Effects of compounds 5f, 5g, 5h and 4i on the HeLa cell cycle distribution after incubation for 24 (left panels) and 48 h (right panels).

<p>Gray and black columns indicate 3 and 10 μM, respectively. * and ** denote p < 0.05 and p < 0.01, respectively, as compared with the control cells.</p

Effects of 5f and 5g (10 μM) on the expression of phosphorylated (upper panel) and total (lower panel) stathmin protein in HeLa cells after incubation for 48 h, determined by western blot analysis.

<p>Results are mean values ± SEM of the data on two separate measurements, n = 6. ns indicates p > 0.05, *** indicates p < 0.001 as compared with the untreated control cells. Panels below are representative membrane pictures.</p

Effects of compounds 5f and 5g on the Bax/Bcl-2 ratio after incubation of HeLa cells for 24 h.

<p>* and ** denote p < 0.05 and p < 0.01, respectively, as compared with the control condition.</p

Induction of caspase-3, caspase-8 and caspase-9 activities after incubation with compounds 5f and 5g for 24 h.

<p>White, gray and black columns denote 3, 10 and 30 μM of the given agent. * and ** denote p < 0.05 and p < 0.01, respectively, as compared with the control condition.</p

Expression of CDK1, cyclin B1, cyclin B2 and cdc25B at the mRNA level after incubation with 3 (gray columns) or 10 (black columns) μM of compounds 5f or 5g.

<p>*, ** and *** denote p < 0.05, p < 0.01 and p < 0.001, respectively, as compared with the control condition.</p

Synthesis of steroidal 16α-triazoles by CuAAc.

<p>Synthesis of steroidal 16α-triazoles by CuAAc.</p

Fluorescent microscopy images of Hoechst 33258-PI double staining.

<p>Two separate pictures from the same field were taken for the two markers. MRC5 cells were treated with vehicle (control), or with <b>5f</b> and <b>5g</b> at the indicated concentrations. The blue fluorescence (left panels) indicates Hoechst 33258, and the red fluorescence (right panels) is a consequence of PI accumulation. The bar in the PI control picture indicates 100 μm.</p

Calculated IC₅₀ values of the structural combination (hybrid compounds) and experimental combination of the corresponding thymoquinone and protoflavone building blocks on the U-87 cells.

Calculated IC50 values of the structural combination (hybrid compounds) and experimental combination of the corresponding thymoquinone and protoflavone building blocks on the U-87 cells.</p

Data_Sheet_1_Antiproliferative Properties of Newly Synthesized 19-Nortestosterone Analogs Without Substantial Androgenic Activity.pdf

<p>19-Nortestosterone C-17 epimers with prominent antiproliferative properties have been previously described. In our present study, five novel 17α-19-nortestosterones (3−7) were synthesized to increase their beneficial biological activities with no associated undesired hormonal effects. The compounds were screened by a viability assay against a panel of human adherent gynecological cancer cell lines. Three of the tested derivatives (3−5) exhibited a remarkable inhibitory effect on the proliferation of HeLa cells with IC₅₀ values lower than that of our reference agent cisplatin (CIS). These three active agents also displayed considerable cancer selectivity as evidenced by their weaker growth inhibitory effect on non-cancerous fibroblast cells compared to CIS. The most potent newly synthesized 17α-chloro derivative (3) was selected for additional experiments in order to characterize its mechanism of action. Since nandrolone (19-nortestosterone, 1) is a structural analog with selective antiproliferative action on cervical carcinoma cells, it was utilized as a positive control in these studies. A lactate dehydrogenase (LDH) assay demonstrated a moderate cytotoxic effect of the test compounds. Cell cycle disturbance and the elevation of the hypodiploid population elicited by the test agents were detected by flow cytometry following propidium staining. The proapoptotic effects of the tested steroids were confirmed by fluorescent microscopy and a caspase-3 activity assay. Treatment-related caspase-9 activation without a substantial change in caspase-8 activity indicates the induction of the intrinsic apoptotic pathway. The selected agents directly influence the rate of tubulin assembly as evidenced by a polymerization assay. Yeast-based reporter gene assay revealed that the androgenic activity of the novel 19-nortestosterone derivative 3 is by multiple orders of magnitude weaker than that of the reference agent 1. Based on the behavior of the examined compounds it can be concluded that a halogen substitution of the 19-nortestosterone scaffold at the 17α position may produce compounds with unique biological activities. The results of the present study support that structurally modified steroids with negligible hormonal activity are a promising basis for the research and development of novel anticancer agents.</p

S1 File -

We describe herein the synthesis of eight new ester-coupled hybrid compounds from thymoquinone and protoflavone building blocks, and their bioactivity testing against multiple cancer cell lines. Among the hybrids, compound 14 showed promising activities in all cell lines studied. The highest activities were recorded against breast cancer cell lines with higher selectivity to MDA-MB-231 as compared to MCF-7. Even though the hybrids were found to be completely hydrolysed in 24 h under cell culture conditions, compound 14 demonstrated a ca. three times stronger activity against U-87 glioblastoma cells than a 1:1 mixture of its fragments. Further, compound 14 showed good tumour selectivity: it acted 4.4-times stronger on U-87 cells than on MRC-5 fibroblasts. This selectivity was much lower, only ca. 1.3-times, when the cells were co-treated with a 1:1 mixture of its non-coupled fragments. Protoflavone-thymoquinone hybrids may therefore serve as potential new antitumor leads particularly against glioblastoma.</div