Alterations of receptors and insulin-like growth factor binding proteins in senescent cells.

The knowledge about cellular senescence expands dynamically, providing more and more conclusive evidence of its triggers, mechanisms, and consequences. Senescence-associated secretory phenotype (SASP), one of the most important functional traits of senescent cells, is responsible for a large extent of their context-dependent activity. Both SASP's components and signaling pathways are well-defined. A literature review shows, however, that a relatively underinvestigated aspect of senescent cell autocrine and paracrine activity is the change in the production of proteins responsible for the reception and transmission of SASP signals, i.e., receptors and binding proteins. For this reason, we present in this article the current state of knowledge regarding senescence-associated changes in cellular receptors and insulin-like growth factor binding proteins. We also discuss the role of these alterations in senescence induction and maintenance, pro-cancerogenic effects of senescent cells, and aging-related structural and functional malfunctions.

Mechanisms of carboplatin- and paclitaxel-dependent induction of premature senescence and pro-cancerogenic conversion of normal peritoneal mesothelium and fibroblasts.

Carboplatin (CPT) and paclitaxel (PCT) are the optimal non-surgical treatment of epithelial ovarian cancer (EOC). Although their growth-restricting influence on EOC cells is well known, their impact on normal peritoneal cells, including mesothelium (PMCs) and fibroblasts (PFBs), is poorly understood. Here, we investigated whether, and if so, by what mechanism, CPT and PCT induce senescence of omental PMCs and PFBs. In addition, we tested whether PMC and PFB exposure to the drugs promotes the development of a pro-cancerogenic phenotype. The results showed that CPT and PCT induce G2/M growth arrest-associated senescence of normal peritoneal cells and that the strongest induction occurs when the drugs act together. PMCs senesce telomere-independently with an elevated p16 level and via activation of AKT and STAT3. In PFBs, telomeres shorten along with an induction of p21 and p53, and their senescence proceeds via the activation of ERK1/2. Oxidative stress in CPT + PCT-treated PMCs and PFBs is extensive and contributes causatively to their premature senescence. Both PMCs and PFBs exposed to CPT + PCT fuel the proliferation, migration, and invasion of established (A2780, OVCAR-3, SKOV-3) and primary EOCs, and this activity is linked with an overproduction of multiple cytokines altering the cancer cell transcriptome and controlled by p38 MAPK, NF-κB, STAT3, Notch1, and JAK1. Collectively, our findings indicate that CPT and PCT lead to iatrogenic senescence of normal peritoneal cells, which paradoxically and opposing therapeutic needs alters their phenotype towards pro-cancerogenic. It cannot be excluded that these adverse outcomes of chemotherapy may contribute to EOC relapse in the case of incomplete tumor eradication and residual disease initiation. © 2023 The Pathological Society of Great Britain and Ireland.

Serum starvation-based method of ovarian cancer cell dormancy induction and termination in vitro.

Awakening and growth reinitiation by dormant cells may contribute to epithelial ovarian cancer (EOC) relapse. The links between these phenomena are loose because of the limited stock of compelling models of EOC dormancy. Here, we show a simple and convenient dormancy research protocol based on serum starvation. This study was conducted on established EOC cell lines A2780, OVCAR-3, and SKOV-3, as well as on primary EOC cells. Cell growth arrest and proliferation were monitored by assessing the Ki67 antigen, PKH26 fluorescence, and cell cycle distribution. In addition, cells were tested for ERK1/2/p38 MAPK activity ratio, apoptosis, and senescence. The study showed that 72-h serum starvation induces G0/G1 growth arrest of a significant fraction of cells, accompanied by reduced Ki67 and ERK1/2/p38 MAPK activity ratio, without signs of apoptosis or cellular senescence. Moreover, providing cells with 72 h of a medium enriched in 5% serum allows the culture to regain its proliferative potential. At the same time, we attempted to induce and terminate dormancy with Mitomycin C addition and withdrawal, which were unsuccessful. In conclusion, serum starvation is a convenient way to reliably induce dormancy in EOC cells, allowing them to be efficiently awakened for further mechanistic research in vitro.

An integrative review of nonobvious puzzles of cellular and molecular cardiooncology.

Oncologic patients are subjected to four major treatment types: surgery, radiotherapy, chemotherapy, and immunotherapy. All nonsurgical forms of cancer management are known to potentially violate the structural and functional integrity of the cardiovascular system. The prevalence and severity of cardiotoxicity and vascular abnormalities led to the emergence of a clinical subdiscipline, called cardiooncology. This relatively new, but rapidly expanding area of knowledge, primarily focuses on clinical observations linking the adverse effects of cancer therapy with deteriorated quality of life of cancer survivors and their increased morbidity and mortality. Cellular and molecular determinants of these relations are far less understood, mainly because of several unsolved paths and contradicting findings in the literature. In this article, we provide a comprehensive view of the cellular and molecular etiology of cardiooncology. We pay particular attention to various intracellular processes that arise in cardiomyocytes, vascular endothelial cells, and smooth muscle cells treated in experimentally-controlled conditions in vitro and in vivo with ionizing radiation and drugs representing diverse modes of anti-cancer activity.

Correction: Pakula et al. Deciphering the Molecular Mechanism of Spontaneous Senescence in Primary Epithelial Ovarian Cancer Cells. Cancers 2020, 12, 296.

In the original publication [...].

Pro-cancerogenic effects of spontaneous and drug-induced senescence of ovarian cancer cells in vitro and in vivo: a comparative analysis.

BACKGROUND: Clinical outcomes of cancer cell senescence are still elusive. Here, we reveal and compare pro-cancerous activity of spontaneously and drug-inducible senescent ovarian cancer cells. Experiments were performed on tumors and tumor-derived primary epithelial ovarian cancer cells (pEOCs) that were obtained from chemotherapy-naïve patients and from patients who received carboplatin (CPT) and paclitaxel (PCT) before cytoreduction. RESULTS: The analysis of tumors showed that senescent cancer cells are present in patients from both groups, albeit most frequently and covering a greater area in tissues from chemotherapy-positive women. This in vivo senescence of pEOCs translated to an expression of senescence markers in early-passage cells in vitro. A conditioned medium from senescent pEOCs fueled the cancer progression, including adhesion of non-senescent pEOCs to normal peritoneal cells, and their increased proliferation, migration, invasion, and EMT. Senescent pEOCs' secretome promoted angiogenic activity of vascular endothelium, induced senescence of normal peritoneal cells, reprogrammed their secretome towards hypersecretion of cancer-promoting proteins, and stimulated motility of cancer cells subjected to a mesothelium- and fibroblast-derived medium. The most striking finding was, however, that spontaneously senescent pEOCs supported all the above pro-cancerous effects more efficiently than drug-inducible senescent cells, which was plausibly related to augmented release of several cancer spread mediators by these cells. The prevalence of spontaneously senescent pEOCs was most evident in experiments on mice when they were able, unlike the drug-inducible cells, to promote the development of drug-sensitive i.p. xenografts. CONCLUSIONS: Our study shows that spontaneous senescence of pEOCs should be treated as an independent pathogenetic factor of cancer progression.

The functional multipotency of transforming growth factor ß signaling at the intersection of senescence and cancer.

The transforming growth factor ß (TGF-ß) family of cytokines comprises a group of proteins, their receptors, and effector molecules that, in a coordinated manner, modulate a plethora of physiological and pathophysiological processes. TGF-ß1 is the best known and plausibly most active representative of this group. It acts as an immunosuppressant, contributes to extracellular matrix remodeling, and stimulates tissue fibrosis, differentiation, angiogenesis, and epithelial-mesenchymal transition. In recent years, this cytokine has been established as a vital regulator of organismal aging and cellular senescence. Finally, the role of TGF-ß1 in cancer progression is no longer in question. Because this protein is involved in so many, often overlapping phenomena, the question arises whether it can be considered a molecular bridge linking some of these phenomena together and governing their reciprocal interactions. In this study, we reviewed the literature from the perspective of the role of various TGF-ß family members as regulators of a complex mutual interplay between senescence and cancer. These aspects are then considered in a broader context of remaining TGF-ß-related functions and coexisting processes. The main narrative axis in this work is centered around the interaction between the senescence of normal peritoneal cells and ovarian cancer cells. The discussion also includes examples of TGF-ß activity at the interface of other normal and cancer cell types.

Where does cellular senescence belong in the pathophysiology of ovarian cancer?

Although ovarian cancer is the leading cause of death from gynecological malignancies, there are still some issues that hamper accurate interpretation of the complexity of cellular and molecular events underlying the pathophysiology of this disease. One of these is cellular senescence, which is the process whereby cells irreversibly lose their ability to divide and develop a phenotype that fuels a variety of age-related diseases, including cancer. In this review, various aspects of cellular senescence associated with intraperitoneal ovarian cancer metastasis are presented and discussed, including mechanisms of senescence in normal peritoneal mesothelial cells; the role of senescent mesothelium in ovarian cancer progression; the effect of drugs commonly used as first-line therapy in ovarian cancer patients on senescence of normal cells; mechanisms of spontaneous senescence in ovarian cancer cells; and, last but not least, other pharmacologic strategies to induce senescence in ovarian malignancies. Collectively, this study shows that cellular senescence is involved in several aspects of ovarian cancer pathobiology. Proper understanding of this phenomenon, particularly its clinical relevance, seems to be critical for oncology patients from both therapeutic and prognostic perspectives.

Primary high-grade serous ovarian cancer cells are sensitive to senescence induced by carboplatin and paclitaxel in vitro.

BACKGROUND: Various types of normal and cancer cells undergo senescence in response to carboplatin and paclitaxel, which are considered the gold standard treatments in ovarian cancer management. Surprisingly, the effect of these drugs on ovarian cancer cell senescence remained unknown. METHODS: The experiments were conducted on primary high-grade serous ovarian cancer cells. Molecular markers of senescence were evaluated using cytochemistry and immunofluorescence. Cell cycle distribution was analyzed using flow cytometry. Expression of cyclins and signaling pathways was tested using western blot. Telomere length and telomerase activity were measured using qPCR, and the colocalization of telomeres with DNA damage foci using immuno-FISH. Oxidative stress-related parameters were quantified using appropriate fluorescence probes. Production of cancerogenic agents was analyzed using qPCR and ELISA. RESULTS: Carboplatin applied with paclitaxel induces senescence of ovarian cancer cells in vitro. This activity was reflected by permanent G2/M growth arrest, a high fraction of cells expressing senescence biomarkers (SA-ß-Gal and γ-H2A.X), upregulated expression of p16, p21, and p53 cell cycle inhibitors, and decreased expression of cyclin B1. Neither telomere length nor telomerase activity changed in the senescent cells, and the majority of DNA damage was localized outside telomeres. Moreover, drug-treated cancer cells exhibited increased production of STAT3 protein, overproduced superoxide and peroxides, and increased mitochondrial mass. They were also characterized by upregulated ANG1, CCL11, IL-6, PDGF-D, TIMP-3, TSP-1, and TGF-ß1 at the mRNA and/or protein level. CONCLUSIONS: Our findings imply that conventional chemotherapy may elicit senescence in ovarian cancer cells, which may translate to the development of a cancer-promoting phenotype, despite the inability of these cells to divide.

Patient-Specific Variables Determine the Extent of Cellular Senescence Biomarkers in Ovarian Tumors In Vivo.

The mechanisms and clinical significance of the cellular senescence of tumor cells are a matter of ongoing debate. Recently, the triggers and molecular events underlying spontaneous, replicative senescence of primary epithelial ovarian cancer cells were characterized. In this study, we reanalyzed tumors obtained from ovarian cancer patients with respect to the expression of the senescence biomarkers SA-ß-Gal and γ-H2A.X and the proliferative antigen Ki67. The results showed that the tumors displayed strong heterogeneity with respect to the expression of analyzed markers. The expression of SA-ß-Gal and γ-H2A.X in the oldest patients (61-85 y.o.) was significantly higher than in the younger age groups. Conversely, the area of Ki67-positive cancer cells was greater in younger individuals. At the same time, there was a positive correlation between SA-ß-Gal expression and calendar age in FIGO III-IV and malignant ascites-positive patients. The γ-H2A.X positively correlated with age in the whole group, FIGO III-IV, and ascites-positive patients. Ki67 levels correlated negatively with the age of patients among those same groups. Collectively, our study indicated that organismal aging may determine the development of the senescence phenotype in ovarian tumors, particularly in patients with advanced disease and those accumulating malignant ascites.

Molecular Biology of Ovarian Cancer: From Mechanisms of Intraperitoneal Metastasis to Therapeutic Opportunities.

Ovarian cancer (OC) is one of the most frequent malignancies of the female genital tract, and is still the leading cause of death from gynecological tumors [...].

Malignant Ascites Promote Adhesion of Ovarian Cancer Cells to Peritoneal Mesothelium and Fibroblasts.

Although malignant ascites (MAs) are known to contribute to various aspects of ovarian cancer progression, knowledge regarding their role in the adhesion of cancer cells to normal peritoneal cells is incomplete. Here, we compared the effect of MAs and benign ascites (BAs) on the adhesion of A2780 and OVCAR-3 cancer cells to omentum-derived peritoneal mesothelial cells (PMCs) and peritoneal fibroblasts (PFBs). The results showed that MAs stimulated the adhesion of A2780 and OVCAR-3 cells to PMCs and PFBs more efficiently than did BAs, and the strongest binding occurred when both cancer and normal cells were exposed to the fluid. Intervention studies showed that MAs-driven adhesion of A2780 cells to PMCs/PFBs depends on the presence of TGF-ß1 and HGF, whereas binding of OVCAR-3 cells was mediated by TGF-ß1, GRO-1, and IGF-1. Moreover, MAs upregulated α5ß1 integrin expression on PFBs but not on PMCs or cancer cells, vimentin expression in all cells tested, and ICAM-1 only in cancer cells. When integrin-linked kinase was neutralized in PMCs or PFBs, cancer cell adhesion to PMCs and PFBs decreased. Collectively, our report shows that MAs may contribute to the early stages of ovarian cancer metastasis by modulating the proadhesive interplay between normal and cancer cells.

Aging correlates with lower threshold cycle values for RdRP/RdRP+S genes during molecular detection of SARS-CoV-2.

RT-qPCR is the most reliable molecular method for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we analyzed results of RT-qPCR obtained for 3044 patients diagnosed as SARS-CoV-2-positive using four different molecular tests utilizing five RNA sequences. The analysis showed that patients' age inversely correlates with threshold cycle (Ct) values for RdRP gene (LightMix® Modular Wuhan CoV RdRP-gene by Roche Diagnostics) and RdRP+S genes (MutaPLEX® Coronavirus RT-PCR kit by Immundiagnostic). At the same time, there was no correlation between age and Ct values for E, N, and ORF1ab genes. When patients were grouped by age, mean Ct values for RdRP gene in older patients were significantly lower compared with younger individuals. Collectively, our report indicates that older SARS-CoV-2-infected individuals exhibit higher viremia at diagnosis than younger patients, which may reflect impaired functioning of their immune response and predispose to more severe disease and worse prognosis.

Diverse functional responses to high glucose by primary and permanent hybrid endothelial cells in vitro.

Various types of human endothelial cells, including human umbilical vein endothelial cells (HUVECs) and the established hybrid EAhy926 cells, are used in experimental research. Here, we compared the biological properties of HUVECs and EAhy926 cells under normal (5 mM) and high glucose (30 mM; HG) conditions. The results showed that HG induced cellular senescence and a stronger DNA damage response in HUVECs than in EAhy926 cells. The magnitude of oxidative stress elicited in HUVECs by HG was also greater than that elicited in their established counterparts. Both endothelial cell types promoted the progression of breast (MCF7), ovarian (OVCAR-3), and lung (A549) cancer cells; however, the effects elicited by HG-treated HUVECs on adhesion (MCF7, OVCAR-3), proliferation (OVCAR-3), and migration (OVCAR-3) were more pronounced. Finally, HG stimulated the production of a higher number of proangiogenic agents in HUVECs than in EAhy926 cells. Collectively, our study shows that the functional properties of primary and established endothelial cells exposed to HG differ substantially, which seems to result from the higher sensitivity of the former to this stressor. The interchangeability of both types of endothelial cells in biomedical research should be considered with great care to avoid losing some biological effects due to the choice of cells with higher stress tolerance.

Nontraditional systems in aging research: an update.

Research on the evolutionary and mechanistic aspects of aging and longevity has a reductionist nature, as the majority of knowledge originates from experiments on a relatively small number of systems and species. Good examples are the studies on the cellular, molecular, and genetic attributes of aging (senescence) that are primarily based on a narrow group of somatic cells, especially fibroblasts. Research on aging and/or longevity at the organismal level is dominated, in turn, by experiments on Drosophila melanogaster, worms (Caenorhabditis elegans), yeast (Saccharomyces cerevisiae), and higher organisms such as mice and humans. Other systems of aging, though numerous, constitute the minority. In this review, we collected and discussed a plethora of up-to-date findings about studies of aging, longevity, and sometimes even immortality in several valuable but less frequently used systems, including bacteria (Caulobacter crescentus, Escherichia coli), invertebrates (Turritopsis dohrnii, Hydra sp., Arctica islandica), fishes (Nothobranchius sp., Greenland shark), reptiles (giant tortoise), mammals (blind mole rats, naked mole rats, bats, elephants, killer whale), and even 3D organoids, to prove that they offer biogerontologists as much as the more conventional tools. At the same time, the diversified knowledge gained owing to research on those species may help to reconsider aging from a broader perspective, which should translate into a better understanding of this tremendously complex and clearly system-specific phenomenon.

Gender as a factor affecting NK cell activity in patients successfully treated for chronic hepatitis C with direct-acting antivirals.

Chronic hepatitis C (CHC) affects the activity of natural killer (NK) cells, but successful interferon- free treatment partially restores it. The goal of this study was to assess whether gender influences NK functionality. We examined 21 post-menopausal women and 24 men with CHC who were treated with direct-acting antivirals (DAA) and 33 healthy volunteers. Using flow cytometry, we analysed KIR2DS4, NKG2D, NKp30, KIR2DL2/DL3, NKG2A and TRAIL on the surface of NK cells. Intracellular granzyme B was also assessed and serum CXCL10 was quantified via ELISA. Overall, patients with CHC had higher expression of KIR2DS4, NKG2A, and NKp30 relative to the control group. Further, CHC patients had a lower percentage of NK cells among lymphocytes relative to the control group. After treatment, KIR2DS4, KIR2DL2/DL, NKG2A, TRAIL and NKp30 on NK cells were decreased whilst the percentage of NK cells and the expression of granzyme B and NKG2D increased. Prior to treatment, serum CXCL10 was elevated, but it was inhibited post-treatment. We observed gender-specific differences in the expression of KIR2DL2/DL3 (higher in women) and NKp30 (elevated in men) compared to CHC/control groups. After treatment, KIR2DL2/DL3, NKp30 and CXCL10 dropped only in the female group while granzyme B increased in the male group. In conclusion, the response of NK cells among men and women of post-menopausal ages with CHC differs. Our research may lead to more studies on the different nature of female and male immune systems in the context of HCV infection and treatment.

Impact of chronic HCV treatment on quality of life of patients with metabolic disorders in context of immunological disturbances.

Chronic viral hepatitis C (CHC) and its complications have a negative effect on patient's quality of life. We evaluated the impact of a successful interferon-free treatment on the quality of life of patients with obesity and metabolic disorders in the context of immunological disturbances. Twenty overweight or obese (BMI > 25) patients with CHC were tested before the therapy and after a successful treatment regimen. After the therapy, patient's emotional well-being improved (p = 0.02), while physical well-being remained unchanged. There was a decrease of patient's liver fibrosis and an increase of steatosis along with body mass. Among HCV-infected individuals, the expression of toll-like receptor 3 (TLR3) on lymphocytes was higher than in the control group (p = 0.03), but it decreased (p = 0.001) after the treatment. There was also a decrease of the intensity of immunofluorescence of FoxP3+ after the treatment (p = 0.04). Our study showed an improvement in mental aspects of patient's quality of life after the treatment. Unfortunately, probably due to rapid immunological changes, patient's BMI, serum cholesterol levels and hepatic steatosis have a tendency to increase and may lead to cardiovascular and other complications, like hepatocellular carcinoma.

Deciphering the Molecular Mechanism of Spontaneous Senescence in Primary Epithelial Ovarian Cancer Cells.

Spontaneous senescence of cancer cells remains a puzzling and poorly understood phenomenon. Here we comprehensively characterize this process in primary epithelial ovarian cancer cells (pEOCs). Analysis of tumors from ovarian cancer patients showed an abundance of senescent cells in vivo. Further, serially passaged pEOCs become senescent after a few divisions. These senescent cultures display trace proliferation, high expression of senescence biomarkers (SA--Gal, -H2A.X), growth-arrest in the G1 phase, increased level of cyclins D1, D2, decreased cyclin B1, up-regulated p16, p21, and p53 proteins, eroded telomeres, reduced activity of telomerase, predominantly non-telomeric DNA damage, activated AKT, AP-1, and ERK1/2 signaling, diminished JNK, NF-B, and STAT3 pathways, increased formation of reactive oxygen species, unchanged activity of antioxidants, increased oxidative damage to DNA and proteins, and dysfunctional mitochondria. Moreover, pEOC senescence is inducible by normal peritoneal mesothelium, fibroblasts, and malignant ascites via the paracrine activity of GRO-1, HGF, and TGF-1. Collectively, pEOCs undergo spontaneous senescence in a mosaic, telomere-dependent and telomere-independent manner, plausibly in an oxidative stress-dependent mechanism. The process may also be activated by extracellular stimuli. The biological and clinical significance of pEOC senescence remains to be explored.

Mechanisms and significance of therapy-induced and spontaneous senescence of cancer cells.

In contrast to the well-recognized replicative and stress-induced premature senescence of normal somatic cells, mechanisms and clinical implications of senescence of cancer cells are still elusive and uncertain from patient-oriented perspective. Moreover, recent years provided multiple pieces of evidence that cancer cells may undergo senescence not only in response to chemotherapy or ionizing radiation (the so-called therapy-induced senescence) but also spontaneously, without any external insults. Since the molecular nature of the latter process is poorly recognized, the significance of spontaneously senescent cancer cells for tumor progression, therapy effectiveness, and patient survival is purely speculative. In this review, we summarize the most up-to-date research regarding therapy-induced and spontaneous senescence of cancer cells, by delineating the most important discoveries regarding the occurrence of these phenomena in vivo and in vitro. This review provides data collected from studies on various cancer cell models, and the narration is presented from the broader perspective of the most critical findings regarding the senescence of normal somatic cells.

Senescence-related deterioration of intercellular junctions in the peritoneal mesothelium promotes the transmesothelial invasion of ovarian cancer cells.

Mechanisms of transmesothelial invasion of ovarian cancer are still poorly understood. Here we examined whether this phenomenon may be determined by an expression of intercellular junctions in peritoneal mesothelial cells (PMCs). Analysis of ovarian tumors showed that cancer cells are localized below an intact layer of PMCs. The PMCs located near the invaded cancer cells displayed low expression of connexin 43, E-cadherin, occludin, and desmoglein, as well as expressed SA-ß-Gal, a marker of senescence. Experiments in vitro showed that senescent PMCs exhibited decreased levels of the four tested intercellular junctions, and that the invasion of ovarian cancer cells through the PMCs increased proportionally to the admixture of senescent cells. Intervention studies showed that the expression of connexin 43, E-cadherin, occludin, and desmoglein in senescent PMCs could be restored upon the blockade of p38 MAPK, NF-κB, AKT, JNK, HGF, and TGF-ß1. When these molecules were neutralized, the efficiency of the transmesothelial cancer cell invasion was diminished. Collectively, our findings show that the integrity of the peritoneal mesothelium, which is determined by the expression of junctional proteins, is critical for the invasion of ovarian cancer. They also indicate a mechanism by which senescent PMCs may promote the invasive potential of cancer cells.