Expression Levels Of PARP Research Articles

Imatinib inhibits oral squamous cell carcinoma by suppressing the PI3K/AKT/mTOR signaling pathway.

Oral squamous cell carcinoma (OSCC) is a prevalent oral and maxillofacial cancer with high mortality as OSCC cells readily invade tissues and metastasize to cervical lymph nodes. Although imatinib exhibits potential anticancer and remarkable clinical activities that therapeutically affect several cancer types, its specific impact on OSCC has yet to be fully explored. Therefore, this study investigated the potential anticancer effect of imatinib on OSCC cells and the underlying mechanisms. The Cell Counting Kit-8 was used to determine the impact of imatinib on cell viability. Then, morphological cell proliferation analysis was conducted to examine how imatinib impacted OSCC cell growth. Moreover, OSCC cell migration was determined through wound-healing assays, and colony formation abilities were investigated through the soft agar assay. Lastly, the effect of imatinib on OSCC cell apoptosis was verified with flow cytometry, and its inhibitory mechanism was confirmed through Western blot. Our results demonstrate that imatinib effectively inhibited OSCC cell proliferation and significantly curtailed OSCC cell viability in a time- and concentration-dependent manner. Furthermore, imatinib suppressed migration and colony formation while promoting OSCC cell apoptosis by enhancing p53, Bax, and PARP expression levels and reducing Bcl-2 expression. Imatinib also inhibited the PI3K/AKT/mTOR signaling pathway and induced OSCC cell apoptosis, demonstrating the potential of imatinib as a treatment for oral cancer.

In vitro and in vivo killing effects of methionine enkephalin on osteosarcoma

ObjectiveThis study aimed to investigate the underlying regulatory effects of methionine enkephalin (MENK) on osteosarcoma. MethodsThe Cell Counting Kit-8 assay, clone formation, wound healing, transwell assay, and flow cytometry were performed to measure the effects of MENK on the proliferation, migration, invasion, and apoptosis of MG-63 and Saos-2 cells. Opiate growth factor receptor expression (OGFr) in cells was stably knocked down using siRNA. A tumor model was established by inoculating MG-63 cells into mice. Flow cytometry was performed to identify alterations in mice bone marrow, spleen, and tumor tissue immune cells. The phenotype of tumor-associated macrophages was determined using immunohistochemistry. After OGFr knockdown or/and treatment with MENK, Bax, Bcl-2, caspase 3, caspase 9, and PARP expression levels were characterized using qRT-PCR, western blot, and WES, respectively. ResultsMENK could significantly inhibit the proliferation, invasion, and migration of MG-63 and Saos-2, arrest the cell cycle in the G0/G1 phase, upregulate Bax, caspase 3, caspase 9, and PARP expression, and downregulate Bcl-2 expression. Tumor size and weight were lower in the MENK group than those in the control group. MENK-treated mice exhibited a reduced ratio of CD11b + Gr-1 + myeloid-derived suppressor cells. MENK increased the ratio of M1-type macrophages and decreased the proportion of M2-type macrophages in tumor tissue. Furthermore, the level of TNF-α significantly increased while that of IL-10 decreased in MENK-treated mice. The effect of MENK could be partly reversed by OGFr knockdown. ConclusionMENK reduces the abundance of myeloid-derived suppressor cells, induces M1 polarization of macrophages, and exhibits an inhibitory effect on osteosarcoma.

Automatic Segmentation of Magnetic Resonance Images of Severe Patients with Advanced Liver Cancer and the Molecular Mechanism of Emodin-Induced Apoptosis of HepG2 Cells under the Deep Learning.

To improve the accuracy of clinical diagnosis of severe patients with advanced liver cancer and enhance the effect of chemotherapy treatment, the U-Net model was optimized by introducing the batch normalization (BN) layer and the dropout layer, and the segmentation training and verification of the optimized model were realized by the magnetic resonance (MR) image data. Subsequently, HepG2 cells were taken as the research objects and treated with 0, 10, 20, 40, 60, 80, and 100 μmol/L emodin (EMO), respectively. The methyl thiazolyl tetrazolium (MTT) method was used to explore the changes in cell viability, the acridine orange (AO)/ethidium bromide (EB) and 4′,6-diamidino-2-phenylindole (DAPI) were used for staining, the Annexin V fluorescein isothiocyanate (FITC)/propidium iodide (PI) (Annexin V-FITC/PI) was adopted to detect the apoptosis after EMO treatment, and the Western blot (WB) method was used with the purpose of exploring the changes in protein expression levels of PARP, Bcl-2, and p53 in the cells after treatment. It was found that compared with the original U-Net model, the introduction of the BN layer and the dropout layer can improve the robustness of the U-Net model, and the optimized U-Net model had the highest dice similarity coefficient (DSC) (98.45%) and mean average precision (MAP) (0.88) for the liver tumor segmentation.

Brassica juncea polysaccharides induce apoptosis of colorectal cancer cells via mitochondrial- and caspasedependent apoptosis pathways

Purpose: To determine the effect of polysaccharides from Gleditsia on apoptosis of colorectal cancer cells, and the mechanism involved.Methods: Polysaccharides were extracted from Lycium barbarum, and their concentration was more than 85 %. Then, DLD-1 cells were cultured in medium with the polysaccharides at concentrations of 75 and 150 μg/mL. Cell proliferation was determined with MTT (3-(4,5)-dimethylthiahiazo (-z-y1)-3,5- diphenytetrazoliumromide) assay and colony formation assay, while apoptosis was determined with flow cytometry. Changes in MMP were measured flow cytometrically. The protein levels of PARP, Bcl-2, Bax, and caspases 3 and 9 were determined with Western blot assay.Results: Cell viability decreased time-dependently. Compared with control without polysaccharide exposure, cell viability, colony forming cells, % apoptosis, red: green fluorescence ratio, and bcl-2 expression were significantly and concentration-dependently decreased, while the expression levels of PARP, Bax, caspase-3 and caspase-8 were significantly increased (p < 0.05).Conclusion: These results indicate that the polysaccharides suppressed apoptosis of colorectal cancer cells by inhibiting the mitochondrial and caspase-dependent apoptosis pathways. Gleditsia polysaccharide may be used as an adjuvant therapy for colorectal cancer.

Peptide T8 isolated from yak milk residue ameliorates H2O2-induced oxidative stress through Nrf2 signaling pathway in HUVEC cells

This study was aimed to explore the anti-oxidative effect of active peptide (T8) isolated from yak milk residue on H 2 O 2 -induced damage in HUVEC cells. The results showed that peptide T8 increased superoxide dismutase (SOD) and glutathione reductase (GR) activities while decreased malondialdehyde (MDA) content and ROS of HUVEC cells in the H 2 O 2 -induced damage model. Flow cytometry assays showed that peptide T8 reduced H 2 O 2 -induced cell apoptosis. The protein expression levels of PARP and cleaved-Caspase 9 was increased by Western blot analysis in the H 2 O 2 -induced HUVEC cells. The ratio of Bax/Bcl-2 was increased, suggesting peptide T8 inhibits H 2 O 2 -induced in mitochondrial-dependant death. The protein expression levels of p-Nrf2 was increased in HUVEC cells and the expressions of Nrf2 target genes Keap1, Nrf2, HO-1 and NQO1 were modulated by peptide T8 treatments. Blocking Nrf2 activation by inhibitor ML385, the protective effect of peptide T8 on the H 2 O 2 -induced injury was partially reversed. Our results indicate that peptide T8 ameliorate H 2 O 2 -induced oxidative stress via Nrf2 pathway in HUVEC cells. • The strong antioxidative peptide T8 was isolated from yak milk residue. • Peptide T8 reduced H 2 O 2 -induced damage in HUVEC cells. • Peptide T8 regulated antioxidation-related gene expressions via Nrf2 pathway.

Bortezomib enhances the anti-cancer effect of the novel Bruton's tyrosine kinase inhibitor (BGB-3111) in mantle cell lymphoma expressing BTK.

BGB-3111, a novel Bruton’s tyrosine kinase (BTK) inhibitor, shows promising anti-cancer effects in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), mantle cell lymphoma (MCL), and Waldenstrom macroglobulinemia (WM). This study aimed to investigate the anti-cancer effects of BGB-3111 combined with bortezomib (BTZ) against the BTK-expressing MCL. We found that BTK, which was overexpressed in 59.4% of patients with MCL, was mainly characterized by high Ki67 and elevated MIPI scores. BGB-3111 strongly inhibited cell proliferation, induced cell cycle arrest in the G1/G0-phase, and promoted cell apoptosis in the MCL cells expressing BTK. BGB-3111 provides better safety than another BTK inhibitor, ibrutinib as ibrutinib inhibits the inducible T-cell kinase (ITK) as an off-target effect but BGB-3111 does not inhibit ITK. Low doses of BTZ enhanced the anti-cancer effect induced by the low dose of BGB-3111 by downregulating the expression levels of PARP and Bcl-2 and increasing the expression levels of cleaved PARP and cleaved caspase-9. In addition, low doses of BGB-3111, but not of BTZ, inhibited BTK phosphorylation. However, low-doses of BTZ strengthened the anti-cancer effect induced by the low-doses of BGB-3111 via synergistically suppressing the IκBα and P65 phosphorylation. Taken together, our findings validate that BGB-3111 is a novel and effective BTK inhibitor for MCL-expressing BTK. Hence, it can be harnessed as a potential therapeutic strategy through a combinatorial treatment comprising low-dose BGB-3111 and low-dose BTZ to gain strong anti-cancer effects and better safety for MCL patients.

PARP1 Is Overexpressed in Hematological Malignant Cell Lines: A Framework for Experimental Oncology.

Experimental oncology commonly uses cells as oncological models, providing a framework for the testing of drugs, and investigation of cytotoxicity, mutagenesis and carcinogenesis. Investigations into poly-ADP-ribose polymerase 1 (PARP1) inhibition have become ever more relevant due to its approval as a therapeutic option for tumors with BRCA1/2 DNA repair-associated mutation and the seemingly high PARP expression levels in some tumor subtypes. In this study, we aimed to determine PARP1 gene expression of different hematological cancer-derived cell lineages and compare them to that of normal cell lines. PARP1 gene expression in seven different neoplastic lineages, representing three different hematological disorders (chronic myeloid leukemia, Burkitt lymphoma and acute lymphoblastic leukemia), was quantified by quantitative real-time polymerase chain reaction. All hematological malignant lineages in this study overexpressed PARP1 when compared to the normal cell line MRC-5, with Burkitt's lymphoma cells having the highest expression values (fold change: 93). Overexpression of PARP1 in hematological malignant lineages is a finding of crucial importance to future studies exploring possible cellular oncogenic pathways and supports investigations into the effectiveness of PARP1 inhibitors against hematological disorders.

The Ibr-7 derivative of ibrutinib radiosensitizes pancreatic cancer cells by downregulating p-EGFR

BackgroundRadiotherapy is one of the main treatments for pancreatic cancer, but radiation resistance limits its clinical application. As a result, novel therapeutic agents to improve radiosensitivity are urgently needed. This study aimed to investigate the effect of Ibr-7 (a derivative of ibrutinib) on the radiosensitivity of human pancreatic cancer cells.MethodsThe effect of Ibr-7 on pancreatic cancer cell proliferation was detected by CCK-8 assays. Radiosensitivity was assessed by clonogenic formation assays. Cell cycle and cell apoptosis were analysed by flow cytometry. DNA damage was evaluated by immunofluorescence analysis. The expression levels of PARP, Cleaved caspase 3, p-EGFR and EGFR were determined by western blot.ResultsIbr-7 showed an anti-proliferative effect on PANC-1 and Capan2 cells in a dose- and time-dependent manner. Ibr-7 (2 μmol/L) enhanced the effect of radiation on PANC-1 and Capan2 cells. Further findings showed that this combination enhanced G2/M phase arrest and increased cell apoptosis. Additional molecular mechanism studies revealed that the expression of p-EGFR was decreased by Ibr-7 alone or in combination with radiation. Overexpression of p-EGFR reversed the cell apoptosis induced by Ibr-7 combined with radiation. Moreover, the expression of γ-H2AX was significantly decreased in the Ibr-7 plus radiation group.ConclusionsOur study indicated the potential application of Ibr-7 as a highly effective radiosensitizer for the treatment of pancreatic cancer cells.

Paris Saponin VII Induces Apoptosis and Cell Cycle Arrest in Erythroleukemia Cells by a Mitochondrial Membrane Signaling Pathway.

Leukemia is considered a top-listed ailment, according to WHO, which contributes to the death of a major population of the world every year. Paris Saponin VII (PS), a saponin which was isolated from the roots of Trillium kamtschaticum, from our group, was reported to provide hemostatic, cytotoxic and antimicrobial activities. However, its molecular mechanism underlying the anti-proliferative effects remains unclear. Thus, this study hypothesized to assess that mechanism in PS treated HEL cells. The MTT assay was used to analyze the PS inhibited cell viability in the HEL cells. We further found that PS could induce S phase cell cycle arrest through flow cytometry as well as the western blot analysis of intrinsic and extrinsic apoptotic molecules. The MTT assay showed the IC50 concentration of PS as 0.667μM. The study revealed that PS treatment inhibits cell proliferation dose-dependently. It further caused mitochondrial membrane potential changes by PS treatment. Mechanistic protein expression revealed a dose-dependent upsurge for Bid and Bim molecules, while Bcl2 and PARP expression levels were significantly (P<0.05) down-regulated in PS treated HEL cells resulting in caspase -3 release and increased the Bim levels upon 24h of incubation. These findings indicate that PS possesses an excellent anti-leukemic activity via the regulation of the mitochondrial pathway, leading to S phase cell cycle arrest and caspase-dependent apoptosis, suggesting it as a potential alternative chemotherapeutic agent for leukemia patients.

Aloin induces apoptosis via regulating the activation of MAPKs signaling pathway in human gastric cancer cells in vitro

To investigate the effect of aloin on apoptosis of human gastric cancer cells and explore the molecular mechanism. Gastric cancer MKN-28 and HGC-27 cells were cultured routinely in 1640 medium supplemented with 10% fetal bovine serum and 10% non-essential amino acids (for HGC-27 cells) and treated with different concentrations of aloin for different durations. The cell viability, cell nuclear morphology, and apoptotic rate of the cells were detected using CCK-8 assay, DAPI staining and AnnexinV-FITC/PI, respectively; Western blotting was used to detect the expression levels of PARP, procaspase 3 and the phosphorylation of p38, ERK and JNK. The cells were treated with specific inhibitors of p38, ERK and JNK, and the inhibitory effects on these pathways were detected with Western blotting; DAPI staining was used to detect the effects of inhibitors on apoptosis of gastric cancer cells. Aloin dose-dependently inhibited the viability and induced apoptosis of HGC-27 and MKN-28 cells. Alion treatment obvious enhanced the phosphorylation of p38 and JNK but decreased ERK phosphorylation in the cells. Blocking ERK activation with the ERK inhibitor obviously enhanced aloin-induced cell apoptosis, where inhibiting p38 and JNK activation partly reversed alion-induced apoptosis in the cells. Aloin induces apoptosis of human gastric cancer cells in vitro by activating p38 and JNK signaling pathways and inhibiting ERK signaling pathway.

Pyramidatine (Z88) Sensitizes Vincristine-Resistant Human Oral Cancer (KB/VCR) Cells to Chemotherapeutic Agents by Inhibition of P-glycoprotein.

Multi-drug resistance (MDR) remains a major impediment in cancer therapy. A major goal for scientists is to discover more effective compounds that are able to circumvent MDR and simultaneously have minimal adverse side effects. In the present study, we aim to determine the anti-MDR effects of pyramidatine (Z88), a cinnamic acid-derived bisamide compound isolated from the leaves of Aglaia perviridis, on KB/VCR (vincristineresistant human oral cancer cells) and MCF-7/ADR (adriamycin-resistant human breast adenocarcinoma) cells. Cell viability and average resistant fold (RF) of Z88 were examined by Cell Counting Kit-8 (CCK-8) assay. Flow cytometry, western blot, RT-PCR, Rhodamine 123 accumulation assay and P-glycoprotein (P-gp) ATPase assay were used to demonstrate the anti-MDR activity and mechanism of Z88. The average RF of Z88 is 0.09 and 0.51 in KB/VCR and MCF-7/ADR cells. A CCK-8 assay showed that Z88 could enhance the cytotoxicity of VCR toward KB/VCR cells. A FACS analysis revealed that Z88 could enhance the VCR-induced apoptosis as well as G2/M arrest in a dose-dependent manner in KB/VCR cells. Western blot results showed that the expression levels of PARP, Bax, and cyclin B1 all increased after treatment with 0.2 µmol/L (µM) of VCR combined with 10 µM of Z88 for 24 h in KB/VCR cells. Z88 also could enhance the accumulation of rhodamine 123. Further studies showed that Z88 could inhibit the verapamil stimulated Pgp ATPase activity. Additionally, qPCR detection and western blot assays revealed that Z88 could decrease the expression of P-gp at both RNA and protein level. Z88 exerted potent anti-MDR activity in vitro and its mechanisms are associated with dualinhibition of the function and expression of P-gp. These findings encourage efforts to develop more effective reversal agents to circumvent MDR based on Z88.

Effects of disodium cantharidinate on dendritic cells of patients with bladder carcinoma

The present study explored the effects of disodium cantharidinate (DC) on the peripheral blood-derived dendritic cells of patients with bladder carcinoma. The peripheral blood mononuclear cells from the 15 cases of urinary bladder carcinoma of middle and advanced stage were separated, and dendritic cells were prepared. The morphological changes of dendritic cells were observed. Flow cytometry was used to detect the expression levels of CD1a and CD83 on dendritic cell surface. MTT assay was utilized to measure the proliferation ability of allogeneic lymphocyte stimulated by DC. Annexin V-FITC/propidium iodide (PI) double staining flow cytometry method was carried out to detect cell apoptosis after treatment with DC. The changes in caspase-3 and PARP expression levels were investigated by western blot method. The high-dose DC resulted in a significant increase in the expressions of dendritic cell phenotyptic molecules CDla and CD83 as compared to control group. In addition, the proliferation index of allogenic lymphocyte stimulated by DC was significantly higher than that of control group. Moreover, MTT assay showed significant inhibition of the growth of BIU-87 cells. After 24 h of DC treatment, double staining flow cytometry confirmed the ability of DC to induce cell apoptosis. Further, western blot method showed a significant elevation of caspase-3 and PARP protein expression after DC treatment. In conclusion, DC treatment could induce dendritic cell maturation of patient with carcinoma of urinary bladder and promote its functional changes. Furthermore, DC was able to inhibit the proliferation of cell BIU-87 and also has the ability to induce cell apoptosis.

Neocarzinostatin, Aptamer Conjugates for Targeting EpCAM-positive Tumor Cells.

The aim of this study was to investigate the role of Neocarzinostatin (NCS) conjugated with epithelial cell adhesion molecule (EpCAM) aptamer in EpCAM-positive cancer cells. NCS is an antitumor antibiotic protein chromophore that has the ability to cleave double stranded DNA and can be used as a potential drug for the treatment of EpCAM-positive cancers. EpCAM aptamer is an oligonucleotide ligand that binds specifically to EpCAM, a protein overexpressed in tumor cells. NCS was conjugated with EpCAM aptamer using Sulfo-Succinimidyl 6-(3-(2-pyridyldithio) - propionamide hexanoate) LC-(SPDP) cross-linker to deliver it to EpCAM-positive tumor cells. The conjugates were characterized using polyacrylamide gel electrophoresis (PAGE) and high-performance liquid chromatography (HPLC). Flow cytometry was used to study the binding efficiency of the aptamer and the conjugates in cancer cells. The effect of the conjugate on cancer cells was studied using propidium iodide (PI) to analyze the cell cycle phase changes. The apoptosis assay was performed using the IC50 concentration of NCS. Microarrays were performed to study the gene level changes in cancer cells upon treatment with NCS and the conjugate. Flow cytometry revealed significant binding of aptamer and conjugate in the MCF-7 and WERI-Rb1 cell lines. Briefly, 62% in MCF and 30% in WERI-Rb1 cells with conjugate treated cells (p<0.005). The cell-cycle analysis indicated G2 phase arrest in MCF-7 cells and S phase arrest in WERI-Rb1 cells (p<0.005). Microarray analysis showed differentially expressed genes involved in cell cycle, DNA damage, and apoptosis. The BrDU assay and the apoptosis assay showed that the expression of BrDU was reduced in conjugate-treated cells and the PARP levels were increased confirming the double stranded DNA breaks (p<0.005). In MCF-7 and WERI-Rb1 cells, most of the cells underwent necrosis (p<0.005). The EpCAM aptamer conjugated NCS showed specificity to EpCAM-positive cells. The effect of the conjugates on cancer cells were impressive as the conjugate arrested the cell cycle and promoted apoptosis and necrosis. The high levels of PARP expression confirmed the DNA breaks upon conjugate treatment. Our study demonstrates that the NCS conjugated with EpCAM can be targeted to cancer cells sparing normal cells.

Anticancer bioactive peptides suppress human colorectal tumor cell growth and induce apoptosis via modulating the PARP-p53-Mcl-1 signaling pathway.

We have reported novel anticancer bioactive peptides (ACBPs) that show tumor-suppressive activities in human gastric cancer, leukemia, nasopharyngeal cancer, and gallbladder cancer. In this study, we investigated the effects of ACBPs on human colorectal cancer and the underlying mechanisms. Cell growth and apoptosis of human colorectal tumor cell line HCT116 were measured using cell proliferation assay and flow cytometry, respectively. The expression levels of PARP, p53 and Mcl1A were assessed with Western blotting and immunohistochemistry. For evaluation of the in vivo antitumor activity of ACBPs, HCT116 xenograft nude mice were treated with ACBPs (35 μg/mL, ip) for 10 days. Treatment of HCT116 cells with ACBPs (35 μg/mL) for 4-6 days significantly inhibited the cell growth. Furthermore, treatment of HCT116 cells with ACBPs (35 μg/mL) for 6-12 h significantly enhanced UV-induced apoptosis, increased the expression of PARP and p53, and decreased the expression of Mcl-1. Administration of ACBPs did not change the body weight of HCT116 xenograft nude mice, but decreased the tumor growth by approximately 43%, and increased the expression of PARP and p53, and decreased the expression of Mcl-1 in xenograft mouse tumor tissues. Administration of ACBPs inhibits human colorectal tumor cell growth and induces apoptosis in vitro and in vivo through modulating the PARP-p53-Mcl-1 signaling pathway.

The effects of PEP-1-FK506BP on dry eye disease in a rat model.

As FK506 binding proteins (FK506BPs) are known to play an important role in the regulation of a variety of biological processes related to cell survival, this study was designed to examined the protective effects of FK506 binding protein 12 (FK506BP) on low humidity air flow induced dry eye in a rat model using transduced PEP-1-FK506BP. After the topical application of PEP-1-FK506BP, tear volumes were markedly increased and significant prevention of cornea damage was observed compared with dry eye rats. Further, immunohistochemical analysis demonstrated that PEP-1-FK506BP markedly prevented damage to the cornea, the bulbar conjunctiva, and the palpebral conjunctiva epithelial lining compared with dry eye rats. In addition, caspase-3 and PARP expression levels were found to be decreased. These results demonstrated that topical application of PEP-1-FK506BP significantly ameliorates dry eye injury in an animal model. Thus, we suggest that PEP-1-FK506BP can be developed as a new ophthalmic drop to treat dry eye diseases. [BMB Reports 2015; 48(3): 153-158]

Effect of the regimen of Gaoshan Hongjingtian on the mechanism of poly(ADP-ribose) polymerase regulation of nuclear factor kappa B in the experimental diabetic retinopathy

Background Poly(ADP-ribose) polymerase (PARP) plays an important role in the death of retinal capillary cells in diabetic retinopathy (DR) partly via its regulation of nuclear factor kappa B (NF-κB). The current study investigated the effect of the regimen of Gaoshan Hongjingtian (RG) on the mechanism of PARP regulation of NF-κB, and demonstrated the possible impact of the RG and Gaoshan Hongjingtian (Rhodiola sachalinensis, RS) on diabetic retinopathy. Methods Wistar rats were made diabetic by administering streptozotocin. They were then assigned to three groups at random. After 2 months, the three groups of these diabetic rats were treated with RS or RG, or untreated. Analyses of expression levels of PARP, NF-κB, and intercellular adhesion molecule-1 (ICAM-1) in the retinas of rats in different groups were performed by Western blotting and immunohistochemical assays, and mRNA levels of NF-κB and ICAM-1 were determined by real-time polymerase chain reaction (PCR). In addition, the basement membranes of capillaries in the rats' retinas were observed using electron microscopy, and diabetes-induced capillary degeneration (ghost pericytes and acellular capillaries) were quantitated. Results From the third month after the injection of streptozotocin, the diabetic rats were given daily RG, RS or tap water separately. The diabetic rats failed to gain weight compared with normal age-matched rats, whereas their glycated hemoglobin levels were significantly increased. After 5 months, the mRNA levels of NF-κB and ICAM-1 and the protein expression of PARP, NF-κB, and ICAM-1 were significantly increased in the retinas of diabetic rats in the untreated group compared with the nondiabetic controls. After 8 months, the number of degenerated retinal capillaries (ghost pericytes and acellular capillaries) was significantly increased in the diabetic rats in the untreated group compared with normal age-matched rats. RG and RS inhibited diabetes-induced over-expression of PARP, NF-κB, and ICAM-1 in the retinas of diabetic rats at the end of 5-month diabetic duration. Treatment using RG and RS significantly inhibited increases in the number of acellular capillaries and pericyte ghosts and suppressed the basement membrane thickening in the retinas of rats with diabetes for 8 months compared with the control diabetic rats. Conclusions These results indicate that PARP plays an important role in the pathogenesis of diabetic retinopathy. RS and RG may have acted on the mechanism of PARP regulation of NF-κB, which suppressed the expression of NF-κB and ICAM-1, and led to the inhibition of retinal capillary degeneration.

Atorvastatin delays progression of pancreatic lesions to carcinoma by regulating PI3/AKT signaling in p48Cre/+ LSL-KrasG12D/+ mice.

Pancreatic cancer is the one of most common causes of cancer deaths and has the worst prognosis. Clinical observational studies suggest that statins may reduce the risk of pancreatic cancer. The chemopreventive efficacy of the statin atorvastatin (Lipitor(®)) and the role of the phosphatidyl-inositol 3-kinase (PI3/AKT) signaling pathway were evaluated for the progression of pancreatic intraepithelial neoplasms (PanINs) to pancreatic ductal adenocarcinoma (PDAC) in conditional p48(Cre/+) -LSL-Kras(G12D/+) transgenic mice. Six-week-old male p48(Cre/+) -LSL-Kras(G12D/+) (20/group) mice were fed AIN-76A diets containing 0, 200 and 400 ppm atorvastatin for 35 weeks. At termination, pancreata were evaluated histopathologically for PanINs and PDAC, and for various PI3/AKT signaling markers, and inflammatory cytokines, by immunohistochemistry/immunohistoflourscence, ELISA, Western blotting and/or reverse transcription-PCR methods. Control diet-fed mice showed 85% incidence of PDAC; whereas, mice fed with atorvastatin showed PDAC incidence of 65 and 35%, respectively (p < 0.0001). Similarly, significant suppression of PanIN-3 (22.6%) was observed in mice fed 400 ppm atorvastatin. Importantly, pancreata from atorvastatin-treated mice were ∼68% free from ductal lesions. Furthermore, pancreas of mice administered with atorvastatin had significantly reduced expressions levels of PCNA, p2X7, p-ERK, RhoA, cyclin D1, survivin, Akt, pAKT, β-catenin, cyclin E, cdK2 and caveolin-1. Also, atorvastatin-treated mice had shown dose-dependent suppression of inflammatory cytokines and a significant increase in tunnel-positive cells, p21 and PARP expression levels in pancreas. Atorvastatin significantly delays the progression of PanIN-1 and -2 lesions to PanIN-3 and PDAC by modulating PI3/AKT signal molecules in a preclinical model, suggesting potential clinical benefits of statins for high-risk pancreatic cancer patients.

Abstract 2484: Aurora B silencing attenuates radiation resistance through PARP and identification of a missense mutation in aurora B as a determinant of radiosensitization in gliomas

Abstract Purpose: Aurora B (Aur B), a mitotic protein located on chromosome 17p13.1, is critical for proper chromosome segregation and changes in chromosome 17p are quite common in glioblastoma multiforme (GBM). Upregulation of Aur B induces multicellularity and fosters polyploidy. Aur B reaches maximum levels later in mitosis and regulates the chromosomal passenger complex progression. It has been reported that Aur B expression markedly correlates with short survival of GBM patients and may be a prognostic factor. Targeting Aur B is an ideal strategy, since it is active during mitosis and non-proliferating cells would not be adversely affected. Materials and Methods: The cell lines used for this study include both established and primary GBM cell lines. Radiation experiments were conducted using gammacell irradiator. DNA damage was estimated using comet assay. Apoptosis, cell cycle and multinucleated polyploid cells were estimated using flow cytometry. Gene silencing and overexpression were carried out using established protocols. Results and Discussion: Aur B expression increases after radiation in GBM cells and not in normal human astrocytes as determined both at transcriptional and translational levels. The radiosensitivity of cells lines was estimated by clonogenic survival assay. Through sequencing Aur B, we discovered mutations at the 885(C) and 893(T) residues in the GBM cell lines. The 885(C) was a silent mutation and 893(T) leads to an amino acid change from methionine (M) to threonine (T). This missense mutation leads to a new phospho site. The GBM cell lines tested had heterozygous and homozygous deletions. The radiosensitivity assay conducted on these cell lines shows a differential response with regards to the nature of mutation. The cell lines which had homozygous deletions on both sites 885(C) and 893(T) exhibit an increased radioresistance under in vitro conditions when compared to one of them being a heterozygote. Further, we show that Aur B silencing inhibits cytokinesis leading to 4N or 8N DNA content in p53 functional cell lines. The endoreduplicated cells due to Aur-B inhibition leads to the loss of viability in p53 mutant/null cell lines. Overexpression of Aur B increases the DNA repair enzyme PARP and scaffolding protein XRCC1 after radiation, which are critical for the activation and recruitment of Base Excision Repair. Whereas, silencing Aur B decreases the PARP expression levels. Conclusion: In GBM cell lines, we report that Aur B knockdown sensitizes cells to radiation and over expression of Aur B leads to radioresistance. Thus far, one of the prime targets in mediating this effect was found to be PARP, since silencing and forced induction of Aur B correlates with expression levels of PARP. For the first time, we have shown that the mutational status of Aur B alters the radiation sensitivity. Further in vivo studies are underway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2484. doi:10.1158/1538-7445.AM2011-2484

Poly(ADP-Ribose) Polymerase Inhibition Down-Regulates Expression of Metastasis-Related Genes in CT26 Colon Carcinoma Cells

Objectives: The current study was designed to test the hypothesis that inhibition of poly(ADP-ribose) polymerase in colorectal cancer mediates down-regulation of metastasis-related gene expression through the regulation of nuclear factor-κB (NF-κB) activity. Methods: Mouse colon carcinoma cells (CT26) were treated with and without the PARP inhibitor 5-aminoisoquinolin-1(2H)-one hydrochloride (5-AIQ). We investigated adhesion, migration and invasion of differently treated CT26 cells. In addition, the expression levels of PARP, NF-κB, integrin β₁, MMP-9 and MMP-2 as well as the activities of NF-κB, MMP-9 and MMP-2 were determined by Western blot, electrophoretic mobility gel shift assay and zymography, respectively. Results: Inhibition ofPARP attenuated the adhesion of CT26 cells to the extracellular matrix and their migration and invasion through Matrigel. In addition, the results of Western blot showed that the expression levels of PARP, NF-κB, integrin β₁, MMP-9 and MMP-2 were reduced in 5-AIQ-treated CT26 cells; the activities of NF-κB, MMP-9 and MMP-2 were also suppressed. Conclusions: Inhibition of PARP down-regulates the expression of metastasis-related genes in mouse colon carcinoma cells. This could be, at least in part, through the regulation of NF-κB activity, but the precise mechanisms of action remain to be elucidated.

44. Combination of Oncolytic Measles Virus Strains and Radiation Therapy Has Synergistic Activity in the Treatment of Glioblastoma Multiforme

Background: Glioblastoma Multiforme (GBM), the most frequent primary brain tumor in adults, is one of the most lethal malignancies with a median survival of 12-16 months. We have previously shown that oncolytic measles virus derivatives expressing soluble human carcinoembryonic antigen (MV-CEA) have significant antitumor activity against GBM cell lines and xenografts. Here we tested the hypothesis that the combination of RT with MV-CEA would have synergistic activity against gliomas.Methods: MTS (Promega) and clonogenic assays were performed in U87 and U251glioma cell lines (ATCC) to assess the impact of the different treatment modalities on cell proliferation and survival. One-step viral growth curves, Western blots to assess expression of viral proteins, TUNEL assays, immunostaining for Fas/caspase-8 and western immunobloting for caspase-8, caspase-9, caspase-3 and PARP were employed to investigate the mechanism of the synergistic interaction between MV-CEA and RT in GBM cell lines.Results: MTS and clonogenic assays revealed a synergistic interaction between MV-CEA and RT in GBM cells. The optimal combination index for the RT/MV-CEA combination was 0.524 for U87 (0.01MOI/10GY) and 0.529 for U251 (0.05MOI/7.5GY), indicating strong synergy. Morphologic changes of U251 and U87 cells in response to the combination treatment demonstrated significant augmentation of cytopathic effect. In clonogenic assays, colony formation was significantly decreased in the MV-CEA/RT group as compared to either MV-CEA or RT alone. One-step viral growth curves demonstrated increased MV-CEA propagation by at least 2 logs in MV-CEA/RT combination treated cells, as compared to glioma cells treated with single agent MV-CEA. Changes in CEA levels and expression of viral N protein were also consistent with viral titer changes. TUNEL staining showed increased positivity in RT/MV-CEA group as compared to MV-CEA or RT alone. Furthermore, Western blot analysis demonstrated an increase in activated caspase-8, caspase-3 and PARP expression levels after combination treatment. The pan-caspase inhibitor Z-VAD-FMK completely protected cells from MV/RT induced cleavage of PARP, while the caspase-8 inhibitor (Z-IETD-FMK) partially protected glioma cells from MV-CEA/RT induced cleavage of PARP, and the caspase-9 inhibitor Z-IEHD-FMK showed no protection.Conclusions: Our results indicate that there is significant synergy between MV-CEA and RT in vitro. The synergistic effect of the combination appears to be due to increased viral bust size and increase in apoptotic death. This latter effect is mostly mediated via the extrinsic caspase 8 pathway. Experiments in primary glioma lines and animal experiments are ongoing. These results could have immediate translational implications in glioma therapy. Background: Glioblastoma Multiforme (GBM), the most frequent primary brain tumor in adults, is one of the most lethal malignancies with a median survival of 12-16 months. We have previously shown that oncolytic measles virus derivatives expressing soluble human carcinoembryonic antigen (MV-CEA) have significant antitumor activity against GBM cell lines and xenografts. Here we tested the hypothesis that the combination of RT with MV-CEA would have synergistic activity against gliomas. Methods: MTS (Promega) and clonogenic assays were performed in U87 and U251glioma cell lines (ATCC) to assess the impact of the different treatment modalities on cell proliferation and survival. One-step viral growth curves, Western blots to assess expression of viral proteins, TUNEL assays, immunostaining for Fas/caspase-8 and western immunobloting for caspase-8, caspase-9, caspase-3 and PARP were employed to investigate the mechanism of the synergistic interaction between MV-CEA and RT in GBM cell lines. Results: MTS and clonogenic assays revealed a synergistic interaction between MV-CEA and RT in GBM cells. The optimal combination index for the RT/MV-CEA combination was 0.524 for U87 (0.01MOI/10GY) and 0.529 for U251 (0.05MOI/7.5GY), indicating strong synergy. Morphologic changes of U251 and U87 cells in response to the combination treatment demonstrated significant augmentation of cytopathic effect. In clonogenic assays, colony formation was significantly decreased in the MV-CEA/RT group as compared to either MV-CEA or RT alone. One-step viral growth curves demonstrated increased MV-CEA propagation by at least 2 logs in MV-CEA/RT combination treated cells, as compared to glioma cells treated with single agent MV-CEA. Changes in CEA levels and expression of viral N protein were also consistent with viral titer changes. TUNEL staining showed increased positivity in RT/MV-CEA group as compared to MV-CEA or RT alone. Furthermore, Western blot analysis demonstrated an increase in activated caspase-8, caspase-3 and PARP expression levels after combination treatment. The pan-caspase inhibitor Z-VAD-FMK completely protected cells from MV/RT induced cleavage of PARP, while the caspase-8 inhibitor (Z-IETD-FMK) partially protected glioma cells from MV-CEA/RT induced cleavage of PARP, and the caspase-9 inhibitor Z-IEHD-FMK showed no protection. Conclusions: Our results indicate that there is significant synergy between MV-CEA and RT in vitro. The synergistic effect of the combination appears to be due to increased viral bust size and increase in apoptotic death. This latter effect is mostly mediated via the extrinsic caspase 8 pathway. Experiments in primary glioma lines and animal experiments are ongoing. These results could have immediate translational implications in glioma therapy.