Justyna Mikuła-Pietrasik

Senescent Peritoneal Mesothelial Cells Promote Ovarian Cancer Cell Adhesion : The Role of Oxidative Stress-Induced Fibronectin

Adhesion of ovarian cancer cells to the peritoneal mesothelium is a key step in the malignant progression of the disease. In an in vitro study, we showed that the adherence of ovarian cancer cells (of the OVCAR-3, SKOV-3, and A2780 cell lines) to senescent human omentum-derived peritoneal mesothelial cells (HOMCs) was greater than to early passage cells. The process was mediated primarily by the increased interaction of the alpha5beta1 integrin on cancer cells with HOMC-associated fibronectin (FN). In comparison with early passage HOMCs, senescent cells exhibited increased FN mRNA expression levels and produced significantly more FN. To assess the effect of senescence-associated oxidative stress on FN release, HOMCs were rendered senescent by exposure to an oxidant, tert-butyl hydroperoxide. Treatment with tert-butyl hydroperoxide resulted in a significant increase in HOMC FN mRNA and protein expression levels. The effect of oxidative stress on FN synthesis was found to be mediated by transforming growth factor-beta1, whose signaling pathway was controlled at upstream and downstream levels by p38 MAPK. The activity of p38 MAPK increased markedly in senescent HOMCs. Treatment of HOMCs with antioxidants significantly attenuated senescence-associated increases in p38 MAPK activity, production of both transforming growth factor-beta1 and FN, and ovarian cancer cell adhesion. These data indicate that oxidative stress that accompanies senescence may increase FN production by HOMCs and thus facilitate binding and dissemination of ovarian cancer cells.

Oxidative stress-dependent increase in ICAM-1 expression promotes adhesion of colorectal and pancreatic cancers to the senescent peritoneal mesothelium.

Intercellular adhesion molecule‐1 (ICAM‐1) has been implicated in adhesion of colorectal and pancreatic cancer cells (of the SW480 and PSN‐1 line, respectively) to the peritoneal mesothelium. It has been demonstrated that ICAM‐1 expression increases with senescence in some cell types, however, the significance of this phenomenon in the context of malignant dissemination remains elusive. In this report we show that the adherence of SW480 and PSN‐1 cells to senescent human omentum‐derived mesothelial cells (HOMCs) in vitro is greater than to early‐passage cells and that the effect is mediated by ICAM‐1. Senescent HOMCs display increased expression of ICAM‐1 mRNA and cell surface protein. The development of this phenotype is related to increased oxidative stress in senescent cells. The augmented ICAM‐1 expression in HOMCs can be reduced by culturing cells with antioxidants; in contrast, exposure of HOMCs to an oxidant, t‐BHP, leads to cellular senescence and increased ICAM‐1 expression. The effect is partly mediated by activation of p38 MAPK and AP‐1 signaling pathways. Finally, culture of HOMCs in the presence of a strong antioxidant, PBN, significantly reduces the senescence‐associated increase in SW480 and PSN‐1 cancer cell binding. These results indicate that increased oxidative stress and increased expression of ICAM‐1 in senescent HOMCs may facilitate peritoneal adhesion of selected colorectal and pancreatic cancers.

Oxidative stress-mediated early senescence contributes to the short replicative life span of human peritoneal mesothelial cells

The replicative life span of cells in culture is thought to be determined by the gradually rising pool of senescent cells rather than by the simultaneous loss of proliferative capacity by all cells in the population. We found that early-passage cultures of human peritoneal mesothelial cells (HPMCs) contained a significant fraction of senescent-like cells. Furthermore, early-passage populations with a high percentage of senescent cells had a reduced subsequent life span in culture compared with populations consisting of the same number of apparently young cells but containing no senescent cells. The exposure of early-passage HPMCs to the conditioned medium from cultures containing senescent cells resulted in the retardation of growth and the induction of senescence-associated beta-galactosidase (SA-beta-Gal). This effect could be partly reduced by neutralizing TGF-beta1 activity. The timely treatment with N-tert-butyl-alpha-phenylnitrone (PBN) reduced oxidative stress, the number of early senescent cells, TGF-beta1 secretion, and ultimately extended the population life span. The effect was evident only when PBN was introduced at a very early, but not at a late, phase of tissue culture history. These results indicate that a sudden onset of senescence in early-passage HPMCs is related to oxidative stress and may influence the replicative life span of the population as a whole.

Resveratrol inhibits ovarian cancer cell adhesion to peritoneal mesothelium in vitro by modulating the production of α5β1 integrins and hyaluronic acid

OBJECTIVE Resveratrol (Res) is known to inhibit adhesion of numerous malignancies though its effect on an adherence of ovarian cancer cells to peritoneal mesothelium remains undefined. METHODS To address this issue, ovarian cancer cells (A2780, OVCAR-3, SKOV-3) were subjected to Res (10, 50, 100 μM), and then their adhesion to omentum-derived human peritoneal mesothelial cells (HPMCs) was assayed. RESULTS The study showed that Res inhibits adhesion of all ovarian cancer cell lines investigated. More importantly, this effect was evident either when cancer cells were directly treated with Res (cell-dependent activity) or when intact cancer cells were pretreated with conditioned medium (CM) generated by their counterparts subjected to Res (medium-dependent activity). Cell-dependent activity of Res has been recognized to be linked with decreased level of cellular α5β1 integrins which decreased functionality corresponds with reduced efficiency of cancer cell adhesion. Medium-related effects have been, in turn, associated with up-regulated secretion of soluble HA to environment (CM). The experiments with exogenous HA revealed the inverse relation between HA concentration in CM and cancer cell adhesion. When the CM from cells subjected with Res (with elevated HA) was supplemented with hyaluronidase, the restoration of cell adhesive capabilities occurred. CONCLUSIONS Our studies evidenced that Res affects ovarian cancer cell adhesion to HPMCs by decreasing cellular α5β1 integrin level and by increasing the secretion of HA to environment.

Resveratrol and its synthetic derivatives exert opposite effects on mesothelial cell-dependent angiogenesis via modulating secretion of VEGF and IL-8/CXCL8

We examined the effect of resveratrol (RVT) and its two derivatives (3,3′,4,4′-tetrahydroxy-trans-stilbene and 3,3′,4,4′,5,5′-hexahydroxy-trans-stilbene) on human peritoneal mesothelial cell (HPMC)-dependent angiogenesis in vitro. To this end, angiogenic activity of endothelial cells (HUVEC, HMVEC, and HMEC-1) was monitored upon their exposure to conditioned medium (CM) from young and senescent HPMCs treated with stilbenes or to stilbenes themselves. Results showed that proliferation and migration of endothelial cells were inhibited in response to indirect (HPMC-dependent) or direct RVT activity. This effect was associated with decreased secretion of VEGF and IL-8/CXCL8 by HPMCs treated with RVT, which confirmed the experiments with recombinant forms of these angiogenic agents. Angiogenic activity of endothelial cells treated with CM from HPMCs exposed to RVT analogues was more effective. Improved migration was particularly evident in cells exposed to CM from senescent HPMCs. Upon direct treatment, RVT derivatives stimulated proliferation (but not migration) of HUVECs, and failed to affect the behaviour of HMVEC and HMEC-1 cells. These compounds stimulated production of VEGF and IL-8/CXCL8 by HPMCs. Studies with neutralizing antibodies against angiogenic factors revealed that augmented angiogenic reactions of endothelial cells exposed to CM from HPMC treated with RVT analogues were related to enhanced production of VEGF and IL-8/CXCL8. Collectively, these findings indicate that RVT and its synthetic analogues divergently alter the secretion of the angiogenic factors by HPMCs, and thus modulate HPMC-dependent angiogenic responses in the opposite directions. This may have implications for the attempts of practical employment of the stilbenes for treatment of pathologies proceeding with abnormal vascularisation of the peritoneal tissue.

Vulnerability to oxidative stress and different patterns of senescence in human peritoneal mesothelial cell strains

Both the ascites fluid-derived mesothelial cell line LP-9 and primary cultures of human omentum-derived mesothelial cells (HOMCs) are commonly used in experimental studies. However, they seem to have a different replicative potential in vitro. In the present study, we have attempted to determine the causes of this discrepancy. HOMCs were found to divide fewer times and enter senescence earlier than LP-9 cells. This effect was coupled with earlier increases in the expression of senescence-associated-beta-galactosidase and cell cycle inhibitors p16INK4a and p21WAF1. Moreover, almost 3 times as many early-passage HOMCs as LP-9 cells bore senescence-associated DNA damage foci. In sharp contrast to LP-9 cells, the foci present in HOMCs localized predominantly outside the telomeres, and the HOMC telomere length did not significantly shorten during senescence. Compared with LP-9 cells, HOMCs were found to enter senescence with significantly lower levels of lipofuscin and damaged DNA, and markedly decreased glutathione contents. In addition, early-passage HOMCs generated significantly more reactive oxygen species either spontaneously or in response to exogenous oxidants. These results indicate that compared with LP-9 cells, HOMCs undergo stress-induced telomere-independent premature senescence, which may result from increased vulnerability to oxidative DNA injury.

The double-edged sword of long non-coding RNA: The role of human brain-specific BC200 RNA in translational control, neurodegenerative diseases, and cancer.

The complexity of eukaryotic organisms involves the regulation of gene expression through DNA-protein, RNA-DNA, RNA-RNA, and RNA-protein interactions. The role of RNA molecules in the regulation of genes in higher species has become even more evident with the discovery that about 97% of transcription products are represented by non-protein coding RNAs (ncRNAs) including short ncRNAs and long ncRNAs (lncRNAs). In addition to the well-characterized role of ncRNAs in different physiological cellular processes, numerous studies have also indicated the crucial roles of ncRNAs in neurological diseases and cancer. Although involvement of short ncRNA in those pathologies has already been well documented, there is only scarce evidence to show the participation of lncRNAs. One of the examples of lncRNAs is BC200 RNA, which plays an important role in the regulation of dendritic protein expression. Mislocalization and overexpression of BC200 RNA leads to inadequate RNA delivery to the synapses and results in neurodegenerative processes of Alzheimers disease and neoplastic changes in various groups of tissues. In this review, we summarize the current state of art in the field of the biological significance of lncRNAs, with particular attention paid to the physiological and pathophysiological role of BC200 RNA.

Peritoneal mesothelium promotes the progression of ovarian cancer cells in vitro and in a mice xenograft model in vivo

The role of mesothelial cells in the intraperitoneal spread of ovarian cancer is still elusive. In particular, it is unclear whether these cells constitute a passive barrier preventing cancer cell progression or perhaps act as an active promoter of this process. In this report we show that omental human peritoneal mesothelial cells (HPMCs) stimulate adhesion and proliferation of ovarian cancer cells (A2780, OVCAR-3, SKOV-3). The latter was associated with the paracrine activity of GRO-1, IL-6, and IL-8 released to the environment by HPMCs. Furthermore, the growth dynamics of ovarian cancer xenografts produced in response to i.p. injection of ovarian cancer cells together with HPMCs was remarkably greater than for implantation of cancer cells alone. A layer of peritoneal mesothelium was consistently present in close proximity to the tumor mass in every xenograft model. In conclusion, our results indicate that HPMCs play a supporting role in the intraperitoneal invasiveness of ovarian malignancy, whose effect may be attributed to their ability to stimulate adhesion and proliferation of cancer cells.

Colorectal cancer-promoting activity of the senescent peritoneal mesothelium

Gastrointestinal cancers metastasize into the peritoneal cavity in a process controlled by peritoneal mesothelial cells (HPMCs). In this paper we examined if senescent HPMCs can intensify the progression of colorectal (SW480) and pancreatic (PSN-1) cancers in vitro and in vivo. Experiments showed that senescent HPMCs stimulate proliferation, migration and invasion of SW480 cells, and migration of PSN-1 cells. When SW480 cells were injected i.p. with senescent HPMCs, the dynamics of tumor formation and vascularization were increased. When xenografts were generated using PSN-1 cells, senescent HPMCs failed to favor their growth. SW480 cells subjected to senescent HPMCs displayed up-regulated expression of transcripts for various pro-cancerogenic agents as well as increased secretion of their products. Moreover, they underwent an epithelial-mesenchymal transition in the Smad 2/3-Snail1-related pathway. The search for mediators of senescent HPMC activity showed that increased SW480 cell proliferation was stimulated by IL-6, migration by CXCL8 and CCL2, invasion by IL-6, MMP-3 and uPA, and epithelial-mesenchymal transition by TGF-β1. Secretion of these agents by senescent HPMCs was increased in an NF-κB- and p38 MAPK-dependent mechanism. Collectively, our findings indicate that in the peritoneum senescent HPMCs may create a metastatic niche in which critical aspects of cancer progression become intensified.

Bystander senescence in human peritoneal mesothelium and fibroblasts is related to thrombospondin-1-dependent activation of transforming growth factor-β1.

Senescence bystander effect refers to a phenomenon in which senescent cells elicit the development of senescence phenotype in their nearby young counterparts. In this paper we examined the mechanism of senescence bystander effect triggered by senescent human peritoneal mesothelial cells (HPMCs) in proliferating HPMCs and peritoneal fibroblasts (HPFBs). The results showed that conditioned medium (CM) derived from senescent HPMCs elicited a senescence response (growth inhibition coupled with increased expression of senescence-associated β-galactosidase and accumulation of histone γ-H2A.X) in either early-passage HPMCs or HPFBs. Samples of CM from senescent HPMCs contained increased amounts of numerous soluble mediators of which only transforming growth factor-β1 (TGF-β1) was able to induce senescence phenotype in the both types of peritoneal cells, likely through an induction of reactive oxygen species (ROS) and p38 mitogen-activated protein kinase (MAPK). At the same time, senescent HPMCs released increased amounts of thrombospondin-1 (TSP-1), a major activator of TGF-β1. Significantly, TSP-1 itself was unable to induce senescence phenotype in HPMCs or in HPFBs. The experiments employing anti-TSP-1 antibodies and specific TSP-1 blocking peptide revealed that neutralization of TSP-1 in CM prevented TGF-β1-dependent development of senescence phenotype. Collectively, our findings indicate that senescent HPMCs exhibit senescence-promoting activity toward neighboring young cells (HPMCs and HPFBs), and this effect is, at least partly, related to TSP-1-dependent activation and further ROS- and p38 MAPK-related activity of TGF-β1.