer stem-like cells（CSLCs）were enriched by the sphere formation assay and the effect of chetomin on the sphere-forming ability of CSLCs was examined.The effect of chetomin on the percentage of cell percentage labeling positive for acetaldehyde dehydrogenase（ALDH⁺）was analyzed by flow cytometry,and the effect of chetomin on the apoptosis of CSLCs was examined by Western blot.Further,the effect of chetomin on normal cell viability was detected by MTT assay in normal tissue cell lines such as BEAS-2B,RPE and HT-22.Finally,to further validate the in vitro experimental findings,the antitumor activity of chetomin was determined in a NOD-SCID mouse xenograft model implanted with H1299 human lung cancer cells（5×10⁶cells/mouse）.When the tumor volume reached approximately50-150 mm³,chetomin（50 or 100 mg/kg）was orally administered daily for 3 weeks in H1299 xenograft model.At the termination of the experiment,the tumor growth of mice in chetomin group was compared with that in control group by observing the subcutaneous tumor volume and weight,and indicated tumor markers expression in lung tumor tissue from mice by IHC analysis.Results:1.Chetomin inhibits NSCLC cell growth by inhibiting cell proliferation and inducing cell apoptosisMTT assay showed that the IC₅₀value of chetomin for the above NSCLC cells ranged from 1-10μM.Additionally,MTT assay also revealed that micromolar concentrations of chetomin could dose-dependently inhibit NSCLC cell viability,and chetomin at 1μM,5μM and 10μM were selected for subsequent NSCLC parental cell experiments.Plate clone formation assay indicated that chetomin could also dose-dependently suppressed NSCLC cell proliferation.Moreover,while the percentage of G0/G1 phase cells in NSCLC cells was shown to be increased after chetomin intervention by cell cycle analysis,chetomin was demonstrated to enhance the cleavage of apoptotic proteins PARP and Caspase-3 as evidenced in Western blot analysis.2.Chetomin suppresses NSCLC stemness phenotype by inhibiting CSLCs proliferation and inducing apoptosisIHC results showed that the expression of stem cell markers CD133 and Sox2 was increased in NSCLC tumor tissues after drug resistance compared with that before chemoresistance,and the expression of stem cell markers in H1299 cells could be modulated by chetomin intervention as shown in Western blot analysis.Under the serum-free suspension culture condition,the H1299 cell line was able to form stable cell spheres after 3-5 days,and these cell spheres were shown to be positive for the stemness markers CD133 and ALDH1 by immunofluorescence assay,indicating successful enrichment of H1299-CSLCs.Based on the IC₅₀values of chetomin in NSCLC cell spheroid formation assay,nanomolar concentrations of chetomin was used in the following experiments regarding stemness phenotype investigation.Chetomin at this dose level was further to be found to inhibit the spheroid formation efficiency in a variety of NSCLC cell lines including H1299 cells.The results of serum-free suspension sphere formation assay indicated that the sphere-forming ability of H1299,H460,H226B,H460/R,and H226B/R cells could be dose-dependently decreased by2-week continuous incubation with chetomin from 1 n M to 10 n M.Flow cytometric analysis revealed that chetomin reduced the proportion of ALDH⁺cells in H1299 cells and enhanced the cleavage of the apoptotic protein PARP and Caspase-3.3.Chetomin has a minor inhibitory effect on cell growth in normal cell linesThe effect of chetomin on cell viability in normal cell lines such as BEAS-2B,RPE and HT-22,was additionally detected by MTT assay,and it was found that chetomin at micromolar level concentration exhibited minor inhibitory effect on the cell growth in the above normal cell lines.4.Chetomin effectively inhibits tumor growth in a NOD-SCID mouse xenograft modelH1299 xenograft model was successfully constructed.Compared with the control group,the tumor volume and tumor weight of mice in experimental group were reduced after 3 weeks of continuous intervention with chetomin.IHC results of lung tumor tissues from mice revealed that the expression of apoptotic protein Caspase-3 was increased and the expression of microvascular density marker CD34 was decreased in chetomin group compared with that in control group.Notably,there was no significant difference in body weight of mice in chetomin group when compared to that in control group during chetomin administration.Conclusion:Chetomin inhibits the growth and stemness phenotype of NSCLC cells,and the antitumor activity of chetomin is confirmed in in vivo experiments in mice.