Recent Approaches Encompassing the Phenotypic Cell Heterogeneity for Anticancer Drug Efficacy Evaluation

Despite the advancements in biomarker-based personalized cancer therapy, the inadequacy of molecular and genetic profiling in identifying effective drug combinations was defined in most cases. Drug resistance remains a major limitation of the current predictive oncology. Emerging reports indicate that the success of anticancer therapy is usually limited by intratumoral heterogeneity which is not captured by the existing cancer cell biomarker-based approaches. Cell heterogeneity, not only genetic but also phenotypic, is considered to be the root cause of resistance to anticancer treatment, cancer progression, and the presence of cancer stem cells. Therefore, functional testing of live cells representing various cell types within the tumor exposed to potential therapies is needed for identification of effective drug combinations. Here we look at the different existing model systems, including ex vivo models of the patient’s tumor cells, 2D/3D in vitro cultures/cocultures, patient-derived cellular organoids, single-cell models, ex vivo tumor platforms containing tumor microenvironment and extracellular matrix, etc., scoping at drug efficacy evaluation and solving the problem of cancer resistance

Investigation of protein kinase MAPK and AKT crosstalk and dependencies on extracellular contacts in lung cancer-derived cell models.

This thesis focuses on the molecular signaling pathways that regulate cell functions. Mitogen-activated protein kinase (MAPK) and PI3K/AKT signaling molecules are responsible for cell proliferation, regulation of cell death, and participate in signal transduction from extracellular contacts. MAPK and AKT are also involved in drug-induced signaling, and disruption of these signaling pathways is common in cancer cells and is associated with resistance to chemotherapy. Unfortunately, the knowledge about regulation and interactions of the signaling pathways is incomplete yet, and there are no anticancer drugs fully successful in restoring the balance of vital signaling. A promising strategy for cancer treatment is the combination of conventional chemotherapy with targeted drugs, such as protein kinase inhibitors, to manipulate the signals induced by conventional drugs. Therefore, another topic addressed in this work is the resistance of cancer cells to treatment. We have chosen several primary non-small cell lung cancer cell lines, a commercial A549 lung adenocarcinoma cell line, and healthy, stem-like Myo cell line as the subjects of our study, as well as models of inhibition of the extracellular contacts, in combination with the use of various protein kinase inhibitors (PI3K – LY294002, MEK – PD98059, p38 – SB203580, JNK –SP600125, etc.) and approved drugs (MEK inhibitor selumetinib, AKT inhibitor capivasertib, etc.). We investigated whether/how MAPK and AKT participate in the death of adherent cells in "weightless condition", i.e., in the anoikis process, as well as the interdependence of MAPK and AKT activity when the cells are treated with kinase inhibitors and the conventional drug cisplatin. Our studies suggest that, irrespective of cell origin/cell type, the basal phosphorylation level of the protein kinases studied is dependent on extracellular contacts. Among other results, we also showed that despite differences in cell phenotype or genotype, inhibition of the PI3K/AKT pathway promoted activation of the MAP kinase ERK and, vice versa, inhibition of the MEK/ERK pathway increased the level of phosphorylated AKT. This phenomenon was confirmed in both control and cisplatin-treated cells, also using different inhibitors of these kinases. Importantly, the ERK-AKT interaction is dependent on cell-substratum contacts and/or the kinase FAK. Since the activity of signaling molecules regulating cell fate is a principal factor in predicting cell behavior as well as response to therapy, this work highlights the dependence of the self-regulation and the response to the targeted drugs on the cellular state, proposing different molecular strategies for compact (tumor) and circulating cancer cells

Ethics of human therapeutic cloning

The article discusses the ethical problems of human cloning for therapeutic purposes. The therapeutic cloning of man today is restricted only by the incompleteness of technology. We are rapidly approaching the start of the cloning of people for "therapeutic purposes", i.e. organs, vitally necessary for sick persons, will begin to be grown "in vivo" or under laboratory conditions. However, this noble purpose is not justifiable from a moral, Christian point of view first of all because it is impossible to d o this without killing already begun life and requires the nonreturnable destruction of a developing person to get the organs. Often artificial insemination is also needed for this as well as unethical methods of acquiring ova. At the present time, in order to clone an organ for a sick person it is necessary to murder three live persons: the unborn donor of the ovum, the embryo, and the final clone of the patient

Cell death and its different modes: history of understanding and current trends

Discussions about what is life continue to struggle; there are pros and cons for whether a virus is alive. However, an opposite thing – cell death – appears to be tantamount important and equally not-easygoing to define. Nevertheless, our current knowledge about eukaryotic cell death has made a long way and resulted in a fruitful outcome: starting from three types of cell death (type I, II and III which are mainly applicable to eukaryotic cells of organisms from the biological kingdom animalia) in 1970s, Nomenclature Committee on Cell Death has named already twelve cell death forms in 2018, including the above mentioned apoptosis, autophagy and necrosis among them. How the scientific attitude towards cellular demise evolved and various aspects of different cell death modes are reviewed in this article

The role and efficacy of JNK inhibition in inducing lung cancer cell death depend on the concentration of cisplatin /

Toxicity and the emergence of resistance are the main challenges in cancer treatment. The optimal dose of cisplatin, one of the most widely used chemotherapeutic anticancer drugs, is currently being widely debated. Furthermore, the dose-dependent molecular mechanisms of its action are poorly understood. To assess the role of protein kinase JNK (cJun N-terminal kinase) signaling in lung cancer treatment, we combined small-molecule JNK inhibitors and cisplatin. Wild-type p53 (tumor suppressor transcription factor TP53) and mutated RAS-bearing lung adenocarcinoma cell line A549 was used as a model in our studies. Here, we demonstrate cisplatin concentration-dependent opposing roles of JNK in killing cancer cells: a cell-protective role at low cisplatin concentrations and an apoptosis-promoting (or neutral) role at high concentrations. Time- and dose-dependent activation of pro-survival protein kinase AKT and TP53 was shown, with similar activation dynamics in cells exposed to different (low and high) cisplatin concentrations. Selective inhibition of AKT and activation of TP53 (expression and phosphorylation) led to a decrease in cell survival, indicating their involvement in cisplatin-induced cell death regulation. The activation levels of TP53 and AKT in cisplatin-treated A549 cells after cotreatment with the JNK inhibitor SP600125 correlated with their role in regulating cell death. TP53 and AKT were proposed as signaling proteins mediating the outcome of JNK inhibition in A549 cells exposed to different concentrations of cisplatin. Our findings suggest that a combination of stress kinase JNK inhibition and low-dose cisplatin, together with manipulation of drug-induced signaling, could be considered as a promising treatment strategy for certain lung cancers

DataSheet_1_Crosstalk between protein kinases AKT and ERK1/2 in human lung tumor-derived cell models.pdf

There is no doubt that cell signaling manipulation is a key strategy for anticancer therapy. Furthermore, cell state determines drug response. Thus, establishing the relationship between cell state and therapeutic sensitivity is essential for the development of cancer therapies. In the era of personalized medicine, the use of patient-derived ex vivo cell models is a promising approach in the translation of key research findings into clinics. Here, we were focused on the non-oncogene dependencies of cell resistance to anticancer treatments. Signaling-related mechanisms of response to inhibitors of MEK/ERK and PI3K/AKT pathways (regulators of key cellular functions) were investigated using a panel of patients’ lung tumor-derived cell lines with various stemness- and EMT-related markers, varying degrees of ERK1/2 and AKT phosphorylation, and response to anticancer treatment. The study of interactions between kinases was the goal of our research. Although MEK/ERK and PI3K/AKT interactions are thought to be cell line-specific, where oncogenic mutations have a decisive role, we demonstrated negative feedback loops between MEK/ERK and PI3K/AKT signaling pathways in all cell lines studied, regardless of genotype and phenotype differences. Our work showed that various and distinct inhibitors of ERK signaling – selumetinib, trametinib, and SCH772984 – increased AKT phosphorylation, and conversely, inhibitors of AKT – capivasertib, idelalisib, and AKT inhibitor VIII – increased ERK phosphorylation in both control and cisplatin-treated cells. Interaction between kinases, however, was dependent on cellular state. The feedback between ERK and AKT was attenuated by the focal adhesion kinase inhibitor PF573228, and in cells grown in suspension, showing the possible role of extracellular contacts in the regulation of crosstalk between kinases. Moreover, studies have shown that the interplay between MEK/ERK and PI3K/AKT signaling pathways may be dependent on the strength of the chemotherapeutic stimulus. The study highlights the importance of spatial location of the cells and the strength of the treatment during anticancer therapy.</p