Potential of Orlistat to induce apoptotic and antiangiogenic effects as well as inhibition of fatty acid synthesis in breast cancer cells.

Breast cancer as most often women's cancer is the second cause of mortality worldwide. Research interest increased in testing non-standard drugs to suppress breast cancer progression and become significant supplements in anticancer therapy. The anti-obesity drug Orlistat showed significant ability for modulation of cancer cell metabolism via antiproliferative, proapoptotic, antiangiogenic, antimetastatic, and hypolipidemic effects. The anticancer potential of Orlistat was evaluated by cytotoxicity (MTT assay), type of cell death (AO/EB double staining), determination of redox status parameters (superoxide, hydrogen peroxide, lipid peroxidation, reduced glutathione), and total lipid levels with colorimetric methods, as well on angiogenesis-related (VEGF, MMP-9, CXCR4/CXCL12) and fatty acid synthesis-related (ACLY, ACC, FASN) parameters on gene and protein levels (immunocytochemistry and qPCR). Based on obtained results Orlistat induces significant cytotoxic, proapoptotic, and anti-angiogenic effects in MDA-MB-231, MDA-MB-468 and MCF-7 breast cancer cells, without significant cytotoxic effects on normal MRC-5 cells. It decreased total lipid levels and changed redox status parameters and cancer cell metabolism via suppression of genes and proteins involved and fatty acid synthesis. Based on showed, Orlistat may be an important supplement in antiangiogenic therapy against breast cancer with no side effects on normal cells, making it a good candidate for future clinical trials.

1,2,3,4-Tetrahydroisoquinoline Derivatives as a Novel Deoxyribonuclease I Inhibitors.

Herein we report an assessment of 24 1,2,3,4-tetrahydroisoquinoline derivatives for potential DNase I (deoxyribonuclease I) inhibitory properties in vitro. Four of them inhibited DNase I with IC50 values below 200 µM. The most potent was 1-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolin-1-yl)propan-2-one (2) (IC50 =134.35±11.38 µM) exhibiting slightly better IC50 value compared to three other active compounds, 2-[2-(4-fluorophenyl)-1,2,3,4-tetrahydroisoquinolin-1-yl]-1-phenylethan-1-one (15) (IC50 =147.51±14.87 µM), 2-[2-(4-fluorophenyl)-1,2,3,4-tetrahydroisoquinolin-1-yl]cyclohexan-1-one (18) (IC50 =149.07±2.98 µM) and 2-[6,7-dimethoxy-2-(p-tolyl)-1,2,3,4-tetrahydroisoquinolin-1-yl]cyclohexan-1-one (22) (IC50 =148.31±2.96 µM). Cytotoxicity assessment of the active DNase I inhibitors revealed a lack of toxic effects on the healthy cell lines MRC-5. Molecular docking and molecular dynamics simulations suggest that interactions with Glu 39, His 134, Asn 170, Tyr 211, Asp 251 and His 252 are an important factor for inhibitors affinity toward the DNase I. Observed interactions would be beneficial for the discovery of new active 1,2,3,4-tetrahydroisoquinoline-based inhibitors of DNase I, but might also encourage researchers to further explore and utilize potential therapeutic application of DNase I inhibitors, based on a versatile role of DNase I during apoptotic cell death.

Synthesis and anticancer potential of novel 5,6-oxygenated and/or halogenated steroidal d-homo lactones.

A series of 5,6-modified steroidal d-homo lactones, comprising of halogenated and/or oxygenated derivatives, was synthesized and evaluated for potential anticancer properties. Preparation of many of these compounds involved investigating alternative synthetic pathways. In silico ADME testing was performed for both novel and some previously synthesized compounds. Calculated physicochemical properties were in accordance with the Lipinski, Veber, Egan, Ghose and Muegge criteria, suggesting the potential of these molecules as orally active agents. Cytotoxicity of the synthesized steroid derivatives was tested on six tumor and one normal human cell line. None of the investigated derivatives was toxic to non-cancerous MRC-5 control cells. Most of the compounds showed significant cytotoxicity against the treated cancer cell lines. Most notably, the 3ß,5α,6ß-trihydroxy derivative exhibited strong cytotoxicity against multiple cell lines (MCF-7, MDA-MB-231 and HT-29), with the highest effect observed for lung adenocarcinoma (A549) cells, for which this steroid was more cytotoxic than all of the three commercial chemotherapeutic agents used as reference compounds. Molecular docking suggests the 3ß,5α,6ß-trihydroxy derivative could bind the EGFR tyrosine kinase domain with high affinity, providing a potential mechanism for its cytotoxicity via inhibition of EGFR signaling. The most active compounds were further studied for their potential to induce apoptosis by the double-staining fluorescence method; where the 5α,6ß-dibromide, 5α,6ß-dichloride and 3ß,5α,6ß-triol induced apoptotic changes in all three treated cell lines: MDA-MB-231, HT-29 and A549. To predict interactions with nuclear steroidal receptors, affinity for the ligand binding domains of ERα, ERß and AR was measured using a yeast-based fluorescence assay. The 5ß,6ß-epoxide, dibromide and 5α-hydroxy-3,6-dioxo derivatives showed affinity for ERα, while the 5α-fluoro-6ß-hydroxy and 3ß-acetoxy-5α,6ß-dihydroxy derivatives were identified as ERß ligands. None of the tested compounds showed affinity for AR. Structure-activity relationships of selected compounds were also examined.

Synthesis, characterization, HSA/DNA interactions and antitumor activity of new [Ru(η6-p-cymene)Cl2(L)] complexes.

Three new ruthenium(II) complexes were synthesized from different substituted isothiazole ligands 5-(methylamino)-3-pyrrolidine-1-ylisothiazole-4-carbonitrile (1), 5-(methylamino)-3-(4-methylpiperazine-1-yl)isothiazole-4-carbonitrile (2) and 5-(methylamino)-3-morpholine-4-ylisothiazole-4-carbonitrile (3): [Ru(η6-p-cymene)Cl2(L1)]·H2O (4), [Ru(η6-p-cymene)Cl2(L2)] (5) and [Ru(η6-p-cymene)Cl2(L3)] (6). All complexes were characterized by IR, UV-Vis, NMR spectroscopy, and elemental analysis. The molecular structures of all ligands and complexes 4 and 6 were determined by an X-ray. The results of the interactions of CT-DNA (calf thymus deoxyribonucleic acid) and HSA (human serum albumin) with ruthenium (II) complexes reveal that complex 4 binds well to CT-DNA and HSA. Kinetic and thermodynamic parameters for the reaction between complex and HSA confirmed the associative mode of interaction. The results of Quantum mechanics (QM) modelling and docking experiments toward DNA dodecamer and HSA support the strongest binding of the complex 4 to DNA major groove, as well as its binding to IIa domain of HSA with the lowest ΔG energy, which agrees with the solution studies. The modified GOLD docking results are indicative for Ru(p-cymene)LCl··(HSA··GLU292) binding and GOLD/MOPAC(QM) docking/modelling of DNA/Ligand (Ru(II)-N(7)dG7) covalent binding. The cytotoxic activity of compounds was evaluated by MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) assay. Neither of the tested compounds shows activity against a healthy MRC-5 cell line while the MCF-7 cell line is the most sensitive to all. Compounds 3, 4 and 5 were about two times more active than cisplatin, while the antiproliferative activity of 6 was almost the same as with cisplatin. Flow cytometry analysis showed the apoptotic death of the cells with a cell cycle arrest in the subG1 phase.

New A-homo lactam D-homo lactone androstane derivative: Synthesis and evaluation of cytotoxic and anti-inflammatory activities in vitro.

This paper describes the synthesis of a new A-homo lactam D-homo lactone androstane derivative from dehydroepiandrosterone. To evaluate the impact of the introduction of nitrogen in the parental scaffold on biological activity, a new androstane enamide-type lactam derivative was prepared and characterized. The new compound as well as starting compounds were screened for cytotoxic, anti-angiogenic and anti-inflammatory activities using several human cancer cell lines (MCF-7, MDA-MB-231, PC3, CEM, G-361, HeLa), endothelial (HUVEC) and non-tumour (MRC-5 and BJ) cell lines. Strong cytotoxic and anti-inflammatory activity with a broad therapeutical window was demonstrated by the A-homo lactam D-homo lactone androstane derivative. The induction of apoptosis in treated PC3 cultures was confirmed using apoptotic morphology screening and a fluorescent double-staining method. New A-homo lactam D-homo lactone androstane derivative induced apoptosis more than the tested reference compounds, Formestane and Doxorubicin. An in silico ADME analysis showed that the compounds possess drug-like properties.

The effect of the androstane lung cancer inhibitor content on the cell-selective toxicity of hydroxyapatite-chitosan-PLGA nanocomposites.

An androstane (17ß-hydroxy-17α-picolyl-androst-5-en-3ß-yl-acetate (derivative A)) cancer inhibitor was successfully captured in a carrier made of nano-sized hydroxyapatite (HAp) coated with chitosan-PLGA polymer blends (Ch-PLGA). In our previous studies, we demonstrated that it was convenient to use spherical HAp/Ch-PLGA carriers as vehicles to target the lungs following intravenous administration. In this study, we used emulsification and subsequent freeze-drying to load the spherical HAp/Ch-PLGA carriers with varying contents of the derivative A, in order to examine the selective toxicity towards cancerous/healthy lung cells. The XRD and FT-IR techniques confirmed the drug loading process, and the content of the poorly water soluble derivative A was estimated directly via the DSC technique. The particles were spherical in shape with the d50 distribution varying between 167 and 231 nm, whereas the content of the derivative A ranged from 6.5 to 19.3 wt%. Cell-selective cytotoxicity was examined simultaneously on two cell lines: human lung carcinoma (A549 ATCC CCL 185) and human lung fibroblasts (MRC-5 ATCC CCL 171). All particles exhibited nearly three times larger cytotoxicity towards cancer cells (A549) than towards healthy cells (MRC5), where the particles with the derivative A content of 6.5 wt% allowed for the viability of healthy cells >80%. Ninety-six hours after the treatment of cells with particles with different contents of derivative A (after incubation and recovery), recovery was faster in damaged healthy cells than in cancerous cells.

Identification of d-seco modified steroid derivatives with affinity for estrogen receptor α and ß isoforms using a non-transcriptional fluorescent cell assay in yeast.

Synthesis and biological evaluation of steroidal derivatives with anticancer properties is an active area of drug discovery. Here we measured the relative affinities of d-seco modified steroidal derivatives for estrogen receptor α, estrogen receptor ß or androgen receptor ligand binding domains using an optimized non-transcriptional fluorescent cell assay in yeast. Ligand binding domains of steroid receptors were expressed in-frame with yellow fluorescent protein in the yeast Saccharomyces cerevisiae. Addition of known steroid ligands to yeast expressing the appropriate cognate receptor results in increased fluorescence intensity, enabling estimation of receptor binding affinities in a dose-response and time-dependent manner. Relative binding affinities of d-seco modified steroidal derivatives 1-4 were then evaluated using this yeast system by live cell fluorimetry and fluorescence microscopy, coupled with in vitro cytotoxicity and in silico molecular docking studies. d-Seco estratriene derivative 2displayed strong affinity for both estrogen receptor α and ß ligand binding domains and negligible affinity for the androgen receptor ligand binding domain. Compound 2 also showed moderate cytotoxicity against estrogen receptor positive MCF-7 breast adenocarcinoma cells. In addition to identification of new ligands for steroid receptors, this assay could also be used to filter out compounds with potential for off-target interactions with steroid receptors during the early stages of compound screening.

Selective anticancer activity of hydroxyapatite/chitosan-poly(d,l)-lactide-co-glycolide particles loaded with an androstane-based cancer inhibitor.

In an earlier study we demonstrated that hydroxyapatite nanoparticles coated with chitosan-poly(d,l)-lactide-co-glycolide (HAp/Ch-PLGA) target lungs following their intravenous injection into mice. In this study we utilize an emulsification process and freeze drying to load the composite HAp/Ch-PLGA particles with 17ß-hydroxy-17α-picolyl-androst-5-en-3ß-yl-acetate (A), a chemotherapeutic derivative of androstane and a novel compound with a selective anticancer activity against lung cancer cells. 1H NMR and 13C NMR techniques confirmed the intact structure of the derivative A following its entrapment within HAp/Ch-PLGA particles. The thermogravimetric and differential thermal analyses coupled with mass spectrometry were used to assess the thermal degradation products and properties of A-loaded HAp/Ch-PLGA. The loading efficiency, as indicated by the comparison of enthalpies of phase transitions in pure A and A-loaded HAp/Ch-PLGA, equaled 7.47wt.%. The release of A from HAp/Ch-PLGA was sustained, neither exhibiting a burst release nor plateauing after three weeks. Atomic force microscopy and particle size distribution analyses were used to confirm that the particles were spherical with a uniform size distribution of d50=168nm. In vitro cytotoxicity testing of A-loaded HAp/Ch-PLGA using MTT and trypan blue dye exclusion assays demonstrated that the particles were cytotoxic to the A549 human lung carcinoma cell line (46±2%), while simultaneously preserving high viability (83±3%) of regular MRC5 human lung fibroblasts and causing no harm to primary mouse lung fibroblasts. In conclusion, composite A-loaded HAp/Ch-PLGA particles could be seen as promising drug delivery platforms for selective cancer therapies, targeting malignant cells for destruction, while having a significantly lesser cytotoxic effect on the healthy cells.

Androstane derivatives induce apoptotic death in MDA-MB-231 breast cancer cells.

Biological investigation was conducted to study in vitro antiproliferative and pro-apoptotic potential of selected 17α-picolyl and 17(E)-picolinylidene androstane derivatives. The antiproliferative impact was examined on six human tumor cell lines, including two types of breast (MCF-7 and MDA-MB-231), prostate (PC3), cervical (HeLa), colon (HT 29) and lung cancer (A549), as well as one normal fetal lung fibroblasts cell line (MRC-5). All derivatives selectively decreased proliferation of estrogen receptor negative MDA-MB-231 breast cancer cells after 48 h and 72 h treatment and compounds showed time-dependent activity. We used this cell line to investigate cell cycle modulation and apoptotic cell death induction by flow cytometry, expression of apoptotic proteins by Western blot and apoptotic morphology by visual observation. Tested androstane derivatives affected the cell cycle distribution and induced apoptosis and necrosis. Compounds had different and specific mode of action, depending on derivative type and exposure time. Some compounds induced significant apoptosis measured by Annexin V test compared to reference compound formestane. Higher expression of pro-apoptotic BAX, downregulation of anti-apoptotic Bcl-2 and cleavage of PARP protein were confirmed in almost all treated samples, but the lack of caspase-3 activation suggested the induction of apoptosis in caspase-independent manner. More cells with apoptotic morphology were observed in samples after prolonged treatment. Structure-activity relationship analysis was performed to find correlations between the structure variations of investigated derivatives and observed biological effects. Results of this study showed that some of the investigated androstane derivatives have good biomedical potential and could be candidates for anticancer drug development.

Microwave assisted synthesis and biomedical potency of salicyloyloxy and 2-methoxybenzoyloxy androstane and stigmastane derivatives.

A convenient microwave assisted solvent free synthesis as well as conventional synthesis of salicyloyloxy and 2-methoxybenzoyloxy androstane and stigmastane derivatives 7-19 from appropriate steroidal precursors 1-6 and methyl salicylate is reported. The microwave assisted synthesis in most cases was more successful regarding reaction time and product yields. It was more environmentally friendly too, compared to the conventional method. The antioxidant activity and cytotoxicity of the synthesized derivatives were evaluated in a series of in vitro tests, as well as their inhibition potency exerted on hydroxysteroid dehydrogenase enzymes (Δ(5)-3ßHSD, 17ßHSD2 and 17ßHSD3). All of the tested compounds were effective in OH radical neutralization, particularly compounds 9, 11 and 14, which exhibited about 100-fold stronger activity than commercial antioxidants BHT and BHA. In DPPH radical scavenging new compounds were effective, but less than reference compounds. 2-Methoxybenzoyl ester 10 exhibited strong cytotoxicity against MDA-MB-231 cells. Most compounds inhibited growth of PC-3 cells, where salicyloyloxy stigmastane derivative 15 showed the best inhibition potency. Compounds 9, 10 and 11 were the best inhibitors of 17ßHSD2 enzyme. X-ray structure analysis and molecular mechanics calculations (MMC) were performed for the best cytotoxic agents, compounds 10 and 15. A comparison of crystal and MMC structures of compounds 10 and 15 revealed that their molecules conformations are stable even after releasing of the influence of crystalline field and that the influence of crystal packing on molecular conformation is not predominant.

Synthesis and cytotoxic evaluation of novel pyrimidine deoxyapiothionucleosides.

The synthesis of novel pyrimidine deoxyapiothionucleosides of D- and L-series was realized following application of a versatile and high-yielding scheme, which utilized inexpensive L- and D-arabinose as starting materials, respectively, and which makes use of a regio- and stereo-selective Pummerer rearrangement reaction for the coupling of the nucleobase with the thiosugar moiety. Some of the targeted compounds have shown selective cytotoxic effects (with IC50<10 µM) against specific cancer cell lines. All of the tested compounds had no cytotoxic effect on the normal cell line tested.

Synthesis and cytotoxic activity of some 17-picolyl and 17-picolinylidene androstane derivatives.

New 17-picolyl and 17-picolinylidene androstane derivatives, 3-10, 15, 18, 19, 22 and 23, were synthesized starting from 17α-picolyl-androst-5-en-3ß,17ß-diol (1) and 17(Z)-picolinylidene-androst-5-en-3ß-ol (2). Reaction of 1 with m-chloroperoxybenzoic acid gives 5α,6α-epoxy N-oxide derivative 3, or, with Jones reagent, 3,6-dione derivative 4; while 17α-picolyl-androst-5-en-3ß,4α,17ß-triol (5) or 3ß,4ß,17ß-triol (6) derivatives are obtainable from 1 using SeO(2) in dioxane. Base-catalyzed tosyl group elimination from 7 or 9 affords AB conjugated derivatives 8 and 10. Oppenauer oxidation of 1 and 2 yields 4-en-3-one derivatives 11 and 12, which, with H(2)O(2) in 4 M NaOH, affords 4α,5α and 4ß,5ß-epoxides 13, 14, 16 and 17. New 4-methoxy-3-keto derivatives 15 and 18 were obtained from 13 and 14, or, with methanol in 4 M NaOH, from 16 and 17. Reduction of 11 with NaBH(4) gives 22, which was then acetylated to obtain 23. All new derivatives were screened for antitumor activity against human breast adenocarcinoma ER+, MCF-7; human breast adenocarcinoma ER-, MDA-MB-231; prostate cancer AR-, PC-3; human cervix carcinoma, HeLa; and colon cancer, HT-29 cells; as well as one human non-tumor cell line, MRC-5. Compounds 3, 5, 6, 8, 10, 18, 19 and 22 exhibited significant antitumor activity against MDA-MB-231 breast cancer cells; while 5, 6 and 10 also showed strong cytotoxicity against HT-29. Only compound 19 exhibited significant activity against MCF-7 breast cancer cells. No compounds displayed cytotoxicity against non-tumor MRC-5 cells.

Synthesis and antitumor activity of new D-seco and D-homo androstane derivatives.

Starting from 3beta-hydroxy-17-oxo-16,17-secoandrost-5-ene-16-nitrile (1), the new 16,17-secoandrostane derivatives 4-9 were synthesized. On the other hand, 3beta-hydroxy-17-oxa-D-homoandrost-5-ene-16-one (10) yielded the new d-homo derivatives 12, 13 and 15. In vitro antiproliferative activity of selected compounds against three tumor cell lines (human breast adenocarcinoma ER+, MCF-7, human breast adenocarcinoma ER-, MDA-MB-231, prostate cancer AR-, PC-3, and normal fetal lung fibroblasts, MRC-5) was evaluated. Compounds 3 and 12 showed strong antiproliferative activity against PC-3 cells, the IC(50) values being 2 microM and 0.55 microM, respectively. Compounds 6 (10 microM) and 14 (9 microM) showed moderate activity against MDA-MB-231 cells. The synthesized compounds 1-3, 5-8, 10 and 12-15 were not toxic to normal fetal lung fibroblasts cells, MRC-5.

Synthesis and biological evaluation of some new A,B-ring modified steroidal D-lactones.

Starting from the D-homo lactones of androst-4-en-3-one 3 and 4, prepared from 1 and 2, the new 17a homolactones 5-12, 14 and 15, were synthesized. The 4-hydroxy compounds 9 and 10 were obtained through the reaction of 4alpha,5alpha- (5 and 7) and 4beta,5beta- (6 and 8) epoxides with formic acid. The epoxides 5 and 6 were prepared from compound 3, and epoxides 7 and 8 from compound 4 by oxidation with H(2)O(2) under basic conditions. Compound 1 served as a starting substance for obtaining lactones 11-13. Oxidation of compound 1 with m-chloroperbenzoic acid yielded 11 and 12, but compound 13 gave 14. Compound 15 was obtained from 13 by oxidation with H(2)O(2) under basic conditions. The structures of epoxides 6 and 14 were confirmed by X-ray structural analysis. Cytotoxic activity against three tumor cell lines (human breast adenocarcinoma ER+, MCF-7, human breast adenocarcinoma ER-, MDA-MB-231, and prostate cancer PC3) was evaluated. Compounds 6 and 14 showed strong activity against PC3, the IC(50) being 10.6 and 2.2 microM, respectively, whereas compounds 3 and 8 showed strong activity against MDA-MB-231 (IC(50) is 9.3 and 3.6 microM, respectively). Aromatase inhibition assay showed that the tested compounds 9, 10, and 14 possess lower activity compared to formestane.