PTEN‐ and p53‐mediated apoptosis and cell cycle arrest by FTY720 in gastric cancer cells and nude mice

FTY720, a new immunosuppressant, derived from ISP‐1, has been studied for its putative anti‐cancer properties in the recent years. In this study, we have reported that FTY720 greatly inhibited gastric cancer cell proliferation for the first time, and found this effect was associated with G1 phase cell cycle arrest and apoptosis. Results from our Western blotting and Real‐time PCR showed that FTY720 induced obvious PTEN expression in a p53‐independent way, consistent with a substantial decrease in p‐Akt and MDM2. FTY720 dramatically increased the expression of Cip1/p21, p27, and BH3‐only proteins through the accumulation of p53 by PTEN‐mediated inhibition of the PI3K/Akt/MDM2 signaling. Suppression of PTEN expression with siRNA significantly reduced the p53 and p21 levels and activated Akt, resulting in decreased apoptosis and increased cell survival. Furthermore, we have observed an additive effect of FTY720 in killing gastric cancer cells when in combination with Cisplatin, partly through PTEN‐mediated Akt/MDM2 inhibition. In vivo study has also shown that tumor growth was significantly suppressed after FTY720 treatment. In conclusion, our results suggest that FTY720 induces a significant increase of PTEN, which inhibits p‐Akt and MDM2, and then increases the level of p53, thereby inducing G1 phase arrest and apoptosis. We have characterized a novel immunosuppressant, for the first time, which shows potential anti‐tumor effects on gastric cancer by PTEN activation through p53‐independent mechanism, especially in combination with Cisplatin. This PTEN target‐based therapy is worth further investigation and warrants clinical evaluation. J. Cell. Biochem. 111: 218–228, 2010. © 2010 Wiley‐Liss, Inc.     

Cold stress‐induced ferroptosis involves the ASK1‐p38 pathway

A wide variety of cell death mechanisms, such as ferroptosis, have been proposed in mammalian cells, and the classification of cell death attracts global attention because each type of cell death has the potential to play causative roles in specific diseases. However, the precise molecular mechanisms leading to cell death are poorly understood, particularly in ferroptosis. Here, we show that continuous severe cold stress induces ferroptosis and the ASK1‐p38 MAPK pathway in multiple cell lines. The activation of the ASK1‐p38 pathway is mediated by critical determinants of ferroptosis: MEK activity, iron ions, and lipid peroxide. The chemical compound erastin, a potent ferroptosis inducer, also activates the ASK1‐p38 axis downstream of lipid peroxide accumulation and leads to ASK1‐dependent cell death in a cell type‐specific manner. These lines of evidence provide mechanistic insight into ferroptosis, a type of regulated necrosis.     

Overexpressed poly(ADP‐ribose) polymerase delays the release of rat cells from p53‐mediated G1 checkpoint

We have previously reported that in cells ectopically expressing temperature‐sensitive p53^135val mutant, p53 formed tight complexes with poly(ADP‐ribose) polymerase (PARP). At elevated temperatures, p53^135val protein, adopting the mutant phenotype, was localized in the cytoplasm and sequestered the endogenous PARP. To prove whether an excess of p53^135val protein led to this unusual intracellular distribution of PARP, we have established cell lines overexpressing p53^135val + c‐Ha‐ras alone or in combination with PARP. Interestingly, immunostaining revealed that PARP is sequestered in the cytoplasm by mutant p53 in cells overexpressing both proteins. Simultaneous overexpression of PARP had no effect on temperature‐dependent cell proliferation and only negligibly affected the kinetics of p53‐mediated G₁ arrest. However, if the cells were completely growth arrested at 32°C and then shifted up to 37°C, coexpressed PARP dramatically delayed the reentry of transformed cells into the cell cycle. Even after 72 h at 37°C the proportion of S‐phase cells was reduced to 20% compared to those expressing only p53^135val + c‐Ha‐ras. The coexpressed PARP stabilized wt p53 protein and its enzymatic activity was necessary for stabilization. J. Cell. Biochem. 80:85–103, 2000. © 2000 Wiley‐Liss, Inc.     

MicroRNA-590-3P suppresses cell survival and triggers breast cancer cell apoptosis via targeting sirtuin-1 and deacetylation of p53

Downregulation of microRNA-590-3p (miR-590-3p) is a frequently occurring, nonphysiological event which is observed in several human cancers, especially breast cancer. However, the significance of miR-590-3p still remain unclear in the progression of this disease. This study explored the role of miR-590-3p in apoptosis of breast cancer cells. Gene expression of miR-590-3p, Sirtuin-1 (SIRT1), Bcl-2 associated X protein (BAX), and p21 was evaluated with real-time polymerase chain reaction (PCR) and SIRT1 protein expression was assessed by Western blot analysis in breast cancer cell lines. Bioinformatics analysis and luciferase reporter assay were used to evaluate targeting of SIRT1 messenger RNA (mRNA) by miR-590-3p. Cells were transfected with miR-590-3p mimic and inhibitor and their effects on the expression and activity of SIRT1 were evaluated. The effects of miR-590-3p upregulation on the acetylation of p53 as well as cell viability and apoptosis were assessed by Western blot analysis, WST-1 assay, and flow cytometry, respectively. miR-590-3p expression was considerably downregulated in breast cancer cells which was accompanied by upregulation of SIRT1 expression. SIRT1 was recognized as a direct target for miR-590-3p in breast cancer cells and its protein expression and activity was dramatically inhibited by the miR-590-3p. In addition, there was an increase in p53 and its acetylated form that ultimately led to upregulation of BAX and p21 expression, suppression of cell survival, and considerable induction of apoptosis in breast cancer cells. These findings suggest that miR-590-3p exerts tumor-suppressing effects through targeting SIRT1 in breast cancer cells, which makes it a potential therapeutic target for developing more efficient treatments for breast cancer.     

Phospholipase C ε‐1 inhibits p53 expression in lung cancer

The pathogenesis of lung cancer is to be further investigated. Recent reports indicate that phospholipase C ε‐1 (PLCE1) is a critical molecule involved in tumour growth. This study aims to investigate the role of PLCE1 in the regulation of apoptosis in lung cancer cells. In this study, the surgically removed non‐small‐cell lung cancer (NSCLC) tissue was collected from 36 patients. Single NSCLC cells were prepared from the tissue, in which immune cells of CD3⁺, CD11c⁺, CD19⁺, CD68⁺ and CD14⁺ were eliminated by magnetic cell sorting. The expression of PLCE1 and p53 was assessed by quantitative real‐time polymerase chain reaction and Western blotting. Apoptosis of NSCLC cells was analysed by flow cytometry. The results showed that, in cultured NSCLC cells, high levels of PLCE1 and low levels p53 were detected; the two molecules showed a negative correlation (p < 0.01). The addition of anti‐PLCE1 antibody increased the expression of p53 in NSCLC cells, which increased the frequency of apoptotic NSCLC cells. We conclude that NSCLC cells express high levels of PLCE1, which suppresses the expression of p53 in NSCLC cells. PLCE1 can be a therapeutic target of NSCLC. Copyright © 2013 John Wiley & Sons, Ltd.     

Selenium activates p53 and p38 pathways and induces caspase-independent cell death in cervical cancer cells

The mechanisms of sodium selenite-induced cell death in cervical carcinoma cells were studied during 24 h of exposure in the HeLa Hep-2 cell line. Selenite at the employed concentrations of 5 and 50 μmol/L produced time- and dose-dependent suppression of DNA synthesis and induced DNA damage which resulted in phosphorylation of histone H2A.X. These effects were influenced by pretreatment of cells with the SOD/catalase mimetic MnTMPyP or glutathione-depleting buthionine sulfoximine, suggesting the significant role of selenite-generated oxidative stress. Following the DNA damage, selenite activated p53-dependent pathway as evidenced by the appearance of phosphorylated p53 and accumulation of p21 in the treated cells. Concomitantly, selenite activated p38 pathway but its effect on JNK was very weak. p53- and p38-dependent signaling led to the accumulation of Bax protein, which was preventable by specific inhibitors of p38 (SB 203580) and p53 (Pifithrin-α). Mitochondria in selenite-treated cells changed their dynamics (shape and localization) and released AIF and Smac/Diablo, which initiated caspase-independent apoptosis as confirmed by the caspase-3 activity assay and the low effect of caspase inhibitors z-DEVD-fmk and z-VAD-fmk on cell death. We conclude that selenite induces caspase-independent apoptosis in cervical carcinoma cells mostly by oxidative stress-mediated activation of p53 and p38 pathways, but other selenite-mediated effects, in particular mitochondria-specific ones, are also involved.     

Expression and clinicopathological significance of lipin-1 in human breast cancer and its association with p53 tumor suppressor gene

Breast cancer (BC) is an important cause of female cancer-related death. It has recently been demonstrated that metabolic disorders including lipid metabolism are a hallmark of cancer cells. Lipin-1 is an enzyme that displays phosphatidate phosphatase activity and regulates the rate-limiting step in the pathway of triglycerides and phospholipids synthesis. The objective of this study was to evaluate lipin-1 expression, its prognostic significance, and its correlation with p53 tumor suppressor in patients with BC. In this study, 55 pairs of fresh samples of BC and adjacent noncancerous tissue were used to analyze lipin-1, using quantitative real-time polymerase chain reaction and immunohistochemistry (IHC) staining. The expression of other clinicopathological variables and p53 was also examined using IHC technique. The cell migration was studied in MCF-7 and MDA-MB231 cells following the inhibition of lipin-1 by propranolol. Our results show that the relative expression of lipin-1 messenger RNA was significantly higher in BC tissues compared with the adjacent normal tissue and its inhibition reduced cell migration in cancer cells. This upregulation was negatively correlated with histological grade of tumor and p53 status (p = .001 and p = .034) respectively and positively correlated with the tumor size (p = .006). Our results also seem to indicate that the high lipin-1 expression is related to a good prognosis in patients with BC. The expression of lipin-1 may be considered as a novel independent prognostic factor. The inhibition of lipin-1 may also have therapeutic significance for patients with BC. The correlation between lipin-1 and p53 confirms the role of p53 in the regulation of lipid metabolism in cancer cells.     

miR‐515‐5p controls cancer cell migration through MARK4 regulation

Here, we show that miR‐515‐5p inhibits cancer cell migration and metastasis. RNA‐seq analyses of both oestrogen receptor receptor‐positive and receptor‐negative breast cancer cells overexpressing miR‐515‐5p reveal down‐regulation of NRAS, FZD4, CDC42BPA, PIK3C2B and MARK4 mRNAs. We demonstrate that miR‐515‐5p inhibits MARK4 directly 3′ UTR interaction and that MARK4 knock‐down mimics the effect of miR‐515‐5p on breast and lung cancer cell migration. MARK4 overexpression rescues the inhibitory effects of miR‐515‐5p, suggesting miR‐515‐5p mediates this process through MARK4 down‐regulation. Furthermore, miR‐515‐5p expression is reduced in metastases compared to primary tumours derived from both in vivo xenografts and samples from patients with breast cancer. Conversely, miR‐515‐5p overexpression prevents tumour cell dissemination in a mouse metastatic model. Moreover, high miR‐515‐5p and low MARK4 expression correlate with increased breast and lung cancer patients' survival, respectively. Taken together, these data demonstrate the importance of miR‐515‐5p/MARK4 regulation in cell migration and metastasis across two common cancers.     

TNIP1‐mediated TNF‐α/NF‐κB signalling cascade sustains glioma cell proliferation

As a malignant tumour of the central nervous system, glioma exhibits high incidence and poor prognosis. Although TNIP1 and the TNF‐α/NF‐κB axis play key roles in immune diseases and inflammatory responses, their relationship and role in glioma remain unknown. Here, we revealed high levels of TNIP1 and TNF‐α/NF‐κB in glioma tissue. Glioma cell proliferation was activated with TNF‐α treatment and showed extreme sensitivity to the TNF receptor antagonist. Furthermore, loss of TNIP1 disbanded the A20 complex responsible for IκB degradation and NF‐κB nucleus translocation, and consequently erased TNFα‐induced glioma cell proliferation. Thus, our investigation uncovered a vital function of the TNIP1‐mediated TNF‐α/NF‐κB axis in glioma cell proliferation and provides novel insight into glioma pathology and diagnosis.     

Chemokine receptor CXCR2 is transactivated by p53 and induces p38‐mediated cellular senescence in response to DNA damage

Mammalian cells may undergo permanent growth arrest/senescence when they incur excessive DNA damage. As a key player during DNA damage response (DDR), p53 transactivates an array of target genes that are involved in various cellular processes including the induction of cellular senescence. Chemokine receptor CXCR2 was previously reported to mediate replicative and oncogene‐induced senescence in a DDR and p53‐dependent manner. Here, we report that CXCR2 is upregulated in various types of cells in response to genotoxic or oxidative stress. Unexpectedly, we found that the upregulation of CXCR2 depends on the function of p53. Like other p53 target genes such as p21, CXCR2 is transactivated by p53. We identified a p53‐binding site in the CXCR2 promoter that responds to changes in p53 functional status. Thus, CXCR2 may act downstream of p53. While the senescence‐associated secretory phenotype (SASP) exhibits a kinetics that is distinct from that of CXCR2 expression and does not require p53, it reinforces senescence. We further showed that the cellular senescence caused by CXCR2 upregulation is mediated by p38 activation. Our results thus demonstrate CXCR2 as a critical mediator of cellular senescence downstream of p53 in response to DNA damage.     

Insulin‐like growth factor‐1 regulates the SIRT1‐p53 pathway in cellular senescence

Cellular senescence, which is known to halt proliferation of aged and stressed cells, plays a key role against cancer development and is also closely associated with organismal aging. While increased insulin‐like growth factor (IGF) signaling induces cell proliferation, survival and cancer progression, disrupted IGF signaling is known to enhance longevity concomitantly with delay in aging processes. The molecular mechanisms involved in the regulation of aging by IGF signaling and whether IGF regulates cellular senescence are still poorly understood. In this study, we demonstrate that IGF‐1 exerts a dual function in promoting cell proliferation as well as cellular senescence. While acute IGF‐1 exposure promotes cell proliferation and is opposed by p53, prolonged IGF‐1 treatment induces premature cellular senescence in a p53‐dependent manner. We show that prolonged IGF‐1 treatment inhibits SIRT1 deacetylase activity, resulting in increased p53 acetylation as well as p53 stabilization and activation, thus leading to premature cellular senescence. In addition, either expression of SIRT1 or inhibition of p53 prevented IGF‐1‐induced premature cellular senescence. Together, these findings suggest that p53 acts as a molecular switch in monitoring IGF‐1‐induced proliferation and premature senescence, and suggest a possible molecular connection involving IGF‐1‐SIRT1‐p53 signaling in cellular senescence and aging.     

Suppression of Polo like kinase 1 (PLK1) by p21(Waf1) mediates the p53-dependent prevention of caspase-independent mitotic death

Polo-like kinase 1 (Plk1) plays key roles in many aspects of mitosis. We have previously shown that induction of p21^Waf1 by p53 is responsible for protection of cells against adriamycin-induced polyploidy formation and mitotic catastrophe. Here we show that adriamycin treatment suppressed Plk1 expression in a p53- and p21^Waf1-dependent manner. Ablation of p21^Waf1 inhibited the adriamycin-induced p53 activation, and this inhibition was alleviated by knockdown of Plk1, suggesting that p21^Waf1-dependent suppression of Plk1 expression is responsible for maintaining p53 activation during stress response. Plk1 associated with p53 and disrupted its interaction with target gene promoters in cells treated with adriamycin. Overexpression of Plk1 inhibited the p53-mediated prevention of caspase-independent mitotic death, but not polyploidy formation, in adriamycin-treated cells. Together our results indicate that suppression of Plk1 by p21^Waf1 is responsible for p53-dependent protection against adriamycin-induced caspase-independent mitotic death.     

Extracellular NAMPT/visfatin causes p53 deacetylation via NAD production and SIRT1 activation in breast cancer cells

Visfatin, which is secreted as an adipokine and cytokine, has been implicated in cancer development and progression. In this study, we investigated the NAD‐producing ability of visfatin and its relationship with SIRT1 (silent information regulator 2) and p53 to clarify the role of visfatin in breast cancer. MCF‐7 breast cancer cells were cultured and treated with visfatin. SIRT1 activity was assessed by measuring fluorescence intensity from fluoro‐substrate peptide. To investigate the effect of visfatin on p53 acetylation, SDS‐PAGE followed by western blotting was performed using specific antibodies against p53 and its acetylated form. Total NAD was measured both in cell lysate and the extracellular medium by colorimetric method. Visfatin increased both extracellular and intracellular NAD concentrations. It also induced proliferation of breast cancer cells, an effect that was abolished by inhibition of its enzymatic activity. Visfatin significantly increased SIRT1 activity, accompanied by induction of p53 deacetylation. In conclusion, the results show that extracellular visfatin produces NAD that causes upregulation of SIRT1 activity and p53 deacetylation. These findings explain the relationship between visfatin and breast cancer progression.