A novel series of substituted 1,2,3-triazoles as cancer stem cell inhibitors: Synthesis and biological evaluation.

An alarming increase in global death toll resulting from cancer incidents, particularly due to multidrug resistance and reduced efficacy as a consequence of target mutations, has compelled us to look for novel anticancer agents. Cancer stem cells (CSCs), contributing majorly to the chemoresistance and tumor relapse, seem to the main culprits. In the present investigation, new chemical entities (NCEs) belonging to four novel chemical series (A: 4'-allyl-2'-methoxyphenoxymethyl-1,2,3-triazoles; B: 4'-acetamidophenoxymethyl-1,2,3-triazoles; C: naphthalene-1'-yloxymethyl-1,2,3-triazoles, and D: naphthalene-2'-yloxymethyl-1,2,3-triazoles) were synthesized via Copper (I)-catalyzed alkyne-azide cycloaddition reaction and evaluated for in vitro anticancer activity. A total of 30 NCEs (39-68) were screened at 10 µM concentration in cell viability assay against cancer cell lines such as breast (MDA-MB-231), prostate (PC-3), glioma (U87 MG), along with cervical (SiHa) and lung (A549). The NCEs from Series C (56-60) and D (61-68) were more potent than those in Series A (39-45) and Series B (46-55) at the tested concentration. Furthermore, NCEs with >80% inhibition at 10 µM were evaluated for dose response. A total of five NCEs, 48, 56, 61, 65 and 66, were further assessed in soft-agar assay and found to be relatively potent (IC50 < 10 µM). Finally, the hits were screened in sphere assay to identify potential CSC inhibitors against mammospheres (MDA-MB-231) and prostatospheres (PC-3). More so, the hits were also evaluated to understand in vitro cytotoxicity against normal cells using mouse embryonic fibroblast cell line (NIH/3T3) and human peripheral blood mononuclear cells (hPBMCs). Overall, hits 56 and 61 exhibited potent anticancer as well as CSC inhibitory activities with notably less toxicity toward NIH/3T3 and hPBMCs. On the whole, our arduous study led to the identification of potential hits with anticancer and CSC inhibitory activities, with minimal or no toxicity to normal cells.

New Conjugates of Polyhydroxysteroids with Long-Chain Fatty Acids from the Deep-Water Far Eastern Starfish Ceramaster patagonicus and Their Anticancer Activity.

Four new conjugates, esters of polyhydroxysteroids with long-chain fatty acids (1-4), were isolated from the deep-water Far Eastern starfish Ceramaster patagonicus. The structures of 1-4 were established by NMR and ESIMS techniques as well as chemical transformations. Unusual compounds 1-4 contain the same 5α-cholestane-3ß,6ß,15α,16ß,26-pentahydroxysteroidal moiety and differ from each other in the fatty acid units: 5'Z,11'Z-octadecadienoic (1), 11'Z-octadecenoic (2), 5'Z,11'Z-eicosadienoic (3), and 7'Z-eicosenoic (4) acids. Previously, only one such steroid conjugate with a fatty acid was known from starfish. After 72 h of cell incubation, using MTS assay it was found that the concentrations of compounds 1, 2, and 3 that caused 50% inhibition of growth (IC50) of JB6 Cl41 cells were 81, 40, and 79 µM, respectively; for MDA-MB-231 cells, IC50 of compounds 1, 2, and 3 were 74, 33, and 73 µM, respectively; for HCT 116 cells, IC50 of compounds 1, 2, and 3 were 73, 31, and 71 µM, respectively. Compound 4 was non-toxic against tested cell lines even in three days of treatment. Compound 2 (20 µM) suppressed colony formation and migration of MDA-MB-231 and HCT 116 cells.

Unusual Polyhydroxylated Steroids from the Starfish Anthenoides laevigatus, Collected off the Coastal Waters of Vietnam.

Four new polyhydroxylated steroids 1-4 were isolated along with two previously known related steroids 5 and 6 from the methanolic extract of the starfish Anthenoides laevigatus collected off the coastal waters of Vietnam. Structures of new compounds were substantially elucidated by one-dimensional (1D) and two-dimensional (2D) NMR spectroscopy and HRESIMS techniques. Heptaol 1 and hexaol 2 contain the common 5α-cholestane skeleton, while hexaol 3 and heptaol 4 have the rare among starfish steroid compounds 5ß-cholestane skeleton. Compounds 1, 5, and 6 do not show cytotoxic effects against normal JB6 Cl41 and cancer HT-29 and MDA-MB-231 cells, however they inhibit cell proliferation and colony formation of cancer HT-29 and MDA-MB-231 cells.

Substituted chloroacetamides as potential cancer stem cell inhibitors: Synthesis and biological evaluation.

Cancer kills, irrespective of geographical and cultural origin. Novel modalities for treating cancer are desperately needed. Cancer stem cells (CSCs), main culprits behind chemoresistance and tumor relapse, are one of the few logical choices. Herein, we report the synthesis and biological evaluation of small molecules with chloroacetamide war-head. These molecules were screened for viability against various breast, prostate, and oral cancer cell lines using MTT and soft-agar assays. Further, promising hits were screened in sphere-forming assay with the aim of discovering potential anti-CSC agents. Our optimism yielded four hits inhibiting self-renewal of cancer cells with stem-like characters in vitro. Finally, the hits were evaluated for in vitro toxicity against human peripheral blood mononuclear cells and mouse embryonic fibroblast cell line. Overall, these preliminary investigations yielded three hits exhibiting promising anti-CSC potential with little or no toxicity against normal cells.

In Vitro Self-Renewal Assays for Brain Tumor Stem Cells.

Early development of human organisms relies on stem cells, a population of non-specialized cells that can divide symmetrically to give rise to two identical daughter cells, or divide asymmetrically to produce one identical daughter cell and another more specialized cell. The capacity to undergo cellular divisions while maintaining an undifferentiated state is termed self-renewal and is responsible for the maintenance of stem cell populations during development. In addition, self-renewal plays a crucial role in the homeostasis of developed organism through replacement of defective cells.Similar to their non-malignant counterparts, it has been postulated that tumor cells follow a differentiation hierarchy, with the least differentiated cells termed cancer stem cells (CSCs) at the apex. These tumor stem cells possess the ability to self-renew, have a higher capacity to initiate tumor growth when xenografted into an animal model, and can recapitulate the cell heterogeneity of the tumor they originate from. Hence, further investigation of mechanisms governing the self-renewal in cancer can lead to development of novel therapies targeting CSCs.In this chapter, we described the soft agar assay and the limiting dilution assay (LDA) as two easy-to-implement and inexpensive assays to measure the stemness properties of brain tumor stem cells (BTSCs). These techniques constitute useful tools for the preclinical evaluation of therapeutic strategies targeting BTSCs clonogenicity.

Two New Steroidal Monoglycosides, Anthenosides A1 and A2, and Revision of the Structure of Known Anthenoside A with Unusual Monosaccharide Residue from the Starfish Anthenea aspera.

Two new polyhydroxysteroidal glycosides, anthenosides A1 (1) and A2 (2), and one previously known steroidal glycoside anthenoside A (3) were isolated from extract of the tropical starfish Anthenea aspera. Structures of 1⁻3 were determined by analysis of the spectroscopic data as well as chemical transformations. As a result, the structure of anthenoside A has been revised and the structures of 1 and 2 were established. Glycosides 1⁻3 contain a 2-acetamido-2-deoxy-4-O-methyl-ß-d-glucopyranosyl residue, found in the starfish steroidal glycosides for the first time. All the isolated compounds slightly inhibited cell viability of human cancer T-47D cells and did not show cytotoxic effects against RPMI-7951 cells. Glycoside 1 slightly inhibited colony formation of human cancer RPMI-7951 cells by 16% while compound 2 decreased the number of colonies of T-47D cells by 40%.

In Vitro Long-Term Proliferation Assays to Study Antiproliferative Effects of PARP Inhibitors on Cancer Cells.

Cell proliferation assays are an important component of small molecule inhibitor screens for cancer therapies. An important but often overlooked variable involves the timing and timeframe of inhibitor treatment. Whereas many traditional chemotherapeutics kill or inhibit proliferation on the timeframe of hours or in a few days of treatment, more targeted therapies that affect other cancer-relevant pathways, including differentiation or cell stress responses, can take longer, often several days to weeks to impact cellular growth and survival. Many poly(ADP-ribose) polymerases (PARPs) are involved in cellular stress pathways; therefore, phenotypic effects of PARP inhibition are often only observed with long-term inhibitor treatment. Here we summarize several assays for analyzing long-term proliferation of both adherent and suspension cells, relying either on growth in two-dimensional tissue culture or on systems than enable growth in 3D.

Tumorigenicity Evaluation of Umbilical Cord Blood-derived Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) have been identified in multiple types of tissue and exhibit characteristic self-renewal and multi-lineage differentiation abilities. However, the possibility of oncogenic transformation after transplantation is concerning. In this study, we investigated the tumorigenic potential of umbilical cord blood-derived MSCs (hUCB-MSCs) relative to MRC-5 and HeLa cells (negative and positive controls, respectively) both in vitro and in vivo. To evaluate tumorigenicity in vitro, anchorage-independent growth was assessed using the soft agar colony formation assay. hUCB-MSCs and MRC-5 cells formed few colonies, while HeLa cells formed a greater number of larger colonies, indicating that hUCB-MSCs and MRC-5 cells do not have anchorage-independent proliferation potential. To detect tumorigenicity in vivo, hUCB-MSCs were implanted as a single subcutaneous injection into BALB/c-nu mice. No tumor formation was observed in mice transplanted with hUCB-MSCs or MRC-5 cells based on macroand microscopic examinations; however, all mice transplanted with HeLa cells developed tumors that stained positive for a human gene according to immunohistochemical analysis. In conclusion, hUCB-MSCs do not exhibit tumorigenic potential based on in vitro and in vivo assays under our experimental conditions, providing further evidence of their safety for clinical applications.

Genotoxic and cell-transforming effects of titanium dioxide nanoparticles.

The in vitro genotoxic and the soft-agar anchorage independent cell transformation ability of titanium dioxide nanoparticles (nano-TiO2) and its microparticulated form has been evaluated in human embryonic kidney (HEK293) and in mouse embryonic fibroblast (NIH/3T3) cells. Nano-TiO2 of two different sizes (21 and 50 nm) were used in this study. The comet assay, with and without the use of FPG enzyme, the micronucleus assay and the soft-agar colony assay were used. For both the comet assay and the frequency of micronuclei a statistically significant induction of DNA damage, was observed at the highest dose tested (1000 µg/mL). No oxidative DNA damage induction was observed when the comet assay was complemented with the use of FPG enzyme. Furthermore, long-term exposure to nano-TiO2 has also proved to induce cell-transformation promoting cell-anchorage independent growth in soft-agar. Results were similar for the two nano-TiO2 sizes. Negative results were obtained when the microparticulated form of TiO2 was tested, indicating the existence of important differences between the microparticulated and nanoparticulated forms. As a conclusion it should be indicated that the observed genotoxic/tranforming effects were only detected at the higher dose tested (1000 µg/mL) what play down the real risk of environmental exposures to this nanomaterial.

Zinc oxide nanoparticles: genotoxicity, interactions with UV-light and cell-transforming potential.

The in vitro genotoxic and the soft agar anchorage independent cell transformation ability of zinc oxide nanoparticles (NPs) and its bulky forms have been evaluated in human embryonic kidney (HEK293) and in mouse embryonic fibroblast (NIH/3T3) cells, either alone or in combination with UVB-light. The comet assay, with and without the use of FPG and Endo III enzymes, the micronucleus assay and the soft-agar colony assay were used. For the comet assay a statistically significant induction of DNA damage, with and without the enzymes, were observed up of 100µg/mL. ZnO NPs were able to increase significantly the frequency of micronuclei, and similar results were observed in the cell transformation assay where such NPs were able to induce cell-anchorage independent growth. These effects were observed at doses up 100µg/mL. Although UVB-light was able to induce genotoxic damage and cell-anchorage growth, a significant antagonist interaction effect was observed in combination with ZnO NPs. These in vitro results, obtained with the selected cell lines, contribute to increase our genotoxicity database on the ZnO NPs effects as well as to open the discussion about their risk in photo-protection sun screens.