Cancer Drug Resistance: Targeting Proliferation or Programmed Cell Death.

The development of resistance to chemotherapy is one of the main problems for effective cancer treatment. Drug resistance may result from disturbances in two important physiological processes-cell proliferation and cell death. Importantly, both processes characterize alterations in cell metabolism, the level of which is often measured using MTT/MTS assays. To examine resistance to chemotherapy, different cancer cell lines are usually used for the in vitro modulation of developing resistance. However, after the creation of resistant cell lines, researchers often have difficulty in starting investigations of the mechanisms of insensitivity. In the first stage, researchers should address the question of whether the drug resistance results from a depression of cell proliferation or an inhibition of cell death. To simplify the choice of research strategy, we have suggested a combination of different approaches which reveal the actual mechanism. This combination includes rapid and high-throughput methods such as the MTS test, the LIVE/DEAD assay, real-time cell metabolic analysis, and Western blotting. To create chemoresistant tumor cells, we used four different cancer cell lines of various origins and utilized the most clinically relevant pulse-selection approach. Applying a set of methodological approaches, we demonstrated that three of them were more capable of modulating proliferation to avoid the cytostatic effects of anti-cancer drugs. At the same time, one of the studied cell lines developed resistance to cell death, overcoming the cytotoxic action.

Synthetic Design and Biological Evaluation of New p53-MDM2 Interaction Inhibitors Based on Imidazoline Core.

The use of p53-MDM2 inhibitors is a prospective strategy in anti-cancer therapy for tumors expressing wild type p53 protein. In this study, we have applied a simple approach of two-step synthesis of imidazoline-based alkoxyaryl compounds, which are able to efficiently inhibit p53-MDM2 protein-protein interactions, promote accumulation of p53 and p53-inducible proteins in various cancer cell lines. Compounds 2l and 2k cause significant upregulation of p53 and p53-inducible proteins in five human cancer cell lines, one of which possesses overexpression of MDM2.

Nonresonant CARS Imaging of Porous and Solid Silicon Nanoparticles in Human Cells.

Coherent anti-Stokes Raman scattering (CARS), a nonlinear optical method for rapid visualization of biological objects, represents a progressive tool in biology and medicine to explore cells and tissue structures in living systems and biopsies. In this study, we report efficient nonresonant CARS imaging of silicon nanoparticles (SiNPs) in human cells as a proof of concept. As both bulk and porous silicon exhibit a high third-order nonlinear susceptibility, χ(3), which is responsible for the CARS intensity, it is possible to visualize the SiNPs without specific labels. Porous and solid SiNPs were obtained from layers of porous and nonporous silicon nanowires and mesoporous silicon. Electron microscopy and Raman spectroscopy showed that porous SiNPs consisted of ∼3 nm silicon nanocrystals (nc-Si) and pores, whereas solid nanoparticles comprised ∼30 nm nc-Si. All types of SiNPs were nontoxic at concentrations up to 500 µg/mL after 24 h of incubation with cells. We demonstrated that although nc-Si possesses a distinguished narrow Raman band of about 520 cm-1, it is possible to detect a high CARS signal from SiNPs in the epi-direction even in a nonresonant regime. 3D CARS images showed that all types of studied SiNPs were visualized as bright spots inside the cytoplasm of cells after 3-6 h of incubation because of the contrast provided by the high third-order nonlinear susceptibility of SiNPs, which is 1 × 104 to 1 × 105 times higher than that of water and typical biological media. Overall, CARS microscopy can provide localization of SiNPs within biological structures at the cellular level and can be a powerful tool for in vitro monitoring of silicon-based drug delivery systems or use SiNPs as labels to monitor various bioprocesses inside living cells.

Optical Monitoring of the Biodegradation of Porous and Solid Silicon Nanoparticles.

Silicon nanoparticles (SiNP) are currently of great interest, especially in biomedicine, because of their unique physicochemical properties combined with biodegradability. SiNPs can be obtained in various ways and can have either a non-porous solid (sol-) or porous (por-) structure. In this work, we carry out detailed optical monitoring of sol- and por-SiNP biodegradation using Raman and photoluminescence (PL) micro-spectroscopy. SiNPs were obtained by ultrasound grinding of sol- or por-silicon nanowires, created by silver-assisted chemical etching of crystalline Si with different doping levels. In this case, sol-SiNPs consist of nanocrystals 30 nm in size, while por-SiNPs consist of small 3 nm nanocrystals and 16 nm pores. Both SiNPs show low in vitro cytotoxicity towards MCF-7 and HEK293T cells up to 800 µg/mL. The appearance of the F-band (blue-yellow) PL, as well as a decrease in the intensity of the Raman signal, indicate the gradual dissolution of the sol-SiNPs during 20 days of incubation. At the same time, the rapid dissolution of por-SiNP within 24 h is identified by the quenching of their S-band (red) PL and the disappearance of the Raman signal. The obtained results are important for development of intelligent biodegradable drug delivery systems based on SiNPs.

Bak and Bcl-xL Participate in Regulating Sensitivity of Solid Tumor Derived Cell Lines to Mcl-1 Inhibitors.

BH3 mimetics represent a promising tool in cancer treatment. Recently, the drugs targeting the Mcl-1 protein progressed into clinical trials, and numerous studies are focused on the investigation of their activity in various preclinical models. We investigated two BH3 mimetics to Mcl-1, A1210477 and S63845, and found their different efficacies in on-target doses, despite the fact that both agents interacted with the target. Thus, S63845 induced apoptosis more effectively through a Bak-dependent mechanism. There was an increase in the level of Bcl-xL protein in cells with acquired resistance to Mcl-1 inhibition. Cell lines sensitive to S63845 demonstrated low expression of Bcl-xL. Tumor tissues from patients with lung adenocarcinoma were characterized by decreased Bcl-xL and increased Bak levels of both mRNA and proteins. Concomitant inhibition of Bcl-xL and Mcl-1 demonstrated dramatic cytotoxicity in six of seven studied cell lines. We proposed that co-targeting Bcl-xL and Mcl-1 might lead to a release of Bak, which cannot be neutralized by other anti-apoptotic proteins. Surprisingly, in Bak-knockout cells, inhibition of Mcl-1 and Bcl-xL still resulted in pronounced cell death, arguing against a sole role of Bak in the studied phenomenon. We demonstrate that Bak and Bcl-xL are co-factors for, respectively, sensitivity and resistance to Mcl-1 inhibition.

Mcl-1 as a "barrier" in cancer treatment: Can we target it now?

During the last two decades, the study of Mcl-1, an anti-apoptotic member of the Bcl-2 family, attracted researchers due to its important role in cancer cell survival and tumor development. The significance of Mcl-1 protein in resistance to chemotherapeutics makes it an attractive target in cancer therapy. Here, we discuss the diverse possibilities for indirect Mcl-1 inhibition through its downregulation, for example, via targeting for proteasomal degradation or blockage of translation and transcription. We also provide an overview of the direct blocking of protein-protein interactions with pro-apoptotic Bcl-2 family proteins, including examples of the most promising regulators of Mcl-1 and selective BH3-mimetics, which at present are under clinical evaluation. Moreover, several approaches for the co-targeting of Mcl-1 and other proteins (e.g., CDKs) are also presented. In addition, we highlight the broad spectrum of problems that accompanied the discovery and development of effective Mcl-1 inhibitors.

2,4,5-Tris(alkoxyaryl)imidazoline derivatives as potent scaffold for novel p53-MDM2 interaction inhibitors: Design, synthesis, and biological evaluation.

Imidazoline-based small molecule inhibitors of p53-MDM2 interaction intended for the treatment of p53 wild-type tumors are the promising structures for design of anticancer drugs. Based on fragment approach we have investigated a key role of substituents in cis-imidazoline core for biological activity of nutlin family compounds. Although the necessity of the substituents in the phenyl rings of cis-imidazoline has been shown, there are no studies in which the replacements of a halogen by other substituents have been investigated. A series of simple cis-imidazoline derivatives containing halogen, hydroxy and alkoxy-substituents were synthesized. The biological activity of the compounds was studied using assays of cytotoxicity (MTT) and p53 level. It was found that the hydroxyl-derivatives were not cytotoxic whereas the alkoxy analogues were the same or more active as halogen-substituted compounds in cell viability test. The synthesized alkoxy derivatives induced an increase of p53 level and did not promote necrotic cell death in the concentration up to 40 µM.