Polyploidy formation in cancer cells: How a Trojan horse is born.

Ploidy increase has been shown to occur in different type of tumors and participate in tumor initiation and resistance to the treatment. Polyploid giant cancer cells (PGCCs) are cells with multiple nuclei or a single giant nucleus containing multiple complete sets of chromosomes. The mechanism leading to formation of PGCCs may depend on: endoreplication, mitotic slippage, cytokinesis failure, cell fusion or cell cannibalism. Polyploidy formation might be triggered in response to various genotoxic stresses including: chemotherapeutics, radiation, hypoxia, oxidative stress or environmental factors like: air pollution, UV light or hyperthermia. A fundamental feature of polyploid cancer cells is the generation of progeny during the reversal of the polyploid state (depolyploidization) that may show high aggressiveness resulting in the formation of resistant disease and tumor recurrence. Therefore, we propose that modern anti-cancer therapies should be designed taking under consideration polyploidization/ depolyploidization processes, which confer the polyploidization a hidden potential similar to a Trojan horse delayed aggressiveness. Various mechanisms and stress factors leading to polyploidy formation in cancer cells are discussed in this review.

Hypoxia, but Not Normoxia, Reduces Effects of Resveratrol on Cisplatin Treatment in A2780 Ovarian Cancer Cells: A Challenge for Resveratrol Use in Anticancer Adjuvant Cisplatin Therapy.

Natural compounds, such as resveratrol (Res), are currently used as adjuvants for anticancer therapies. To evaluate the effectiveness of Res for the treatment of ovarian cancer (OC), we screened the response of various OC cell lines to the combined treatment with cisplatin (CisPt) and Res. We identified A2780 cells as the most synergistically responding, thus optimal for further analysis. Because hypoxia is the hallmark of the solid tumor microenvironment, we compared the effects of Res alone and in combination with CisPt in hypoxia (pO2 = 1%) vs. normoxia (pO2 = 19%). Hypoxia caused an increase (43.2 vs. 5.0%) in apoptosis and necrosis (14.2 vs. 2.5%), reactive oxygen species production, pro-angiogenic HIF-1α (hypoxia-inducible factor-1α) and VEGF (vascular endothelial growth factor), cell migration, and downregulated the expression of ZO1 (zonula occludens-1) protein in comparison to normoxia. Res was not cytotoxic under hypoxia in contrast to normoxia. In normoxia, Res alone or CisPt+Res caused apoptosis via caspase-3 cleavage and BAX, while in hypoxia, it reduced the accumulation of A2780 cells in the G2/M phase. CisPt+Res increased levels of vimentin under normoxia and upregulated SNAI1 expression under hypoxia. Thus, various effects of Res or CisPt+Res on A2780 cells observed in normoxia are eliminated or diminished in hypoxia. These findings indicate the limitations in using Res as an adjuvant with CisPt therapy in OC.

Heme Oxygenase-1 Has a Greater Effect on Melanoma Stem Cell Properties Than the Expression of Melanoma-Initiating Cell Markers.

Melanoma-initiating cells (MICs) contribute to the tumorigenicity and heterogeneity of melanoma. MICs are identified by surface and functional markers and have been shown to display cancer stem cell (CSC) properties. However, the existence of MICs that follow the hierarchical CSC model has been questioned by studies showing that single unselected melanoma cells are highly tumorigenic in xenotransplantation assays. Herein, we characterize cells expressing MIC markers (CD20, CD24, CD133, Sca-1, ABCB1, ABCB5, ALDHhigh) in the B16-F10 murine melanoma cell line. We use flow cytometric phenotyping, single-cell sorting followed by in vitro clonogenic assays, and syngeneic in vivo serial transplantation assays to demonstrate that the expression of MIC markers does not select CSC-like cells in this cell line. Previously, our group showed that heme-degrading enzyme heme oxygenase-1 (HO-1) can be upregulated in melanoma and increase its aggressiveness. Here, we show that HO-1 activity is important for non-adherent growth of melanoma and HO-1 overexpression enhances the vasculogenic mimicry potential, which can be considered protumorigenic activity. However, HO-1 overexpression decreases clone formation in vitro and serial tumor initiation in vivo. Thus, HO-1 plays a dual role in melanoma, improving the progression of growing tumors but reducing the risk of melanoma initiation.

Hypoxia-Mediated Decrease of Ovarian Cancer Cells Reaction to Treatment: Significance for Chemo- and Immunotherapies.

Hypoxia, a common factor ruling the microenvironment composition, leads to tumor progression. In this hypoxic context, cytokines and cells cooperate to favor cancer development and metastasis. Tumor hypoxia is heterogeneously distributed. Oxygen gradients depend on the vicinity, functionality of blood vessels, and oxygen ability to diffuse into surrounding tissues. Thus, the vasculature state modulates the microenvironment of the tumor cells. Cells sense and react to small variations in oxygen tension, which explains the lack of tumor cells' unicity in their reaction to drugs. Ovarian cancers are highly hypoxia-dependent, ascites worsening the access to oxygen, in their reactions to both chemotherapy and new immunotherapy. Consequently, hypoxia affects the results of immunotherapy, and is thus, crucial for the design of treatments. Controlling key immunosuppressive factors and receptors, as well as immune checkpoint molecule expression on tumor, immune and stromal cells, hypoxia induces immunosuppression. Consequently, new approaches to alleviate hypoxia in the tumor microenvironment bring promises for ovarian cancer immunotherapeutic strategies. This review focuses on the effects of hypoxia in the microenvironment and its consequences on tumor treatments. This opens the way to innovative combined treatments to the advantage of immunotherapy outcome in ovarian cancers.

Effect of crossing C57BL/6 and FVB mouse strains on basal cytokine expression.

C57BL/6 is the most often used laboratory mouse strain. However, sometimes it is beneficial to cross the transgenic mice on the C57BL/6 background to the other strain, such as FVB. Although this is a common strategy, the influence of crossing these different strains on homeostatic expression of cytokines is not known. Here we have investigated the differences in the expression of selected cytokines between C57BL/6J and C57BL/6JxFVB mice in serum and skeletal muscle. We have found that only few cytokines were altered by crossing of the strains. Concentrations of IL5, IL7, LIF, MIP-2, and IP-10 were higher in serum of C57BL/6J mice than in C57BL/6JxFVB mice, whereas concentration of G-CSF was lower in C57BL/6J. In the skeletal muscle only the concentration of VEGF was higher in C57BL/6J mice than in C57BL/6JxFVB mice. Concluding, the differences in cytokine expression upon crossing C57BL/6 and FVB strain in basal conditions are not profound.

[Stem cells and senescence]. / Komórki macierzyste a starzenie.

Stem cells are undifferentiated cells that can differentiate into specialized cells, that build the whole body. These rare cells are required for homeostasis and tissue replacement throughout the human lifespan, and appear to be characterized by a few specific physiological and biochemical properties, particularly the capacity for self-renewal. Recent studies suggest that stem cells may undergo senescence, what plays a crucial role in organismal aging. Importantly, both senescence and apoptosis are anti-cancer mechanisms that counteract neoplastic transformation of stem cells. On the other hand, mechanisms that suppress the development of cancer may also induce an unwanted consequence: a decline in the number and functional alterations of stem cells with advancing age. These functional changes reflect harmful effects of age on the genome, epigenome, and proteome of stem cells. Some of which arise cell independently and others which are imposed by an age-related change in the local milieu or systemic environment. Remarkably, some of the changes, particularly epigenomic and proteomic ones, are potentially reversible, and both environmental (e.g. caloric restrictions, hypoxia) and genetic interventions can lead to inducible pluripotency. Here, we discuss recent discoveries in the field of senescence of stem cells. These findings have profound implications, not only for our understanding of stem cells' biology and organismal aging, but also for stem cell-based regenerative medicine and stem cell-based therapy of age-related diseases.

Risk factors of using late-autophagy inhibitors: Aspects to consider when combined with anticancer therapies.

Cancer resistance to therapy is still an unsolved scientific and clinical problem. In 2022, the hallmarks of cancer have been expanded to include four new features, including cellular senescence. Therapy-induced senescence (TIS) is a stressor-based response to conventional treatment methods, e.g. chemo- and radiotherapy, but also to non-conventional targeted therapies. Since TIS reinforces resistance in cancers, new strategies for sensitizing cancer cells to therapy are being adopted. These include macroautophagy as a potential target for inhibition due to its potential cytoprotective role in many cancers. The mechanism of late-stage autophagy inhibitors is based on blockage of autophagolysosome formation or an increase in lysosomal pH, resulting in disrupted cargo degradation. Such inhibitors are relevant candidates for increasing anticancer therapy effectiveness. In particular, 4-aminoquoline derivatives: chloroquine/hydroxychloroquine (CQ/HCQ) have been tested in multiple clinical trials in combination with senescence-inducing anti-cancer drugs. In this review, we summarize the properties of selected late-autophagy inhibitors and their role in the regulation of autophagy and senescent cell phenotype in vitro and in vivo models of cancer as well as treatment response in clinical trials on oncological patients. Additionally, we point out that, although these compounds increase the effectiveness of treatment in some cases, their practical usage might be hindered due to systemic toxicity, hypoxic environment, dose- ant time-dependent inhibitory effects, as well as a possible contribution to escaping from TIS.

Stromal cell-derived factor 1 promotes angiogenesis via a heme oxygenase 1-dependent mechanism.

Stromal cell-derived factor 1 (SDF-1) plays a major role in the migration, recruitment, and retention of endothelial progenitor cells to sites of ischemic injury and contributes to neovascularization. We provide direct evidence demonstrating an important role for heme oxygenase 1 (HO-1) in mediating the proangiogenic effects of SDF-1. Nanomolar concentrations of SDF-1 induced HO-1 in endothelial cells through a protein kinase C zeta-dependent and vascular endothelial growth factor-independent mechanism. SDF-1-induced endothelial tube formation and migration was impaired in HO-1-deficient cells. Aortic rings from HO-1(-/-) mice were unable to form capillary sprouts in response to SDF-1, a defect reversed by CO, a byproduct of the HO-1 reaction. Phosphorylation of vasodilator-stimulated phosphoprotein was impaired in HO-1(-/-) cells, an event that was restored by CO. The functional significance of HO-1 in the proangiogenic effects of SDF-1 was confirmed in Matrigel plug, wound healing, and retinal ischemia models in vivo. The absence of HO-1 was associated with impaired wound healing. Intravitreal adoptive transfer of HO-1-deficient endothelial precursors showed defective homing and reendothelialization of the retinal vasculature compared with HO-1 wild-type cells following ischemia. These findings demonstrate a mechanistic role for HO-1 in SDF-1-mediated angiogenesis and provide new avenues for therapeutic approaches in vascular repair.

High Hemin Concentration Induces Escape from Senescence of Normoxic and Hypoxic Colon Cancer Cells.

Hemoglobin from either red meat or bowel bleeding may promote oxidative stress and increase the risk of colorectal cancer (CRC). Additionally, solid cancers or their metastases may be present with localized bruising. Escape from therapy-induced senescence (TIS) might be one of the mechanisms of tumor re-growth. Therefore, we sought to study whether hemin can cause escape from TIS in CRC. To induce senescence, human colon cancer cells were exposed to a chemotherapeutic agent irinotecan (IRINO). Cells treated with IRINO exhibited common hallmarks of TIS. To mimic bleeding, colon cancer cells were additionally treated with hemin. High hemin concentration activated heme oxygenase-1 (HO-1), induced escape from TIS and epithelial-to-mesenchymal transition, and augmented progeny production. The effect was even stronger in hypoxic conditions. Similar results were obtained when TIS cells were treated with another prooxidant agent, H2O2. Silencing of antioxidative enzymes such as catalase (CAT) or glutathione peroxidase-1 (GPx-1) maintained colon cancer cells in a senescent state. Our study demonstrates that a high hemin concentration combined with an increased activity of antioxidative enzymes, especially HO-1, leads to escape from the senescence of colon cancer cells. Therefore, our observations could be used in targeted anti-cancer therapy.

Mechanisms of Chemotherapy-Induced Neurotoxicity.

Since the first clinical trials conducted after World War II, chemotherapeutic drugs have been extensively used in the clinic as the main cancer treatment either alone or as an adjuvant therapy before and after surgery. Although the use of chemotherapeutic drugs improved the survival of cancer patients, these drugs are notorious for causing many severe side effects that significantly reduce the efficacy of anti-cancer treatment and patients' quality of life. Many widely used chemotherapy drugs including platinum-based agents, taxanes, vinca alkaloids, proteasome inhibitors, and thalidomide analogs may cause direct and indirect neurotoxicity. In this review we discuss the main effects of chemotherapy on the peripheral and central nervous systems, including neuropathic pain, chemobrain, enteric neuropathy, as well as nausea and emesis. Understanding mechanisms involved in chemotherapy-induced neurotoxicity is crucial for the development of drugs that can protect the nervous system, reduce symptoms experienced by millions of patients, and improve the outcome of the treatment and patients' quality of life.

Role of heme oxygenase-1 in human endothelial cells: lesson from the promoter allelic variants.

OBJECTIVE: Heme oxygenase-1 (HO-1) is an antioxidative, antiinflammatory, and cytoprotective enzyme that is induced in response to cellular stress. The HO-1 promoter contains a (GT)n microsatellite DNA, and the number of GT repeats can influence the occurrence of cardiovascular diseases. We elucidated the effect of this polymorphism on endothelial cells isolated from newborns of different genotypes. METHODS AND RESULTS: On the basis of HO-1 expression, we classified the HO-1 promoter alleles into 3 groups: short (S) (most active, GT < or = 23), medium (moderately active, GT=24 to 28), and long (least active, GT > or = 29). The presence of the S allele led to higher basal HO-1 expression and stronger induction in response to cobalt protoporphyrin, prostaglandin-J(2), hydrogen peroxide, and lipopolysaccharide. Cells carrying the S allele survived better under oxidative stress, a fact associated with the lower concentration of oxidized glutathione and more favorable oxidative status, as determined by measurement of the ratio of glutathione to oxidized glutathione. Moreover, they proliferated more efficiently in response to vascular endothelial growth factor A, although the vascular endothelial growth factor-induced migration and sprouting of capillaries were not influenced. Finally, the presence of the S allele was associated with lower production of some proinflammatory mediators, such as interleukin-1beta, interleukin-6, and soluble intercellular adhesion molecule-1. CONCLUSIONS: The (GT)n promoter polymorphism significantly modulates a cytoprotective, proangiogenic, and antiinflammatory function of HO-1 in human endothelium.

Escape From Cisplatin-Induced Senescence of Hypoxic Lung Cancer Cells Can Be Overcome by Hydroxychloroquine.

Chemotherapy is the commonly used treatment for advanced lung cancer. However, it produces side effects such as the development of chemoresistance. A possible responsible mechanism may be therapy-induced senescence (TIS). TIS cells display increased senescence-associated ß-galactosidase (SA-ß-gal) activity and irreversible growth arrest. However, recent data suggest that TIS cells can reactivate their proliferative potential and lead to cancer recurrence. Our previous study indicated that reactivation of proliferation by TIS cells might be related with autophagy modulation. However, exact relationship between both processes required further studies. Therefore, the aim of our study was to investigate the role of autophagy in the senescence-related chemoresistance of lung cancer cells. For this purpose, human and murine lung cancer cells were treated with two commonly used chemotherapeutics: cisplatin (CIS), which forms DNA adducts or docetaxel (DOC), a microtubule poison. Hypoxia, often overlooked in experimental settings, has been implicated as a mechanism responsible for a significant change in the response to treatment. Thus, cells were cultured under normoxic (~19% O2) or hypoxic (1% O2) conditions. Herein, we show that hypoxia increases resistance to CIS. Lung cancer cells cultured under hypoxic conditions escaped from CIS-induced senescence, displayed reduced SA-ß-gal activity and a decreased percentage of cells in the G2/M phase of the cell cycle. In turn, hypoxia increased the proliferation of lung cancer cells and the proportion of cells proceeding to the G0/G1 phase. Further molecular analyses demonstrated that hypoxia inhibited the prosenescent p53/p21 signaling pathway and induced epithelial to mesenchymal transition in CIS-treated cancer cells. In cells treated with DOC, such effects were not observed. Of importance, pharmacological autophagy inhibitor, hydroxychloroquine (HCQ) was capable of overcoming short-term CIS-induced resistance of lung cancer cells in hypoxic conditions. Altogether, our data demonstrated that hypoxia favors cancer cell escape from CIS-induced senescence, what could be overcome by inhibition of autophagy with HCQ. Therefore, we propose that HCQ might be used to interfere with the ability of senescent cancer cells to repopulate following exposure to DNA-damaging agents. This effect, however, needs to be tested in a long-term perspective for preclinical and clinical applications.

A new in vitro model applied 90Y microspheres to study the effects of low dose beta radiation on colorectal cancer cell line in various oxygenation conditions.

We propose a new in vitro model to assess the impact of 90Y-microspheres derived low-dose beta radiation on colorectal cancer cell line under various oxygenation conditions that mimic the tumor environment. Cancer cells (HCT116) proliferation was assessed using Alamar Blue (AB) assay after 48, 72, and 96 h. FLUKA code assessed changes in cancer cell populations relative to the absorbed dose. In normoxia, mitochondrial activity measured by Alamar Blue after 48-72 h was significantly correlated with the number of microspheres (48 h: r = 0.87 and 72 h: r = 0.89, p < 0.05) and absorbed dose (48 h: r = 0.87 and 72 h: r = 0.7, p < 0.05). In hypoxia, the coefficients were r = 0.43 for both the number of spheres and absorbed dose and r = 0.45, r = 0.47, respectively. Impediment of cancer cell proliferation depended on the absorbed dose. Doses below 70 Gy could reduce colorectal cancer cell proliferation in vitro. Hypoxia induced a higher resistance to radiation than that observed under normoxic conditions. Hypoxia and radiation induced senescence in cultured cells. The new in vitro model is useful for the assessment of 90Y radioembolization effects at the micro-scale.

Effect of Heme Oxygenase-1 on Melanoma Development in Mice-Role of Tumor-Infiltrating Immune Cells.

OBJECTIVE: Heme oxygenase-1 (HO-1) is a cytoprotective, proangiogenic and anti-inflammatory enzyme that is often upregulated in tumors. Overexpression of HO-1 in melanoma cells leads to enhanced tumor growth, augmented angiogenesis and resistance to anticancer treatment. The effect of HO-1 in host cells on tumor development is, however, hardly known. METHODS AND RESULTS: To clarify the effect of HO-1 expression in host cells on melanoma progression, C57BL/6xFvB mice of different HO-1 genotypes, HO-1+/+, HO-1+/-, and HO-1-/-, were injected with the syngeneic wild-type murine melanoma B16(F10) cell line. Lack of HO-1 in host cells did not significantly influence the host survival. Nevertheless, in comparison to the wild-type counterparts, the HO-1+/- and HO-1-/- males formed bigger tumors, and more numerous lung nodules; in addition, more of them had liver and spleen micrometastases. Females of all genotypes developed at least 10 times smaller tumors than males. Of importance, the growth of primary and secondary tumors was completely blocked in HO-1+/+ females. This was related to the increased infiltration of leukocytes (mainly lymphocytes T) in primary tumors. CONCLUSIONS: Although HO-1 overexpression in melanoma cells can enhance tumor progression in mice, its presence in host cells, including immune cells, can reduce growth and metastasis of melanoma.

Histone deacetylase inhibitors exert anti-tumor effects on human adherent and stem-like glioma cells.

BACKGROUND: The diagnosis of glioblastoma (GBM), a most aggressive primary brain tumor with a median survival of 14.6 months, carries a dismal prognosis. GBMs are characterized by numerous genetic and epigenetic alterations, affecting patient survival and treatment response. Epigenetic mechanisms are deregulated in GBM as a result of aberrant expression/activity of epigenetic enzymes, including histone deacetylases (HDAC) which remove acetyl groups from histones regulating chromatin accessibility. Nevertheless, the impact of class/isoform-selective HDAC inhibitors (HDACi) on glioma cells, including glioma stem cells, had not been systematically determined. RESULTS: Comprehensive analysis of the public TCGA dataset revealed the increased expression of HDAC 1, 2, 3, and 7 in malignant gliomas. Knockdown of HDAC 1 and 2 in human GBM cells significantly decreased cell proliferation. We tested the activity of 2 new and 3 previously described HDACi with different class/isoform selectivity on human GBM cells. All tested compounds exerted antiproliferative properties on glioma cells. However, the HDACi 1 and 4 blocked proliferation of glioblastoma cells leading to G2/M growth arrest without affecting astrocyte survival. Moreover, 1 and 4 at low micromolar concentrations displayed cytotoxic and antiproliferative effects on sphere cultures enriched in glioma stem cells. CONCLUSIONS: We identified two selective HDAC inhibitors that blocked proliferation of glioblastoma cells, but did not affect astrocyte survival. These new and highly effective inhibitors should be considered as promising candidates for further investigation in preclinical GBM models.

Zinc protoporphyrin IX, a heme oxygenase-1 inhibitor, demonstrates potent antitumor effects but is unable to potentiate antitumor effects of chemotherapeutics in mice.

BACKGROUND: HO-1 participates in the degradation of heme. Its products can exert unique cytoprotective effects. Numerous tumors express high levels of HO-1 indicating that this enzyme might be a potential therapeutic target. In this study we decided to evaluate potential cytostatic/cytotoxic effects of zinc protoporphyrin IX (Zn(II)PPIX), a selective HO-1 inhibitor and to evaluate its antitumor activity in combination with chemotherapeutics. METHODS: Cytostatic/cytotoxic effects of Zn(II)PPIX were evaluated with crystal violet staining and clonogenic assay. Western blotting was used for the evaluation of protein expression. Flow cytometry was used to evaluate the influence of Zn(II)PPIX on the induction of apoptosis and generation of reactive oxygen species. Knock-down of HO-1 expression was achieved with siRNA. Antitumor effects of Zn(II)PPIX alone or in combination with chemotherapeutics were measured in transplantation tumor models. RESULTS: Zn(II)PPIX induced significant accumulation of reactive oxygen species in tumor cells. This effect was partly reversed by administration of exogenous bilirubin. Moreover, Zn(II)PPIX exerted potent cytostatic/cytotoxic effects against human and murine tumor cell lines. Despite a significant time and dose-dependent decrease in cyclin D expression in Zn(II)PPIX-treated cells no accumulation of tumor cells in G1 phase of the cell cycle was observed. However, incubation of C-26 cells with Zn(II)PPIX increased the percentage of cells in sub-G1 phase of the cells cycle. Flow cytometry studies with propidium iodide and annexin V staining as well as detection of cleaved caspase 3 by Western blotting revealed that Zn(II)PPIX can induce apoptosis of tumor cells. B16F10 melanoma cells overexpressing HO-1 and transplanted into syngeneic mice were resistant to either Zn(II)PPIX or antitumor effects of cisplatin. Zn(II)PPIX was unable to potentiate antitumor effects of 5-fluorouracil, cisplatin or doxorubicin in three different tumor models, but significantly potentiated toxicity of 5-FU and cisplatin. CONCLUSION: Inhibition of HO-1 exerts antitumor effects but should not be used to potentiate antitumor effects of cancer chemotherapeutics unless procedures of selective tumor targeting of HO-1 inhibitors are developed.

Heme oxygenase-1 in tumors: is it a false friend?

Heme oxygenase-1 (HO-1) catalyzes the oxidation of heme to biologically active products: carbon monoxide (CO), biliverdin, and ferrous iron. It participates in maintaining cellular homeostasis and plays an important protective role in the tissues by reducing oxidative injury, attenuating the inflammatory response, inhibiting cell apoptosis, and regulating cell proliferation. HO-1 is also an important proangiogenic mediator. Most studies have focused on the role of HO-1 in cardiovascular diseases, in which its significant, beneficial activity is well recognized. A growing body of evidence indicates, however, that HO-1 activation may play a role in carcinogenesis and can potently influence the growth and metastasis of tumors. HO-1 is very often upregulated in tumor tissues, and its expression is further increased in response to therapies. Although the exact effect can be tissue specific, HO-1 can be regarded as an enzyme facilitating tumor progression. Accordingly, inhibition of HO-1 can be suggested as a potential therapeutic approach sensitizing tumors to radiation, chemotherapy, or photodynamic therapy.

Bafilomycin A1 triggers proliferative potential of senescent cancer cells in vitro and in NOD/SCID mice.

Anticancer therapies that induce DNA damage tend to trigger senescence in cancer cells, a process known as therapy-induced senescence (TIS). Such cells may undergo atypical divisions, thus contributing to tumor re-growth. Accumulation of senescent cancer cells reduces survival of patients after chemotherapy. As senescence interplays with autophagy, a dynamic recycling process, we sought to study whether inhibition of autophagy interferes with divisions of TIS cells. We exposed human colon cancer HCT116 cells to repeated cycles of a chemotherapeutic agent - doxorubicin (doxo) and demonstrated induction of hallmarks of TIS (e.g. growth arrest, hypertrophy, poliploidization and secretory phenotype) and certain properties of cancer stem cells (increased NANOG expression, percentages of CD24+ cells and side population). Colonies of small and highly proliferative progeny appeared shortly after drug removal. Treatment with bafilomycin A1 (BAF A1), an autophagy inhibitor, postponed short term in vitro cell re-population. It was associated with reduction in the number of diploid and increase in the number of poliploid cells. In a long term, a pulse of BAF A1 resulted in reactivation of autophagy in a subpopulation of HCT116 cells and increased proliferation. Accordingly, the senescent HCT116 cells treated with BAF A1 when injected into NOD/SCID mice formed tumors, in contrast to the controls.Our results suggest that senescent cancer cells that appear during therapy, can be considered as dormant cells that contribute to cancer re-growth, when chemotherapeutic treatment is stopped. These data unveil new mechanisms of TIS-related cancer maintenance and re-population, triggered by a single pulse of BAF A1 treatment.

Effects of 15d-PGJ(2) on VEGF-induced angiogenic activities and expression of VEGF receptors in endothelial cells.

15-Deoxy-Delta(12,14)-prostaglandin-J(2) (15d-PGJ(2)) upregulates expression of vascular endothelial growth factor (VEGF), but may inhibit angiogenesis. We found that 15d-PGJ(2) (1-10muM) attenuated all VEGF-induced angiogenic activities in human umbilical vein endothelial cells (HUVEC). It blocked almost completely cell proliferation, potently reduced migration, assembly into tube-like network on matrigel, and growth of capillaries into collagen gel. 15d-PGJ(2) inhibited expression of VEGFR-1 and VEGFR-2 receptors both at mRNA and protein levels. This inhibition, however, was transient (observed after 6-12h, but not after 24h) and weak (20-30%), and could not fully explain inhibition of response to VEGF. Accordingly, proliferation was inhibited when 15d-PGJ(2) was added 24h after VEGF or in cells stimulated with basic fibroblast growth factor. Interestingly, 15d-PGJ(2) decreased activities of c-jun and c-myc in HUVEC and overexpression of c-myc attenuated its antiproliferative effects. This suggests that inhibition of this transcription factor by 15d-PGJ(2) contributes to decrease in angiogenic response.