miRNA-149 targets PARP-2 in endometrial epithelial and stromal cells to regulate the trophoblast attachment process.

Embryo implantation is a highly complex process involving many regulatory factors, including several micro RNAs (miRNAs/miRs). One miRNA present in the stromal cells of normal endometrium is miR-149, which targets poly (ADP-ribose) polymerase 2 (PARP-2), a gene involved in endometrial receptivity for trophoblast implantation. However, the precise role of miR-149 in the endometrial receptivity during blastocyst implantation is still unknown. We studied miR-149-dependent PARP-2 regulation during trophoblast attachment to endometrial epithelial cells. Using FISH, we found that miR-149 is expressed in mouse endometrial epithelial and stromal cells at implantation and inter-implantation sites. Endometrial receptivity for embryo implantation and attachment is inhibited by the upregulation of miR-149 in the endometrium. Our RT-PCR analysis revealed downregulation of miR-149 in the implantation region of the uterus during the receptive stage (Day 5, 0500 h, p.c.) in the mouse. Under in-vitro conditions, miR-149 overexpression in human endometrial epithelial cells (hEECs) abrogated the human trophoblastic cells spheroid and mouse blastocyst attachment. Subsequently, miR-149 also regulates transformed human endometrial stromal cell (T-hESCs) decidualization by downregulating PARP-2 and upregulating caspase-8 proteins. Overexpression of miR-149 in hEECs and downregulated PARP-2 protein expression, reconfirming that PARP-2 is a downstream target of miR-149 in endometrial cells as well. miR-149 is also able to alter the expression of caspase-8, another PARP-2 regulator. In conclusion, our data indicate that miR-149 is one of the regulators of endometrial receptivity and decidualization for trophoblast implantation, and it exerts the effects by acting on the downstream targets PARP-2 and caspase-8.

Molecular and clinical characterization of PARP9 in gliomas: A potential immunotherapeutic target.

BACKGROUND: Glioma is a primary malignancy of the central nervous system (CNS). As biomedicine advances, an efficient molecular target is urgently needed for the diagnosis and treatment of glioma. Meanwhile, several studies have demonstrated that glioma development is closely related to immunity. PARP9 is an inactive mono-ADP-ribosyltransferase belonging to the poly-ADP ribosyltransferase (ARTD) family. In this article, we aimed to reveal the relationship between PARP9 and glioma and explore the potential prognostic value and immunotherapeutic targetability of PARP9 in glioma. METHODS: PARP9 transcript levels were analyzed with TCGA and GEO databases. The clinicopathological information of patients with glioma in the TCGA database and gene expression profiles were analyzed to determine the relationship between the expression of PARP9 and clinicopathologic characteristics. Kaplan-Meier survival analysis, univariate Cox regression analysis, and multivariate Cox regression analysis were used for survival analysis. Gene set enrichment analysis (GSEA) and gene set variation analysis (GSVA) were used for bioinformatics analysis. Correlation analysis explored the relationships between PARP9, infiltrating inflammatory immune cells, and immune checkpoint molecules. RESULTS: PARP9 is highly expressed in glioma, and high expression of PARP9 is associated with poor prognosis and advanced clinicopathological features. Bioinformatics analysis showed that some immune-related pathways were closely associated with high expression of PARP9. Correlation analysis indicated that PARP9 was closely related to inflammatory and immune responses, high immune cell infiltration, and immune checkpoint molecules. CONCLUSIONS: PARP9 may serve as an unfavorable prognosis predictor for glioma and a potential immunotherapeutic target.

Expression of poly-ADP-ribose polymerase (PARP) in endometrial adenocarcinoma: Prognostic potential.

BACKGROUND: In the United States endometrial carcinoma is the most common female gynecologic malignancy. An average of more than 60,000 new cases of endometrial carcinomas have been diagnosed yearly over the past 5 years, with a higher incidence occurring in the central Appalachian states of Ohio and West Virginia. In the U.S., the national average of newly diagnosed endometrial carcinomas is 26.8 in every 100,000 women, while in the states of Ohio and West Virginia the average is 30.5 and 31.1 in every 100,000 women, respectively. This notable increase in the incidence of endometrial carcinomas may be due a variety of elevated risk factors including but not limited to: tobacco use, obesity, and genetic predisposition of the predominant demographic. The American Cancer Society estimates that approximately 55,000 new cases of endometrial carcinoma will be diagnosed in 2020 yet, this disease is widely considered understudied and under-represented in mainstream cancer research circles. METHODS: The aim of this study was to quantitate the co-expression of two DNA repair proteins poly-ADP-ribose polymerase 1 and 2 (Parp-1 and Parp-2) by enzyme- linked immuno-sorbent assay (ELISA) in 60 endometrioid endometrial tumor samples and compare their expression to matched non-malignant endometrial tissue from the same corresponding donors from central Appalachia. RESULTS: We found that Parp-1 was significantly overexpressed in endometrial carcinoma relative to corresponding normal tissue. This overexpression implicates Parp inhibition therapy as a possible treatment for the disease. Our results also found a protective effect of native Parp-2 expression in non-malignant endometrial tissue with each 1 ng/mL increase in PARP-2 concentration in normal tissue was associated with a 10 % reduction in the hazard of tumor progression (HR = 0.90; p = 0.039) and a 21 % reduction in the hazard of death (HR = 0.79; p = 0.044). CONCLUSIONS: This study demonstrated the over-expression of the druggable target Parp-1 in endometrial adenocarcinoma and observed a strong negative correlation of native Parp-2 expression and disease progression via the quantification of the Parp proteins using enzyme- linked immuno-sorbent assay (ELISA) assays.

Murine Coronavirus Infection Activates the Aryl Hydrocarbon Receptor in an Indoleamine 2,3-Dioxygenase-Independent Manner, Contributing to Cytokine Modulation and Proviral TCDD-Inducible-PARP Expression.

The aryl hydrocarbon receptor (AhR) is a cytoplasmic receptor/transcription factor that modulates several cellular and immunological processes following activation by pathogen-associated stimuli, though its role during virus infection is largely unknown. Here, we show that AhR is activated in cells infected with mouse hepatitis virus (MHV), a coronavirus (CoV), and contributes to the upregulation of downstream effector TCDD-inducible poly(ADP-ribose) polymerase (TiPARP) during infection. Knockdown of TiPARP reduced viral replication and increased interferon expression, suggesting that TiPARP functions in a proviral manner during MHV infection. We also show that MHV replication induced the expression of other genes known to be downstream of AhR in macrophages and dendritic cells and in livers of infected mice. Further, we found that chemically inhibiting or activating AhR reciprocally modulated the expression levels of cytokines induced by infection, specifically, interleukin 1ß (IL-1ß), IL-10, and tumor necrosis factor alpha (TNF-α), consistent with a role for AhR activation in the host response to MHV infection. Furthermore, while indoleamine 2,3-dioxygenase (IDO1) drives AhR activation in other settings, MHV infection induced equal expression of downstream genes in wild-type (WT) and IDO1-/- macrophages, suggesting an alternative pathway of AhR activation. In summary, we show that coronaviruses elicit AhR activation by an IDO1-independent pathway, contributing to upregulation of downstream effectors, including the proviral factor TiPARP, and to modulation of cytokine gene expression, and we identify a previously unappreciated role for AhR signaling in CoV pathogenesis.IMPORTANCE Coronaviruses are a family of positive-sense RNA viruses with human and agricultural significance. Characterizing the mechanisms by which coronavirus infection dictates pathogenesis or counters the host immune response would provide targets for the development of therapeutics. Here, we show that the aryl hydrocarbon receptor (AhR) is activated in cells infected with a prototypic coronavirus, mouse hepatitis virus (MHV), resulting in the expression of several effector genes. AhR is important for modulation of the host immune response to MHV and plays a role in the expression of TiPARP, which we show is required for maximal viral replication. Taken together, our findings highlight a previously unidentified role for AhR in regulating coronavirus replication and the immune response to the virus.

Sodium selenite induces apoptosis and inhibits autophagy in human synovial sarcoma cell line SW982 in vitro.

The present study aimed to examine the effects of sodium selenite on the SW982 human synovial sarcoma cell line in relation to cell viability, apoptosis and autophagy. The results indicated that sodium selenite reduced cell viability and induced apoptosis by activating caspase­3 and members of the poly (ADP­ribose) polymerase and Bcl­2 protein families in SW982 cells. Furthermore, autophagy was also suppressed by sodium selenite treatment in SW982 cells, and apoptosis was upregulated in cells co­treated with sodium selenite and the autophagy inhibitor 3­methyladenine. By contrast, apoptosis was downregulated when sodium selenite was combined with rapamycin, an inducer of autophagy. The results indicated that autophagy may protect cells from the cytotoxicity of sodium selenite. The present study results demonstrated that sodium selenite induced apoptosis and inhibited autophagy and autophagy­protected cells from death by antagonizing sodium selenite­induced apoptosis in SW982 cells in vitro.

5-aminolevulinic acid mediated photodynamic therapy inhibits survival activity and promotes apoptosis of A375 and A431 cells.

OBJECTIVES: The purpose of this study was to investigate the effects of 5-aminolaevulinic acid mediated photodynamic therapy (ALA-PDT) on the survival activity and apoptosis of human melanoma cell line A375 and non-melanoma skin carcinoma cell line A431 cells. The mechanism for cellular apoptosis was explored. METHODS: The cell survival activity was determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and the proportion of apoptotic cells was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The expression levels of Bcl-2, Bax, caspase-3, caspase-8 and caspase-9 protein were assessed by western blot. The subcellular localization of cytochrome c was comparatively investigated by immunohistochemistry between pre-ALA-PDT and post- ALA-PDT. RESULTS: ALA-PDT significantly inhibited the survival activity of A375 cells and A431 cells in a dose- and time-dependent manner. The optimum inhibition efficiencies for A375 cells and A431 cells were obtained at 0.6 mM ALA at 4 h and 8 h after ALA-PDT, respectively. The phenomena of apoptosis were observed in ALA-PDT treated cells by TUNEL assay. The apoptotic rates of A375 cells and A431 cells were 90.0% and 61.5% at 6 h after ALA-PDT, respectively. Apoptosis induced by ALA-PDT involved in down-regulation of Bcl-2 protein, up-regulation of Bax protein and cleaved-PARP protein. It was observed that the expression of cleaved- caspase-3, caspase-8 and caspase-9 proteins in A375 cells and A431 cells gradually increased in 2 h and 4 h but decreased at 4-6 h and 6-8 h after ALA-PDT, respectively. In apoptosis cells immunohistochemical localization show that cytochrome C diffused from the mitochondria into the cytosol. CONCLUSION: ALA-PDT could significantly inhibit the survival activity of A375 and A431 cells. The apoptosis induced by ALA-PDT in A375 and A431 cells was related to the caspase-dependent death-receptor pathway and Cytochrome c-dependent mitochondrial pathway.

Arsenic, Cadmium, and Lead Like Troglitazone Trigger PPARγ-Dependent Poly (ADP-Ribose) Polymerase Expression and Subsequent Apoptosis in Rat Brain Astrocytes.

We previously demonstrated that arsenic, cadmium, and lead mixture at environmentally relevant doses induces astrocyte apoptosis in the developing brain. Here, we investigated the mechanism and contribution of each metal in inducing the apoptosis. We hypothesized participation of transcription factor, peroxisome proliferator-activated receptor gamma (PPARγ), reported to affect astrocyte survival. We treated cultured rat astrocytes with single metals and their combinations and performed apoptosis assay and measured PPARγ expression levels. We found that cadmium demonstrated maximum increase in PPARγ as well as apoptosis, followed by arsenic and then lead. Interestingly, we observed that the metals mimicked PPARγ agonist, troglitazone, and enhanced PPARγ-transcriptional activity. Co-treatment with PPARγ-siRNA or PPARγ-antagonist, GW9662, suppressed the astrocyte apoptosis, suggesting a prominent participation of PPARγ in metal(s)-induced astrocyte loss. We explored PPARγ-transcriptional activity and identify its target gene in apoptosis, performed in silico screening. We spotted PPARγ-response elements (PPREs) within poly(ADP-ribose) polymerase (PARP) gene, and through gel-shift assay verified metal(s)-mediated increased PPARγ binding to PARP-PPREs. Chromatin-immunoprecipitation and luciferase-reporter assays followed by real-time PCR and Western blotting proved PPRE-mediated PARP expression, where cadmium contributed most and lead least, and the effects of metal mixture were comparable with troglitazone. Eventually, dose-dependent increased cleaved-PARP/PARP ratio confirmed astrocyte apoptosis. Additionally, we found that PPARγ and PARP expressions were c-Jun N-terminal kinases and cyclin-dependent kinase5-dependent. In vivo treatment of developing rats with the metals corroborated enhanced PPARγ-dependent PARP and astrocyte apoptosis, where yet again cadmium contributed most. Overall, our study enlightens a novel PPARγ-dependent mechanism of As-, Cd-, and Pb-induced astrocyte apoptosis.

Effect of irinotecan on HMGB1, MMP9 expression, cell cycle, and cell growth in breast cancer (MCF-7) cells.

Irinotecan is a natural alkaloid agent widely used in cancer therapy. High-mobility group protein B1 as a non-histone chromosomal protein plays a fundamental role in gene expression and inflammation. In this study, the effect of irinotecan on high-mobility group protein B1 and MMP9 content, gene expression, cell cycle, and cell growth in human breast cancer cells (MCF-7) was investigated. The cells were exposed to various concentrations of irinotecan and the viability determined by trypan blue exclusion and 3-(4,5-dimethylthiazal-2-yl)-2,5-diphenyltetrazolium bromide assays. High-mobility group B proteins were extracted from the control and drug-treated cells and analyzed by immunoblot. High-mobility group protein B1 and MMP9 messenger RNA expression was studied by reverse transcription polymerase chain reaction. The results demonstrated reduction of cell viability upon increasing irinotecan concentration, up-regulated high-mobility group protein B1 gene expression, and down-regulated MMP9 mRNA. Although the content of high-mobility group protein B1 was decreased in chromatin extract upon drug action, no high-mobility group protein B1 release to extracellular space was detected by immunoblot analysis. Irinotecan decreased H3K9 acetylation and increased poly ADP-ribose polymerase fragmentation to 89 kDa and anion superoxide production suggesting induction of apoptosis in these cells. Propidium iodide staining of the cells 24 h after the drug treatment revealed arrest of the cells in S-phase. From the results, it is concluded that overexpression of high-mobility group protein B1 in the presence of irinotecan precedes breast cancer cells into apoptosis and in this response the binding of irinotecan to chromatin or high-mobility group protein B1 may condense/aggregate chromatin, preventing high-mobility group protein B1 release from chromatin.

Combination treatment using DDX3 and PARP inhibitors induces synthetic lethality in BRCA1-proficient breast cancer.

Triple-negative breast cancers have unfavorable outcomes due to their inherent aggressive behavior and lack of targeted therapies. Breast cancers occurring in BRCA1 mutation carriers are mostly triple-negative and harbor homologous recombination deficiency, sensitizing them to inhibition of a second DNA damage repair pathway by, e.g., PARP inhibitors. Unfortunately, resistance against PARP inhibitors in BRCA1-deficient cancers is common and sensitivity is limited in BRCA1-proficient breast cancers. RK-33, an inhibitor of the RNA helicase DDX3, was previously demonstrated to impede non-homologous end-joining repair of DNA breaks. Consequently, we evaluated DDX3 as a therapeutic target in BRCA pro- and deficient breast cancers and assessed whether DDX3 inhibition could sensitize cells to PARP inhibition. High DDX3 expression was identified by immunohistochemistry in breast cancer samples of 24% of BRCA1 (p = 0.337) and 21% of BRCA2 mutation carriers (p = 0.624), as compared to 30% of sporadic breast cancer samples. The sensitivity to the DDX3 inhibitor RK-33 was similar in BRCA1 pro- and deficient breast cancer cell lines, with IC50 values in the low micromolar range (2.8-6.6 µM). A synergistic interaction was observed for combination treatment with RK-33 and the PARP inhibitor olaparib in BRCA1-proficient breast cancer, with the mean combination index ranging from 0.59 to 0.62. Overall, we conclude that BRCA pro- and deficient breast cancers have a similar dependency upon DDX3. DDX3 inhibition by RK-33 synergizes with PARP inhibitor treatment, especially in breast cancers with a BRCA1-proficient background.

The Prognostic Value of BRCA1 and PARP Expression in Epithelial Ovarian Carcinoma: Immunohistochemical Detection.

BRCA1/2 mutation status in epithelial ovarian cancer (EOC) presently relies on genetic testing which is resource consuming. Immunohistochemistry is cheap, fairly reproducible, and may identify gene product alterations due to both germline and somatic mutations and other defects along the BRCA gene pathway (BRCAness phenomenon), which is important when treatment with poly (adenosine-diphosphate-ribose) polymerase (PARP) inhibitors is considered. The aim of this study was to investigate immunohistochemical detection of BRCA1 and PARP expression in EOC and their possible prognostic relevance. Tumor tissue from 170 patients with EOC was stained immunohistochemically with BRCA1 and PARP antibodies. Semiquantitative analyses were performed to determine loss of, equivocal, and retained BRCA1 and high versus low PARP protein expression. These parameters were analyzed for relation with patient and clinicopathologic characteristics and overall survival. BRCA1 expression was reduced in 21.2 % of the tumors and 36.5% showed high PARP expression. No correlation between the 2 parameters or between PARP and clinicopathologic features was found. Overall survival was significantly increased in the BRCA1-reduced and equivocal groups [median survival 2.4 y (95% CI, 1.6-6.6) and 4.9 y (95 % CI, 2.3-6.7) vs. 1.5 y (95% CI, 1.3-1.9); P=0.0002]. Multivariate analysis confirmed these findings; hazard ratio=0.53 (95% CI, 0.34-0.81; P=0.0037; loss of BRCA1 expression). In conclusion, immunohistochemical BRCA1 expression in EOC holds considerable prognostic information, whereas PARP expression did not influence the outcome. The results call for validation in prospective trials.

Resveratrol enhances the efficacy of sorafenib mediated apoptosis in human breast cancer MCF7 cells through ROS, cell cycle inhibition, caspase 3 and PARP cleavage.

Despite advances in diagnosis and treatment options, breast cancer is one of the main causes of cancer related death among women worldwide. Present study is aimed to preliminarily evaluate our hypothesis that the combination of resveratrol (RSV), a natural antioxidant, and lower dose of sorafenib (SF), a multi-kinase inhibitor and a component of ERK1/2 (extracellular signal-regulated kinase 1/2) pathway, would augment apoptosis in human breast cancer MCF7 cells. MCF7 cellexpressions s were treated with RSV, SF and their combination. MTT (3-[4,5-dimethylthiazol-2-yl] -2, 5-diphenyl-tetrazolium bromide) assay, DNA fragmentation assay, Hoechst33342, H2DCFDA (2', 7'-Dichlorodihydrofluorescein diacetate), Rhodamine123 staining, and Western Blot to detect different signaling protein expressions, were conducted to test the hypothesis. Combination of RSV and SF showed higher cytotoxicity on MCF7 cells than their individual treatment. Results from morphology change, Hoechst33342 staining, and DNA fragmentation suggested higher apoptosis data in the combinational treatment. Intracellular ROS (reactive oxygen species) levels, p53 and Bax/Bcl2 expressions, and decrease in mitochondrial membrane potential were also higher in the combinational treatment. Up-regulation of apaf-1, cl. caspase 9, cl. caspase 3 and cl. PARP (poly (ADP-Ribose) polymerase) were also noticed, while the expressions of cyclinD1 and cyclinB1 were decreased in the combinational group. The increase in apoptosis and signaling protein expressions with RSV and SF combinational treatment were increased over time. The combination of RSV and lower dose of SF at 6µM showed enhanced apoptotic activity than SF alone. Therefore, RSV can be considered as a neo-adjuvant to improve SF efficacy in breast cancer treatment.

Carbon ions of different linear energy transfer (LET) values induce apoptosis & G2 cell cycle arrest in radio-resistant melanoma cells.

BACKGROUND & OBJECTIVES: The main goal when treating malignancies with radiation is to deprive tumour cells of their reproductive potential. One approach is to induce tumour cell apoptosis. This study was conducted to evaluate the ability of carbon ions ( [12] C) to induce apoptosis and cell cycle arrest in human HTB140 melanoma cells. METHODS: In this in vitro study, human melanoma HTB140 cells were irradiated with the 62 MeV/n carbon ( [12] C) ion beam, having two different linear energy transfer (LET) values: 197 and 382 keV/µm. The dose range was 2 to 16 Gy. Cell viability was estimated by the sulforhodamine B assay seven days after irradiation. The cell cycle and apoptosis were evaluated 48 h after irradiation using flow cytometry. At the same time point, protein and gene expression of apoptotic regulators were estimated using the Western blot and q-PCR methods, respectively. RESULTS: Cell viability experiments indicated strong anti-tumour effects of [12] C ions. The analysis of cell cycle showed that [12] C ions blocked HTB140 cells in G2 phase and induced the dose dependent increase of apoptosis. The maximum value of 21.8 per cent was attained after irradiation with LET of 197 keV/µm at the dose level of 16 Gy. Pro-apoptotic effects of [12] C ions were confirmed by changes of key apoptotic molecules: the p53, Bax, Bcl-2, poly ADP ribose polymerase (PARP) as well as nuclear factor kappa B (NFκB). At the level of protein expression, the results indicated significant increases of p53, NFκB and Bax/Bcl-2 ratio and PARP cleavage. The Bax/Bcl-2 mRNA ratio was also increased, while no change was detected in the level of NFκB mRNA. INTERPRETATION & CONCLUSIONS: The present results indicated that anti-tumour effects of [12] C ions in human melanoma HTB140 cells were accomplished through induction of the mitochondrial apoptotic pathway as well as G2 arrest.

Induction of Poly(ADP-ribose) Polymerase in Mouse Bone Marrow Stromal Cells Exposed to 900 MHz Radiofrequency Fields: Preliminary Observations.

Background. Several investigators have reported increased levels of poly(ADP-ribose) polymerase-1 (PARP-1), a nuclear enzyme which plays an important role in the repair of damaged DNA, in cells exposed to extremely low dose ionizing radiation which does not cause measurable DNA damage. Objective. To examine whether exposure of the cells to nonionizing radiofrequency fields (RF) is capable of increasing messenger RNA of PARP-1 and its protein levels in mouse bone marrow stromal cells (BMSCs). Methods. BMSCs were exposed to 900 MHz RF at 120 µW/cm(2) power intensity for 3 hours/day for 5 days. PARP-1 mRNA and its protein levels were examined at 0, 0.5, 1, 2, 4, 6, 8, and 10 hours after exposure using RT-PCR and Western blot analyses. Sham-exposed (SH) cells and those exposed to ionizing radiation were used as unexposed and positive control cells. Results. BMSCs exposed to RF showed significantly increased expression of PARP-1 mRNA and its protein levels after exposure to RF while such changes were not observed in SH-exposed cells. Conclusion. Nonionizing RF exposure is capable of inducing PARP-1.

Miz-1 promotes the proliferation of esophageal cancer cells via suppression of p21 and release of p21-arrested cyclin D1.

Tumorigenesis results from various types of dysregulation of oncogenes and tumor suppressors that influence cellular proliferation, differentiation, apoptosis and senescence. The transcription factor Myc-interacting zinc finger protein 1 (Miz-1) can either activate or repress gene expression in concert with binding partners including the Myc oncoprotein in several types of tumors. Known target genes of these complexes encode the cyclin­dependent kinase inhibitors such as cdkn2b (p15) and cdkn1a (p21). In the present study, we showed that the silencing of Miz-1 expression, through shRNA in a lentiviral vector, influenced various biological processes in two types of esophageal carcinoma cell lines. Silencing of the expression of Miz-1 inhibited cell proliferation and promoted apoptosis in vitro. Loss of Miz-1 reduced the migration ability in esophageal carcinoma cells. High expression of p21 and downregulation of cyclin D1 accompanied the knockdown of Miz-1. Our data demonstrated that esophageal cancer has a cell cycle arrest pathway via Miz-1, p21 and cyclin D1.

Sinuleptolide inhibits proliferation of oral cancer Ca9-22 cells involving apoptosis, oxidative stress, and DNA damage.

OBJECTIVE: Sinuleptolide, a soft corals-derived bioactive norditerpenoid, is a marine natural product with a potent anti-inflammatory effect. We evaluate the potential anti-oral cancer effects of sinuleptolide and investigate the possible mechanisms involved. DESIGNS: Cell viability, cell cycle, apoptosis, reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and DNA damage analyses were performed. RESULTS: In a cell viability assay, we found that sinuleptolide is dose-responsively antiproliferative against oral gingival cancer Ca9-22 cells but less harmful to normal human gingival fibroblast (HGF-1) cells (P<0.001). In cell cycle analysis, sinuleptolide induced subG1 accumulation at a higher dose and led to G2/M arrest of Ca9-22 cells (P<0.005). Apoptosis was significantly increased in sinuleptolide-treated Ca9-22 cells based on annexin V and poly(ADP-ribose) polymerase (PARP) expressions (P<0.05-0.0001). Based on flow cytometer analysis, sinuleptolide also induced the generation of ROS and decreased MMP in a dose-responsive manner (P<0.05-0.0001). DNA damage increased dose-responsively after sinuleptolide treatments (P < 0.001) based on comet and γH2AX assays. CONCLUSION: Sinuleptolide can induce an antiproliferation of oral cancer Ca9-22 cells involving apoptosis, oxidative stress and DNA damage, suggesting that sinuleptolide represents a potential chemotherapeutic drug for oral cancer treatment.

APR-246 (PRIMA-1(MET)) strongly synergizes with AZD2281 (olaparib) induced PARP inhibition to induce apoptosis in non-small cell lung cancer cell lines.

APR-246 (PRIMA-1(Met)) is able to bind mutant p53 and restore its normal conformation and function. The compound has also been shown to increase intracellular ROS levels. Importantly, the poly-[ADP-ribose] polymerase-1 (PARP-1) enzyme plays an important role in the repair of ROS-induced DNA damage. We hypothesize that by blocking this repair with the PARP-inhibitor AZD2281 (olaparib), DNA damage would accumulate in the cell leading to massive apoptosis. We observed that APR-246 synergistically enhanced the cytotoxic response of olaparib in TP53 mutant non-small cell lung cancer cell lines, resulting in a strong apoptotic response. In the presence of wild type p53 a G2/M cell cycle block was predominantly observed. NOXA expression levels were significantly increased in a TP53 mutant background, and remained unchanged in the wild type cell line. The combined treatment of APR-246 and olaparib induced cell death that was associated with increased ROS production, accumulation of DNA damage and translocation of p53 to the mitochondria. Out data suggest a promising targeted combination strategy in which the response to olaparib is synergistically enhanced by the addition of APR-246, especially in a TP53 mutant background.

Effects of Sodium Fluoride on Lipid Peroxidation and PARP, XBP-1 Expression in PC12 Cell.

This study aims to clarify the molecular mechanism of fluorine exposure that leads to nerve injury. PC12 cells were treated with fluorine at different concentrations (0.5, 1.0, 1.5, and 2.0 mM). Cytoactivity was detected at different time points (2, 4, 6, 8, 12, 24, and 48 h). After 2 h, DCF was used to detect and mark the level of reactive oxygen species (ROS) within cells. After 24 h, cellular metamorphosis was observed using an inverted microscope. After 2 h, Hoechst-33342 was used to detect apoptosis. After 24 h, Western blot analysis was performed to detect apoptosis-related poly (ADP-ribose) polymerase (PARP) protein, p-elF, and expression of the endoplasmic reticulum stress-related X-box binding protein 1 (XBP-1). The results showed that Fluorine exposure resulted in a reduction of cell viability, which was negatively correlated with fluorine dose. Within certain fluorine exposure duration, the ROS level within the cell and the apoptotic level are linearly related to fluorine exposure level. XBP-1 and PARP protein are sensitive to variations in fluorine concentration, which indicates that oxidative stress from fluorine exposure can lead to apoptosis. XBP-1 and PARP may be the key proteins during the entire process. These results provide a valid basis for fluorine-induced free radical injury theory.

The SMAD2/3 pathway is involved in hepaCAM-induced apoptosis by inhibiting the nuclear translocation of SMAD2/3 in bladder cancer cells.

The aim of this study was to explore the correlation between hepatocyte cell adhesion molecule (hepaCAM) and SMAD family member 2/3 (SMAD2/3) in bladder carcinoma, and the involvement of the SMAD2/3 pathway in hepaCAM-induced tumor apoptosis. Immunohistochemistry was used to measure hepaCAM and p-SMAD2/3 protein levels in bladder cancer tissues. Flow cytometry and Hoechst staining were used to study the effect of hepaCAM on cellular apoptosis. Western blot was employed to determine the expression of hepaCAM and SMAD2/3/caspase pathway molecules using a hepaCAM overexpression adenovirus, a caspase inhibitor (Z-VAD-FMK), and a SMAD2/3 activator (transforming growth factor (TGF)-ß1), respectively. Translocation of p-SMAD2/3 was measured by immunofluorescence and western blot. HepaCAM proteins were significantly decreased (P < 0.05), while p-SMAD2/3 proteins were remarkably increased (P < 0.05) in bladder carcinoma compared to adjacent tissues. However, the low hepaCAM and high p-SMAD2/3 were not statistically associated with clinicopathological characteristics of the patients. A negative linear correlation between hepaCAM and p-SMAD2/3 was observed according to Pearson analysis (r = -0.712/-0.724, P = 0.008/0.011). Overexpression of hepaCAM activated caspase 3/8/9 and downregulated poly-ADP ribose polymerase (PARP) and p-SMAD2/3. Treatment of bladder cancer cells with Z-VAD-FMK + hepaCAM significantly downregulated procaspase 3/8/9 and PARP and induced cellular apoptosis, compared with that using Z-VAD-FMK alone. Similarly, combined treatment of TGF-ß1 + hepaCAM significantly downregulated p-SMAD2/3, procaspase 3/8/9, and PARP and induced apoptosis of bladder cancer cells, compared with TGF-ß1 alone. Overexpression of hepaCAM prevented the p-SMAD2/3 translocation from the cytoplasm to the nucleus in bladder cancer cells BIU-87 and T24. Our findings uncover that the p-SMAD2/3 pathway is critical for hepaCAM-induced cancer cell apoptosis and provide valuable insights for current and future Ad-hepaCAM and p-SMAD2/3 clinical trials.

Decreased DNA repair gene XRCC1 expression is associated with radiotherapy-induced acute side effects in breast cancer patients.

DNA repair plays a critical role in response to ionizing radiation (IR) and developing of radiotherapy induced normal tissue reactions. In our study, we investigated the association of radiotherapy related acute side effects, with X-ray repair cross complementing group 1 (XRCC1) and Poly (ADP-ribose) polymerase 1 (PARP1) DNA repair gene expression levels, their changes in protein expression and DNA damage levels in breast cancer patients. The study included 40 women with newly diagnosed breast cancer; an experimental case group (n=20) with acute side effects and the control group (n=20) without side effects. For gene and protein expression analysis, lymphocytes were cultured for 72 h and followed by in vitro 2 Gray (Gy) gamma-irradiation. For detection of DNA damage levels, lymphocytes were irradiated with in vitro 2 Gy gamma-rays and followed by incubation for 72 h. XRCC1 mRNA and protein expression levels were significantly higher in controls than in experimental cases (P=0.020). In terms of DNA damage levels, an increased frequency of micronucleus (MN) was observed in experimental cases versus controls, but this association was not significant (P=0.206). We also observed a significant negative correlation between MN frequency and XRCC1 protein levels in experimental (r=-0.469, P=0.037) vs control (r=-0.734, P<0.001). Our results suggested that decreased XRCC1 expression levels might be associated with the increased risk of therapeutic IR-related acute side effects in patients with breast cancer.

Ginkgetin exerts growth inhibitory and apoptotic effects on osteosarcoma cells through inhibition of STAT3 and activation of caspase-3/9.

Osteosarcoma is composed of tumor osteoblasts and bone-like tissues, with malignant tumors originating from osteogenesis organization. Osteosarcoma is a primary malignant bone tumor. Invasion and metastasis of osteosarcoma affect the prognosis of patients. However, effective therapeutic treatments remain to be identified. The aim of the present study was to investigate the possible inhibitory and apoptotic effects of ginkgetin in osteosarcoma cells. 3.3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays were used to determine the effect ginkgetin exerted on the growth of osteosarcoma cells. Flow cytometry was used to determine cell apoptosis. STAT3 protein expression and activation of caspase-3/9 were measured using western blot analysis and the MTT and LDH assays, respectively. The results showed that ginkgetin inhibited cell growth and induced cell cytotoxicity in osteosarcoma cells in a dose-dependent manner. Treatment with ginkgetin significantly activated the apoptosis of osteosarcoma cells in a concentration-dependent manner. The anticancer activity of ginkgetin significantly inhibited STAT3 and promoted caspase-3/9 activation in osteosarcoma cells. The findings demonstrated that ginkgetin exerts growth inhibitory and apoptotic effects on osteosarcoma cells through the inhibition of STAT3 and activation of caspase-3/9.