Proportion Of Senescent Cells Research Articles

Stem Cells Derived from Human Exfoliated Deciduous Teeth Functional Assessment: Exploring the Changes of Free Fatty Acids Composition during Cultivation.

The metabolic regulation of stemness is widely recognized as a crucial factor in determining the fate of stem cells. When transferred to a stimulating and nutrient-rich environment, mesenchymal stem cells (MSCs) undergo rapid proliferation, accompanied by a change in protein expression and a significant reconfiguration of central energy metabolism. This metabolic shift, from quiescence to metabolically active cells, can lead to an increase in the proportion of senescent cells and limit their regenerative potential. In this study, MSCs from human exfoliated deciduous teeth (SHEDs) were isolated and expanded in vitro for up to 10 passages. Immunophenotypic analysis, growth kinetics, in vitro plasticity, fatty acid content, and autophagic capacity were assessed throughout cultivation to evaluate the functional characteristics of SHEDs. Our findings revealed that SHEDs exhibit distinctive patterns of cell surface marker expression, possess high self-renewal capacity, and have a unique potential for neurogenic differentiation. Aged SHEDs exhibited lower proliferation rates, reduced potential for chondrogenic and osteogenic differentiation, an increasing capacity for adipogenic differentiation, and decreased autophagic potential. Prolonged cultivation of SHEDs resulted in changes in fatty acid composition, signaling a transition from anti-inflammatory to proinflammatory pathways. This underscores the intricate connection between metabolic regulation, stemness, and aging, crucial for optimizing therapeutic applications.

Effects of blood components of preoperative autologous blood donation on functions of myelopoietic stem cells

ABSTRACTObjectiveTo explore the effects of preoperative autologous whole blood donation and autologous pure red blood cell (RBC) donation on hematopoietic stem cells (HSC), clarify the effects of transfusion by different blood components on HSC, and improve the treatment effect of autotransfusion.MethodsForty healthy male New Zealand rabbits were divided into five groups (n = 8) at random: control (Group A), surgery alone (Group B), surgery + blood sampling (Group C), surgery + preoperative autologous whole blood autotransfusion (Group D), and surgery + preoperative autologous pure RBC autotransfusion (Group E). The rabbits’ bone marrow was collected before surgery (T1), 6 h after surgery (T2), and 24 h after surgery (T3) to detect the CD34+ cell count, the reactive oxygen species (ROS) concentration, the ratio of senescent cells, and the expression of HSC-related proteins (p53 and p21).ResultsCompared with groups A and B, the percentage of CD34+ cells in groups D and E at each time point was significantly increased, while the proportion of senescent cells, ROS, p53 and p21 were significantly decreased (P<0.05). Compared with Group C, the percentage of CD34+ cells at T2 and T3 rose significantly, while the ratio of senescent cells, the ROS concentration, and the content of p53 and p21 declined significantly in Groups D and E (P < 0.05). Compared with Group D, the ratio of senescent cells at T2 declined significantly, the percentage of CD34+ cells at T3 rose significantly, and the ROS concentration at T2 and T3 declined significantly in Group E (P < 0.05).ConclusionFrom the perspective of HSC, collection and transfusion of pure RBC is more beneficial to postoperative recovery of patients than whole blood transfusion.

Artesunate alleviates 5-fluorouracil-induced intestinal damage by suppressing cellular senescence and enhances its antitumor activity

BackgroundColorectal cancer (CRC) is one of the most prevalent diagnosed malignancies and one of the leading causes of cancer-related deaths worldwide. 5-Fluorouracil (5-FU) and its combination regimen are commonly used as primary chemotherapeutic agents for advanced CRC. Intestinal mucositis is one of the most frequent side effects of 5-FU. Artesunate (Arte) is derived from the wormwood plant Artemisia annua. Arte is not only effective against malaria but also diabetes, atherosclerosis, inflammation, and other conditions. The mechanism by which 5-FU damages the intestinal tract is unclear, and there is no standard treatment for diarrhea caused by 5-FU. Therefore, it is critical to discover novel and promising therapeutic drugs for 5-FU side effect treatment.MethodsThe morphology and expression of genes and proteins associated with the aging of HUVECs, HIECs, and intestinal tissues were compared to the those of the control group. The cell lines and tissues were evaluated by SA-β-Gal staining, Western blotting, and RT‒qPCR. HIEC and HCT116 cell viability was assessed in vitro by a CCK-8 assay and in vivo by a subcutaneous tumor mouse assay. Tumor cell proliferation and apoptosis was evaluated by immunohistochemistry.ResultsHere, we report that Arte alleviates the adverse side effects caused by 5-FU in intestinal tissue, and that 5-FU-induced intestinal damage is associated with drug-induced chemical inflammation and an increase in the proportion of senescent cells. Arte decreases the ratio of SA-β-Gal-positive cells and downregulated the expression of aging-related proteins (p53, p16) and aging-related genes (p53, p21). Mechanistically, Arte relieves intestinal injury by inhibiting mTOR expression, which is associated with the regulation of aging. Moreover, Arte suppresses the p38MAPK and NF-κB signaling pathways, which are related to inflammation regulation. In addition, the combined therapy of Arte plus 5-FU significantly decreases cancer cell viability in vitro. Arte and 5-FU synergistically reduce the growth of colorectal cancer (CRC) xenografts in vivo.ConclusionsOverall, our findings point to the crucial treatment effect of Arte on inflammation, intestinal cell senescence, and CRC cell proliferation and offer a new option for CRC treatment.

7-Ketocholesterol accelerates pancreatic β-cell senescence by inhibiting the SIRT1/CDK4–Rb–E2F1 signaling pathway

ABSTRACT Pancreatic β-cell dysfunction is a key factor in the development of type 2 diabetes. Pancreatic β-cell senescence accelerates abnormal glucose metabolism, which decreases insulin secretion and cell regeneration ability, eventually leading to diabetes. A cholesterol oxidation product, 7-ketocholesterol (7-KC) can affect pancreatic β-cell function. However, its role in pancreatic β-cell senescence has not been reported. We investigated the role of 7-KC in pancreatic β-cell senescence and its underlying molecular mechanism in MIN6 cells. MIN6 cells were treated with 25 μmol/L 7-KC for 24 h and the proportion of senescent cells was detected based on senescence-associated β-galactosidase (SA-β-gal) activity. The cell cycle, DNA damage, and the senescence-associate secretory phenotype (SASP) and protein expression were detected by flow cytometry, immunofluorescence, and western blotting, respectively. 7-KC can significantly increase SA-β-gal activity, promoted G0/G1 arrest, DNA damage, and interleukin-1β expression in MIN6 cells and significantly inhibited insulin synthesis. Further studies indicated that 7-KC induced β-cell senescence by inhibiting the SIRT1/CDK4–Rb – E2F1 signaling pathway.

Residual Foci of DNA Damage Response Proteins in Relation to Cellular Senescence and Autophagy in X-Ray Irradiated Fibroblasts.

DNA repair (DNA damage) foci observed 24 h and later after irradiation are called "residual" in the literature. They are believed to be the repair sites for complex, potentially lethal DNA double strand breaks. However, the features of their post-radiation dose-dependent quantitative changes and their role in the processes of cell death and senescence are still insufficiently studied. For the first time in one work, a simultaneous study of the association of changes in the number of residual foci of key DNA damage response (DDR) proteins (γH2AX, pATM, 53BP1, p-p53), the proportion of caspase-3 positive, LC-3 II autophagic and SA-β-gal senescent cells was carried out 24-72 h after fibroblast irradiation with X-rays at doses of 1-10 Gy. It was shown that with an increase in time after irradiation from 24 h to 72 h, the number of residual foci and the proportion of caspase-3 positive cells decrease, while the proportion of senescent cells, on the contrary, increases. The highest number of autophagic cells was noted 48 h after irradiation. In general, the results obtained provide important information for understanding the dynamics of the development of a dose-dependent cellular response in populations of irradiated fibroblasts.

EIF5A2 specifically regulates the transcription of aging-related genes in human neuroblastoma cells

BackgroundPost-transcriptional regulation plays a critical role in controlling biological processes such as aging. Previous studies have shown that eukaryotic initiation factor 5A (EIF5A) might play a crucial role in aging. It is unknown whether EIF5A2, a second isoform of EIF5A, could impact aging through post-transcriptional regulation.MethodsIn the present study, EIF5A2 overexpression (EIF5A2-OE) was induced in SH-SY5Y cells. RNA-seq, bioinformatics analysis and RT-qPCR validation experiments were then performed to explore the molecular mechanism of EIF5A2-mediated transcriptional regulation. Cell viability, proportion of senescent cells and the cell cycle were respectively determined by Cell Counting Kit-8, SA-β‑galactosidase and flow cytometry to evaluate the cell senescence.ResultsA total of 190 downregulated and 126 upregulated genes related to EIF5A2-OE were identified. Genes closely related to cellular aging processes such as unfolded protein response (UPR), cell adhesion and calcium signaling pathway were under global transcriptional regulation. Moreover, EIF5A2-OE promoted the viability of SH-SY5Y cells and reduced cell senescence in vitro. Among 30 genes with the most significant expression differences in EIF5A2-OE cells, we identified eight genes, including ASNS, ATF3, ATF4, CEBPB, DDIT3, HERPUD1, HSPA5 and XBP1, enriched in the UPR. Through EIF5A2-tanscription factors (TFs)-targets regulation network in EIF5A2-OE cells, we found three TFs, BHLHE40, RHOXF1 and TBX20, that targeted at these eight UPR-related genes. Verification test via the published database of human glial cell tissue showed only BHLHE40 and RHOXF1 were significantly associated with EIF5A2.ConclusionsOur findings suggest that EIF5A2 may alleviate cell senescence in vitro and mediate UPR-related genes via specific TFs. Thus, EIF5A2 could function as a regulator of aging via the regulation of transcription, which greatly expands the current understanding of the mechanisms of EIF5A2-mediated gene regulation.

Abstract P2-06-01: Parp inhibition sensitizes brca deficient cancer cell lines and tumors to clinical x-ray and proton irradiation

Abstract Purpose Triple negative breast cancers (TNBC) are more often found among BRCA gene mutation carriers; even among non-mutation carriers, TNBCs are thought to carry a phenotype of BRCAness. Inhibitors of Poly(adenosine diphosphate-ribose) polymerase (PARPi) have improved survival outcomes for both advanced and early stage breast cancer associated with a BRCA mutation, as well as BRCA mutated ovarian and prostate cancers. Radiotherapy (RT) is a widely used treatment to locally control cancers, but it is unclear how different types of RT, including clinical x-rays and protons, interact with PARPi and how these affect antitumor immunity. Protons induce more clustered DNA lesions, including clustered double and single strand breaks (DSBs and SSBs) and clustered base damages, than x-rays due to their higher ionization density. If not properly repaired, DSBs can generate micronuclei (MN), which activate the cyclic GMP-AMP synthase (cGAS) and stimulator of interferon (IFN) genes (STING) (cGAS-STING) pathway, leading to antitumor immunity. Residual DNA damage also induces cellular senescence, which is a known tumor suppressor mechanism. In this study we investigated the effects of combining a PARPi with x-rays or protons on clonogenic cell survival, residual DNA damage, senescence, cGAS co-localization with MN and delay in tumor growth in the context of BRCA1 mutation. Methods In vitro, BRCA1 deficient (HCC1937 and MDA-MB-436) and proficient (HCC1937-BRCA and MDA-MB-436-BRCA) breast cancer cell lines were treated with a PARPi (Olaparib, 0.1 - 5.0 μM) and irradiated with 6 MV x-rays or protons (9.9 keV/um) with doses from 0.5 to 5 Gy. HCC1937-BRCA was gifted from Dr. Stecklein (The University of Kansas Cancer Center) and MDA-MB-436-BRCA was gifted from Dr. Johnson (Fox Chase Cancer Center). We then quantified clonogenic cell survival, gH2AX and 53BP1 foci at 24 h after irradiation, MN number and MN-cGAS co-localization (cGAS+ MN) at 24 and 74 h after irradiation, and RT-induced senescence. In vivo, we used a preclinical TNBC mouse model (4T1 tumors on the leg of BALB/c mice) to assess survival and tumor growth delay on animals treated with DMSO, PARPi alone (Olaparib, 100 mg/kg), DMSO+x-rays, and PARPi+x-rays. Experiments with protons are ongoing. PARPi was administrated via oral gavage 2 h prior to irradiation (1 × 11 Gy). Results Survival fraction was lower after protons compared to x-rays (RBED10%&amp;gt;1) in HCC1937-BRCA, HCC1937 and MDA-MB-436 cells. Regardless of the radiation type, survival fraction was lower for HCC1937 compared to HCC1937-BRCA cells. PARPi treatment appears to result in greater radiosensitization of cells exposed to protons than those exposed to x-rays as assessed by clonogenic cell survival. The number of MN per nucleus was higher in HCC1937 than HCC1937-BRCA at 24 and 72 h after protons and x-rays at both 2 and 5 Gy, and this effect that was amplified with PARPi vs. without PARPi. Moreover, the number of cGAS+ MN was greater after protons than x-rays for HCC1937 but not HCC1937-BRCA at 24 and 72 h. The proportion of senescent cells for HCC1937-BRCA was higher (1.4 fold) after PARPi+protons compared to PARPi+x-rays. In vivo, x-rays lead to a delay in tumor growth, which was higher for the PARPi+x-rays group compared to PARPi alone or DMSO+x-rays groups. Conclusion In vitro, a PARPi treatment in BRCA1 mutated cell lines leads to a greater sensitivity to radiation compared to their counterparts with recovered BRCA1 function. This sensitivity is higher after protons compared to x-rays (survival fraction and number of micronuclei). In vivo, PARPi treatment combined with x-rays lead to higher delay in tumor growth compared to x-rays alone. These preliminary data are promising results on the effect of radiation combined with PARPi on BRCA1 mutated models. Citation Format: Mariam Ben Kacem, Scott J. Bright, Broderick X Turner, David B Flint, Mandira Manandhar, David Martinus, Gabriel O Sawakuchi, Simona F Shaitelman. Parp inhibition sensitizes brca deficient cancer cell lines and tumors to clinical x-ray and proton irradiation [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-06-01.

Improved Survival and Regeneration of Irradiated Mouse Neural Stem Cells after Co-Culturing with Non-Irradiated Mouse Neural Stem Cells or Mesenchymal Stem Cells from the Adipose Tissue.

We studied the effect of neural stem cells (NSC) and mesenchymal stem cells (MSC) from mouse adipose tissue on survival, clonogenic activity, and senescence of NSC after exposure to γ-radiation. It was found that survival and clonogenic activity of NSC irradiated in doses of 1 and 2 Gy was enhanced when irradiated cells were co-cultured with non-irradiated NSC and MSC in permeable Transwell inserts. The proportion of senescent NSC (cells with high β-galactosidase activity) increased with increasing irradiation dose. Co-culturing with non-irradiated NSC in 3 days after irradiation in a dose of 1 Gy led to a decrease in the proportion of senescent cells among irradiated NSC. Factors secreted by NSC and MSC can become the basis for the development of means for prevention and treatment of damage to brain cells resulting from radiation therapy of head and neck cancer.

Contribution of senescence in human endometrial stromal cells during proliferative phase to embryo receptivity†.

Successful assisted reproductive technology pregnancy depends on the viability of embryos and endometrial receptivity. However, the literature has neglected effects of the endometrial environment during the proliferative phase on implantation success or failure. Human endometrial stromal cells (hESCs) were isolated from endometrial tissues sampled at oocyte retrieval during the proliferative phase from women undergoing infertility treatment. Primary hESC cultures were used to investigate the relationship between stemness and senescence induction in this population and embryo receptivity. Patients were classified as receptive or non-receptive based on their pregnancy diagnosis after embryo transfer. Biomarkers of cellular senescence and somatic stem cells were compared between each sample. hESCs from non-receptive patients exhibited significantly higher (P < 0.01) proportions of senescent cells, mRNA expressions of CDKN2A and CDKN1A transcripts (P < 0.01), and expressions of genes encoding the senescence-associated secretory phenotype (P < 0.05). hESCs from receptive patients had significantly higher (P < 0.01) mRNA expressions of ABCG2 and ALDH1A1 transcripts. Our findings suggest that stemness is inversely associated with senescence induction in hESCs and, by extension, that implantation failure in infertility treatment may be attributable to a combination of senescence promotion and disruption of this maintenance function in this population during the proliferative phase of the menstrual cycle. This is a promising step towards potentially improving the embryo receptivity of endometrium. The specific mechanism by which implantation failure is prefigured by a loss of stemness among endometrial stem cells, and cellular senescence induction among hESCs, should be elucidated in detail in the future.

P16 deficiency attenuates intervertebral disc degeneration by adjusting oxidative stress and nucleus pulposus cell cycle.

The cell cycle regulator p16 is known as a biomarker and an effector of aging. However, its function in intervertebral disc degeneration (IVDD) is unclear. In this study, p16 expression levels were found to be positively correlated with the severity of human IVDD. In a mouse tail suspension (TS)-induced IVDD model, lumbar intervertebral disc height index and matrix protein expression levels were reduced significantly were largely rescued by p16 deletion. In TS mouse discs, reactive oxygen species levels, proportions of senescent cells, and the senescence-associated secretory phenotype (SASP) were all increased, cell cycling was delayed, and expression was downregulated for Sirt1, superoxide dismutase 1/2, cyclin-dependent kinases 4/6, phosphorylated retinoblastoma protein, and transcription factor E2F1/2. However, these effects were rescued by p16 deletion. Our results demonstrate that p16 plays an important role in IVDD pathogenesis and that its deletion attenuates IVDD by promoting cell cycle and inhibiting SASP, cell senescence, and oxidative stress.

MiR‑20b inhibits the senescence of human umbilical vein endothelial cells through regulating the Wnt/β‑catenin pathway via the TXNIP/NLRP3 axis.

Endothelial cell senescence is closely related to the occurrence of cardiovascular diseases and microRNAs (miRNAs/miRs) are considered as therapeutic targets for cardiovascular disease. The current study aimed to investigate the role of miR-20b in the senescence process of endothelial cells and its underlying mechanism. Cell viability, proportion of senescent cells and the cell cycle were respectively determined by Cell Counting Kit-8, SA-β-galactosidase and flow cytometry. The relative expressions of mRNA and protein were detected by reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. The possible target genes and binding sites of miR-20b were predicted using Targetscan and further verified by dual luciferase reporter assay. The present study found that H2O2 inhibited cell viability, caused cell cycle arrest in G1 phase, decreased miR-20b level and induced cell senescence. Moreover, high expression of miR-20b promoted cell viability and reduced H2O2-induced cell senescence, whereas low expression of miR-20b produced the opposite effects. Thioredoxin interacting protein (TXNIP) was predicted as a target gene for miR-20b and knockdown of TXNIP increased cell viability, inhibited cell senescence, reduced the expression of p16, p21, TXNIP, NLR family pyrin domain containing 3 (NLRP3) and cleaved Caspase-1 and reversed the promoting effects of the miR-20b inhibitor and H2O2 on cell senescence. Furthermore, the knockdown of TXNIP inhibited the Wnt/β-catenin pathway. The finding reveals that high expression of miR-20b inhibits the senescence of human umbilical vein endothelial cells through regulating the Wnt/β-catenin pathway via the TXNIP/NLRP3 axis.

MicroRNA-29b promotes cellular senescence of colorectal tumors by targeting tribbles pseudokinase 2

Objective To investigate the mechanism of microRNA (miRNA, miR)-29b regulation of tribbles pseudokinase 2 (TRIB2) in colorectal tumors. Methods The online database was used to analyze the miRNAs that bind to the 3’Untranslated Region (UTR) of TRIB2, and the highest scored miR-29b was selected for study. The eukaryotic cell transfection technique was used to interfere with the expression of miR-29b in colorectal tumor cells; Western blotting and real-time quantitative polymerase chain reaction (Real-time PCR) was used to detect the difference in protein and mRNA expression of TRIB2; the position of miR-29b binding on TRIB2-3’untranslated regions (3’UTR) was confirmed by luciferase reporter gene assay; β-galactosidase staining was used to detect effects of miR-29b on colorectal cancer cell senescence. All data were quantified as Mean±SD. Two-tailed Student’s t-test was used to evaluate the differences between two groups. All statistical analyses were performed using SPSS 24.0 (SPSS Inc.). Results In SW48 and SW480 cell lines overexpressing miR-29b, the mRNA levels of TRIB2 decreased to (63.468±4.154)% and (43.145±7.523)% (t=5.351, P 0.05). In the cancer cells overexpressing of miR-29b, the proportion of senescent cells increased from 41.473% to 62.085% (χ2=19.731, P<0.01), and overexpression of TRIB2 reduced the proportion to 46.866% (χ2=12.031, P<0.01). Conclusion miR-29b can inhibits TRIB2 expression and promote the senescence of colorectal tumors, and it can be used as a potential therapeutic target for colorectal cancer. Key words: MicroRNA-29b; Tribbles pseudokinase 2; Colorectal cancer; Cell senescence

Doxorubicin-Induced Cancer Cell Senescence Shows a Time Delay Effect and Is Inhibited by Epithelial-Mesenchymal Transition (EMT).

BackgroundSenescence is a natural barrier for the body to resist the malignant transformation of its own cells. This work investigated the senescence characteristics of cancer cells in vitro.Material/MethodsHuman cervical cancer HeLa cells were treated with different concentrations of doxorubicin for 3 days, with or without subsequent extended culture in drug-free medium for 6 days. Senescent cell ratios between these 2 culture schemes were calculated. Expression of 2 senescence-associated secretory factors, IL-6 and IL-8, were detected by RT-PCR and ELISA. Doxorubicin treatment induced epithelial-mesenchymal transition in cancer cells. The proportions of senescent cells in epithelial-like and mesenchymal-like sub-groups were calculated. Doxorubicin-treated HeLa cells were stained with Vimentin antibody and sorted by flow cytometry. Senescent cell marker p16INK4a and IL-8 expression in Vimentin-high and Vimentin-low cells were detected by Western blot.ResultsWe found that less than 1% of HeLa cells showed senescence phenotype after treatment with doxorubicin for 3 days. However, the proportion of senescent cells was significantly increased when the doxorubicin-treated cells were subsequently cultured in drug-free medium for another 6d. RT-PCR and ELISA results showed that this prolonged culture method could further improve the expression of IL-6 and IL-8. We also found that the senescent cells were mainly epithelial-like type and few presented mesenchymal-like shape. p16INK4a and IL-8 expression were decreased in cell fraction with higher Vimentin expression.ConclusionsOur results suggested the existence of time delay effect in doxorubicin-induced senescence of HeLa cells, and epithelial-mesenchymal transition may resist doxorubicin-induced cell senescence.

Protective effects of human induced pluripotent stem cell-derived exosomes on high glucose-induced injury in human endothelial cells.

Exosomes are a family of extracellular vesicles that are secreted from almost all types of cells and are associated with cell-to-cell communication. The present study was performed to investigate the effects of human induced pluripotent stem cell-derived exosomes (hiPSC-exo) on cell viability, capillary-like structure formation and senescence in endothelial cells exposed to high glucose. Exosomes were isolated from the conditional medium of hiPSCs and confirmed by transmission electron microscopy, nanoparticle tracking analysis and western blot analysis using Alix and cluster of differentiation-63 as markers. hiPSC-exo were labeled with PKH26 for tracking, and it was determined that spherical exosomes, with a typical cup-shape, were absorbed by human umbilical vascular endothelial cells (HUVECs). Cultured HUVECs were treated with high glucose (33 mM) with or without hiPSC-exo (20 µg/ml) for 48 h, and cell viability, capillary tube formation and senescence were assessed. When exposed to high glucose, viability and tube formation in HUVECs was significantly reduced (P<0.0001), whereas the proportion of senescent cells was higher compared with that in control HUVECs (P<0.0001). Furthermore, hiPSC-exo restored cell viability and capillary-like structure formation, and reduced senescence in HUVECs exposed to high glucose (P<0.0001). However, hiPSC-exo had minimal effects on normal HUVECs. These findings suggest that stem cell-derived exosomes are able to promote cell proliferation, enhance capillary-like structure formation and reduce senescence in endothelial cells exposed to high glucose.

VEGFR2 Signaling Prevents Colorectal Cancer Cell Senescence to Promote Tumorigenesis in Mice With Colitis

Senescence prevents cellular transformation. We investigated whether vascular endothelial growth factor (VEGF) signaling via its receptor, VEGFR2, regulates senescence and proliferation of tumor cells in mice with colitis-associated cancer (CAC). CAC was induced in VEGFR2(ΔIEC) mice, which do not express VEGFR2 in the intestinal epithelium, and VEGFR2(fl/fl) mice (controls) by administration of azoxymethane followed by dextran sodium sulfate. Tumor development and inflammation were determined by endoscopy. Colorectal tissues were collected for immunoblot, immunohistochemical, and quantitative polymerase chain reaction analyses. Findings from mouse tissues were confirmed in human HCT116 colorectal cancer cells. We analyzed colorectal tumor samples from patients before and after treatment with bevacizumab. After colitis induction, VEGFR2(ΔIEC) mice developed significantly fewer tumors than control mice. A greater number of intestinal tumor cells from VEGFR2(ΔIEC) mice were in senescence than tumor cells from control mice. We found VEGFR2 to activate phosphatidylinositol-4,5-bisphosphate-3-kinase and AKT, resulting in inactivation of p21 in HCT116 cells. Inhibitors of VEGFR2 and AKT induced senescence in HCT116 cells. Tumor cell senescence promoted an anti-tumor immune response by CD8(+) T cells in mice. Patients whose tumor samples showed an increase in the proportion of senescent cells after treatment with bevacizumab had longer progression-free survival than patients in which the proportion of senescent tumor cells did not change before and after treatment. Inhibition of VEGFR2 signaling leads to senescence of human and mouse colorectal cancer cells. VEGFR2 interacts with phosphatidylinositol-4,5-bisphosphate-3-kinase and AKT to inactivate p21. Colorectal tumor senescence and p21 level correlate with patient survival during treatment with bevacizumab.

Abstract A63: A stroma-based 15 gene profile for prostate cancer suggests increased DNA methylation and senescence in the stroma of patients with poor prognosis

Abstract Biomarkers are needed to address overtreatment that occurs for the majority of prostate cancer patients that would not die of the disease but receive radical treatment. Barriers to biomarker discover include the polyclonal/multifocal nature of prostate tumors and the cell-type heterogeneity between patient samples. Tumor-adjacent stroma (tumor microenvironment) is much less affected by these problems but exhibit hundreds of gene expression changes compared to normal stroma (1). We performed Affymetrix gene expression profiles of tumor-adjacent stroma for asymptomatic organ-confined disease with negative surgical margins. We identified a set of 115 probe sets for which the expression levels were significantly correlated with time-to-relapse following prostatectomy. Next, we compared expression in patients that chemically relapsed shortly after prostatectomy (&amp;lt; 1 year) versus patients that did not relapse in the first four years after prostatectomy. This comparison yielded 131 differentially expressed microarray probe sets. 19 probe sets (15 genes) were common between the two approaches, a significant degree of overlap (p &amp;lt; 0.0001). Using these 19 probe sets as input, we developed a PAM (Predicative Analysis of Microarrays)-based classifier by training on the expression profiles of samples containing stroma near tumor; 9 rapid relapse patient samples and 9 indolent patient samples. We then tested (validated) the classifier on array data from 47 independent samples containing 90% or more stroma. The classifier predicted the risk status of patients with an average accuracy of 87%. This is the first general tumor microenvironment-based prognostic classifier. Notably of the 19 probe sets, 15 are down-regulated in poor outcome disease. Moreover of the 4 upregulated probe sets in poor outcome disease are transcripts of p53 (2) and its target genes, p21 and GADD45A. These features suggest an increased proportion of senescent cells with increased gene silencing (2,3) in the stroma, consistent with a substantially increased stressful environment in the tumor-adjacent microenvironment of of the subset of prostate cancers with poor outcome. 1. Jia, . et al. Canc. Res. 2011;71 :2476-2487. 2. Gabai, V. et al. Oncogene. 2010 ;2129 :1952-1962. 3. Banerjee, J. et al. Oncogene 2013 ; Epub., PMC accession no. 24141771. Citation Format: Zhenyu Jia, Farah Rahmatpanah, Xin Chen, Waldemar Lernhardt, Yipeng Wang, Xiao-Qin Xia, Anne Sawyers, Michael McClelland, Dan Mercola. A stroma-based 15 gene profile for prostate cancer suggests increased DNA methylation and senescence in the stroma of patients with poor prognosis. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A63. doi:10.1158/1538-7445.CHTME14-A63

Inhibition of VEGF induces cellular senescence in colorectal cancer cells

Vascular endothelial growth factor (VEGF) inhibitors, such as bevacizumab, have improved outcomes in metastatic colorectal cancer (CRC). Recent studies have suggested that VEGF can delay the onset of cellular senescence in human endothelial cells. As VEGF receptors are known to be upregulated in CRC, we hypothesized that VEGF inhibition may directly influence cellular senescence in this disease. In our study, we observed that treatment with bevacizumab caused a significant increase (p < 0.05) in cellular senescence in vitro in several CRC cells, such as MIP101, RKO, SW620 and SW480 cells, compared to untreated or human IgG-treated control cells. Similar results were also obtained from cells treated with a VEGFR2 kinase inhibitor Ki8751. In vivo, cellular senescence was detected in MIP101 tumor xenografts from 75% of mice treated with bevacizumab, while cellular senescence was undetectable in xenografts from mice treated with saline or human IgG (p < 0.05). Interestingly, we also observed that the proportion of senescent cells in colon cancer tissues obtained from patients treated with bevacizumab was 4.4-fold higher (p < 0.01) than those of untreated patients. To understand how VEGF inhibitors may regulate cellular senescence, we noted that among the two important regulators of senescent growth arrest of tumor cells, bevacizumab-associated increase in cellular senescence coincided with an upregulation of p16 but appeared to be independent of p53. siRNA silencing of p16 gene in MIP101 cells suppressed bevacizumab-induced cellular senescence, while silencing of p53 had no effect. These findings demonstrate a novel antitumor activity of VEGF inhibitors in CRC, involving p16.

Sex-specific compromised bone healing in female rats might be associated with a decrease in mesenchymal stem cell quantity

Introduction The clinically known importance of patient sex as a major risk factor for compromised bone healing is poorly reflected in animal models. Consequently, the underlying cellular mechanisms remain elusive. Because mesenchymal stem cells (MSCs) are postulated to regulate tissue regeneration and give rise to essential differentiated cell types, they may contribute to sex-specific differences in bone healing outcomes. Methods We investigated sex-specific variations in bone healing and associated differences in MSC populations. A 1.5 mm osteotomy gap in the femora of 8 male and 8 female 12-month-old Sprague–Dawley rats was stabilized by an external fixator. Healing was analyzed in terms of biomechanical testing, bridging and callus size over time (radiography at 2, 4, and 6 weeks after surgery), and callus volume and geometry by μCT at final follow-up. MSCs were obtained from bone marrow samples of an age-matched group of 12 animals (6 per gender) and analyzed for numbers of colony-forming units (CFUs) and their capacity to differentiate and proliferate. The proportion of senescent cells was determined by β-galactosidase staining. Results Sex-specific differences were indicated by a compromised mechanical competence of the callus in females compared with males (maximum torque at failure, p = 0.028). Throughout the follow-up, the cross-sectional area of callus relative to bone was reduced in females ( p ≤ 0.01), and the bridging of callus was delayed ( p 2weeks = 0.041). μCT revealed a reduced callus size ( p = 0.003), mineralization ( p = 0.003) and polar moment of inertia ( p = 0.003) in female animals. The female bone marrow contained significantly fewer MSCs, represented by low CFU numbers in both femora and tibiae ( p femur = 0.017, p tibia = 0.010). Functional characteristics of male and female MSCs were similar. Conclusion Biomechanically compromised and radiographically delayed bone formation were distinctive in female rats. These differences were concomitant with a reduced number of MSCs, which may be causative for the suboptimal bone healing.

In situ ageing of fine beech roots ( Fagus sylvatica) assessed by transmission electron microscopy and electron energy loss spectroscopy: description of microsites and evolution of polyphenolic substances

Root biomass is quantitatively and qualitatively important in most ecosystems, but its contribution to the pool of organic matter in the soil is not clear. This work was designed to specify root ageing on an ultrastructural scale by transmission electron microscopy combined with microanalysis by electron energy loss spectroscopy. This approach is very suitable for studying the soil/plant interface, and for semi-quantitative analysis of the evolution of polyphenolic substances during root evolution. Three root segments were studied according to a gradient of root senescence: the apical and basal segments of the mycorrhiza and the mycorrhiza-carrier root. Each segment contained a certain proportion of senescent cells, some of which were of fungal origin, and this proportion increased as the root aged. In the three segments, the soil/plant interfaces were differentiated, and the micro-organisms observed in situ were described. Senescent root cells contained many polyphenolic substances and our results showed that these substances were, according to the root segment, differently associated with Ca, N and Si. When all these ultrastructural data are correlated with more global data, they can be usefully applied to root cell physiology, microbiology and pedology. This approach makes it possible to specify the evolution of organic matter in situ in soils whatever its origin.

Age-related loss of proliferative activity of human vascular smooth muscle cells in culture

This work studied the proliferation activity in cultures of vascular smooth muscle cells (SMC) from individuals of different ages. The cells derived from arteries of 12 donors of both sexes from 45 to 91 years of age. The main parameter considered was the ‘proliferation rate’ (cells grown per day in the different culture passages) taking into account the age of the donor. No significant relationship between age of the donor and the cell life in proliferation was found. On the contrary, the mean time of passage duration for reaching the maximum of proliferation as well as its ‘efficiency’ (maximum of proliferation rate registered/mean time of passage duration) show a statistically significant dependence on the age of the donor. Furthermore, the proliferation rate measured in each passage is statistically significant related to donor age. The regressions obtained show a similar negative slope (VC 4%). Considering the first five culture passages, the regression crosses the x-axis at the age of 105.6±11.7 years. This age in which no proliferative activity of human SMC would be expected lies near the limit of maximum life potential for human beings. Our results suggest that with advancing donor age there is an increasing number of senescent SMC either primarily transferred or appeared in the culture. Vascular SMC of individuals whose life is near the end would almost be all senescent and therefore show extremely low proliferation rates in the culture. If the proliferative activity of arterial SMC is a condition for atherogenesis, the proportion of senescent cells would be inversely related to the propensity of developing the atheroma because of the inability of these cells to divide.