Protein Expression Of Poly ADP-ribose Polymerase Research Articles

Curzerene suppresses hepatocellular carcinoma progression through the PI3K/AKT/MTOR pathway.

Hepatocellular carcinoma (HCC) is one of the most aggressive cancers worldwide. Curzerene is a sesquiterpene and component of Curcuma rhizomes and has anti-tumor and anti-inflammatory properties. The study aimed to investigate the effects of curzerene on the malignant phenotypes and tumor growth in HCC. Various concentrations of curzerene were used to treat human HCC cells (Huh7 and HCCLM3). Cell viability, apoptosis, cell cycle, invasion, and migration were detected by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, flow cytometry, Transwell, and wound healing assays. Cell cycle-, apoptosis-, and signaling pathway-related proteins were analyzed by Western blot analysis. A mouse xenograft model was established to analyze the anti-tumor effects of curzerene in vivo. Curzerene repressed the proliferation, invasion, and migration of Huh7 and HCCLM3 cells. Curzerene also induced G2/M cycle arrest and cell apoptosis. Curzerene downregulated the CDK1, cyclin B1, PCNA, Bcl-2, matrix metallopeptidases (MMP)2, and MMP9 protein expression and upregulated the Bax, cleaved caspase3, and cleaved poly ADPribose polymerase protein expression in HCC cells. Curzerene restrained the phosphorylation of PI3K, AKT, and the Mammalian target of rapamycin (mTOR) in Huh7 and HCCLM3 cells. The in vivo data revealed that curzerene inhibited HCC tumor growth and decreased the expression of phosphorylated mTOR in xenograft mouse models. Curzerene inhibited cell malignancy in vitro and tumor growth in vivo in HCC, suggesting that curzerene may be a candidate agent for anti-HCC therapy.

Protective Effects of Baicalein on Lipopolysaccharide-Induced AR42J PACs through Attenuation of Both Inflammation and Pyroptosis via Downregulation of miR-224-5p/PARP1.

Baicalein has been used to treat inflammation-related diseases; nevertheless, its specific mechanism of action is unclear. Therefore, we examined the protective effects of baicalein on lipopolysaccharide-induced damage to AR42J pancreatic acinar cells (PACs) and determined its mechanism of action for protection. An in vitro cell model of acute pancreatitis (AP) was established using lipopolysaccharide (LPS) (1 mg/L)-induced PACs (AR42J), and the relative survival rate was determined using the 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT) technique. Flow cytometry was applied to evaluate the apoptotic rates of AR42J PACs. The RNA and protein expression of miR-224-5p, poly ADP-ribose polymerase-1 (PARP1), nuclear transcription factor-κB65 (NF-κB65), phospho-kappa B alpha(p-IκB-α), interleukin(IL)-18R, NOD-like receptor thermal protein domain-associated protein 3 (NLRP3), gasdermin D (GSDMD), apoptosis-associated speck-like protein containing a CARD (ASC), and caspase-1 was detected based on the WB and RT-PCR assays. IL-1β, IL-6, IL-18, and TNF-α expression levels in AR42J cells were measured via ELISA method. The cell morphology was examined using the AO/EB method. The experiment confirmed a significant increase in the activity of AR42J cells treated with various doses of baicalein. Moreover, IL-1β, IL-6, TNF-α, and IL-18 expression levels in AR42J cells were dramatically reduced (P < 0.05), while miR-224-5p level was obviously enhanced. The protein and gene expression of PARP1, NF-κB65, p-IκB-α, IL-18R, GSDMD, ASC, NLRP3, and caspase-1 was obviously decreased (P < 0.05). Apoptosis in AR42J cells was significantly reduced with significant improvement in cell morphology. Baicalein may significantly alleviate LPS-induced AR42J PAC damage by inhibiting the inflammatory response and pyroptosis. Its mode of action might be linked to higher miR-224-5p expression, which inhibits the PARP1/NF-κB and NLPR3/ASC/caspase-1/GSDMD pathways.

Effect of digestion product of royal jelly protein on SGC-7901 gastric cancer cell

Royal jelly used for larvae and queens has many health-promoting properties such as spatial memory improvement, antibacterial activity and antioxidant capacity. However, the anticancer ability of the royal jelly is not unknown. In this study, effect of the royal jelly protein on SGC-7901 gastric cancer cell was investigated, and the key factors including the cell morphological, the colony formation, the proliferation, the cycle, and the expression of p53 and poly (ADP-ribose) polymerase-1 (PARP-1) proteins were analyzed. Result showed royal jelly protein was excellent in inhibiting the growth of SGC-7901gastric cancer cell. After treating with the digestion product of royal jelly protein (0.05 mg/mL～0.20 mg/mL), the morphological of gastric cancer cell significantly shrink, and both of density and quantity (1000～491) of gastric cancer cell significantly (p < 0.05) decreased. The proliferation of gastric cancer was strongest inhibited by 62.84 % of 0.20 mg/mL royal jelly protein. The expression of p53 and PARP-1 protein of gastric cancer cell was respectively enhanced (0.29～0.46) and reduced (0.51～0.42). Moreover, the higher content of royal jelly protein (0.05 mg/mL～ 0.2 mg/mL), the stronger inhibit ability of 45 SGC-7901 gastric cancer cell. In conclusion, royal jelly protein can be used as a potential food in adjunctive therapy for gastric cancer.

Mollugin ameliorates murine allergic airway inflammation by inhibiting Th2 response and M2 macrophage activation

Mollugin, isolated from Rubia cordifolia L, is a pharmacological compound with anti-inflammatory activity. This study aimed to investigate whether mollugin protects mice against shrimp tropomyosin (ST)-induced allergic airway inflammation. Mice were sensitized with ST combined with Al(OH)3 administered intraperitoneally (i.p.) once weekly for 3 wk followed by ST challenge for 5 d. Mice were i.p.-administered daily with mollugin for 7 d. Results showed that mollugin attenuated ST-induced infiltration of eosinophils and epithelial mucus secretion in the lung tissues and suppressed lung eosinophil peroxidase activity. Additionally, mollugin lowered the Th2 cytokine, IL-4 and IL-5, production and downregulated the mRNA levels of Il-4, Il-5, Il-13, eotaxin, Ccl-17, Muc5ac, arginase-1, Ym-1, and Fizz-1 in the lung tissues. Network pharmacology was employed to predict core targets, and the molecular docking approach was used to verify the compound targets. The results of the molecular docking study of mollugin into p38 MAPK or poly(ADP-ribose) polymerase 1 (PARP1) binding sites revealed that its mechanism was possibly similar to that of SB203580 (a p38 MAPK inhibitor) or olaparib (a PARP1 inhibitor). Immunohistochemistry analysis revealed that mollugin mitigated ST-induced elevation of arginase-1 expression and macrophage levels in the lungs and bronchoalveolar lavage fluid, respectively. Furthermore, arginase-1 mRNA level and phosphorylation of p38 MAPK were inhibited in IL-4-stimulated peritoneal macrophages. In ST-stimulated mouse primary splenocytes, mollugin notably inhibited IL-4 and IL-5 production and downregulated PARP1 and PAR protein expression. According to our findings, mollugin ameliorated allergic airway inflammation by inhibiting Th2 response and macrophage polarization.

Hyperthermia Improves Doxorubicin-Based Chemotherapy by Activating Mitochondrial Apoptosis in Bladder Cancer

Purpose: Although intravesical chemotherapy has several antitumoral benefits, it can also have severe side effects. The development of novel therapeutic approaches for bladder cancer (BC) is thus warranted. Hyperthermia (HT) is a widely applicable adjuvant therapy in various cancers. Therefore, this study investigated the effect of HT on improving the chemosensitivity of BC. Materials and Methods: The BC cell lines 5637 and T24 were cultured and treated with HT (43°C) for 24 h. Then, cell viability and survival were assessed using resazurin reagent and colony formation assay, respectively. Western blot assay was used to analyze the levels of Bax, Bcl-2, cleaved caspase-3, and cleaved poly (ADP-ribose) polymerase (PARP) protein expression. Mitochondria degradation was observed by MitoTracker Green staining. Results: In BC cells, HT co-administered with various concentrations of doxorubicin significantly inhibited cell viability and survival. Moreover, HT combined with doxorubicin promoted mitochondrial apoptosis, which caused Bax upregulation and Bcl-2 downregulation. Levels of cleaved caspase-3 and PARP protein expression were also elevated after co-treatment. Conclusion: Taken together, HT improved the chemosensitivity of BC cells to doxorubicin. HT combined with chemotherapy further activated mitochondrial apoptosis in BC cells. The findings suggested that HT may serve as a potential adjunctive treatment for BC that is ready to be applied clinically.

Borealin-Derived Peptides as Survivin-Targeting Cancer Imaging and Therapeutic Agents.

Survivin is overexpressed in most cancer cells but is rarely expressed in normal adult tissues. It is associated with poor prognosis and resistance to radiation therapy and chemotherapy. In this study, we designed and synthesized borealin-derived small peptides (Bor peptides) to function as survivin-targeting agents for the diagnosis and treatment of cancers. These peptides exhibited binding affinities for recombinant human survivin (Kd = 49.6-193 nM), with Bor65-75 showing the highest affinity (Kd = 49.6 nM). Fluorescence images of fluorescein isothiocyanate-labeled Bor65-75 showed its co-localization with survivin expression in the human pancreatic cancer cell line, MIA PaCa-2. In the WST-1 assay, cell penetrable nona-d-arginine-conjugated Bor65-75 (r9-Bor65-75) inhibited the growth of MIA PaCa-2 cells and MDA-MB-231 cells (89 and 88% inhibition at 10 μM, respectively), whereas it had almost no effect on the human mammary epithelial cell line, MCF-10A, that inherently does not have high survivin expression. Flow cytometry with annexin V and propidium iodide staining revealed that r9-Bor65-75 induced apoptosis in MIA PaCa-2 cells in a dose-dependent manner. An increase in cleaved poly ADP-ribose polymerase protein expression was observed in MIA PaCa-2 cells exposed to r9-Bor65-75 by western blotting, suggesting that r9-Bor65-75 inhibits cell proliferation by inducing apoptosis. In vivo, r9-Bor65-75 significantly suppressed tumor growth in MIA PaCa-2 xenograft mice, without any marked weight loss. Hence, Bor peptides are promising candidates for the development of cancer imaging and anticancer agents targeting survivin.

The Prognostic Relevance of Poly (ADP-Ribose) Polymerase Expression in Ovarian Cancer Tissue of Wild Type and BRCA-Mutation Carrier Patients.

(1) Background: The mechanism of platinum resistance in ovarian cancer is not fully clarified, but the properly functioning DNA repair mechanism can counteract the effect of conventional anticancer treatment. The objective of our study was to evaluate the expression of an important DNA repair enzyme, the Poly (ADP-Ribose) Polymerase (PARP) expression in epithelial ovarian cancer (EOC) tissues depending on BRCA status and to assess its relationship with platinum resistance. (2) Methods: Immunostaining to highlight PARP protein expression was performed using a rabbit polyclonal anti-PARP antibody. The intensity and distribution of immunostaining were assessed by light. Somatic BRCA1 or BRCA2 mutation carriers were identified with bidirectional sequencing of DNA from archived tumor tissue, if the test could not be performed due to technical reasons from tumor cells, the sequencing was done from peripheral blood cells to identify germline mutation carriers. The median progression-free survival (PFS) was generated for each semiquantitative group of PARP expression among chemotherapy-naive cases at the time of PARP immunohistochemistry. (3) Results: In the overall population, negative PARP immunohistochemistry predicted significant PFS (20.1 vs. 11.9 months, p = 0.001) and OS (49 vs. 114 months, p = 0.014) benefit. Genotype-stratified subgroup analysis in BRCA-negative cases confirmed the role of PARP positivity indicating an unfavorable prognosis in the entire population (relapsed 73.91% vs. 92%; OR: 4.06; p = 0.04). In the cases of the subgroup carrying the BRCA mutation, the presence of PARP expression was not associated with less favorable relapse rates, but with marginal significance for overall survival predicted a lower chance of survival (OS more than 32 months 72.73% vs. 35%; OR: 0.2; p = 0.05). (4) Conclusion: The BRCA wild type patients with strong expression of PARP enzymes before the first set of chemotherapy have a poor prognosis.

The Prognostic Value of PARP Expression in High-Grade Epithelial Ovarian Cancer

In an attempt to clarify the prognostic relevance of poly (ADP-ribose) polymerase (PARP) expression, we analysed the clinical data of 86 high-grade epithelial ovarian cancer (EOC) cases in which PARP immunohistochemistry results were available. Immunostaining to highlight PARP protein expression was performed using a Leica Bond MAX Immunostainer (Leica Microsystems, Wetzlar, Germany). We applied a rabbit polyclonal anti-PARP antibody (ab6079 330, Abcam, Cambridge, UK) for the specific reaction. The intensity and distribution of immunostaining were assessed by light microscopy (Leica DM2500 microscope, DFC 420 camera, and Leica Application Suite V3 software; Leica) and evaluated with a four-grade (0–3+) system. The median progression-free survival (PFS) was generated for each semiquantitative group of PARP expression among chemotherapy-naive cases at the time of PARP immunohistochemistry. Eighty-six cases were chemotherapy-naive at the time of PARP immunohistochemistry, and 41 cases showed no PARP expression. Forty-five cases showed intermediate or high PARP expression. The median PFS among patients in the PARP-negative group was 16 months (interquartile range; IQR 10.7–35.9 months), and the median PFS of patients in the PARP-positive group was 12 months (IQR 6.1–21.8 months). The difference was significant according to the log-rank test (p = 0.01). The median overall survival (OS) of patients in the PARP-negative group was 65 months (IQR 43.6–110.8 months), and the median OS of patients in the PARP-positive group was 52 months (IQR 36.9–66.7 months). The difference was significant according to the log-rank test (p = 0.028). Multiple comparisons confirmed that PARP expression results in a significant difference in PFS and OS achieved by first-line Taxol-carboplatin chemotherapy. The lack of PARP expression assessed by immunohistochemistry may predict improved PFS in ovarian cancer patients after adjuvant platinum-based chemotherapy.

Targeted PARP Inhibition Combined with FGFR1 Blockade is Synthetically Lethal to Malignant Cells in Patients with Pancreatic Cancer.

The role and therapeutic promise of poly-ADP ribose polymerase (PARP) inhibitors in anticancer chemotherapy are increasingly being explored, particularly in adjuvant or maintenance therapy, considering their low efficacy as monotherapy agents and their potentiating effects on concurrently administered contemporary chemotherapeutics. Against the background of increasing acquired resistance to FGFR1 inhibitors and our previous work, which partially demonstrated the caspase-3/PARP-mediated antitumor and antimetastatic efficacy of PD173074, a selective FGFR1 inhibitor, against ALDH-high/FGFR1-rich pancreatic ductal adenocarcinoma (PDAC) cells, we investigated the probable synthetic lethality and therapeutic efficacy of targeted PARP inhibition combined with FGFR1 blockade in patients with PDAC. Using bioinformatics-based analyses of gene expression profiles, co-occurrence and mutual exclusivity, molecular docking, immunofluorescence staining, clonogenicity, Western blotting, cell viability or cytotoxicity screening, and tumorsphere formation assays, we demonstrated that FGFR1 and PARP co-occur, form a complex, and reduce survival in patients with PDAC. Furthermore, FGFR1 and PARP expression was upregulated in FGFR1 inhibitor (dasatinib)-resistant PDAC cell lines SU8686, MiaPaCa2, and PANC-1 compared with that in sensitive cell lines Panc0403, Panc0504, Panc1005, and SUIT-2. Compared with the limited effect of single-agent olaparib (PARP inhibitor) or PD173074 on PANC-1 and SUIT-2 cells, low-dose combination (olaparib + PD173074) treatment significantly, dose-dependently, and synergistically reduced cell viability, upregulated cleaved PARP, pro-caspase (CASP)-9, cleaved-CASP9, and cleaved-CASP3 protein expression, and downregulated Bcl-xL protein expression. Furthermore, combination treatment markedly suppressed the clonogenicity and tumorsphere formation efficiency of PDAC cells regardless of FGFR1 inhibitor-resistance status and enhanced RAD51 and γ-H2AX immunoreactivity. In vivo studies have shown that both early and late initiation of combination therapy markedly suppressed tumor xenograft growth and increase in weight, although the effect was more pronounced in the early initiation group. In conclusion, FGFR1 inhibitor-resistant PDAC cells exhibited sensitivity to PD173074 after olaparib-mediated loss of PARP signaling. The present FGFR1/PARP-mediated synthetic lethality proof-of-concept study provided preclinical evidence of the feasibility and therapeutic efficacy of combinatorial FGFR1/PARP1 inhibition in human PDAC cell lines.

Abstract 4300: Overexpression of PARP is an independent prognostic marker for poor survival in Middle Eastern breast cancer and its inhibition can be enhanced with embelin co-treatment

Abstract Patients with aggressive breast cancer (BC) subtypes usually don’t have favorable prognosis despite the improvement in treatment modalities. These cancers still remain a major cause of morbidity and mortality in females. This has fostered a major effort to discover actionable molecular targets to treat these patients. Poly ADP ribose polymerase (PARP) is one of these molecular targets that are under comprehensive investigation for treatment of such tumors. However, its role in the pathogenesis of BC from Middle Eastern ethnicity has not yet explored. Therefore, we examined the expression of PARP protein in a large cohort of over 1000 Middle Eastern BC cases by immunohistochemistry. Correlation with clinico-pathological parameters were performed. Nuclear PARP overexpression was observed in 44.7% of all BC cases and was significantly associated with aggressive clinico-pathological markers. Interestingly nuclear PARP overexpression was an independent predictor of poor prognosis. PARP overexpression was also directly associated with XIAP overexpression, with PARP and XIAP co-expression in 15.8% (159/1008) of our cases. We showed that combined inhibition of PARP by olaparib and XIAP by embelin significantly and synergistically inhibited cell growth and induced apoptosis in BC cell lines. Finally, co-treatment of olaparib and embelin regressed BC xenograft tumor growth in nude mice. Our results revealed the role of PARP in Middle Eastern BC pathogenesis and prognosis. Furthermore, our data support the potential clinical development of combined inhibition of PARP and XIAP, which eventually could extend the utility of olaparib beyond BRCA deficient cancer. Citation Format: Maqbool Ahmed, Abdul K. Siraj, Poyil Pratheeshkumar, Sandeep Parvathareddy, Sasidharan Padmaja Divya, Fouad Al-Dayel, Asma Tulbah, Dahish Ajarim, Khawla S. Al-Kuraya. Overexpression of PARP is an independent prognostic marker for poor survival in Middle Eastern breast cancer and its inhibition can be enhanced with embelin co-treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4300.

Poly(ADP-ribose) polymerase 1 induces cardiac fibrosis by mediating mammalian target of rapamycin activity.

Cardiac fibrosis is involved in nearly all forms of heart diseases and is characterized by excessive deposition of extracellular matrix proteins by cardiac fibroblasts (CFs). We and others have reported the possibility of poly(ADP-ribose) polymerase 1 (PARP1), the founding subtype of the PARPs enzyme family, as a novel therapeutic target of heart diseases. The cardiac fibrotic induction of mammalian target of rapamycin (mTOR) is mainly due to collagen expression, Smad3- and p53/JNK-mediated apoptosis. However, the possible link between PARP1 and mTOR in the progression of cardiac fibrosis remains unclear. In this study, PARP1 protein expression, and the activity of mTOR and its three target substrates (p70 ribosomal S6 Kinase 1, eukaryotic initiation factor 4E--binding protein 1, and UNC-51-like kinase 1) were augmented; meanwhile, the nicotinamide adenine dinucleotide (NAD) content was significantly reduced in the process of cardiac fibrosis in vivo and in vitro. Sprague-Dawley rats were intraperitoneally injected with 3-aminobenzamide (3AB) (20 mg/kg/d; a well-established PARP1 inhibitor) or rapamycin (Rapa; 1 mg/kg/d; used for mTOR inhibition) 7 days after abdominal aortic constriction (AAC) surgery for 6 weeks. Pretreatment of 3AB or Rapa both relieved AAC-caused cardiac fibrosis and heart dysfunction. Overexpression of PARP1 with adenovirus carrying PARP1 gene specifically transduced into the hearts via intramyocardial multipoint injection caused similar myocardial damage. In CFs, preincubation with PARP1 or mTOR inhibitors all blocked TGF-β1 induced cardiac fibrosis. PARP1 overexpression evoked cardiac fibrosis, which could be antagonized by mTOR inhibitors or NAD supplementation in CFs. These results provide novel and compelling evidence that PARP1 exacerbated cardiac fibrosis, which was partially attributed to NAD-dependent activation of mTOR.

Overexpression of PARP is an independent prognostic marker for poor survival in Middle Eastern breast cancer and its inhibition can be enhanced with embelin co-treatment.

Patients with aggressive breast cancer (BC) subtypes usually don’t have favorable prognosis despite the improvement in treatment modalities. These cancers still remain a major cause of morbidity and mortality in females. This has fostered a major effort to discover actionable molecular targets to treat these patients. Poly ADP ribose polymerase (PARP) is one of these molecular targets that are under comprehensive investigation for treatment of such tumors. However, its role in the pathogenesis of BC from Middle Eastern ethnicity has not yet been explored. Therefore, we examined the expression of PARP protein in a large cohort of over 1000 Middle Eastern BC cases by immunohistochemistry. Correlation with clinico-pathological parameters were performed. Nuclear PARP overexpression was observed in 44.7% of all BC cases and was significantly associated with aggressive clinico-pathological markers. Interestingly, nuclear PARP overexpression was an independent predictor of poor prognosis. PARP overexpression was also directly associated with XIAP overexpression, with PARP and XIAP co-expression in 15.8% (159/1008) of our cases. We showed that combined inhibition of PARP by olaparib and XIAP by embelin significantly and synergistically inhibited cell growth and induced apoptosis in BC cell lines. Finally, co-treatment of olaparib and embelin regressed BC xenograft tumor growth in nude mice. Our results revealed the role of PARP in Middle Eastern BC pathogenesis and prognosis. Furthermore, our data support the potential clinical development of combined inhibition of PARP and XIAP, which eventually could extend the utility of olaparib beyond BRCA deficient cancer.

Endonuclease G takes part in AIF-mediated caspase-independent apoptosis in Mycobacterium bovis-infected bovine macrophages

Mycobacterium bovis, the causative agent of bovine tuberculosis encodes different virulence mechanisms to survive inside of host cells. One of the possible outcomes in this host–pathogen interaction is cell death. Previous results from our group showed that M. bovis induces a caspase-independent apoptosis in bovine macrophages with the possible participation of apoptosis inducing factor mitochondria associated 1 (AIFM1/AIF), a flavoprotein that functions as a cell-death regulator. However, contribution of other caspase-independent cell death mediators in M. bovis-infected macrophages is not known. In this study, we aimed to further characterize M. bovis-induced apoptosis, addressing Endonuclease G (Endo G) and Poly (ADP-ribose) polymerase 1 (PARP-1). In order to accomplish our objective, we infected bovine macrophages with M. bovis AN5 (MOI 10:1). Analysis of M. bovis-infected nuclear protein extracts by immunoblot, identified a 15- and 43-fold increase in concentration of mitochondrial proteins AIF and Endo G respectively. Interestingly, pretreatment of M. bovis-infected macrophages with cyclosporine A, a mitochondrial permeability transition pore inhibitor, abolished AIF and Endo G nuclear translocation. In addition, it also decreased macrophage DNA fragmentation to baseline and caused a 26.2% increase in bacterial viability. We also demonstrated that PARP-1 protein expression in macrophages did not change during M. bovis infection. Furthermore, pretreatment of M. bovis-infected bovine macrophages with 3-aminobenzamide, a PARP-1 inhibitor, did not change the proportion of macrophage DNA fragmentation. Our results suggest participation of Endo G, but not PARP-1, in M. bovis-induced macrophage apoptosis. To the best of our knowledge this is the first report associating Endo G with caspase-independent apoptosis induced by a member of the Mycobacterium tuberculosis complex.

IDENTIFICATION OF PARP1 AS A POTENTIAL THERAPEUTIC TARGET IN TRIPLE NEGATIVE AND BASAL-LIKE BREAST CANCER MOLECULAR SUBTYPES AMONG WEST ALGERIAN PATIENTS

Purpose: Breast cancer is partly responsible for the therapeutic failures of tumors with the most worsening prognoses. Increasing understanding of the cellular aberrations inherent to cancer cells was an important step towards therapy individualization. Poly (ADP-ribose) polymerase-1 (PARP1) as a promising novel target in breast cancer was an example of this strategy. However, the biological and clinical significance of PARP1 expression and its associations with outcome remain to be defined. We aimed in present study to explore PARP1 expression in breast cancers samples among west Algerian patients. Materials and Methods: We classified 78 primary breast cancers collected and diagnosed at the Regional Military Hospital of Oran (Algeria) using immunohistochemical biomarkers then; we examined immunohistochemical PARP1 expression and assessed its correlation with clinicopathological parameters and breast cancer molecular subtypes. Results: PARP1 nuclear overexpression was found in (44, 8%) of all infiltrating breast carcinomas. PARP-1 protein overexpression was associated to higher tumor grade (P=0.036), hormone-negative tumors (P=0.005) and estrogen-negative tumors (P=0.003). Moreover, PARP1 was significantly expressed in Triple-negative (P=0.015) and basal-like breast cancers, but was also present in HER2 positive breast cancers (P=0.014). Conclusions: Our data highlights the interest of immunohistochemical biomarkers in breast cancer molecular classification. We found that PARP1 overexpression is associated with triple-negative and basal-like tumors and could be a potential therapeutic in these two pejorative breast cancer subtypes. Furthermore, PARP1 overexpression could be a predictive biomarker of therapeutic resistance in HER2 positive tumors.The assessment of PARP1 protein expression may improve the selection of patients for PARP1 inhibitor therapy.

MicroRNA-124-3p inhibits the progression of congenital hypothyroidism via targeting programmed cell death protein 6.

The incidence of congenital hypothyroidism (CH) in newborn infants ranges from 1 in 3,000 to 1 in 4,000. Previous studies have indicated the neuroprotective role of microRNA (miR)-124-3p, however the expression and role of miR-124-3p in CH remain unclear. Therefore, the present study was performed to investigate the role and precise molecular mechanism of miR-124-3p in CH. Propylthiouracil (50 mg/day) was injected into the stomach of pregnant rats from gestational day 15 until parturition in order to establish a thyroid hypofunction model. Newborn rats were divided into the following four groups: The control group; the thyroid hypofunction group; the miR-124-3p mimic group; and the miR-124-3p negative control group. Reverse transcription-quantitative polymerase chain reaction indicated that miR-124-3p was significantly decreased in the hippocampus of the thyroid hypofunction group compared with the control group. Bioinformatics software was used to predict mRNA targets recognized by miR-124-3p and the programmed cell death protein 6 (PDCD6) 3' untranslated region (UTR) was demonstrated to exhibit the seed sequence of miR-124-3p. The interaction between miRNA-124-3p and PDCD6 was then verified using a dual-luciferase reporter assay system. PDCD6 expression was significantly increased in the hippocampus of rats with CH compared with the control group. Flow cytometry was performed to investigate the effects of miR-124-3p on neuronal cell apoptosis and the results indicated that the apoptosis rate in the thyroid hypofunction group was significantly increased compared with the control group; this increase was reversed by transfection with miR-124-3p mimics. Western blot analysis was used to detect the levels of cleaved poly [ADP-ribose] polymerase (PARP), full-length PARP, caspase-3, B cell lymphoma-2 (Bcl-2) and Bcl-2-associated X protein (Bax) proteins. The results indicated that the expression of cleaved PARP, caspase-3 and Bax protein were significantly increased and the expression of full-length PARP and Bcl-2 protein was significantly decreased compared with the control group. These effects were reversed by miRNA-124-3p mimic transfection. Taken together, the results of the present study demonstrate that miRNA-124-3p serves a protective role in CH via targeting PDCD6.

Anticancer effects of 10-hydroxycamptothecin induce apoptosis of human osteosarcoma through activating caspase-3, p53 and cytochrome c pathways.

In order to evaluate the anticancer effect of 10-hydroxycamptothecin (HCPT) in terms of inducing the apoptosis of human osteosarcoma cells, its apoptosis-inducing molecular mechanisms were investigated. In the present study, the anticancer effects of HCPT were revealed to result in suppressed cell viability, increased cytotoxicity, the induction of apoptosis and an augmented apoptotic nucleolus of human osteosarcoma cells. MG-63 cells were cultured with HCPT (0, 20, 40 and 80 nM) for 24 and 48 h. An MTT assay and a lactate dehydrogenase assay were used to analyze the anticancer effect of HCPT on cell viability and cytotoxicity in MG-63 cells. MG-63 cell apoptosis, and caspase-9 and caspase-3 activity levels were evaluated using flow cytometry and an ELISA. Western blot analysis was used to detect the protein expression levels of p53, poly (ADP-ribose) polymerase-1 (PARP-1), cytochrome c and B cell lymphoma-2 (Bcl-2) in MG-63 cells. The anticancer effects of HCPT were demonstrated to significantly activate the protein expression of p53, PARP-1 and cytochrome c, and suppress Bcl-2 protein expression and promote the activity of caspase-9 and caspase-3 in human osteosarcoma cells. In conclusion, the anticancer effects of HCPT appear to induce the apoptosis of human osteosarcoma cells through the activation of the caspase-3, p53 and cytochrome c pathways.

Poly (ADP-Ribose) Polymerase-1 (PARP-1) Induction by Cocaine Is Post-Transcriptionally Regulated by miR-125b.

Cocaine exposure alters gene expression in the brain via methylation and acetylation of histones along with methylation of DNA. Recently, poly (ADP-ribose) polymerase-1 (PARP-1) catalyzed PARylation has been reported as an important regulator of cocaine-mediated gene expression. In this study, we report that the cellular microRNA “miR-125b” plays a key role for cocaine-induced PARP-1 expression. Acute and chronic cocaine exposure resulted in the downregulation of miR-125b concurrent with upregulation of PARP-1 in dopaminergic neuronal cells and nucleus accumbens (NAc) of mice but not in the medial prefrontal cortex (PFC) or ventral tegmental area (VTA). In silico analysis predicted a binding site of miR-125b in a conserved 3’-untranslated region (3’UTR) of the PARP-1 mRNA. Knockdown and overexpression studies showed that miR-125b levels negatively correlate with PARP-1 protein expression. Luciferase reporter assay using a vector containing the 3’UTR of PARP-1 mRNA confirmed regulation of PARP-1 by miR-125b. Specific nucleotide mutations within the binding site abrogated miR-125b’s regulatory effect on PARP-1 3’UTR. Finally, we established that downregulation of miR-125b and concurrent upregulation of PARP-1 is dependent on binding of cocaine to the dopamine transporter (DAT). Collectively, these results identify miR-125b as a post-transcriptional regulator of PARP-1 expression and establish a novel mechanism underlying the molecular effects of cocaine action.

MKP-1 suppresses PARP-1 degradation to mediate cisplatin resistance.

Understanding the mechanisms of platinum compound resistance, including cisplatin resistance, has important implications for improving cancer treatments. Previous studies identified a potential role for mitogen-activated protein kinase phosphatase-1 (MKP-1) in cisplatin resistance. This work focuses on the regulation of poly(ADP-ribose) polymerase-1 (PARP-1) expression by MKP-1. We found that MKP-1 overexpression stimulates PARP-1 and poly(ADP-ribose) (PAR) protein expression and cisplatin resistance while its downregulation suppresses PARP-1 and PAR protein expression and cisplatin resistance. Silencing MKP-1 promoted PARP-1 ubiquitination, which decreased PARP-1 protein levels. We also found that silencing c-Jun N-terminal kinase 1/2 (JNK1/2) decreased PARP-1 ubiquitination while increasing total PARP-1 protein levels. Furthermore, we showed that acquired cisplatin resistant ovarian cancer cells expressed the high levels of MKP-1 and PARP-1 proteins, and that silencing MKP-1 or PARP-1 increased cisplatin sensitivity in resistant cells. Notably, the pharmacologic inhibition of PARP activity restored cisplatin sensitivity in MKP-1 overexpressing cells. Thus, this work indicates that suppression of JNK1/2 activity by MKP-1 maintains PARP-1 levels and suggests that MKP-1-mediated cisplatin resistance can be bypassed by PARP-1 inhibition.

Protective effect of diethylcarbamazine inhibits NF-κB activation in isoproterenol-induced acute myocardial infarction rat model through the PARP pathway

The present study investigated the protective effect of diethylcarbamazine in inhibiting nuclear factor (NF)-κB activation in isoproterenol‑induced acute myocardial infarction (AMI) rats through the poly ADP ribose polymerase (PARP) pathway. Male albino Wistar rats were injected subcutaneously with isoproterenol (100mg/kg/day) for 2days to induce an AMI model. Diethylcarbamazine (50mg/kg) was administered by gavage for 12days prior to the isoproterenol-induced AMI. It was noted that diethylcarbamazine significantly inhibited AMI‑induced casein kinase and lactate dehydrogenase levels, and reduced the AMI‑induced wet heart weight to body weight ratio in AMI rats. Diethylcarbamazine treatment significantly weakened reactive oxygen species production and reduced the levels of tumor necrosis factor (TNF)‑α, interleukin‑6 and NF‑κB/p65 in AMI rats. Western blotting demonstrated that diethylcarbamazine significantly suppressed the AMI‑induced inducible nitric oxide synthase (iNOS), transforming growth factor (TGF)‑β1, cyclooxygenase‑2 (COX‑2) and PARP protein expression in AMI rats. The results demonstrated that the protective effect of diethylcarbamazine inhibited isoproterenol‑induced AMI through the suppression of inflammation, iNOS, TGF‑β1, COX‑2 and the PARP pathway, and revealed the clinical potential of diethylcarbamazine for therapeutic and clinical applications.

PARP-1 Expression Quantified by [18F]FluorThanatrace: A Biomarker of Response to PARP Inhibition Adjuvant to Radiation Therapy.

Poly (ADP-ribose) polymerase 1 (PARP-1) is the major target of clinical PARP inhibitors and is a potential predictive biomarker for response to therapy. Due to the limited success of PARP inhibitors as monotherapy, investigators have shifted the clinical role of PARP inhibitors to the adjuvant setting. In this study, we evaluate the radiotracer [18F]FluorThanatrace ([18F]FTT) as a marker of PARP expression in vitro and the associated biological implications of PARP-1 expression in PARP inhibitor treatment adjuvant to radiation therapy. SNU-251 (BRCA1-mutant) and SKOV3 (BRCA1-WT) cell lines were evaluated in vitro by using the radiotracer [18F]FTT. Pharmacological binding assays were performed at baseline and were correlated with PARP-1 protein expression measured by Western blot protein analysis. Cell viability and clonogenic assays were used to characterize in vitro cytotoxicity for treatments, including: PARP inhibitors alone, radiation alone, and PARP inhibitor adjuvant to radiation. Western blot protein analysis was used to assess response to treatment by using γH2AX to measure DNA damage and PAR to measure the catalytic inhibition of PARP. [18F]FTT was capable of measuring PARP-1 protein expression in vitro and corresponded to Western blot protein analysis at baseline. The addition of a PARP inhibitor enhanced radiation effects in both cell lines; however, a greater synergy was observed in the SNU-251 cell line that expresses a BRCA1 mutation and homologous recombination deficiency. Western blot protein analysis showed that the addition of a PARP inhibitor adjuvant to radiation increases DNA damage in both cell lines and reduces PARP enzymatic activity as measured by PAR. In this work, we found that PARP-1 expression positively corresponds in vitro to the response of PARP inhibitors in combination with radiation therapy in ovarian cancer.