CtBP determines ovarian cancer cell fate through repression of death receptors.

C-terminal binding protein 2 (CtBP2) is elevated in epithelial ovarian cancer, especially in the aggressive and highly lethal subtype, high-grade serous ovarian cancer (HGSOC). However, whether HGSOC tumor progression is dependent on CtBP2 or its paralog CtBP1, is not well understood. Here we report that CtBP1/2 repress HGSOC cell apoptosis through silencing of death receptors (DRs) 4/5. CtBP1 or 2 knockdown upregulated DR4/5 expression, and triggered autonomous apoptosis via caspase 8 activation, but dependent on cell-type context. Activation of DR4/5 by CtBP1/2 loss also sensitized HGSOC cell susceptibility to the proapoptotic DR4/5 ligand TRAIL. Consistent with its function as transcription corepressor, CtBP1/2 bound to the promoter regions of DR4/5 and repressed DR4/5 expression, presumably through recruitment to a repressive transcription regulatory complex. We also found that CtBP1 and 2 were both required for repression of DR4/5. Collectively, this study identifies CtBP1 and 2 as potent repressors of DR4/5 expression and activity, and supports the targeting of CtBP as a promising therapeutic strategy for HGSOC.

p53-inducible SESTRINs might play opposite roles in the regulation of early and late stages of lung carcinogenesis.

SESTRINs (SESN1-3) are proteins encoded by an evolutionarily conserved gene family that plays an important role in the regulation of cell viability and metabolism in response to stress. Many of the effects of SESTRINs are mediated by negative and positive regulation of mechanistic target of rapamycin kinase complexes 1 and 2 (mTORC1 and mTORC2), respectively, that are often deregulated in human cancers where they support cell growth, proliferation, and cell viability. Besides their effects on regulation of mTORC1/2, SESTRINs also control the accumulation of reactive oxygen species, cell death, and mitophagy. SESN1 and SESN2 are transcriptional targets of tumor suppressor protein p53 and may mediate tumor suppressor activities of p53. Therefore, we conducted studies based on a mouse lung cancer model and human lung adenocarcinoma A549 cells to evaluate the potential impact of SESN1 and SESN2 on lung carcinogenesis. While we observed that expression of SESN1 and SESN2 is often decreased in human tumors, inactivation of Sesn2 in mice positively regulates tumor growth through a mechanism associated with activation of AKT, while knockout of Sesn1 has no additional impact on carcinogenesis in Sesn2-deficient mice. However, inactivation of SESN1 and/or SESN2 in A549 cells accelerates cell proliferation and imparts resistance to cell death in response to glucose starvation. We propose that despite their contribution to early tumor growth, SESTRINs might suppress late stages of carcinogenesis through inhibition of cell proliferation or activation of cell death in conditions of nutrient deficiency.

Sestrin2 is induced by glucose starvation via the unfolded protein response and protects cells from non-canonical necroptotic cell death.

Sestrin2 is a member of a family of stress responsive proteins, which controls cell viability via antioxidant activity and regulation of the mammalian target of rapamycin protein kinase (mTOR). Sestrin2 is induced by different stress insults, which diminish ATP production and induce energetic stress in the cells. Glucose is a critical substrate for ATP production utilized via glycolysis and mitochondrial respiration as well as for glycosylation of newly synthesized proteins in the endoplasmic reticulum (ER) and Golgi. Thus, glucose starvation causes both energy deficiency and activation of ER stress followed by the unfolding protein response (UPR). Here, we show that UPR induces Sestrin2 via ATF4 and NRF2 transcription factors and demonstrate that Sestrin2 protects cells from glucose starvation-induced cell death. Sestrin2 inactivation sensitizes cells to necroptotic cell death that is associated with a decline in ATP levels and can be suppressed by Necrostatin 7. We propose that Sestrin2 protects cells from glucose starvation-induced cell death via regulation of mitochondrial homeostasis.

Sestrin2 facilitates death receptor-induced apoptosis in lung adenocarcinoma cells through regulation of XIAP degradation.

Apoptosis plays a critical physiological role in controlling cell number and eliminating damaged, non-functional and transformed cells. Cancerous cells as well as some types of normal cells are often resistant to cell death induced by pro-inflammatory cytokines through death receptors. This potentially allows cancer cells to evade the control from the immune system and to proceed toward a more malignant stage, although the mechanisms of this evasion are not well established. We have recently identified the stress-responsive Sestrin2 protein as a critical regulator of cell viability under stress conditions. Sestrin2 is a member of a small family of antioxidant proteins and inhibitors of mechanistic Target of Rapamycin Complex 1 (mTORC1) kinase. Down-regulation of Sestrin1/2 leads to genetic instability and accelerates the growth of lung adenocarcinoma xenografts. Here we addressed the potential role of Sestrin2 in regulation of cell death induced by TNFR1 and related Fas and TRAIL receptors in lung adenocarcinoma cells. We found that Sestrin2 silencing strongly inhibits cytokine-induced cell death through a mechanism independent of ROS and mTORC1 regulation. We determined that the X-linked inhibitor of apoptosis protein (XIAP) plays a critical role in the control of cytokine-induced cell death by Sestrin2. Thus our study defines a new, previously unrecognized role of Sestrin2 in the regulation of apoptosis.

Sestrins inhibit mTORC1 kinase activation through the GATOR complex.

The mechanistic target of rapamycin complex 1 (mTORC1) kinase is a sensor of different environmental conditions and regulator of cell growth, metabolism, and autophagy. mTORC1 is activated by Rag GTPases, working as RagA:RagB and RagC:RagD heterodimers. Rags control mTORC1 activity by tethering mTORC1 to the lysosomes where it is activated by Rheb GTPase. RagA:RagB, active in its GTP-bound form, is inhibited by GATOR1 complex, a GTPase-activating protein, and GATOR1 is in turn negatively regulated by GATOR2 complex. Sestrins are stress-responsive proteins that inhibit mTORC1 via activation of AMP-activated protein kinase (AMPK) and tuberous sclerosis complex. Here we report an AMPK-independent mechanism of mTORC1 inhibition by Sestrins mediated by their interaction with GATOR2. As a result of this interaction, the Sestrins suppress mTOR lysosomal localization in a Rag-dependent manner. This mechanism is potentially involved in mTORC1 regulation by amino acids, rotenone, and tunicamycin, connecting stress response with mTORC1 inhibition.

Arsenic trioxide synergizes with everolimus (Rad001) to induce cytotoxicity of ovarian cancer cells through increased autophagy and apoptosis.

Phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin pathway plays a key role in the tumorigenesis of a variety of human cancers including ovarian cancer. However, inhibitors of this pathway such as Rad001 have not shown therapeutic efficacy as a single agent for this cancer. Arsenic trioxide (ATO) induces an autophagic pathway in ovarian carcinoma cells. We found that ATO can synergize with Rad001 to induce cytotoxicity of ovarian cancer cells. Moreover, we identified synergistic induction of autophagy and apoptosis as the likely underlying mechanism that is responsible for the enhanced cytotoxicity. The enhanced cytotoxicity is accompanied by decreased p-AKT levels as well as upregulation of ATG5-ATG12 conjugate and LC3-2, hallmarks of autophagy. Rad001 and ATO can also synergistically inhibit tumors in a xenograft animal model of ovarian cancer. These results thus identify and validate a novel mechanism to enhance and expand the existing targeted therapeutic agent to treat human ovarian cancer.

Combination of Rad001 (everolimus) and propachlor synergistically induces apoptosis through enhanced autophagy in prostate cancer cells.

PI3K/AKT/mTOR pathway plays a key role in the tumorigenesis of many human cancers including prostate cancer. However, inhibitors of this pathway, such as Rad001, have not shown therapeutic efficacy as a single agent. Through a high-throughput screen of 5,000 widely used small molecules, we identified compounds that can synergize with Rad001 to inhibit prostate cancer cells. One of the compounds, propachlor, synergizes with Rad001 to induce apoptosis of castration-resistant prostate cancer cells via enhanced autophagy. This enhanced autophagic cell death is accompanied by increased Beclin1 expression as well as upregulation of Atg5-Atg12 conjugate and LC3-2. Rad001 and propachlor can also synergistically inhibit tumors in a xenograft animal model of prostate cancer. These findings provide a novel direction to develop combination therapies for advanced and metastatic prostate cancer that has failed the currently available therapies.

Overexpression of SKI oncoprotein leads to p53 degradation through regulation of MDM2 protein sumoylation.

Protooncogene Ski was identified based on its ability to transform avian fibroblasts in vitro. In support of its oncogenic activity, SKI was found to be overexpressed in a variety of human cancers, although the exact molecular mechanism(s) responsible for its oncogenic activity is not fully understood. We found that SKI can negatively regulate p53 by decreasing its level through up-regulation of MDM2 activity, which is mediated by the ability of SKI to enhance sumoylation of MDM2. This stimulation of MDM2 sumoylation is accomplished through a direct interaction of SKI with SUMO-conjugating enzyme E2, Ubc9, resulting in enhanced thioester bond formation and mono-sumoylation of Ubc9. A mutant SKI defective in transformation fails to increase p53 ubiquitination and is unable to increase MDM2 levels and to increase mono-sumoylation of Ubc9, suggesting that the ability of SKI to enhance Ubc9 activity is essential for its transforming function. These results established a detailed molecular mechanism that underlies the ability of SKI to cause cellular transformation while unraveling a novel connection between sumoylation and tumorigenesis, providing potential new therapeutic targets for cancer.

The toxicity study on marine low-temperature lysozyme.

Marine low-temperature lysozyme is purified from a marine bacterium. The lysozyme can keep high activity at low-temperature and has broad-spectrum antibiotic reaction. This study was undertaken to investigate the major characteristics, acute and subchronic toxicity of marine low-temperature lysozyme. The relative molecular weight of this lysozyme was determined as approximate 16 kD; its optimum pH value and temperature towards Micrococcus lysodleikticus were pH 6.5 and 35 degrees C, respectively. The lysozyme activity was slightly enhanced by Zn(2+) and Cu(2+) and slightly inhibited by Mn(2+) and Ag(+). The lysozyme showed good compatibility to many common chemical agents such as EDTA (0.1%), KH(2)PO(4) (1.0%), etc. In experiments on acute toxicity, the drug was injected through the tail vein of mice, and intoxication symptoms and date of death were recorded. The 50% lethal dose (LD(50)) of Marine low-temperature lysozyme and 95%, 99% confidence interval (CI) was calculated. The subchronic study was designed to determine whether effects progressed with repeated Marine low-temperature lysozyme exposure. Wistar rats were tested by daily intragastric administration of Marine low-temperature lysozyme at the doses of 1.0; 0.5; 0.25 g/kg bw for 90 days. The LD(50) value of lysozyme was 4530 mg/kg bw; 90 days of Marine low-temperature lysozyme treatment at three doses, and there is no significant difference on blood biochemistry and organ index in drug treatment groups compared to saline treatment group. There is no affirmative pathologic change of all the observed organs in this study. The present results suggest that Marine low-temperature lysozyme can be safely used at the dose of experiment applied.

Role of p53 in antioxidant defense of HPV-positive cervical carcinoma cells following H2O2 exposure.

In HPV-positive cervical carcinoma cells, p53 protein is functionally antagonized by the E6 oncoprotein. We investigated a possible role of p53 in antioxidant defense of HPV-positive cervical cancer cell lines. We found that SiHa cells containing integrated HPV 16 had higher expression of p53 and exhibited the greatest resistant to H2O2-induced oxidative damage, compared with HeLa, CaSki and ME180 cell lines. Downregulation of p53 resulted in the inhibition of p53-regulated antioxidant enzymes and elevated intracellular ROS in SiHa cells. By contrast, the ROS level was not affected in HeLa, CaSki and ME180 cell lines after inhibition of the p53 protein. Under mild or severe H2O2-induced stress, p53-deficient SiHa cells exhibited much higher ROS levels than control SiHa cells. Furthermore, we analyzed cell viability and apoptosis after H2O2 treatment and found that p53 deficiency sensitized SiHa cells to H2O2 damage. Inhibition of p53 resulted in excessive oxidation of DNA; control SiHa cells exhibited a more rapid removal of 8-oxo-7,8-dihydro-2'-deoxyguanosine from DNA compared with p53-deficient SiHa cells exposed to the same level of H2O2 challenge. These data collectively show that endogenous p53 in SiHa cells has an antioxidant function and involves in the reinforcement of the antioxidant defense.

Methylation of p16INK4a is a non-rare event in cervical intraepithelial neoplasia.

The cell cycle inhibitor, p16INK4a may be a useful surrogate biomarker of cervical intraepithelial neoplasia (CIN); however, there is currently no consensus of p16INK4a genetic alterations throughout the multiple step process of CIN. Our goal was to identify the methylation frequency of p16INK4a in each step of CIN that is associated with human papillomavirus (HPV) infection, using several different detection methods of p16INK4a methylation to correlate the data. The present study included a total of 43 patients, including 38 with CIN, and 5 normal patients. Three different methods were used to detect hypermethylation of CpG islands, methylation-specific PCR (MSP) amplification of different primer sets of M1, M2, and M3, pyrosequencing of each forward primer region, and immunohistochemistry of p16INK4a. Analysis of MSP showed that 20 of the 38 CIN patients (52.6%) revealed hypermethylation in at least 1 primer set of the p16INK4a promoter. A complete loss of p16INK4a protein expression was observed in 11 cases (28.9%). There was no observed association of methylation of the p16INK4a gene with either CIN grading (P=0.0698) or HPV status (P=0.2811): specifically 42.9% (3/7) was found in CIN 1, 57.1% (8/14) in CIN 2, and 52.9% (9/17) in CIN 3. In concordance with immunohistochemistry results, hypermethylation of the p16INK4a promoter was significantly correlated with a lack of p16 protein expression (P=0.0145). All positive peaks from pyrosequencing matched the MSP results, which ranged from 6.3% to 24.5%. In conclusion, p16INK4a gene silencing during CIN was not determined to be a particularly rare event; however, it does not correlate with either HPV status or CIN grading.

Comparative proteomics of ovarian epithelial tumors.

We analyzed 12 ovarian epithelial tumors using 2D PAGE-based comparative proteomics to construct intra- and inter-tumoral distance map trees and to discover surrogate biomarkers indicative of an ovarian tumor. The analysis was performed after laser microdissection of 12 fresh-frozen tissue samples, including 4 serous, 5 mucinous, and 3 endometrioid tumors, with correlation with their histopathological characteristics. Ovarian epithelial tumors and normal tissues showed an apparent separation on the distance map tree. Mucinous carcinomas were closest to the normal group, whereas serous carcinomas were located furthest from the normal group. All mucinous tumors with aggressive histology were separated from the low malignant potential (LMP) group. The benign-looking cysts adjacent to the intraepithelial carcinoma (IEC) showed an expression pattern identical to that of the IEC area. The extent of change on the lineages leading to the mucinous and serous carcinoma was 1.98-fold different. The overall gene expression profiles of serous or endometrioid carcinomas appeared to be less affected by grade or stage than by histologic type. The potential candidate biomarkers screened in ovarian tumors and found to be significantly up-regulated in comparison to normal tissues were as follows: NM23, annexin-1, protein phosphatase-1, ferritin light chain, proteasome alpha-6, and NAGK (N-acetyl glucosamine kinase). In conclusion, ovarian mucinous tumors are distinct from other ovarian epithelial tumors. LMP mucinous tumors showing histologically aggressive features belong to mucinous carcinoma on the proteomic basis.

Inhibitory effect of polypeptides from Chlamys farreri on UVB-induced apoptosis and DNA damage in normal human dermal fibroblasts in vitro.

AIM: To investigate the effect of polypeptide from Chlamys farreri (PCF) on ultraviolet B (UVB)-induced apoptosis and DNA damage in cultured normal human dermal fibroblasts. METHODS: MTT assay was used to measure the viability of cells. Measurements of apoptosis and cytosolic free [Ca2+]i were performed with flow cytometry. The comet assay was employed to detect DNA damage in individual cell. RESULTS: PCF (0.25 %-1%) greatly enhanced the proliferative capacity of cultured fibroblasts irradiated by UVB (1.176 x 10(-4) J/cm2) and markedly reduced apoptosis and the level of DNA damage in a concentration-dependent manner. Meanwhile, PCF could decrease the cytosolic free [Ca2+]i (P<0.01, compared with UVB model). CONCLUSION: The inhibitory effect of PCF on UVB-induced photoaging is due to enhanced abating of UVB-injured DNA and UVB-induced apoptosis. Therefore, PCF can resist UV-induced aging development at the initiation stage.

Protective effect of polypeptide from Chlamys farreri on mitochondria in human dermal fibroblasts irradiated by ultraviolet B.

AIM: To study the effect of polypeptide from Chlamys farreri (PCF) on mitochondria of human dermal fibroblasts irradiated by ultraviolet B (UVB) in vitro. METHODS: Malondialdehyde (MDA) and antioxidant enzymes including superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) were determined by biochemical methods. Mitochondrial transmembrane potential was measured by flow cytometry. Ultrastructure of fibroblasts was observed with transmission electron microscope. RESULTS: UVB (1.176 x 10(-4) J/cm(2)) induced mitochondria damage in dermal fibroblast and PCF (0.25%-1%) reduced the damage in a concentration-dependent manner. Furthermore, PCF also concentration-dependently maintained the stability of mitochondrial transmembrane potential. PCF was able to reduce the MDA formation caused by UVB, meanwhile increased the activities of SOD and GSH-PX. The differences among the PCF groups and UVB model group were significant (P<0.05, P<0.01). CONCLUSION: The UVB-induced mitochondria damage was alleviated by PCF in human dermal fibroblasts.