calcein-am and Colonic-Neoplasms

P-glycoprotein (P-gp or MDR1) is an ATP-binding cassette (ABC) transporter. It is involved in the efflux of several anticancer drugs, which leads to chemotherapy failure and multidrug resistance (MDR) in cancer cells. Representative secondary metabolites (SM) including phenolics (EGCG and thymol), terpenoids (menthol, aromadendrene, β-sitosterol-O-glucoside, and β-carotene), and alkaloids (glaucine, harmine, and sanguinarine) were evaluated as potential P-gp inhibitors (transporter activity and expression level) in P-gp expressing Caco-2 and CEM/ADR5000 cancer cell lines. Selected SM increased the accumulation of the rhodamine 123 (Rho123) and calcein-AM (CAM) in a dose dependent manner in Caco-2 cells, indicating that they act as competitive inhibitors of P-gp. Non-toxic concentrations of β-carotene (40μM) and sanguinarine (1μM) significantly inhibited Rho123 and CAM efflux in CEM/ADR5000 cells by 222.42% and 259.25% and by 244.02% and 290.16%, respectively relative to verapamil (100%). Combination of the saponin digitonin (5μM), which also inhibits P-gp, with SM significantly enhanced the inhibition of P-gp activity. The results were correlated with the data obtained from a quantitative analysis of MDR1 expression. Both compounds significantly decreased mRNA levels of the MDR1 gene to 48% (p<0.01) and 46% (p<0.01) in Caco-2, and to 61% (p<0.05) and 1% (p<0.001) in CEM/ADR5000 cells, respectively as compared to the untreated control (100%). Combinations of digitonin with SM resulted in a significant down-regulation of MDR1. Our findings provide evidence that the selected SM interfere directly and/or indirectly with P-gp function. Combinations of different P-gp substrates, such as digitonin alone and together with the set of SM, can mediate MDR reversal in cancer cells.

Topics: Alkaloids; Antineoplastic Agents, Phytogenic; ATP Binding Cassette Transporter, Subfamily B, Member 1; Benzophenanthridines; beta Carotene; Caco-2 Cells; Colonic Neoplasms; Digitonin; Dose-Response Relationship, Drug; Drug Combinations; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Drug Synergism; Fluoresceins; Humans; Isoquinolines; Leukemia; Phenols; Phytochemicals; Phytotherapy; Plant Extracts; Rhodamine 123; RNA, Messenger; Terpenes

Putative cancer stem cell (CSC) populations efflux dyes such as Hoechst 33342 giving rise to side populations (SP) that can be analyzed or isolated by flow cytometry. However, Hoechst 33342 is highly toxic, more so to non-SP cells, and thus presents difficulties in interpreting in vivo studies where non-SP cells appear less tumorigenic than SP cells in immunodeficient mice. We searched for non-toxic dyes to circumvent this problem as well as to image these putative CSCs. We found that the fluorescent dye calcein, a product of intracellular Calcein AM cleavage, is effluxed by a small subpopulation, calcein low population (C(lo)P). This population overlaps with SP and demonstrated long term cell viability, lack of cell stress and proliferation in several cancer cell lines when stained whereas Hoechst 33342 staining caused substantial apoptosis and ablated proliferation. We also found that the effluxed dye D-luciferin exhibits strong UV-fluorescence that can be imaged at cellular resolution and spatially overlaps with Calcein AM. In order to evaluate the hypothesis that p53 loss promotes enrichment of putative CSC populations we used Calcein AM, D-luciferin and Mitotracker Red FM as a counterstain to visualize dye-effluxing cells. Using fluorescence microscopy and flow cytometry we observed increased dye-effluxing populations in DLD-1 colon tumor cells with mutant p53 versus wild-type (WT) p53-expressing HCT116 cells. Deletion of the wild-type p53 or pro-apoptotic Bax genes induced the putative CSC populations in the HCT116 background to significant levels. Restoration of WT p53 in HCT116 p53(-/-) cells by an adenovirus vector eliminated the putative CSC populations whereas a control adenovirus vector, Ad-LacZ, maintained the putative CSC population. Our results suggest it is possible to image and quantitatively analyze putative CSC populations within the tumor microenvironment and that loss of pro-apoptotic and tumor suppressing genes such as Bax or p53 enrich such tumor-prone populations.

Topics: Adenocarcinoma; Aldehyde Dehydrogenase; Aldehyde Dehydrogenase 1 Family; Apoptosis; ATP-Binding Cassette Transporters; bcl-2-Associated X Protein; Benzimidazoles; Benzothiazoles; Biological Transport, Active; Calcium Channel Blockers; Cell Line, Tumor; Colonic Neoplasms; DNA Damage; Flow Cytometry; Fluoresceins; Fluorescent Dyes; Genes, p53; Humans; Isoenzymes; Microscopy, Fluorescence; Neoplasm Proteins; Neoplastic Stem Cells; Prodrugs; Retinal Dehydrogenase; Staining and Labeling; Tumor Suppressor Protein p53

Topics: Adenocarcinoma; ATP-Binding Cassette Transporters; Benzimidazoles; Biological Transport, Active; Cell Line, Tumor; Cell Separation; Colonic Neoplasms; Flow Cytometry; Fluoresceins; Fluorescent Dyes; Humans; Neoplasm Proteins; Neoplastic Stem Cells; Phenotype; Prodrugs; Staining and Labeling