Tumor suppressor and aging biomarker p16(INK4a) induces cellular senescence without the associated inflammatory secretory phenotype

Cellular senescence suppresses cancer by preventing the proliferation of cells that experience potentially oncogenic stimuli. Senescent cells often express p16(INK4a), a cyclin-dependent kinase inhibitor, tumor suppressor, and biomarker of aging, which renders the senescence growth arrest irreversible. Senescent cells also acquire a complex phenotype that includes the secretion of many cytokines, growth factors, and proteases, termed a senescence-associated secretory phenotype (SASP). The SASP is proposed to underlie age-related pathologies, including, ironically, late life cancer. Here, we show that ectopic expression of p16(INK4a) and another cyclin-dependent kinase inhibitor, p21(CIP1/WAF1), induces senescence without a SASP, even though they induced other features of senescence, including a stable growth arrest. Additionally, human fibroblasts induced to senesce by ionizing radiation or oncogenic RAS developed a SASP regardless of whether they expressed p16(INK4a). Cells induced to senesce by ectopic p16(INK4a) expression lacked paracrine activity on epithelial cells, consistent with the absence of a functional SASP. Nonetheless, expression of p16(INK4a) by cells undergoing replicative senescence limited the accumulation of DNA damage and premature cytokine secretion, suggesting an indirect role for p16(INK4a) in suppressing the SASP. These findings suggest that p16(INK4a)-positive cells may not always harbor a SASP in vivo and, furthermore, that the SASP is not a consequence of p16(INK4a) activation or senescence per se, but rather is a damage response that is separable from the growth arrest.     

Endogenous mutagens and the causes of aging and cancer

A very large oxidative damage rate to DNA occurs as part of normal metabolism. In each rat cell the steady-state level is estimated to be about 10⁶ oxidative adducts and about 10⁵ new adducts are formed daily. It is argued that this endogenous DNA damage is a major contributor to aging and the degenerative diseases of aging, such as cancer. The oxidative damage rate in mammalian species with a high metabolic rate, short life span, and high age-specific cancer rate is much higher than the rate in humans, a long-lived creature with a lower metabolic rate and a lower age-specific cancer rate. It is argured that deficiency of micronutrients, such as dietary antioxidants or folate, is a major contributor to human cancer and degenerative diseases.

Understanding the role of mitogenesis in mutagenesis is critical for clarifying the mechanisms of carcinogenesis and interpreting high-dose animal cancer tests. High-dose animal cancer tests have been done mainly on synthetic industrial chemicals, yet almost all of the chemicals humans are exposed to are natural. About half of natural chemicals tested in high-dose animal cancer tests are rodent carcinogens, a finding that is consistent with the view that high-dose tests frequently increase mitogenesis rates. Animals have numerous defenses against toxins that make them very well buffered against low doses of almost all toxins, whether synthetic or natural.     

Up-regulation of eEF1A2 promotes proliferation and inhibits apoptosis in prostate cancer

Background

eEF1A2 is a protein translation factor involved in protein synthesis, which possesses important function roles in cancer development. This study aims at investigating the expression pattern of eEF1A2 in prostate cancer and its potential role in prostate cancer development.

Methods

We examined the expression level of eEF1A2 in 30 pairs of prostate cancer tissues by using RT-PCR and immunohistochemical staining (IHC). Then we applied siRNA specifically targeting eEF1A2 to down-regulate its expression in DU-145 and PC-3 cells. Flow cytometer was used to explore apoptosis and Western-blot was used to detect the pathway proteins of apoptosis.

Results

Our results showed that the expression level of eEF1A2 in prostate cancer tissues was significantly higher compared to their corresponding normal tissues. Reduction of eEF1A2 expression in DU-145 and PC-3 cells led to a dramatic inhibition of proliferation accompanied with enhanced apoptosis rate. Western blot revealed that apoptosis pathway proteins (caspase3, BAD, BAX, PUMA) were significantly up-regulated after suppression of eEF1A2. More importantly, the levels of eEF1A2 and caspase3 were inversely correlated in prostate cancer tissues.

Conclusion

Our data suggests that eEF1A2 plays an important role in prostate cancer development, especially in inhibiting apoptosis. So eEF1A2 might serve as a potential therapeutic target in prostate cancer.     

CXCL12 overexpression and secretion by aging fibroblasts enhance human prostate epithelial proliferation in vitro

The direct relationship between the aging process and the incidence and prevalence of both benign prostatic hyperplasia (BPH) and prostate cancer (PCa) implies that certain risk factors associated with the development of both diseases increase with the aging process. In particular, both diseases share an overly proliferative phenotype, suggesting that mechanisms that normally act to suppress cellular proliferation are disrupted or rendered dysfunctional as a consequence of the aging process. We propose that one such mechanism involves changes in the prostate microenvironment, which 'evolves' during the aging process and disrupts paracrine interactions between epithelial and associated stromal fibroblasts. We show that stromal fibroblasts isolated from the prostates of men 63-81 years of age at the time of surgery express and secrete higher levels of the CXCL12 chemokine compared with those isolated from younger men, and stimulate CXCR4-mediated signaling pathways that induce cellular proliferation. These studies represent an important first step towards a mechanistic elucidation of the role of aging in the etiology of benign and malignant prostatic diseases.     

AaHIV a sodium channel scorpion toxin inhibits the proliferation of DU145 prostate cancer cells

Prostate cancer is the most highly diagnosed cancer in men worldwide. It is characterized by high proliferation, great invasion and metastatic potential. Sodium channel subtypes have been identified as highly expressed in different prostate cancer cell lines. In this study, we have screened the negatively charged fractions of Androctonus australis (Aa) scorpion venom to identify active peptides on DU145 prostate cancer cells proliferation. The most active compound was identified to be the sodium channel peptide AaHIV with an IC₅₀ value of 15 μM. At this concentration, AaHIV had low effect on the adhesion of DU145 cells to fibronectin. When compared to other Na⁺ channel Aa toxins, AaHIV was found to be 2 times more active than AaHI and AaHII on DU145 cells proliferation and slightly less active than AaHII on their adhesion. The three peptides are inactive on DU145 cells migration. AaHIV was found to be 16 times more active than veratridine, asteroidal alkaloid from plants of the lily family widely used as a sodium channel activator. Electrophysiological experiments showed that the AaHIV toxin activates Nav1.6 channel, suggesting that this sodium channel subtype is implicated in the proliferation of DU145 prostate cancer cells.     

Implications of a 5′-nucleotidase inhibitor in human leukemic cells for cellular aging and cancer

5′-Nucleotidase activity of normal human embryonic lung fibroblasts (IMR-90) was found to be inhibited by the homogenates of seven different cell lines originated from patients with different kinds of leukemia and of fresh lymphocytes from a patient with Sezary syndrome (circulating T-cell lymphoma). About 97% of the inhibiting activity was found in the soluble fraction of RPMI 8402 cells, a cell line originated from the lymphocytes of a patient with acute lymphocytic leukemia. This inhibiting activity was not destroyed by dialysis, heating at 56°C for 30 min, nor digestion with RNAase or DNAase. About 85% of the inhibiting activity was destroyed by digestion with papain at 37°C for 1 h and it was destroyed completely by heating at 100°C for 30 min. When the heated (56°C for 30 min) soluble fraction of RPMI 8402 cells was mixed with the homogenate of IMR-90 cells, it had no effect on the activities of alkaline, neutral or acid phosphatases, nor of $\text{N-}\text{acetyl}\text{-β-}\text{d}\text{-glucosaminidase}$ or cytochrome c oxidase of IMR-90 cells. Preincubating the mixed samples for 1, 20 and 45 min, respectively, before adding the substrate, the heated soluble fraction of RPMI 8402 cells did not increase the percentage of inhibition for 5′-nucleotidase of the homogenate of IMR-90 cells. No inhibition of other enzyme activities was observed under similar conditions. These data suggest that the inhibiting activity is due to a protein(s) that is not a protease. The inhibiting activity was found in a single peak after the soluble fraction was fractionated by Sephadex G-100 chromatography and sedimentation centrifugation. The molecular weight of the inhibitor was found to be approx. 35 000 by comparing its retention volume and sedimentation rate with those of proteins of known molecular weight. The present study suggests that the previously reported undetectability of 5′-nucleotidase in permanent cell lines could be due to the presence of a protein inhibitor for 5′-nucleotidase in these human leukemic cell lines. It also supports the hypothesis that the increased 5′-nucleotidase activity in normal senescent cells in vitro may be a control in cellular aging that is missing from leukemic cells in vitro.     

Investigation of cellular movement in the prostate epithelium using an agent-based model

Experimental patterns of epithelial cell proliferation in the prostate suggest that cell movement may play an important role in prostate epithelial homeostasis, and genes known to regulate cell movement are commonly mutated or deleted in prostate carcinomas. However, the nature of cell movement within the prostate epithelium remains unknown. Here, the role of cellular movement in the prostate epithelium was explored by developing an agent-based model of the prostate duct. Prostatic adult stem cells, transit amplifying/intermediate cells (TA/ICs), and luminal cells were individually modeled within a three-dimensional reconstruction of a prostate duct. Different movement behaviors for TA/ICs and luminal cells were assessed by their ability to recreate experimental patterns of prostate cell proliferation and epithelial morphology. Strongly directed TA/IC movement toward the distal region of the prostate duct combined with weakly directed luminal cell movement toward the proximal region of the prostate duct was able to best recreate experimental patterns of prostate proliferation and morphology. The effects on cell mobility from abnormalities in PTEN and thymosin β15 (Tβ15), genes which are commonly altered in prostate cancer, were simulated in the model. These simulations show that altering prostate stem cell movement can dysregulate epithelial homeostasis and lead to excessive cell growth, suggesting that disruption of cell movement may contribute to prostate carcinogenesis.     

Polo-like kinase 1 facilitates loss of Pten tumor suppressor-induced prostate cancer formation

Loss of the tumor suppressor Pten (phosphatase and tensin homolog deleted on chromosome 10) is thought to mediate the majority of prostate cancers, but the molecular mechanism remains elusive. In this study, we demonstrate that Pten-depleted cells suffer from mitotic stress and that nuclear function of Pten, but not its phosphatase activity, is required to reverse this stress phenotype. Further, depletion of Pten results in elevated expression of Polo-like kinase 1 (Plk1), a critical regulator of the cell cycle. We show that overexpression of Plk1 correlates with genetic inactivation of Pten during prostate neoplasia formation. Significantly, we find that elevated Plk1 is critical for Pten-depleted cells to adapt to mitotic stress for survival and that reintroduction of wild-type Pten into Pten-null prostate cancer cells reduces the survival dependence on Plk1. We further show that Plk1 confers the tumorigenic competence of Pten-deleted prostate cancer cells in a mouse xenograft model. These findings identify a role of Plk1 in facilitating loss of Pten-induced prostate cancer formation, which suggests that Plk1 might be a promising target for prostate cancer patients with inactivating Pten mutations.     

Antioxidant activity of melatonin in Chinese hamster ovarian cells: changes in cellular proliferation and differentiation

Melatonin is an endogenously generated molecule with free radical scavenging and antioxidant properties. Here, we studied the antiproliferative role of melatonin and other antioxidants on transformed Chinese hamster ovarian cells. Melatonin reduces cell proliferation in a dose- and time-dependent manner. Natural antioxidants which appear in edible plants including resveratrol and vitamin E mimicked the effect of melatonin. Flow cytometer analysis revealed that melatonin treatment reduces the number of cells in S-phase and increases cells in both G0/G1 and G2/M gaps. In addition, melatonin, as well as trolox, caused a clear morphological change by inducing the cells to become spindle shaped and fibroblast-like. Its effect is a reversible phenomenon that disappeared when melatonin was withdrawn from the culture medium. GSH levels are increased after melatonin treatment but pharmacologically blockade of GSH synthesis did not abolish melatonin's antiproliferative effect. Reduction of cell proliferation and the apparent induction of cell differentiation overlapped with melatonin's ability to change the intracellular redox state of CHO cells. We conclude that the cellular redox state may be involved in cellular transformation caused by antioxidants such as melatonin and trolox.     

The PKB/FOXO switch in aging and cancer

Aging is characterized by the general decline in tissue and body function and the increased susceptibility to age-related pathologies, such as cancer. To maintain optimal tissue and body function, organisms have developed complex mechanisms for tissue homeostasis. Importantly, it is becoming apparent that these same mechanisms when deregulated also result in the development of age-related disease. The build in failsafe mechanisms of homeostasis, which prevent skewing toward disease, themselves contribute to aspects of aging. Thus, longevity is limited by an intrinsic trade-off between optimal tissue function and disease. Consequently, aging and age-related diseases, such as cancer and diabetes are driven by the same genetic determinants. Illustrative in this respect is the insulin/IGF-1 signaling pathway acting through PI3K/PKB and FOXO. Loss of PKB signaling contributes to diabetes, whereas gain of function of PKB drives cancer. Enhanced FOXO activity, at least in model organism contributes to extended lifespan and acts as a tumor suppressive mechanism. Here, we focus on the linkage between PKB and FOXO as a central switch in contributing to tissue homeostasis and age-related diseases in particular cancer. This article is part of a Special Issue entitled: P13K-AKT-FoxO axis in cancer and aging.     

Calcium signaling and cellular senescence

Cellular senescence is a stable cell proliferation arrest induced by a variety of stresses including telomere shortening, oncogene activation and oxidative stress. This process plays a crucial role in many physiopathological contexts, especially during aging when cellular senescence favors development of age-related diseases, shortening lifespan. However, the molecular and cellular mechanisms controlling senescence are still a matter of active research. In the last decade, there has been emerging literature indicating a key involvement of calcium signaling in cellular senescence. In this review we will initially give an account of the direct evidence linking calcium and the regulation of senescence. We will then review our current knowledge on the role of calcium in some senescence-associated features and physiopathological conditions, which will shed light on additional ways in which calcium signaling is implicated in cellular senescence.     

Kallikarein-related peptidase 3 common genetic variant and the risk of prostate cancer

Kallikarein-related peptidase 3 (KLK3) gene polymorphisms seem to play a role in susceptibility to prostate cancer (PC). The purpose of this study was to investigate the association between rs2735839 polymorphism of KLK3 gene and risk of PC in an Iranian population. In this case-control study, rs2735839 was genotyped in 532 patients with PC and 602 controls with benign prostate hyperplasia (BPH) using polymerase chain reaction-restriction fragment length polymorphism assay. The frequency of GG, AG, and AA genotypes of KLK3 polymorphism was 24.6% and 76.2%, 46.6% and 21.7%, and 28.8% and 2.1%, in patients with BPH and PC, respectively (P < 0.001). The frequency of G allele in patients with BPH and PC was 47.9% and 87%, respectively (odds ratio: 7.31; confidence interval: 5.88-9.10; P < 0.001). Patients with AG and GG genotypes had a higher total serum level of prostate-specific antigen (PSA) compared to those with AA genotype (P < 0.001). Patients with this polymorphism had higher risk of tumor with higher grade (P = 0.23), advanced stage (P = 0.11), perineural invasion (P = 0.07), and vascular invasion (P = 0.07) compared to those without it but this difference was not statistically significant. Based on our results, KLK3 gene polymorphism was associated with the risk of PC. Higher levels of PSA in the presence of KLK3 polymorphism in patients with PC indicated that rs2735839 polymorphism could be a risk factor for increased levels of PSA.     

Mitochondrial damage and intralysosomal degradation in cellular aging

Normal mitochondrial respiration is associated with a continuous production of superoxide and hydrogen peroxide, inevitably resulting in minor macromolecular damage. Damaged cellular components are not completely turned over by autophagy and other cellular repair systems, leading to a progressive age-related accumulation of biological “garbage” material, such as defective mitochondria, cytoplasmic protein aggregates and an intralysosomal undegradable material, lipofuscin. These changes primarily affect neurons, cardiac myocytes and other long-lived postmitotic cells that neither dilute this “garbage” by mitotic activity, nor are replaced by newly differentiated cells. Defective mitochondria are insufficient in ATP production and often generate increased amounts of reactive oxygen species, further enhancing oxidative stress. Lipofuscin-loaded lysosomes, in turn, poorly turn over mitochondria that gradually leads to the overload of long-lived postmitotic cells with “garbage” material, decreased adaptability and eventual cell death.     

The effect of aging on micronuclei frequency and proliferation in human peripheral blood lymphocytes

INTRODUCTION:

Increase in the instability of cellular genome with an increasing age is the result of an accumulation of cellular damage and mutations. This instability which might be observed as chromosome damage or chromosome losses can be measured by the micronucleus technique.

AIM:

The aim of this study was to investigate the effect of aging and oxidative stress induced by non-toxic levels of H₂O₂ on micronuclei induction and their relationship to cell proliferation in human peripheral blood lymphocytes.

MATERIALS AND METHODS:

Healthy volunteers with different ages were choosen. Spontaneous and H₂O₂ induced micronuclei frequencies were measured in peripheral blood lymphocytes of 30 volunteers by the micronucleus method.

RESULTS:

Spontaneous micronuclei frequencies increased first then started to decrease after 50 years of age. This biphasic response was significantly higher than micronucleus (MN) frequencies induced by H₂O₂ (P < 0.05), which followed the similar shape of response to increasing ages with lower frequencies. Proliferative capacity of cells either treated with H₂O₂ or not did not differ with an increasing age giving similar responses.

CONCLUSION:

These results indicate biphasic character of chromosome damage; first increase and decrease after 50 years with an increasing age. But this change pattern was not correlated with the steady state of proliferation capacity of cells through an increasing age. Decreases in H₂O₂-induced MN frequencies compared to spontaneous MN frequencies may be inducing an apoptosis by H₂O₂ treatment leading to underscoring damaged cells.     

Increased infiltrated macrophages in benign prostatic hyperplasia (BPH): role of stromal androgen receptor in macrophage-induced prostate stromal cell proliferation

Infiltrated macrophages may play important roles in the development and progression of benign prostatic hyperplasia (BPH), but the underlying mechanisms remain largely unknown. We found increased macrophages infiltration in human and mouse BPH tissues. By establishing a co-culture transwell system, we found increased migration of macrophages and proliferation of prostate stromal cells during co-culture. Importantly, stromal androgen receptor (AR) could enhance the migration of macrophages and macrophage-mediated stromal cell proliferation. We identified CCL3 as an AR downstream player, and found CCL3 levels were notably increased in human and mouse BPH prostates. Ablation of prostate stromal AR in a mouse BPH model significantly reduced CCL3 expression levels in prostates. Consistently, targeting AR via an AR degradation enhancer, ASC-J9§, or neutralization of CCL3 with an antibody, resulted in suppression of macrophage migration and prostate stromal cell growth. Our study provides mechanistic insights on the regulation of prostate stromal cells by macrophages via stromal AR/CCL3 signaling pathways, which could potentially allow the development of therapeutic approaches for battling BPH with persistent inflammation.     

Androgen deficiency in the aging male: benefits and risks of androgen supplementation

There is now convincing evidence that in a subset of aging men, increasing with age, plasma testosterone levels fall below a critical level resulting in hypogonadism. This state of testosterone deficiency has an impact on bone, muscle and brain function and is maybe a factor in the accumulation of visceral fat which again has a significant impact on the cardiovascular risk profile. From the above it follows that androgen replacement to selected men with proven androgen deficiency will have beneficial effects. There is, however a concern that androgen administration to aging men may be harmful in view of effects on prostate disease. Benign prostate hyperplasia (BPH) and prostate cancer are typically diseases of the aging male, steeply increasing with age. But epidemiological studies provide no clues that the levels of circulating androgen are correlated with or predict prostate disease. Similarly, androgen replacement studies in men do not suggest that these men suffer in a higher degree from prostate disease than control subjects. It seems a defensible practice to treat aging men with androgens if and when they are testosterone-deficient, but long-term studies including sufficient numbers of men are needed.     

Age-dependent methylation of ESR1 gene in prostate cancer

The incidence of prostate cancer increases dramatically with age and the mechanism underlying this association is unclear. Age-dependent methylation of estrogen receptor alpha (ESR1) gene has been previously implicated in other cancerous and benign diseases. We evaluated the age-dependent methylation of ESR1 in prostate cancer. The methylation status of ESR1 in 83 prostate cancer samples from patients aged 49 to 77 years (mean age at 67.4 years) was examined using the bisulfite genomic sequencing technique. The samples were divided into three age groups: men aged 60 years and under (n = 14), men aged 61-70 years (n = 40), and men aged over 70 years (n = 29). Overall, ESR1 promoter methylation was detected in 54 out of 83 (65.1%) prostate samples. The methylation rate of ESR1 increased dramatically with age from 50.0% in patients aged 60 years and under to 89.7% for patients aged 70 years and over. Logistic regression analyses revealed that age and Gleason score were the only variables that affect incidence of ESR1 methylation; other clinical factors such as prostate-specific antigen level and clinical stage did not. We also calculated ESR1 methylation density (the percentage of methylated CpGs among all CpGs within the analyzed region) and severity (the percentage of methylated CpG alleles) for each sample analyzed. Multiple regression analyses showed a positive correlation between age and methylation density (beta, 0.35; P, 0.012; 95% CI, 0.26-2.01); while Gleason score was positively associated with methylation severity (beta, 0.45; P, 0.018; 95% CI, 1.04-4.26). These findings suggest that methylation of ESR1 is both age-dependent and tumor differentiation-dependent and age-dependent methylation of ESR1 may represent a mechanism linking aging and prostate cancer.     

Polymorphisms of MLH1 in benign prostatic hyperplasia and sporadic prostate cancer

Mismatch repair is one of several DNA repair pathways of which defects may lead to cancer. We hypothesize that polymorphisms of the MLH1 gene can be a risk factor for benign prostatic hyperplasia (BPH) and prostate cancer. The genetic distribution of MLH1 polymorphisms that lead to amino acid changes at codons 132, 219, 384, and 723 were analyzed in BPH and sporadic prostate cancer patients, and compared to healthy controls from an Asian population. These experiments demonstrate a protective role for the codon 384 variant allele against prostate cancer (P = 0.031) but not BPH when compared to normal controls and furthermore, an inverse association was observed with stage (P = 0.074) and grade (P = 0.056) of cancer. This is the first report that demonstrates a protective effect for the race-related MLH1 polymorphism at codon 384 against prostate cancer and these results are important in understanding their role in this disease.