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.

A matter of new life and cell death: programmed cell death in the mammalian ovary.

BACKGROUND: The mammalian ovary is a unique organ that displays a distinctive feature of cyclic changes throughout the entire reproductive period. The estrous/menstrual cycles are associated with drastic functional and morphological rearrangements of ovarian tissue, including follicular development and degeneration, and the formation and subsequent atrophy of the corpus luteum. The flawless execution of these reiterative processes is impossible without the involvement of programmed cell death (PCD). MAIN TEXT: PCD is crucial for efficient and careful clearance of excessive, depleted, or obsolete ovarian structures for ovarian cycling. Moreover, PCD facilitates selection of high-quality oocytes and formation of the ovarian reserve during embryonic and juvenile development. Disruption of PCD regulation can heavily impact the ovarian functions and is associated with various pathologies, from a moderate decrease in fertility to severe hormonal disturbance, complete loss of reproductive function, and tumorigenesis. This comprehensive review aims to provide updated information on the role of PCD in various processes occurring in normal and pathologic ovaries. Three major events of PCD in the ovary-progenitor germ cell depletion, follicular atresia, and corpus luteum degradation-are described, alongside the detailed information on molecular regulation of these processes, highlighting the contribution of apoptosis, autophagy, necroptosis, and ferroptosis. Ultimately, the current knowledge of PCD aberrations associated with pathologies, such as polycystic ovarian syndrome, premature ovarian insufficiency, and tumors of ovarian origin, is outlined. CONCLUSION: PCD is an essential element in ovarian development, functions and pathologies. A thorough understanding of molecular mechanisms regulating PCD events is required for future advances in the diagnosis and management of various disorders of the ovary and the female reproductive system in general.

The New Face of Autophagy in Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) mainly afflicts adults and accounts for 25% of all new leukemia cases [...].

Prognostic signature based on mitochondria quality control proteins for the prediction of lung adenocarcinoma patients survival.

Lung cancer is the leading cause of cancer mortality worldwide. In recent years, the incidence of lung cancer subtype lung adenocarcinoma (LUAD) has steadily increased. Mitochondria, as a pivotal site of cell bioenergetics, metabolism, cell signaling, and cell death, are often dysregulated in lung cancer cells. Mitochondria maintenance and integrity depend on mitochondrial quality control proteins (MQCPs). During lung cancer progression, the levels of MQCPs could change and promote cancer cell adaptation to the microenvironment and stresses. Here, univariate and multivariate proportional Cox regression analyses were applied to develop a signature based on the level of MQCPs (dimeric form of BNIP3, DRP1, and SIRT3) in tumorous and non-tumorous samples of 80 patients with LUAD. The MQCP signature could be used to separate the patients with LUAD into high- and low-risk groups. Survival analysis indicated that patients in the high-risk group had dramatically shorter overall survival compared with the low-risk patients. Moreover, a nomogram combining clinicopathologic features and the MQCP signature was constructed and validated to predict 1-, 3-, and 5-year overall survival of the patients. Thus, this study presents a novel signature based on MQCPs as a reliable prognostic tool to predict overall survival for patients with LUAD.

Drug toxicity assessment: cell proliferation versus cell death.

Analysis of the toxicity of chemotherapeutic drugs is one of the main tasks of clinical pharmacology. Decreased viability of tumor cells may reflect two important physiological processes, namely the arrest of proliferation associated with disturbances in cellular metabolism or actual cell death. Elucidation of the exact processes mediating a reduction in the number of cells is fundamentally important to establish the mechanisms of drug action. Only the use of a combination of cell biological and biochemical approaches makes it possible to understand these mechanisms. Here, using various lines of tumor cells and a set of methodological approaches, we carried out a detailed comparative analysis and demonstrated the possible ways to overcome the uncertainties in establishing the mechanisms of cell response to the action of chemotherapeutic drugs and their toxicity.

Exploring Caspase Mutations and Post-Translational Modification by Molecular Modeling Approaches.

Apoptosis is a type of programmed cell death that eliminates damaged cells and controls the development and tissue homeostasis of multicellular organisms. Caspases, a family of cysteine proteases, play a key role in apoptosis initiation and execution. The maturation of caspases and their activity is fine-tuned by post-translational modifications in a highly dynamic fashion. To assess the effect of post-translational changes, potential sites are routinely mutated with residues persistent to any modifications. For example, the serine residue is replaced with alanine or aspartic acid. However, such substitutions could alter the caspase active site's conformation, leading to disturbances in catalytic activity and cellular functions. Moreover, mutations of other amino acid residues located in critical positions could also break the structure and functions of caspases and lead to apoptosis perturbation. To avoid the difficulties of employing mutated residues, molecular modeling approaches can be readily applied to estimate the potential effect of amino acid substitutions on caspase structure. The present protocol allows the modeling of both the wild-type caspase and its mutant forms with the biomolecular simulation package (Amber) and supercomputer facilities to test the effect of mutations on the protein structure and function.

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.

A Balance Between Autophagy and Other Cell Death Modalities in Cancer.

Autophagy is an intracellular self-digestive process involved in catabolic degradation of damaged proteins, and organelles, and the elimination of cellular pathogens. Initially, autophagy was considered as a prosurvival mechanism, but the following insights shed light on its prodeath function. Nowadays, autophagy is established as a crucial player in the development of various diseases through interaction with other molecular pathways within a cell. Additionally, disturbance in autophagy is one of the main pathological alterations that lead to resistance of cancer cells to treatment. These autophagy-related pathologies gave rise to the development of new therapeutic drugs. Here, we summarize the current knowledge on the autophagic role in disease pathogenesis, particularly in cancer, and the interplay between autophagy and other cell death modalities in order to combat cancer.

A link between mitotic defects and mitotic catastrophe: detection and cell fate.

Although the phenomenon of mitotic catastrophe was first described more than 80 years ago, only recently has this term been used to explain a mechanism of cell death linked to delayed mitosis. Several mechanisms have been suggested for mitotic catastrophe development and cell fate. Depending on molecular perturbations, mitotic catastrophe can end in three types of cell death, namely apoptosis, necrosis, or autophagy. Moreover, mitotic catastrophe can be associated with different types of cell aging, the development of which negatively affects tumor elimination and, consequently, reduces the therapeutic effect. The effective triggering of mitotic catastrophe in clinical practice requires induction of DNA damage as well as inhibition of the molecular pathways that regulate cell cycle arrest and DNA repair. Here we discuss various methods to detect mitotic catastrophe, the mechanisms of its development, and the attempts to use this phenomenon in cancer treatment.

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.

Anastasis: Return Journey from Cell Death.

For over 20 years, it has been a dogma that once the integrity of mitochondria is disrupted and proapoptotic proteins that are normally located in the intermembrane space of mitochondria appeared in the cytoplasm, the process of cell death becomes inevitable. However, it has been recently shown that upon removal of the death signal, even at the stage of disturbance in the mitochondria, cells can recover and continue to grow. This phenomenon was named anastasis. Here, we will critically discuss the present knowledge concerning the mechanisms of cell death reversal, or development of anastasis, methods for its detection, and what role signaling from different intracellular compartments plays in anastasis stimulation.

Platinum drugs and taxanes: can we overcome resistance?

Cancer therapy is aimed at the elimination of tumor cells and acts via the cessation of cell proliferation and induction of cell death. Many research publications discussing the mechanisms of anticancer drugs use the terms "cell death" and "apoptosis" interchangeably, given that apoptotic pathways are the most common components of the action of targeted and cytotoxic compounds. However, there is sound evidence suggesting that other mechanisms of drug-induced cell death, such as necroptosis, ferroptosis, autophagy, etc. may significantly contribute to the fate of cancer cells. Molecular cross-talks between apoptotic and nonapoptotic death pathways underlie the successes and the failures of therapeutic interventions. Here we discuss the nuances of the antitumor action of two groups of the widely used anticancer drugs, i.e., platinum salts and taxane derivatives. The available data suggest that intelligent interference with the choice of cell death pathways may open novel opportunities for cancer treatment.

Long non-coding RNAs: A view to kill ovarian cancer.

An emerging role of long non-coding RNAs (lncRNAs) in tumor progression has been revealed in the last decade. Through interactions with nucleic acids and proteins, lncRNAs could act as enhancers, scaffolds or decoys for a number of oncoproteins and tumor suppressors. The aberrant lncRNA expression or mutations are often associated with changes in a variety of cellular processes, including proliferation, stress response and cell death. Here, we will focus on the tumor-associated lncRNAs in ovarian cancer according to their contribution to cancer hallmarks, such as intense proliferation, cell death resistance, altered energy metabolism, invasion and metastasis, and immune evasion. Moreover, the potential clinical implications of lncRNAs and their significance for the diagnosis, prognosis and therapy of ovarian cancer will be discussed.

Simple and Efficient Protocol for Subcellular Fractionation of Normal and Apoptotic Cells.

Subcellular fractionation approaches remain an indispensable tool among a large number of biochemical methods to facilitate the study of specific intracellular events and characterization of protein functions. During apoptosis, the best-known form of programmed cell death, numerous proteins are translocated into and from the nucleus. Therefore, suitable biochemical techniques for the subcellular fractionation of apoptotic cells are required. However, apoptotic bodies and cell fragments might contaminate the fractions upon using the standard protocols. Here, we compared different nucleus/cytoplasm fractionation methods and selected the best-suited approach for the separation of nuclear and cytoplasmic contents. The described methodology is based on stepwise lysis of cells and washing of the resulting nuclei using non-ionic detergents, such as NP-40. Next, we validated this approach for fractionation of cells treated with various apoptotic stimuli. Finally, we demonstrated that nuclear fraction could be further subdivided into nucleosolic and insoluble subfractions, which is crucial for the isolation and functional studies of various proteins. Altogether, we developed a method for simple and efficient nucleus/cytoplasm fractionation of both normal and apoptotic cells.

Caspase-2 as a master regulator of genomic stability.

Genomic instability underlies genesis and the development of various types of cancer. During tumorigenesis, cancer initiating cells assume a set of features, which allow them to survive and proliferate. Different mutations and chromosomal alterations promote a selection of the most aggressive cancer clones that worsen the prognosis of the disease. Despite that caspase-2 was described as a protease fulfilling an initiator and an effector function in apoptosis, it has recently been discovered to play an important role in the maintenance of genomic integrity and normal chromosome configuration. This protein is able to stabilize p53 and affect the level of transcription factors, which activates cell response to oxidative stress. Here we focus on the discussion on the mechanism(s) of how caspase-2 regulates genomic stability and decreases tumorigenesis.

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.

Upregulation of Mcl-1S Causes Cell-Cycle Perturbations and DNA Damage Accumulation.

As an important regulator of apoptosis, Mcl-1 protein, a member of the Bcl-2 family, represents an attractive target for cancer treatment. The recent development of novel small molecule compounds has allowed Mcl-1-inhibitory therapy to proceed to clinical trials in cancer treatment. However, the possible adverse effects of either direct inhibition of Mcl-1 or upregulation of Mcl-1S, proapoptotic isoform resulting from alternative splicing of Mcl-1, remain unclear. Here, we investigated changes in Mcl-1S levels during cell cycle and the cell cycle-related functions of Mcl-1 isoforms to address the above-mentioned concerns. It was shown that an anti-mitotic agent monastrol caused accumulation of Mcl-1S mRNA, although without increasing the protein level. In contrast, both mRNA and protein levels of Mcl-1S accrued during the premitotic stages of the normal cell cycle progression. Importantly, Mcl-1S was observed in the nuclear compartment and an overexpression of Mcl-1S, as well as knockdown of Mcl-1, accelerated the progression of cells into mitosis and resulted in DNA damage accumulation. Surprisingly, a small molecule inhibitor of Mcl-1, BH3-mimetic S63845, did not affect the cell cycle progression or the amount of DNA damage. In general, upregulated Mcl-1S protein or genetically inhibited Mcl-1L were associated with the cell cycle perturbations and DNA damage accumulation in normal and cancer cells. At the same time, BH3-mimetic to Mcl-1 did not affect the cell cycle progression, suggesting that direct inhibition of Mcl-1 is devoid of cell-cycle related undesired effects.

Requirement for Serine-384 in Caspase-2 processing and activity.

Caspase-2 is a unique and conservative cysteine protease which plays an important role in several cellular processes including apoptotic cell death. Although the molecular mechanisms of its activation remain largely unclear, a major role belongs to the architecture of the caspase-2 active center. We demonstrate that the substitution of the putative phosphorylation site of caspase-2, Serine-384 to Alanine, blocks caspase-2 processing and decreases its enzymatic activity. Strikingly, in silico analysis using molecular dynamics simulations has shown that Serine-384 is crucially involved in interactions within the caspase-2 active center. It stabilizes Arginine-378, which forms a crucial hydrogen bond with the aspartate residue of a substrate. Hence, Serine-384 is essential for supporting a proper architecture of the active center of caspase-2. Moreover, molecular modeling strongly proved steric inaccessibility of Ser-384 to be phosphorylated. Importantly, a multiple alignment has demonstrated that both Serine-384 and Arg-378 residues are highly conservative across all members of caspase family, which allows us to suggest that this diade is indispensable for caspase processing and activity. Spontaneous mutations in this diade might influence oncosuppressive function of caspases, in particular of caspase-2. Likewise, the mutation of Ser-384 is associated with the development of lung squamous cell carcinoma and adenocarcinoma. Taken together, we have uncovered a central feature of the caspase-2 activation mechanism which is crucial for the regulation of its signaling network.

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.

Saga of Mcl-1: regulation from transcription to degradation.

The members of the Bcl-2 family are the central regulators of various cell death modalities. Some of these proteins contribute to apoptosis, while others counteract this type of programmed cell death, thus balancing cell demise and survival. A disruption of this balance leads to the development of various diseases, including cancer. Therefore, understanding the mechanisms that underlie the regulation of proteins of the Bcl-2 family is of great importance for biomedical research. Among the members of the Bcl-2 family, antiapoptotic protein Mcl-1 is characterized by a short half-life, which renders this protein highly sensitive to changes in its synthesis or degradation. Hence, the regulation of Mcl-1 is of particular scientific interest, and the study of Mcl-1 modulators could aid in the understanding of the mechanisms of disease development and the ways of their treatment. Here, we summarize the present knowledge regarding the regulation of Mcl-1, from transcription to degradation, focusing on aspects that have not yet been described in detail.