Natural Compounds' Activity against Cancer Stem-Like or Fast-Cycling Melanoma Cells

Background Accumulating evidence supports the concept that melanoma is highly heterogeneous and sustained by a small subpopulation of melanoma stem-like cells. Those cells are considered as responsible for tumor resistance to therapies. Moreover, melanoma cells are characterized by their high phenotypic plasticity. Consequently, both melanoma stem-like cells and their more differentiated progeny must be eradicated to achieve durable cure. By reevaluating compounds in heterogeneous melanoma populations, it might be possible to select compounds with activity not only against fast-cycling cells but also against cancer stem-like cells. Natural compounds were the focus of the present study. Methods We analyzed 120 compounds from The Natural Products Set II to identify compounds active against melanoma populations grown in an anchorage-independent manner and enriched with cells exerting self-renewing capacity. Cell viability, cell cycle arrest, apoptosis, gene expression, clonogenic survival and label-retention were analyzed. Findings Several compounds efficiently eradicated cells with clonogenic capacity and nanaomycin A, streptonigrin and toyocamycin were effective at 0.1 µM. Other anti-clonogenic but not highly cytotoxic compounds such as bryostatin 1, siomycin A, illudin M, michellamine B and pentoxifylline markedly reduced the frequency of ABCB5 (ATP-binding cassette, sub-family B, member 5)-positive cells. On the contrary, treatment with maytansine and colchicine selected for cells expressing this transporter. Maytansine, streptonigrin, toyocamycin and colchicine, even if highly cytotoxic, left a small subpopulation of slow-dividing cells unaffected. Compounds selected in the present study differentially altered the expression of melanocyte/melanoma specific microphthalmia-associated transcription factor (MITF) and proto-oncogene c-MYC. Conclusion Selected anti-clonogenic compounds might be further investigated as potential adjuvants targeting melanoma stem-like cells in the combined anti-melanoma therapy, whereas selected cytotoxic but not anti-clonogenic compounds, which increased the frequency of ABCB5-positive cells and remained slow-cycling cells unaffected, might be considered as a tool to enrich cultures with cells exhibiting melanoma stem cell characteristics.

The upper numbers represent those ones used in the present study. The lower numbers (underlined) can be used to get detailed information about a drug from available databases.

Figure S1
A. The influence of natural compounds (5 µM) on viable cell numbers in melanoma (DMBC11 and DMBC12) and leukemia (K562) cell cultures assessed by APA assay. Data are the mean ± SD of two independent experimets performed in triplicates.

Figure S3B
Effects of lower concentrations for the most cytotoxic compounds or of longer exposure for compounds that were ineffective at 30 h.

Figure S4
The influence of natural compounds used at a single concentration of 5 µM on the clonogenic growth of melanoma cells. Cells were incubated in drug-containing medium for 4 h and then they were grown on agar for 14 days in drug-free medium. Cell colonies were stained and counted. Anti-clonogenic activity was expressed as percentage of control treated with vehicle (0.05% DMSO). At least two independent experiments were performed in duplicates. Viability was measured by flow cytometry after PI staining in six different melanoma cell lines DMBC2, DMBC8, DMBC9, DMBC10, DMBC11 and DMBC12. Data expressed as % of control are means ± SD of two independent experiments conducted in triplicates.

Imidazoquinoline
inducer of TLR7-mediated immune cell activation [59]; Rotenone Lonchocarpus nicou Derris elliptica inhibitor of the transfer of electrons from iron-sulfur centers in complex I to ubiquinone in electron transport chain [60];
inhibitor of DNA, RNA and protein synthesis [61];

Teniposide
inhibitor of DNA synthesis that forms a complex with topoisomerase II and DNA [66];