Therapy-induced polyploidization and senescence: Coincidence or interconnection?

Polyploid somatic cells have 'programmed' roles in normal development and stress responses. Transient polyploidy states have been observed in several tumor types at early stages of tumorigenesis. They can give rise to the aneuploidy state which is a common feature of human cancer cells. Similarly, to cancer development, cancer treatment can lead to transient polyploidy. Polyploid giant cells (PGCCs) in cancer are often associated with poor prognosis and disease relapse. Cancer cell senescence- a proliferation arrest accompanied by a set of characteristic markers- induced by therapy is also associated with transient polyploidy formation and cancer relapse. The question is whether therapy-induced senescence (TIS) and therapy induced polyploidy (TIP) are mechanistically or coincidentally connected. This problem needs to be solved rather urgently, because TIS appears to be more common phenomena than originally believed. Another arising question concerns reversibility of cancer cell senescence as a consequence of atypical divisions of polyploid cells. In our review we will try to answer this fundamental question by referring to published literature and to our own studies.

Therapy-Induced Senescent/Polyploid Cancer Cells Undergo Atypical Divisions Associated with Altered Expression of Meiosis, Spermatogenesis and EMT Genes.

Upon anticancer treatment, cancer cells can undergo cellular senescence, i.e., the temporal arrest of cell division, accompanied by polyploidization and subsequent amitotic divisions, giving rise to mitotically dividing progeny. In this study, we sought to further characterize the cells undergoing senescence/polyploidization and their propensity for atypical divisions. We used p53-wild type MCF-7 cells treated with irinotecan (IRI), which we have previously shown undergo senescence/polyploidization. The propensity of cells to divide was measured by a BrdU incorporation assay, Ki67 protein level (cell cycle marker) and a time-lapse technique. Advanced electron microscopy-based cell visualization and bioinformatics for gene transcription analysis were also used. We found that after IRI-treatment of MCF-7 cells, the DNA replication and Ki67 level decreased temporally. Eventually, polyploid cells divided by budding. With the use of transmission electron microscopy, we showed the presence of mononuclear small cells inside senescent/polyploid ones. A comparison of the transcriptome of senescent cells at day three with day eight (when cells just start to escape senescence) revealed an altered expression of gene sets related to meiotic cell cycles, spermatogenesis and epithelial-mesenchymal transition. Although chemotherapy (DNA damage)-induced senescence is indispensable for temporary proliferation arrest of cancer cells, this response can be followed by their polyploidization and reprogramming, leading to more fit offspring.

Improved Autophagic Flux in Escapers from Doxorubicin-Induced Senescence/Polyploidy of Breast Cancer Cells.

The induction of senescence/polyploidization and their role in cancer recurrence is still a poorly explored issue. We showed that MDA-MB-231 and MCF-7 breast cancer cells underwent reversible senescence/polyploidization upon pulse treatment with doxorubicin (dox). Subsequently, senescent/polyploid cells produced progeny (escapers) that possessed the same amount of DNA as parental cells. In a dox-induced senescence/polyploidization state, the accumulation of autophagy protein markers, such as LC3B II and p62/SQSTM1, was observed. However, the senescent cells were characterized by a very low rate of new autophagosome formation and degradation, estimated by autophagic index. In contrast to senescent cells, escapers had a substantially increased autophagic index and transcription factor EB activation, but a decreased level of an autophagy inhibitor, Rubicon, and autophagic vesicles with non-degraded cargo. These results strongly suggested that autophagy in escapers was improved, especially in MDA-MB-231 cells. The escapers of both cell lines were also susceptible to dox-induced senescence. However, MDA-MB-231 cells which escaped from senescence were characterized by a lower number of γH2AX foci and a different pattern of interleukin synthesis than senescent cells. Thus, our studies showed that breast cancer cells can undergo senescence uncoupled from autophagy status, but autophagic flux resumption may be indispensable in cancer cell escape from senescence/polyploidy.

"Mitotic Slippage" and Extranuclear DNA in Cancer Chemoresistance: A Focus on Telomeres.

Mitotic slippage (MS), the incomplete mitosis that results in a doubled genome in interphase, is a typical response of TP53-mutant tumors resistant to genotoxic therapy. These polyploidized cells display premature senescence and sort the damaged DNA into the cytoplasm. In this study, we explored MS in the MDA-MB-231 cell line treated with doxorubicin (DOX). We found selective release into the cytoplasm of telomere fragments enriched in telomerase reverse transcriptase (hTERT), telomere capping protein TRF2, and DNA double-strand breaks marked by γH2AX, in association with ubiquitin-binding protein SQSTM1/p62. This occurs along with the alternative lengthening of telomeres (ALT) and DNA repair by homologous recombination (HR) in the nuclear promyelocytic leukemia (PML) bodies. The cells in repeated MS cycles activate meiotic genes and display holocentric chromosomes characteristic for inverted meiosis (IM). These giant cells acquire an amoeboid phenotype and finally bud the depolyploidized progeny, restarting the mitotic cycling. We suggest the reversible conversion of the telomerase-driven telomere maintenance into ALT coupled with IM at the sub-telomere breakage sites introduced by meiotic nuclease SPO11. All three MS mechanisms converging at telomeres recapitulate the amoeba-like agamic life-cycle, decreasing the mutagenic load and enabling the recovery of recombined, reduced progeny for return into the mitotic cycle.

Curcumin induces multiple signaling pathways leading to vascular smooth muscle cell senescence.

Curcumin, a phytochemical present in the spice named turmeric, and one of the promising anti-aging factors, is itself able to induce cellular senescence. We have recently shown that cells building the vasculature senesced as a result of curcumin treatment. Curcumin-induced senescence was DNA damage-independent; however, activation of ATM was observed. Moreover, neither increased ROS production, nor even ATM were indispensable for senescence progression. In this paper we tried to elucidate the mechanism of curcumin-induced senescence. We analyzed the time-dependence of the level and activity of numerous proteins involved in senescence progression in vascular smooth muscle cells and how inhibition p38 or p38 together with ATM, two proteins involved in canonical signaling pathways, influenced cell senescence. We showed that curcumin was able to influence many signaling pathways of which probably none was dominant and sufficient to induce senescence by itself. However, we cannot exclude that the switch between initiation and progression of senescence is the result of the impact of curcumin on signaling pathways engaging AMPK, ATM, sirtuin 1 and p300 and on their reciprocal interplay. Cytostatic concentration of curcumin induced cellular stress, which exceeded the adaptive response and, in consequence, led to cellular senescence, which is triggered by time dependent activation of several signaling pathways playing diverse roles in different phases of senescence progression. We also showed that activity of ß-glucuronidase, the enzyme involved in deconjugation of the main metabolites of curcumin, glucuronides, increased in senescent cells. It suggests a possible local elevation of curcumin concentration in the organism.

The Role of Curcumin in the Modulation of Ageing.

It is believed that postponing ageing is more effective and less expensive than the treatment of particular age-related diseases. Compounds which could delay symptoms of ageing, especially natural products present in a daily diet, are intensively studied. One of them is curcumin. It causes the elongation of the lifespan of model organisms, alleviates ageing symptoms and postpones the progression of age-related diseases in which cellular senescence is directly involved. It has been demonstrated that the elimination of senescent cells significantly improves the quality of life of mice. There is a continuous search for compounds, named senolytic drugs, that selectively eliminate senescent cells from organisms. In this paper, we endeavor to review the current knowledge about the anti-ageing role of curcumin and discuss its senolytic potential.

Modulatory Effects of Curcumin and Tyrphostins (AG494 and AG1478) on Growth Regulation and Viability of LN229 Human Brain Cancer Cells.

In this study we employed curcumin as a potent adjuvant agent in the treatment of human brain cancer involving selective EGFR kinase inhibitors: tyrphostins AG494 and AG1478. Aim of this work was to evaluate the effect of tested compounds on autocrine growth, cell cycle, and viability of LN229 cells, as well as to assess their proapoptotic and genotoxic properties. Our results showed that all tested compounds significantly inhibited autocrine growth of the investigated cell line in a dose dependent manner. However they are characterized by different kinetics of cell growth inhibition. Suppression of growth by the tyrphostins was completely or partially reversible in contrast to curcumin. Curcumin increased the cytostatic and/or cytotoxic potential of AG494 and AG1478. Tyrphostins did not have genotoxic properties regardless of concentration used, whereas curcumin cytotoxic and genotoxic properties were directly proportional to the concentration. Curcumin significantly increased tyrphostins cytotoxicity. The most promising of the obtained results may be the use of curcumin and tyrphostin AG494 in the treatment of cancer cells. Anticancer effect of the mixture was confirmed by increase of cytotoxic effect, decrease of viability, stimulation of apoptotic procesess, irreversible DNA damage, and decrease of the ROS in the culture of glioblastoma cells.

The vicious circle of stereotypes: Teachers' awareness of and responses to students' gender-stereotypical behaviour.

The study was conducted using a mixed methods approach combining lesson observations and interviews with teachers. A total of 204 hours of observation in 34 classes of 7th and 8th graders (aged 13-14 in the Polish primary school system) were conducted to investigate teachers' behaviour that may exacerbate gender stereotypes and gender bias in the classroom. Moreover, the 25 female teachers conducting the observed lessons were interviewed to identify: (i) teachers' awareness of stereotypical behaviours of girls and boys during classes; (ii) teachers' awareness of possible causes of these behaviours; (iii) teachers' responses to these behaviours, including actions that could deepen gender stereotypes; and (iv) teachers' sensitivity to the gender polarised content of school textbooks. The results of the study show that teachers, although they are aware of the existence of gender stereotypes and declare their willingness to counteract them, tend to strengthen rather than eliminate these stereotypes with the strategies and actions undertaken. They have difficulty recognising possible reasons for the occurrence of stereotypical student behaviour and have little awareness of the gender-polarised content of school textbooks. The results of the study are discussed, inter alia, in light of the concept of the vicious circle of stereotypes and self-fulfilling prophecies in education.

The role of autophagy in escaping therapy-induced polyploidy/senescence.

Autophagy is an evolutionarily conserved process necessary to maintain cell homeostasis in response to various forms of stress such as nutrient deprivation and hypoxia as well as functioning to remove damaged molecules and organelles. The role of autophagy in cancer varies depending on the stage of cancer. Cancer therapeutics can also simultaneously evoke cancer cell senescence and ploidy increase. Both cancer cell senescence and polyploidization are reversible by depolyploidization giving rise to the progeny. Autophagy activation may be indispensable for cancer cell escape from senescence/polyploidy. As cancer cell polyploidy is proposed to be involved in cancer origin, the role of autophagy in polyploidization/depolyploidization of senescent cancer cells seems to be crucial. Accordingly, this review is an attempt to understand the complicated interrelationships between reversible cell senescence/polyploidy and autophagy.

Diversity of the Senescence Phenotype of Cancer Cells Treated with Chemotherapeutic Agents.

It is acknowledged that cancer cells are able to undergo senescence in response to clinically used chemotherapeutics. Moreover, recent years have provided evidence that some drugs can selectively remove senescent cells. Therefore, it is essential to properly identify and characterize senescent cells, especially when it comes to cancer. Senescence was induced in various cancer cell lines (A549, SH-SY-5Y, HCT116, MDA-MB-231, and MCF-7) following treatment with doxorubicin, irinotecan, methotrexate, 5-fluorouracil, oxaliplatin, or paclitaxel. Treatment with tested chemotherapeutics resulted in upregulation of p21 and proliferation arrest without cytotoxicity. A comparative analysis with the use of common senescence markers (i.e., morphology, SA-ß-galactosidase, granularity, secretory phenotype, and the level of double-stranded DNA damage) revealed a large diversity in response to the chemotherapeutics used. The strongest senescence inducers were doxorubicin, irinotecan, and methotrexate; paclitaxel had an intermediate effect and oxaliplatin and 5-fluorouracil did not induce senescence. In addition, different susceptibility of cancer cells to senescence was observed. A statistical analysis aimed at finding any relationship between the senescence markers applied did not show clear correlations. Moreover, increased SA-ß-gal activity coupled with p21 expression proved not to be an unequivocal senescence marker. This points to a need to simultaneously analyze multiple markers, given their individual limitations.

Age equivalence in switch costs for prosaccade and antisaccade tasks.

This study examined age differences in task switching using prosaccade and antisaccade tasks. Significant specific and general switch costs were found for both young and old adults, suggesting the existence of 2 types of processes: those responsible for activation of the currently relevant task set and deactivation of the previously relevant task set and those responsible for maintaining more than 1 task active in working memory. Contrary to the findings of previous research, which used manual response tasks with arbitrary stimulus-response mappings to study task-switching performance, no age-related deficits in either type of switch costs were found. These data suggest age-related sparing of task-switching processes in situations in which memory load is low and stimulus-response mappings are well learned.

The effect of tyrphostins AG494 and AG1478 on the autocrine growth regulation of A549 and DU145 cells.

We employed two selective EGFR tyrosine kinase inhibitors: AG494 (reversible) and AG1478 (irreversible) for growth regulation of human lung (A549) and prostate (DU145) cancer cell lines, cultured in chemically defined DMEM/F12 medium. Both tested tyrphostins significantly inhibited autocrine growth of the investigated cell lines. The action of AG494 was dose dependent, and at highest concentrations led to complete inhibition of growth. AG1478 seemed to be more effective at lower concentrations, but was unable to completely inhibit growth of A549 cells. Inhibition of EGFR kinase activity by AG494 in contrast to AG1478 had no effect on the activity of ERK in both cell lines. Both EGFR's inhibitors induced apoptosis of the investigated lung and prostate cancer cell lines, but the proapoptotic effect of the investigated tyrphostins was greater in A549 than in DU145 cells. The tyrphostins arrested cell growth of DU145 and A549 cells in the G1 phase, similarly to other known inhibitors of EGFR. The influence of AG494 and AG1478 on the activity of two signaling proteins (AKT and ERK) was dependent upon the kind of investigated cells. In the case of DU145 cells, there was an evident decline in enzymatic activity of both kinases (stronger for AG1478), while in A549, only AG1478 effectively inhibited the phosphorylation of Akt. Tyrphostins AG494 and AG1478 are ATP-competitors and are supposed to have a similar mechanism of action, but our results suggest that this is not quite true.