Triterpenoids with modified A-ring as modulators of P-gp-dependent drug-resistance in cancer cells.

Semi-synthetic A-cycle modified triterpenic derivatives with A-cycle condensed with a heterocyclic fragment (compound 1) and fragmented A-ring (compound 2) were tested for cytotoxicity against several tumor cell cultures and doxorubicin (Dox)-resistant cell lines. The equal cytotoxicity of the tested compounds to the parental tumor cell lines (HBL-100, K562) and their resistant subclones (HBL-100/Dox, K562/i-S9) was revealed. The overexpression of ABCB1 (MDR1) gene and P-glycoprotein (P-gp) was confirmed for both resistant subclones of tumor cells. Compounds 1 and 2 were shown to inhibit the ABC-transporter gene expression (MDR1, MRP, MVP, and BCRP) and the transport of well-known P-gp substrate Rhodamine 123 from resistant cells. The docking of triterpenoids 1 and 2 into the drug binding site of P-gp revealed a similarity between the conformation of the tested triterpenoids and that of classical inhibitor verapamil, thus assuming these compounds to be more likely the inhibitors than the substrates of P-gp. Any tested triterpenic derivatives, when combined at non-toxic concentrations with doxorubicin, improved cytotoxic effect of the therapeutic drug against resistant subclones of tumor cells.

The biofilm formation of nontuberculous mycobacteria and its inhibition by essential oils.

Background: Nontuberculous mycobacteria (NTM) form two types of biofilms: Bottom biofilm and pellicle. The spatial distribution of cells between these types of biofilms and their dispersion into the liquid medium depends on the ratio of the nutrient components of the growth medium. The inhibition of biofilm formation by NTM can be achieved through the use of lipophilic compounds, such as essential oils (EOs). Method: The biofilm and pellicle formation of Mycobacterium smegmatis and Mycobacterium avium on four nutrient media under static conditions and in the vapors of six EOs was evaluated by conventional method. The antimycobacterial effect of EOs was also studied by the disc diffusion method. Results: The bottom biofilm and pellicle formation of NTM largely depended on the composition and availability of nutrients. Nutrient media in which NTM form powerful bottom biofilm or pellicle or both have been determined. The growth of studied NTM strains on agar was highly sensitive to the EOs of Scots pine, Atlas cedar, bergamot, and a mixture of EO of different plants. The cultivation of bacteria in the EO vapors also resulted in total suppression of the pellicle for all studied NTM strains. Conclusions: Our data clearly indicate that the carbon-nitrogen ratio is involved in the regulation of the spatial distribution of the biofilm. The preventing effect of EOs vapors, especially the synergistic action of mixture of EOs on the biofilm and pellicle formation by NTMs can be observed.

VapBC and MazEF toxin/antitoxin systems in the regulation of biofilm formation and antibiotic tolerance in nontuberculous mycobacteria.

Background: Mycobacterium smegmatis and other nontuberculous mycobacteria (NTM) are widely distributed in the environment, but a significant increase of NTM infections has taken place in the last few decades. The objective of this study was to determine the role of toxin-antitoxin (TA) vapBC and mazEF systems that act as effectors of persistence in the stress response of NTM. Methods: The growth ability and the biofilm formation of NTM were evaluated by conventional methods. Bacterial cell viability was determined using MTT staining, agar plating, or the method of limiting dilutions. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of antibiotics were estimated using broth and agar dilution methods. Results: Despite a comparable growth dynamics and biofilm formation on solid/liquid interface with the wild type, a M. smegmatis vapBC, mazEF, and vapBC × mazEF deletion mutant produced more abundant pellicle and were more susceptible to heat shock. Significant differences were also found in the resistance wild type of NTM to isoniazid and ciprofloxacin reflected by higher MBC/MIC ratios. The proposed method of cultivation of agar blocks without visible growth after MIC determination into a liquid medium allows us to detect transition of all wild type of NTM strains to a dormant state in the presence of subMICs of isoniazid and ciprofloxacin while all deletion mutants failed to form dormant cells. Conclusion: Our data suggest that both vapBC and mazEF TA systems putatively involved in the heat and antibiotic stress response of NTM via their key role in transition to the dormant state.

Synthesis, cytotoxic evaluation, and molecular docking studies of the semi-synthetic "triterpenoid-steroid" hybrids.

Synthetic transformations of steroids for drug discovery and improvement of drug effectiveness have been an important part of modern medicinal chemistry and pharmaceutical sciences. Pentacyclic triterpenoids, being represented in the nature by various structures and biogenetically related to steroids, can largely expand the spectrum of biologically active steroidal agents via synthesis of the so-called "triterpenoid-steroid" hybrids. In the presented work, the nitrile anion cyclizations of 3,4-secolupane and 3,4-seco-oleanane nitriles and follow-up synthetic transformations of the cyclized products with formation of the gemm-dimethyl-free A ring "triterpenoid-steroid" hybrids were studied. Furthermore, the resulting cyclic compounds were modified at C3, C4, and/or C5 positions of ring A, as well as at C20, C28, and C30 positions of the isopropylidene moiety in the case of lupane triterpenoids. The cytotoxic effect of the synthesized compounds against seven cancer cell lines HEp-2, HCT 116, MS, RD TE32, A549, MCF7, and PC3 was evaluated. The in silico identification of potential anticancer protein targets with regard to the compounds, which were active at micromolar concentrations against tested cell lines, was carried out. The molecular docking studies showed that compound 19, which demonstrated most pronounced cytotoxicity (IC50 0.64-3.17 µM) against all tested cell lines, fits well the active sites of CDK6 and HER2/neu.

Synthesis, modification, and cytotoxic evaluation of 2,3-secotriterpenic ß-ketoesters.

A set of ß-ketoesters was synthesized from 2,3-seco-18αH-oleanane and 2,3-secolupane bromomethyl ketones. Additionally, hydroxy derivatives with the A-seco- or five-membered A ring were obtained as a result of the reduction or of alkaline hydrolysis of acetic acid ß-ketoesters 4, 9. Cytotoxic screening revealed the compound 4 with marked activity (IC50 3.07-3.61 µM) against the HCT 116, MS, RD TE32 cancer cells. The studies of the cytotoxic mechanism enabled elucidating the fact that treatment of the HCT 116 cells with compound 4 for 18 h leads to induction of apoptosis in a dose-dependent manner. This observation was confirmed by registration of chromatin condensation, by the fluorescence increased during Annexin V-FITC staining, and by appearance of a sub-G0 peak in the cell cycle analysis with DAPI. Compound 4 also inhibited migration of cancer cells in the wound healing assay.

Plasma, serum, albumin, and divalent metal ions inhibit the adhesion and the biofilm formation of Cutibacterium (Propionibacterium) acnes.

Adhesion and biofilm formation of human skin bacteria C. acnes on plasma, serum and albumin-coated polystyrene or in the presence of these blood components were studied. The proteins which were pre-adsorbed to polystyrene surface or added to the medium simultaneously with bacterial cells reduced C. acnes adhesion and biofilm formation by 2-5 times to compare to the control. The role of calcium, magnesium and zinc on C. acnes attachment was also assessed. Calcium (1 and 10 mM) had the inhibitory effect on C. acnes adhesion, whereas zinc (1 and 10 mM) diminished the biofilm formation of C. acnes. We also observed that C. acnes cells did not bind to erythrocytes. Thus, we suggest that bacteria C. acnes preferably colonize the plasma-poor environment due to the inhibitory effect of blood components, in particular, albumin, calcium, and zinc.