Sulforaphane-induced apoptosis involves p53 and p38 in melanoma cells

In malignant melanoma complex reprogramming of cell death and survival pathways leads to increased chemoresistance and poor longer-term survival. Sulforaphane (SF) is a promising isothiocyanate compound occurring in cruciferous plants with reported antiproliferative and proapoptotic activity in several tumor cell lines including melanoma. In this work we investigated the effects of SF in several melanoma cell lines and fresh melanoma cultivates. We found that SF is cytotoxic and induces mitochondrial, caspase-dependent apoptosis in our study model, however with lower efficiency in fresh melanoma cultivates. Moreover, our results indicate that in melanoma cell lines and fresh melanoma cultivates SF induces multiple signaling including oxidative stress-mediated activation of DNA-damage response pathway, changes in p38 kinase activity and enhanced expression of Bax and Puma proapoptotic proteins. In addition, in SF-exposed p53-mutant melanoma cells Puma expression seem to be under p38 control and acts as a compensatory proapoptotic mechanism. Conversely, decreased apoptosis in SF-exposed melanoma cultivates might be attributed to Akt-mediated suppression of p38 as well as p53 activity. Together, our results suggest that SF inhibits growth and proliferation and induces mitochondrial apoptosis both in melanoma cell lines as well as in fresh melanoma cultivates. This proapoptotic effect might be enhanced in combination with Akt inhibitors, in particular in melanoma samples. SF is thus commendable for further preclinical testing, both as a single agent as well as in combination regimens.     

Granzyme B-induced cell death involves induction of p53 tumor suppressor gene and its activation in tumor target cells

In this study we investigated the involvement of p53 in cytotoxic T-lymphocyte (CTL)-induced tumor target cell killing mediated by the perforin/granzymes pathway. For this purpose we used a human CTL clone (LT12) that kills its autologous melanoma target cells (T1), harboring a wild type p53. We demonstrated initially that LT12 kills its T1 target in a perforin/granzymes-dependent manner. Confocal microscopy and Western blot analysis indicated that conjugate formed between LT12 and T1 resulted in rapid cytoplasmic accumulation of p53 and its activation in T1 target cells. Cytotoxic assay using recombinant granzyme B (GrB) showed that this serine protease is the predominant factor inducing such accumulation. Furthermore, RNA interference-mediated lowering of the p53 protein in T1 cells or pifithrin-alpha-induced p53-specific inhibition activity significantly decreased CTL-induced target killing mediated by CTL or recombinant GrB. This emphasizes that p53 is an important determinant in granzyme B-induced apoptosis. Our data show furthermore that when T1 cells were treated with streptolysin-O/granzyme B, specific phosphorylation of p53 at Ser-15 and Ser-37 residues was observed subsequent to the activation of the stress kinases ataxia telangiectasia mutated (ATM) and p38K. Treatment of T1 cells with pifithrin-alpha resulted in inhibition of p53 phosphorylation at these residues and in a significant decrease in GrB-induced apoptotic T1 cell death. Furthermore, small interference RNAs targeting p53 was also accompanied by an inhibition of streptolysin-O/granzyme B-induced apoptotic T1 cell death. The present study supports p53 induction after CTL-induced stress in target cells. These findings provide new insight into a potential role of p53 as a component involved in the dynamic regulation of the major pathway of CTL-mediated cell death and may have therapeutic implications.     

Chrysin and Capsaicin induces premature senescence and apoptosis via mitochondrial dysfunction and p53 elevation in Cervical cancer cells

Current studies are focusing on the anti-cancerous properties of natural bioactive compounds, primarily those included in the human diet. These compounds have the potential to alter the redox balance that can hinder cancer cell's growth. In cancer cells, an abnormal rate of ROS production is balanced with higher antioxidant activities, which if not maintained, results in cancer cells being prone to cell death due to oxidative stress. Here, we have analyzed the effects of Chrysin and Capsaicin on the HeLa cells viability and cellular redox signaling. Both these compounds stimulate cellular and mitochondrial ROS overproduction that perturbs the cellular redox state and results in mitochondrial membrane potential loss. Apart from this, these compounds induce cell cycle arrest and induce premature senescence, along with the overexpression of p21, p53, and p16 protein at lower concentration treatment of Chrysin or Capsaicin. Moreover, at higher concentration treatment with these compounds, pro-apoptotic activity was observed with the high level of Bax and cleaved caspase-3 along with suppression of the Bcl-2 protein levels. In-Silico analysis with STITCH v5 also confirms the direct interaction of Chrysin and Capsaicin with target protein p53. This suggests that Chrysin and Capsaicin trigger an increase in mitochondrial ROS, and p53 interaction leading to premature senescence and apoptosis in concentration dependent manner and have therapeutic potential for cancer treatment.     

p53 deficiency rescues the adverse effects of telomere loss and cooperates with telomere dysfunction to accelerate carcinogenesis.

Maintenance of telomere length and function is critical for the efficient proliferation of eukaryotic cells. Here, we examine the interactions between telomere dysfunction and p53 in cells and organs of telomerase-deficient mice. Coincident with severe telomere shortening and associated genomic instability, p53 is activated, leading to growth arrest and/or apoptosis. Deletion of p53 significantly attenuated the adverse cellular and organismal effects of telomere dysfunction, but only during the earliest stages of genetic crisis. Correspondingly, the loss of telomere function and p53 deficiency cooperated to initiate the transformation process. Together, these studies establish a key role for p53 in the cellular response to telomere dysfunction in both normal and neoplastic cells, question the significance of crisis as a tumor suppressor mechanism, and identify a biologically relevant stage of advanced crisis, termed genetic catastrophe.     

Autophagy-independent senescence and genome instability driven by targeted telomere dysfunction

Telomere dysfunction plays a complex role in tumorigenesis. While dysfunctional telomeres can block the proliferation of incipient cancer clones by inducing replicative senescence, fusion of dysfunctional telomeres can drive genome instability and oncogenic genomic rearrangements. Therefore, it is important to define the regulatory pathways that guide these opposing effects. Recent work has shown that the autophagy pathway regulates both senescence and genome instability in various contexts. Here, we apply models of acute telomere dysfunction to determine whether autophagy modulates the resulting genome instability and senescence responses. While telomere dysfunction rapidly induces autophagic flux in human fibroblast cell lines, inhibition of the autophagy pathway does not have a significant impact upon the transition to senescence, in contrast to what has previously been reported for oncogene-induced senescence. Our results suggest that this difference may be explained by disparities in the development of the senescence-associated secretory phenotype. We also show that chromosome fusions induced by telomere dysfunction are comparable in autophagy-proficient and autophagy-deficient cells. Altogether, our results highlight the complexity of the senescence-autophagy interface and indicate that autophagy induction is unlikely to play a significant role in telomere dysfunction-driven senescence and chromosome fusions.     

Drosophila p53 is a structural and functional homolog of the tumor suppressor p53

The importance of p53 in carcinogenesis stems from its central role in inducing cell cycle arrest or apoptosis in response to cellular stresses. We have identified a Drosophila homolog of p53 ("Dmp53"). Like mammalian p53, Dmp53 binds specifically to human p53 binding sites, and overexpression of Dmp53 induces apoptosis. Importantly, inhibition of Dmp53 function renders cells resistant to X ray-induced apoptosis, suggesting that Dmp53 is required for the apoptotic response to DNA damage. Unlike mammalian p53, Dmp53 appears unable to induce a G1 cell cycle block when overexpressed, and inhibition of Dmp53 activity does not affect X ray-induced cell cycle arrest. These data reveal an ancestral proapoptotic function for p53 and identify Drosophila as an ideal model system for elucidating the p53 apoptotic pathway(s) induced by DNA damage.     

Actin-binding doliculide causes premature senescence in p53 wild type cells

Addressing the actin cytoskeleton as future anticancer target can be an innovative chemotherapeutic approach to combat malignancies. Doliculide is a potent stabilizer of actin filaments and can be used as tool and therapeutic lead in cancer research. Though a variety of molecules are known to bind to actin and lead to either its over- or depolymerization little is known about the pharmacological consequences of these effects within the cancer cell. In this work we used p53 wild-type cells to dissect the reaction of these cells towards subtoxic doses of doliculide. We could show that doliculide leads to a transient change in actin cytoskeleton dynamics that are reversible. The cells react towards the treatment with the induction of premature senescence, an established anti-cancer mechanism, in concentrations that are not cytotoxic. Furthermore, we investigated the signaling pathways that are involved in the induction and maintenance of senescence by a pathway directed mRNA PCR-array. This analysis revealed that under doliculide treatment up to 13% of senescence related genes are altered. Taken together, our data provide evidence for an antitumoral potential of actin binding agents in p53 wild type cells and brings the strategy of targeting the actin cytoskeleton closer to clinical application.     

Telomere dysfunction suppresses spontaneous tumorigenesis in vivo by initiating p53-dependent cellular senescence

Dysfunctional telomeres induce p53-dependent cellular senescence and apoptosis, but it is not known which function is more important for tumour suppression in vivo. We used the p53 ( R172P ) knock-in mouse, which is unable to induce apoptosis but retains intact cell-cycle arrest and cellular senescence pathways, to show that spontaneous tumorigenesis is potently repressed in Terc -/- p53 ( R172P ) mice. Tumour suppression is accompanied by global induction of p53, p21 and the senescence marker senescence-associated-beta-galactosidase. By contrast, cellular senescence was unable to suppress chemically induced skin carcinomas. These results indicate that suppression of spontaneous tumorigenesis by dysfunctional telomeres requires the activation of the p53-dependent cellular senescence pathway.     

Mitochondrial dysfunction impairs tumor suppressor p53 expression/function

Recently, mitochondria have been suggested to act in tumor suppression. However, the underlying mechanisms by which mitochondria suppress tumorigenesis are far from being clear. In this study, we have investigated the link between mitochondrial dysfunction and the tumor suppressor protein p53 using a set of respiration-deficient (Res(-)) mammalian cell mutants with impaired assembly of the oxidative phosphorylation machinery. Our data suggest that normal mitochondrial function is required for γ-irradiation (γIR)-induced cell death, which is mainly a p53-dependent process. The Res(-) cells are protected against γIR-induced cell death due to impaired p53 expression/function. We find that the loss of complex I biogenesis in the absence of the MWFE subunit reduces the steady-state level of the p53 protein, although there is no effect on the p53 protein level in the absence of the ESSS subunit that is also essential for complex I assembly. The p53 protein level was also reduced to undetectable levels in Res(-) cells with severely impaired mitochondrial protein synthesis. This suggests that p53 protein expression is differentially regulated depending upon the type of electron transport chain/respiratory chain deficiency. Moreover, irrespective of the differences in the p53 protein expression profile, γIR-induced p53 activity is compromised in all Res(-) cells. Using two different conditional systems for complex I assembly, we also show that the effect of mitochondrial dysfunction on p53 expression/function is a reversible phenomenon. We believe that these findings will have major implications in the understanding of cancer development and therapy.     

Oncogene-induced telomere dysfunction enforces cellular senescence in human cancer precursor lesions

In normal human somatic cells, telomere dysfunction causes cellular senescence, a stable proliferative arrest with tumour suppressing properties. Whether telomere dysfunction-induced senescence (TDIS) suppresses cancer growth in humans, however, is unknown. Here, we demonstrate that multiple and distinct human cancer precursor lesions, but not corresponding malignant cancers, are comprised of cells that display hallmarks of TDIS. Furthermore, we demonstrate that oncogenic signalling, frequently associated with initiating cancer growth in humans, dramatically affected telomere structure and function by causing telomeric replication stress, rapid and stochastic telomere attrition, and consequently telomere dysfunction in cells that lack hTERT activity. DNA replication stress induced by drugs also resulted in telomere dysfunction and cellular senescence in normal human cells, demonstrating that telomeric repeats indeed are hypersensitive to DNA replication stress. Our data reveal that TDIS, accelerated by oncogene-induced DNA replication stress, is a biological response of cells in human cancer precursor lesions and provide strong evidence that TDIS is a critical tumour suppressing mechanism in humans.     

p53 potentiation of tumor cell susceptibility to CTL involves Fas and mitochondrial pathways

In this study, we have investigated the mechanisms used by wild-type p53 (wtp53) to potentiate tumor cell susceptibility to CTL-mediated cell death. We report that wtp53 restoration in a human lung carcinoma cell line Institut Gustave Roussy (IGR)-Heu, displaying a mutated p53, resulted in up-regulation of Fas/CD95 receptor expression associated with an increase of tumor cell sensitivity to the autologous CTL clone, Heu127. However, when IGR-Heu cells were transfected with Fas cDNA, no potentiation to Heu127-mediated lysis was observed, indicating that induction of CD95 is not sufficient to sensitize target cells to CTL killing. Importantly, our data indicate that the effect of wtp53 on the Fas-mediated pathway involves a degradation of short cellular FLICE inhibitory protein resulting in subsequent caspase 8 activation. Furthermore, we demonstrate that wtp53 restoration also resulted in CTL-induced Bid translocation into mitochondria and a subsequent mitochondrial membrane permeabilization leading to cytochrome c release. These results indicate that tumor cell killing by autologous CTL can be enhanced by targeting degranulation-independent mechanisms via restoration of wtp53, a key determinant of apoptotic machinery regulation.     

Endothelial cell telomere dysfunction induces senescence and results in vascular and metabolic impairments

In advanced age, increases in oxidative stress and inflammation impair endothelial function, which contributes to the development of cardiovascular disease (CVD). One plausible source of this oxidative stress and inflammation is an increase in the abundance of senescent endothelial cells. Cellular senescence is a cell cycle arrest that occurs in response to various damaging stimuli. In the present study, we tested the hypothesis that advanced age results in endothelial cell telomere dysfunction that induces senescence. In both human and mouse endothelial cells, advanced age resulted in an increased abundance of dysfunctional telomeres, characterized by activation of DNA damage signaling at telomeric DNA. To test whether this results in senescence, we selectively reduced the telomere shelterin protein telomere repeat binding factor 2 (Trf2) from endothelial cells of young mice. Trf2 reduction increased endothelial cell telomere dysfunction and resulted in cellular senescence. Furthermore, induction of endothelial cell telomere dysfunction increased inflammatory signaling and oxidative stress, resulting in impairments in endothelial function. Finally, we demonstrate that endothelial cell telomere dysfunction-induced senescence impairs glucose tolerance. This likely occurs through increases in inflammatory signaling in the liver and adipose tissue, as well as reductions in microvascular density and vasodilation to metabolic stimuli. Cumulatively, the findings of the present study identify age-related telomere dysfunction as a mechanism that leads to endothelial cell senescence. Furthermore, these data provide compelling evidence that senescent endothelial cells contribute to age-related increases in oxidative stress and inflammation that impair arterial and metabolic function.     

Implication of p53-dependent cellular senescence related gene, TARSH in tumor suppression

A novel target of NESH-SH3 (TARSH) was identified as a cellular senescence related gene in mouse embryonic fibroblasts (MEFs) replicative senescence, the expression of which has been suppressed in primary clinical lung cancer specimens. However, the molecular mechanism underlying the regulation of TARSH involved in pulmonary tumorigenesis remains unclear. Here we demonstrate that the reduction of TARSH gene expression by short hairpin RNA (shRNA) system robustly inhibited the MEFs proliferation with increase in senescence-associated β-galactosidase (SA-β-gal) activity. Using p53^−/− MEFs, we further suggest that this growth arrest by loss of TARSH is evoked by p53-dependent p21^Cip1 accumulation. Moreover, we also reveal that TARSH reduction induces multicentrosome in MEFs, which is linked in chromosome instability and tumor development. These results suggest that TARSH plays an important role in proliferation of replicative senescence and may serve as a trigger of tumor development.     

Neutrophils induce paracrine telomere dysfunction and senescence in ROS‐dependent manner

Cellular senescence is characterized by an irreversible cell cycle arrest as well as a pro‐inflammatory phenotype, thought to contribute to aging and age‐related diseases. Neutrophils have essential roles in inflammatory responses; however, in certain contexts their abundance is associated with a number of age‐related diseases, including liver disease. The relationship between neutrophils and cellular senescence is not well understood. Here, we show that telomeres in non‐immune cells are highly susceptible to oxidative damage caused by neighboring neutrophils. Neutrophils cause telomere dysfunction both in vitro and ex vivo in a ROS‐dependent manner. In a mouse model of acute liver injury, depletion of neutrophils reduces telomere dysfunction and senescence. Finally, we show that senescent cells mediate the recruitment of neutrophils to the aged liver and propose that this may be a mechanism by which senescence spreads to surrounding cells. Our results suggest that interventions that counteract neutrophil‐induced senescence may be beneficial during aging and age‐related disease.     

Mitochondrial dysfunction in breast cancer cells prevents tumor growth

Metformin is a well-established diabetes drug that prevents the onset of most types of human cancers in diabetic patients, especially by targeting cancer stem cells. Metformin exerts its protective effects by functioning as a weak “mitochondrial poison,” as it acts as a complex I inhibitor and prevents oxidative mitochondrial metabolism (OXPHOS). Thus, mitochondrial metabolism must play an essential role in promoting tumor growth. To determine the functional role of “mitochondrial health” in breast cancer pathogenesis, here we used mitochondrial uncoupling proteins (UCPs) to genetically induce mitochondrial dysfunction in either human breast cancer cells (MDA-MB-231) or cancer-associated fibroblasts (hTERT-BJ1 cells). Our results directly show that all three UCP family members (UCP-1/2/3) induce autophagy and mitochondrial dysfunction in human breast cancer cells, which results in significant reductions in tumor growth. Conversely, induction of mitochondrial dysfunction in cancer-associated fibroblasts has just the opposite effect. More specifically, overexpression of UCP-1 in stromal fibroblasts increases β-oxidation, ketone body production and the release of ATP-rich vesicles, which “fuels” tumor growth by providing high-energy nutrients in a paracrine fashion to epithelial cancer cells. Hence, the effects of mitochondrial dysfunction are truly compartment-specific. Thus, we conclude that the beneficial anticancer effects of mitochondrial inhibitors (such as metformin) may be attributed to the induction of mitochondrial dysfunction in the epithelial cancer cell compartment. Our studies identify cancer cell mitochondria as a clear target for drug discovery and for novel therapeutic interventions.     

UVB-induced senescence in human keratinocytes requires a functional insulin-like growth factor-1 receptor and p53

To cope with the frequent exposure to carcinogenic UV B (UVB) wavelengths found in sunlight, keratinocytes have acquired extensive protective measures to handle UVB-induced DNA damage. Recent in vitro and epidemiological data suggest one these protective mechanisms is dependent on the functional status of the insulin-like growth factor-1 receptor (IGF-1R) signaling network in keratinocytes. During the normal UVB response, ligand-activated IGF-1Rs protect keratinocytes from UVB-induced apoptosis; however, as a consequence, these keratinocytes fail to proliferate. This adaptive response of keratinocytes to UVB exposure maintains the protective barrier function of the epidermis while ensuring that UVB-damaged keratinocytes do not replicate DNA mutations. In contrast, when keratinocytes are exposed to UVB in the absence of IGF-1R activation, the keratinocytes are more sensitive to UVB-induced apoptosis, but the surviving keratinocytes retain the capacity to proliferate. This aberrant UVB response represents flawed protection from UVB damage potentially resulting in the malignant transformation of keratinocytes. Using normal human keratinocytes grown in vitro, we have demonstrated that activation of the IGF-1R promotes the premature senescence of UVB-irradiated keratinocytes through increased generation of reactive oxygen species (ROS) and by maintaining the expression of the cyclin-dependent kinase inhibitor p21^CDKN1A. Furthermore, IGF-1R–dependent UVB-induced premature senescence required the phosphorylation of p53 serine 46. These data suggest one mechanism of keratinocyte resistance to UVB-induced carcinogenesis involves the induction of IGF-1R–dependent premature senescence.