Radiosensitivity Of Pancreatic Cancer Cells Research Articles

Hyperthermia Enhances the Radiosensitivity of Pancreatic Cancer Cells by Inhibiting Wnt2B Signaling

Pancreatic cancer (PC) is a highly lethal human malignance. Due to unobvious symptoms at early stage, most of the patients with PC are diagnosed at late stages and lose the chance of surgical resection. Furthermore, PC patients are resistant to chemoradiotherapy and therefore show a dismal survival. Hyperthermia is commonly used as a sensitizer of chemotherapy or radiotherapy for the clinical treatment of human cancers. Our study aimed to investigate whether hyperthermia can improve the radiosensitivity of PC cells and uncover the involved mechanisms. PC cells BxPC3, CFPAC-1 and PANC1 were heated to 43 ℃ 1 h before exposure to ionizing irradiation (IR). The radiosensitivity of PC cells were detected in vitro by colony formation assay, immunofluence analysis and western blotting. The mechanisms studies have been conducted using qRT-PCR analysis, cDNA/siRNA transfection and comet assay. Hyperthermia significantly enhanced the radiosensitivity of PC cells by decreasing their colony formation and increasing DNA damage following IR. By qRT-PCR analysis of Wnt genes expressions, we found Wnt2B was significantly down-regulated in PC-3 cells which were treated with the combination of hyperthermia and IR compared with hyperthermia or IR alone. Functional assays showed that the expression level of Wnt2B was inversely associated with the radiosensitivity of PC-3 cells. Furthermore, we found hyperthermia inhibited the expression of DNA repair proteins such as p-BRCA1 and p-MRE11 in PC cells following IR CONCLUSION: Hyperthermia can significantly enhance the radiosensitivity of PC cells in a Wnt2B signaling-dependent manner.

Hydrogen Peroxide Mediates Pharmacological Ascorbate Induced Radio-Sensitization of Pancreatic Cancer Cells by Enhancing G2-accumulation and Reducing Cyclin B1 Protein Levels.

Pharmacological ascorbate (P-AscH-, high dose, intravenous vitamin C) preferentially sensitizes human pancreas ductal adenocarcinoma (PDAC) cells to radiation-induced toxicity compared to non-tumorigenic epithelial cells. Radiation-induced G2-checkpoint activation contributes to the resistance of cancer cells to DNA damage induced toxicity. We hypothesized that P-AscH- induced radio-sensitization of PDAC cells is mediated by perturbations in the radiation induced activation of the G2-checkpoint pathway. Both non-tumorigenic pancreatic ductal epithelial and PDAC cells display decreased clonogenic survival and increased doubling times after radiation treatment. In contrast, the addition of P-AscH- to radiation increases clonogenic survival and decreases the doubling time of non-tumorigenic epithelial cells but decreasing clonogenic survival and increasing the doubling time of PDAC cells. Results from the mitotic index and propidium iodide assays showed that while the P-AscH- treatments did not affect radiation-induced G2-checkpoint activation, it enhanced G2-accumulation. The addition of catalase reverses the increases in G2-accumulation, indicating a peroxide-mediated mechanism. In addition, P-AscH- treatment of PDAC cells suppresses radiation-induced accumulation of cyclin B1 protein levels. Both translational and post-translational pathways appear to regulate cyclin B1 protein levels after the combination treatment of PDAC cells with P-AscH- and radiation. The protein changes seen are reversed by the addition of catalase suggesting that hydrogen peroxide mediates P-AscH- induced radiation sensitization of PDAC cells by enhancing G2-accumulation and reducing cyclin B1 protein levels.

Disulfiram Alone Functions as a Radiosensitizer for Pancreatic Cancer Both In Vitro and In Vivo.

The prognosis of pancreatic cancer remains very poor worldwide, partly due to the lack of specificity of early symptoms and innate resistance to chemo-/radiotherapy. Disulfiram (DSF), an anti-alcoholism drug widely used in the clinic, has been known for decades for its antitumor effects when simultaneously applied with copper ions, including pancreatic cancer. However, controversy still exists in the context of the antitumor effects of DSF alone in pancreatic cancer and related mechanisms, especially in its potential roles as a sensitizer for cancer radiotherapy. In the present study, we focused on whether and how DSF could facilitate ionizing radiation (IR) to eliminate pancreatic cancer. DSF alone significantly suppressed the survival of pancreatic cancer cells after exposure to IR, both in vitro and in vivo. Additionally, DSF treatment alone caused DNA double-strand breaks (DSBs) and further enhanced IR-induced DSBs in pancreatic cancer cells. In addition, DSF alone boosted IR-induced cell cycle G2/M phase arrest and apoptosis in pancreatic cancer exposed to IR. RNA sequencing and bioinformatics analysis results suggested that DSF could trigger cell adhesion molecule (CAM) signaling, which might be involved in its function in regulating the radiosensitivity of pancreatic cancer cells. In conclusion, we suggest that DSF alone may function as a radiosensitizer for pancreatic cancer, probably by regulating IR-induced DNA damage, cell cycle arrest and apoptosis, at least partially through the CAM signaling pathway.

Catalase Modulates the Radio-Sensitization of Pancreatic Cancer Cells by Pharmacological Ascorbate.

Pancreatic cancer cells (PDACs) are more susceptible to an oxidative insult than normal cells, resulting in greater cytotoxicity upon exposure to agents that increase pro-oxidant levels. Pharmacological ascorbate (P-AscH−), i.e., large amounts given intravenously (IV), generates significant fluxes of hydrogen peroxide (H2O2), resulting in the killing of PDACs but not normal cells. Recent studies have demonstrated that P-AscH− radio-sensitizes PDAC but is a radioprotector to normal cells and tissues. Several mechanisms have been hypothesized to explain the dual roles of P-AscH− in radiation-induced toxicity including the activation of nuclear factor-erythroid 2-related factor 2 (Nrf2), RelB, as well as changes in bioenergetic profiles. We have found that P-AscH− in conjunction with radiation increases Nrf2 in both cancer cells and normal cells. Although P-AscH− with radiation decreases RelB in cancer cells vs. normal cells, the knockout of RelB does not radio-sensitize PDACs. Cellular bioenergetic profiles demonstrate that P-AscH− with radiation increases the ATP demand/production rate (glycolytic and oxidative phosphorylation) in both PDACs and normal cells. Knocking out catalase results in P-AscH− radio-sensitization in PDACs. In a phase I trial where P-AscH− was included as an adjuvant to the standard of care, short-term survivors had higher catalase levels in tumor tissue, compared to long-term survivors. These data suggest that P-AscH− radio-sensitizes PDACs through increased peroxide flux. Catalase levels could be a possible indicator for how tumors will respond to P-AscH−.

The PPARγ Agonist Rosiglitazone Enhances the Radiosensitivity of Human Pancreatic Cancer Cells.

PurposeAs radiation therapy is widely used for the management of pancreatic cancer, identifying novel targets to improve the radiosensitivity of cancer cells is beneficial. Rosiglitazone, a specific peroxisome proliferator-activated receptor γ (PPARγ) agonist, has an inhibitory effect on various types of cancer cells. The purpose of this paper is to investigate the effect of rosiglitazone on the radiosensitivity of pancreatic cancer cells and the potential mechanism.Materials and MethodsPPARγ expression in pancreatic cancer and adjacent tissues was evaluated using immunohistochemistry analysis. The viability, migration and invasion ability of PANC1 and PaTu8988 cells were detected using MTT assay, scratch-wound assay and transwell invasion assay. The effect of rosiglitazone on radiosensitivity of the cells was determined using the clonogenic assay. PANC1 cells were inoculated into BALB/c mice to establish tumors. Microarray was used to investigate changes of genes involved.ResultsHigher PPARγ expression was demonstrated in pancreatic cancer tissues compared with para-carcinoma tissues. Rosiglitazone inhibited the cell viability and enhanced the radiation-induced anti-migration and anti-invasion effect. Rosiglitazone potentiated the radiosensitivity of pancreatic cancer cells and PANC1 xenografts. Microarray analysis revealed that rosiglitazone plus radiation altered the expression of multiple genes and affected multiple pathways.ConclusionRosiglitazone enhances the radiosensitivity of human pancreatic cancer cells in vitro and in vivo via complex mechanisms.

MiR-590-5p Sensitizes Pancreatic Ductal Adenocarcinoma Cells to Radiation by Blocking Autophagy via Targeting ATG3

Background: Resistance against radio therapy is growing concern in treating patients of pancreatic cancer (PC). Improving our depth with regards to mechanisms involved in radio resistance could help in developing strategies for increasing patient's response to this therapy. Here we investigated the role of micro RNAs (miRs) and specifically miR-590-5p in radio-resistance of PC cells. Methods: We successfully developed radio resistant PC cell lines in our lab and subjected them to microarray analysis to compare the levels of miRs with their original parental cell lines. We then transfected the PC cells using miR mimics or inhibitor and performed clonogenic survival assay to study the effects on radiation sensitivity. We also studied the effect of miR-590-5p on autophagy using electron microscopy and Immunoblotting analysis. Luciferase assay was done for identifying mRNA targets of miR-590-5p. Results: The radio resistant PC cells exhibited decreased expression of miR-590-5p with elevated autophagy against the parental cells. The over expression of miR-590-5p inhibited the radiation mediated autophagy and the miR-590-5p inhibitor encouraged autophagy in PC cells. The up-regulation of miR-590-5p enhanced the sensitivity of PC cells towards radiation. We confirmed that ATG-3 as a favorable target of miR-590-5p and was unregulated in radio resistant cells, we also found that ATG-3 was associated with autophagy. MiR-590-5p inhibited radiation-mediated autophagy and enhanced radio sensitivity of PC cells. The results suggested an inverse correlation for miR-590-5p and autophagy in PC tissues. Conclusions: The expression of miR-590-5p in PC cells is associated with elevated levels of autophagy along with ATG-3, which leads to radio resistance; miR-590-5p can be employed to enhance the radio sensitivity of PC cells. Funding Statement: The project was self financed. Declaration of Interests: The authors have no conflict of interest. Ethics Approval Statement: All the patients were educated about the study and informed consent forms were signed before the study. The study was approved from institutional review of research committee at The Second Xiangya Hosipital of Central South University, Changsha, Hunan, China

Effect of miR-449a on radiosensitivity of pancreatic cancer cells

Objective The purpose of this study is to investigate the effect of miR-449a on pancreatic cancer cells and the molecular mechanism. Methods The expression levels of miR-449a in pancreatic cancer cells treated or untreated with radiation was detected by qRT-PCR.High expression of miR-449a was achieved by transfecting miR-449a mimics into SW1990 cells. The cell growth, apoptosis and colony formation ability was assessed by MTT assay, flow cytometry and colony formation assay, respectively. The relationship of miR-449a and Cyclin D1 was determined by the TargetScan and dual luciferase reporter. Immunohistochemistry was used to examine protein levels of Cyclin D1 in pancreatic cancer and normal pancreas tissues. Si-Cyclin D1 was used to detecte the effect of Cyclin D1 on radiosensitivity of pancreatic cancer cells. Results The expression levels of miR-449a in pancreatic cancer cells with radiation treatment were decreased significantly. Mir-449a mimics increased the cell proliferation rates and apoptosis rates obviously, and decreased the colony formation ability in SW1990 cells treated with radiation. Results from the TargetScan and dual luciferase reporter showed that Cyclin D1 was the target of miR-449a. The positive staining rates of Cyclin D1 in pancreatic cancer tissue (85.7%, 30/35) was higher than those in normal pancreas tissue (20%, 2/10). Knockdown of Cyclin D1 enhanced the radiosensitivity of pancreatic cancer cells. Conclusion MiR-449a enhances the radiosensitivity of pancreatic cancer cells by targeting Cyclin D1. Key words: miR-449a; SW1990 cell line; Radiation sensitivity

Abstract 1669: Inhibition of the Rac1/PAK pathway sensitizes pancreatic cancer cells to gamma-irradiation

Abstract Radiation therapy is a staple treatment for pancreatic cancers. However, owing to the intrinsic radioresistance of pancreatic cancer cells, radiation therapy often fails to increase survival of pancreatic cancer patients. Radiation impedes cancer cells by inducing DNA damage, which can activate cell cycle checkpoints. Normal cells possess both G1/S and G2/M checkpoints. However, cancer cells are often defective in G1/S checkpoint due to mutations/alterations in the key regulators of this checkpoint. Accordingly, our results show that normal pancreatic ductal cells respond to ionizing radiation (IR) with activation of both checkpoints whereas pancreatic cancer cells respond to IR with G2/M arrest only. Overexpression/hyperactivation of Rac1 GTPase is detected in the majority of pancreatic cancers. Rac1 plays important roles in survival and Ras mediated transformation. Here, we show that Rac1 and its downstream effector, the PAK kinases, also plays a critical role in the response of pancreatic cancer cells to IR. Inhibition of Rac1/PAK pathway with pharmacological inhibitors (Ehop-016, AZA1, NSC23766, and FRAX597) or a dominant negative mutant of Rac1 (Rac1T17N) blocks activation of the ATM/Chk2 and ATR/Chk1 pathways, abrogates activation of the IR-induced G2/M checkpoint, delays the repair of IR-induced double-stranded DNA breaks, and increases the radiosensitivity of pancreatic cancer cells through induction of apoptosis. These results implicate Rac1 signaling in the survival of pancreatic cancer cells following IR, raising the possibility that this pathway contributes to the intrinsic radioresistance of pancreatic cancer. Citation Format: Michel M. Ouellette, Surinder K. Batra, Ying Yan. Inhibition of the Rac1/PAK pathway sensitizes pancreatic cancer cells to gamma-irradiation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1669.

MEK Inhibitor GSK1120212-Mediated Radiosensitization of Pancreatic Cancer Cells Involves Inhibition of DNA Double-Strand Break Repair Pathways

Purpose: Over 90% of pancreatic adenocarcinoma PC express oncogenic mutant KRAS that constitutively activates the Raf-MEK-MAPK pathway conferring resistance to both radiation and chemotherapy. MEK ...

Abstract B76: Targeting Mcl-1 for radiosensitization of pancreatic cancers

Abstract In order to identify targets whose inhibition may enhance the efficacy of chemoradiation in pancreatic cancer and, thus, improve survival in patients with locally advanced pancreatic cancer, we conducted an siRNA library screen of 8,800 genes. We identified Mcl-1 (myeloid cell leukemia-1), an anti-apoptotic member of the Bcl-2 family, as a target for sensitizing pancreatic cancer cells to gemcitabine-radiation. Thus, we investigated Mcl-1 inhibition by genetic (siRNA) and pharmacological (small molecule) approaches as a radiosensitizing strategy in pancreatic cancer cells. Mcl-1 depletion by siRNA radiosensitized BxPC-3, Panc-1, and MiaPaCa-2 cells with radiation enhancement ratios (RER) of 1.5±0.06, 1.4±0.03, and 1.3±0.01, respectively, and caused Caspase-3 activation and PARP cleavage. We next tested the ability of the recently identified, selective, small molecule inhibitor of Mcl-1, UMI-77 (Abulwerdi F et al., Mol Cancer Ther, 2014), to inhibit the anti-apoptotic functions of Mcl-1 and radiosensitize in pancreatic cancer cells. Consistent with inhibition of Mcl-1 anti-apoptotic function, UMI-77 (1-5uM) caused dissociation of Mcl-1 from the pro-apoptotic protein Bak. Under these same conditions, UMI-77 produced significant radiosensitization in BxPC-3 and Panc-1 cells (RER: 1.5±0.1 and 1.3±0.04, respectively; P<0.05) with minimal radiosensitization of MiaPaCa-2 cells (1.2±0.07). Since Mcl-1 expression levels are highest in BxPC-3 and Panc-1 cells and lowest in MiaPaCa-2 cells, these results suggest that elevated Mcl-1 protein levels may be a biomarker for radiosensitization by Mcl-1 inhibition. In addition, radiosensitization by UMI-77 was associated with Caspase-3 activation, PARP cleavage, and increased sub-G1 DNA content. In contrast, ABT-737, a well-established inhibitor of Bcl-2, Bcl-XL, and Bcl-w, failed to radiosensitize pancreatic cancer cells suggesting the unique importance of Mcl-1 in radiation survival. Taken together, these results validate Mcl-1 as a target for radiosensitization of pancreatic cancer cells and demonstrate the efficacy of small molecules which displace Mcl-1 from Bak/Bax as radiosensitizers in pancreatic cancer. Furthermore, these results support continued investigation of UMI-77 and its analogues in animal models and in combination with gemcitabine-radiation as a strategy for radiation and chemoradiation sensitization of pancreatic cancers. Citation Format: Qiang Zhang, Dongping Wei, Jason Schreiber, Tasneem Kausar, Theodore Lawrence, Yi Sun, Zaneta Nikolovska-Coleska, Meredith A. Morgan. Targeting Mcl-1 for radiosensitization of pancreatic cancers. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B76.

MicroRNA-216a enhances the radiosensitivity of pancreatic cancer cells by inhibiting beclin-1-mediated autophagy

Radioresistance has become a challenge in the treatment of pancreatic cancer, which limits the efficacy and outcomes of radiotherapy in clinical treatment. Autophagy, recognized as an adaptive response to cell stress, has recently been involved in the radioresistance of cancer cells. MicroRNAs (miRNAs) are also involved in the radioresistance of pancreatic cancer cells. In the present study, we established a radioresistant pancreatic cancer cell line and found that miRNA-216a was significantly downregulated whereas the autophagy activity was increased as compared with the control. Forced expression of miR-216a was found to inhibit the expression of beclin-1, a critical autophagic gene, as well as autophagy. Using bioinformatics analysis and the dual-luciferase reporter gene assay, we found that miR-216a directly interacted with 3'-untranslated region (UTR) of beclin-1. Furthermore, the forced expression of miR‑216a inhibited cell growth and colony formation ability and promoted the cell apoptosis of radioresistant pancreatic cancer cells in response to irradiation. By contrast, overexpression of beclin-1 abrogated the effects of miR-216a. Furthermore, miR-216a sensitized xenograft tumor to irradiation treatment and inhibited irradiation-induced autophagy by regulating beclin-1. Collectively, the results demonstrated that miR‑216a enhanced the radiosensitivity of pancreatic cancer cells by inhibiting beclin-1-mediated autophagy, suggesting a promising molecular target for improving the radiotherapy of pancreatic cancer.

Targeting Mcl-1 for Radiosensitization of Pancreatic Cancers

In order to identify targets whose inhibition may enhance the efficacy of chemoradiation in pancreatic cancer, we previously conducted an RNAi library screen of 8,800 genes. We identified Mcl-1 (myeloid cell leukemia-1), an anti-apoptotic member of the Bcl-2 family, as a target for sensitizing pancreatic cancer cells to chemoradiation. In the present study we investigated Mcl-1 inhibition by either genetic or pharmacological approaches as a radiosensitizing strategy in pancreatic cancer cells. Mcl-1 depletion by siRNA produced significant radiosensitization in BxPC-3 and Panc-1 cells in association with Caspase-3 activation and PARP cleavage, but only minimal radiosensitization in MiaPaCa-2 cells. We next tested the ability of the recently identified, selective, small molecule inhibitor of Mcl-1, UMI77, to radiosensitize in pancreatic cancer cells. UMI77 caused dissociation of Mcl-1 from the pro-apoptotic protein Bak and produced significant radiosensitization in BxPC-3 and Panc-1 cells, but minimal radiosensitization in MiaPaCa-2 cells. Radiosensitization by UMI77 was associated with Caspase-3 activation and PARP cleavage. Importantly, UMI77 did not radiosensitize normal small intestinal cells. In contrast, ABT-737, an established inhibitor of Bcl-2, Bcl-XL, and Bcl-w, failed to radiosensitize pancreatic cancer cells suggesting the unique importance of Mcl-1 relative to other Bcl-2 family members to radiation survival in pancreatic cancer cells. Taken together, these results validate Mcl-1 as a target for radiosensitization of pancreatic cancer cells and demonstrate the ability of small molecules which bind the canonical BH3 groove of Mcl-1, causing displacement of Mcl-1 from Bak, to selectively radiosensitize pancreatic cancer cells.

Inhibition of RAC1 GTPase sensitizes pancreatic cancer cells to γ-irradiation.

Radiation therapy is a staple treatment for pancreatic cancer. However, owing to the intrinsic radioresistance of pancreatic cancer cells, radiation therapy often fails to increase survival of pancreatic cancer patients. Radiation impedes cancer cells by inducing DNA damage, which can activate cell cycle checkpoints. Normal cells possess both a G1 and G2 checkpoint. However, cancer cells are often defective in G1 checkpoint due to mutations/alterations in key regulators of this checkpoint. Accordingly, our results show that normal pancreatic ductal cells respond to ionizing radiation (IR) with activation of both checkpoints whereas pancreatic cancer cells respond to IR with G2/M arrest only. Overexpression/hyperactivation of Rac1 GTPase is detected in the majority of pancreatic cancers. Rac1 plays important roles in survival and Ras-mediated transformation. Here, we show that Rac1 also plays a critical role in the response of pancreatic cancer cells to IR. Inhibition of Rac1 using specific inhibitor and dominant negative Rac1 mutant not only abrogates IR-induced G2 checkpoint activation, but also increases radiosensitivity of pancreatic cancer cells through induction of apoptosis. These results implicate Rac1 signaling in the survival of pancreatic cancer cells following IR, raising the possibility that this pathway contributes to the intrinsic radioresistance of pancreatic cancer.

222P - Krüppel Like Factor 10 Modulates Radio-Sensitivity By Transcriptionally Regulating Uv Radiation Resistance Associated Gene in Pancreatic Cancer

ABSTRACT Aim: Deregulation of transforming growth factor (TGF)-beta function is a common feature of pancreatic cancer, rendering it unresponsive to radiotherapy. Kruppel-like factor 10 (KLF10) is an important transcription factor mediating TGF-beta 1 signaling. The aim of this study is to evaluate the mechanisms of Klf10 in regulating radio-sensitivity of pancreatic cancer. Methods: The expression of Klf10 and radio-sensitivity of genetically modulated and radiation selected pancreatic cancer cells were demonstrated by immunoblots and clonogenic assays. Chip-PCR and luciferase promoter assay were used to demonstrate down-stream targets interaction with Klf10. The level of autophagy, apoptosis and DNA damage after radiation was evaluated. Klf10 modulated tumor response to radiation was measured in orthotopic model. Klf10 expression was correlated with pathologic tumor regression in pancreatic cancer patients. Results: Decreasing Klf10 expression was found in Panc-1 cells with increasing radiation resistance. Over-expressing Klf10 suppressed surviving fraction of MiaPaCa cells after radiation. The observation was associated with increased apoptosis, DNA damage, and decreased autophagy. The expression of ultraviolet irradiation resistance-associated gene (UVRAG) was demonstrated to be transcriptionally suppressed by Klf10. In vitro studies demonstrated Klf10 regulated pancreatic cancer radiation sensitivity via UVRAG. In the orthotopic model, up-regulating Klf10 genetically or pharmacologically sensitized pancreatic tumors to radiation. Analysis from pancreatic cancer patients, receiving neoadjuvant chemo-radiotherapy before surgery, showed a significant correlation of high Klf10 expression with good pathologic tumor regression. Conclusions: Our results suggest Klf10 regulates radio-sensitivity of pancreatic cancer cells by transcriptionally suppressing UVRAG expression which modulates autophagy, apoptosis and DNA damage repair after radiation. Klf10 is a potential biomarker of tumor response to radiotherapy and a therapeutic target to pancreatic cancer. Disclosure: All authors have declared no conflicts of interest.

Radiosensitization of Pancreatic Cancer Cells by Metformin through the AMPK Pathway

Pancreatic cancer is relatively radioresistant, however, radiotherapy has been shown to provide efficacy in the treatment of local disease. To increase the effectiveness of radiotherapy in pancreatic cancer, radiosensitizing drugs are under development. In this study, we investigated the radiosensitizing activity of the anti-diabetic drug metformin on pancreatic cancer cells in vitro. We demonstrated that metformin radiosensitized MiaPaCa-2 and Panc1 cells with radiation enhancement ratios (ER) ranging from 1.33-1.45 with metformin concentrations of 30-100 μM, and in addition, we showed that metformin sensitized cells to gemcitabine alone or in combination with radiation treatment. In addition, we found that pancreatic cancer stem cell-like cells showed enhanced radiosensitization in a tumorsphere assay with a REF of 1.66. At these radiosensitizing doses, metformin alone had low toxicity (as shown by >75% clonogenic survival) and did not affect cell cycle. The combination of metformin and radiation yielded greater numbers of γ-H2AX foci after 1 h compared to radiation alone, suggesting increased DNA damage signaling. Examination of the AMPK pathway showed that pharmacological inhibition of AMPK signaling or RNAi of AMPKα1 reversed metformin-mediated radiosensitization. These studies show that metformin radiosensitization of pancreatic cancer cells at micromolar concentration acts through AMPK and may affect DNA damage signaling. The data indicate that metformin may increase the efficacy of radiation therapy for pancreatic cancer.

Radiosensitization of Pancreatic Cancer Cells In Vitro and In Vivo through Poly (ADP-ribose) Polymerase Inhibition with ABT-888

OBJECTIVESTo determine whether poly (ADP-ribose) polymerase-1/2 (PARP-1/2) inhibition enhances radiation-induced cytotoxicity of pancreatic adenocarcinoma in vitro and in vivo, and the mechanism by which this occurs. MethodsPancreatic carcinoma cells were treated with ABT-888, radiation, or both. In vitro cell viability, apoptosis, and PARP activity were measured. Orthotopic xenografts were generated in athymic mice and treated with ABT-888 (25mg/kg), radiation (5Gy), both, or no treatment. Mice were monitored with bioluminescence imaging. RESULTSIn vitro, treatment with ABT-888 and radiation led to higher rates of cell death after 8days (P < .01). Co-treatment with 5Gy and 1, 10 or 100μmol/l of ABT-888 led to dose enhancement factors of 1.29, 1.41 and 2.36, respectively. Caspase activity was not significantly increased when treated with ABT-888 (10 μmol/l) alone (1.28-fold, P = .08), but became significant when radiation was added (2.03-fold, P < .01). PARP activity increased post-radiation and was abrogated following co-treatment with ABT-888. In vivo, treatment with ABT-888, radiation or both led to tumor growth inhibition (TGI) of 8, 30 and 39days, and survival at 60days of 0%, 0% and 40%, respectively. CONCLUSIONSABT-888 with radiation significantly enhanced tumor response in vitro and in vivo. ABT-888 inhibited PAR protein polymerization resulting in dose-dependent feedback up-regulation of PARP and p-ATM suggesting increased DNA damage. This translated into enhancement in TGI and survival with radiation in vivo. In vitro PAR levels correlated with levels of tumor apoptosis suggesting potential as a predictive biomarker. These data are being used to support a Phase I study in locally advanced pancreatic cancer.

The Mechanism of Metformin-Induced Radiosensitization of Pancreatic Cancer Cells

Local control of pancreatic cancer with radiation therapy is poor and the development of radiosensitizers can positively impact treatment. We have previously shown that the anti-diabetic drug metformin radiosensitizes pancreatic cancer cells. Here we investigate the mechanism of metformin induced radiosensitization.

Abstract 2659: Radiosensitization of pancreatic cancer cells in vitro and in vivo through poly (ADP ribose) polymerase-1/2 inhibition

Abstract Background: Poly (ADP ribose) polymerases (PARPs) are DNA-binding proteins involved in DNA repair. PARP inhibition with ABT-888 has resulted in excellent antitumor activity when used with other cytotoxic therapies, including radiation (RT). Herein, we attempt to determine whether PARP-1/2 inhibition enhances RT-induced cytotoxicity of pancreatic cancer in vitro and in vivo, and the mechanism by which this occurs. Methods: Luciferase-expressing pancreatic carcinoma cells (MiaPaCa-2) were treated with ABT-888, RT, combinations thereof, or left untreated. Cell viability was assessed through reduction of resazurin into fluorescent resorufin. Levels of apoptosis were determined by measuring caspase-3/7 activities. PARP activity was determined by measuring PAR protein levels using an elisa. Orthotopic xenografts were generated by injecting 5×105 cells into the pancreatic tail of athymic nude female mice per institutional protocol. Anesthetized mice were imaged for bioluminescence and cone-beam CT was obtained with our small animal linear accelerator. Animals were randomized to receive ABT-888 (25 mg/kg), RT (5 Gy), ABT-888 + RT, or no treatment (saline injection). RT was delivered using bioluminescence image-guided focal treatment. Animals were longitudinally monitored with bi-weekly bioluminescence imaging. All animal experiments were conducted per institutional protocol. Results: Half maximal inhibitory concentration (IC50) of RT was 2 Gy; IC10 for ABT-888 was 10 uM. Treatment with ABT-888 (10 uM) and RT (2 Gy) led to higher rates of cell death 8 days after treatment (P&amp;lt;.01). Co-treatment with 5 Gy and 1, 10 or 100 uM of ABT-888 led to dose enhancement factors of 1.29, 1.41 and 2.36, respectively. Minimal cytotoxicity was noted when cells were treated with ABT-888 alone up to 100 uM. Caspase activity was not significantly increased when treated with ABT-888 (10 uM) alone (1.28 fold, P=.077), but became significant when RT (2 Gy) was added (2.03 fold, P&amp;lt;.01). PARP activity significantly increased 6 hours post-RT; this activity was abrogated following co-treatment with ABT-888. Preliminary in vivo results suggest that treatment with ABT-888 alone does not result in significant tumor growth inhibition (TGI) relative to untreated controls. Additionally, TGI seen with RT is further enhanced following co-treatment with a single dose of ABT-888. Conclusions: ABT-888 appears to be a potent in vitro and in vivo radiosensitizer of MiaPaCa-2 with minimal intrinsic cytotoxicity when used alone. Radiation-induced apoptosis was significantly enhanced by ABT-888, suggesting a synergistic mechanism of interference with DNA repair. In vitro PAR levels correlated with levels of tumor apoptosis suggesting its potential as a predictive clinical biomarker. These data are currently being translated into a phase I clinical trial for patients with locally advanced pancreatic cancer. 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 2659. doi:10.1158/1538-7445.AM2011-2659

Abstract 2657: Radiosensitization of pancreatic cancer cells by combined inhibition of Chk1 and PARP1 using AZD7762 and olaparib

Abstract Small molecule inhibitors targeted to key DNA damage response/repair proteins, such as Chk1 (checkpoint kinase 1) and PARP1 (poly (ADP-ribose) polymerase-1) are being developed to sensitize tumor cells to chemo- and radio- therapy. We have recently shown that inhibition of HRR (homologous recombination repair) by the Chk1/2 inhibitor, AZD7762 (AstraZeneca) is a mechanism of radiosensitization in pancreatic cancer cells. Others have demonstrated that AZD7762 selectively sensitizes p53 mutant tumor cells. PARP inhibitors have demonstrated preferential efficacy as radiation sensitizers in cells with other DNA damage repair defects, including HRR. Thus, we hypothesized that inhibition of HRR (mediated by Chk1 via AZD7762) and PARP1 (via olaparib, AZD2281) would selectively sensitize p53 mutant pancreatic cancer cells to radiation. To test this hypothesis we assessed radiosensitization by clonogenic survival in two p53 mutant pancreatic cancer cell lines, MiaPaCa-2 and MPanc96. Cells were treated with AZD7762 (100nM) and/or olaparib (1uM) for 1 hour before radiation (0-8Gy), and for 23 hours after radiation, followed by processing for clonogenic survival. We verified that treatment with olaparib inhibited poly (ADP-ribose). We found that olaparib or AZD7762 alone produced similar magnitudes of radiosensitization in MiaPaCa-2 cells (1.5 ± 0.1 and 1.5 ± 0.01, respectively). However, the combination of olaparib and AZD7762 produced significantly greater radiosensitization than either agent alone (2.4 ± 0.1, P&amp;lt;0.05), which appeared to be greater than additive. Similarly, in a second cell line, MPanc96, the combination of olaparib and AZD7762 produced significantly greater radiosensitization (3.0 ± 0.4, P&amp;lt;0.05) than either olaparib (1.5 ± 0.1) or AZD7782 alone (2.0 ± 0.1). The combination of olaparib with AZD7762 produced minimal toxicity with clonogenic surviving fractions in MiaPaCa-2 cells of 1.1 ± 0.1 (olaparib), 0.8 ± 0.1 (AZD7762), and 0.7 ± 0.1 (olaparib + AZD7762) and in MPanc96 cells of &amp;gt;1.0 ± 0.2 (olaparib), 0.5 ± 0.1 (AZD7762), and 0.6 ± 0.2 (olaparib + AZD7762). Radiosensitization by olaparib and AZD7762 was accompanied by an increase in γH2AX and S345 Chk1 suggesting that DNA damage was increased under these conditions. Taken together, these findings demonstrate that the combination of olaparib and AZD7762 induces profound radiosensitization of p53 mutant pancreatic cancer cell lines. Furthermore, these studies suggest that inhibition of HRR by Chk1 inhibitors may be a useful strategy for selectively inducing a BRAC1/2 deficient-like phenotype in p53 mutant tumor cells, thus resulting in synergistic radiosensitization when used in combination with PARP1 inhibition. Supported by R01CA78554. 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 2657. doi:10.1158/1538-7445.AM2011-2657

Inhibition of Radiation-Induced DNA Repair and Prosurvival Pathways Contributes to Vorinostat-Mediated Radiosensitization of Pancreatic Cancer Cells

The intrinsic radioresistance of pancreatic cancer (PaCa) is caused by multiple oncogenic signaling pathways. In contrast to combining radiation therapy (RT) with targeted therapeutic agent(s) whose blockade can be circumvented by redundant signaling pathways, we evaluated the combination of RT with a broad-spectrum histone deacetylase inhibitor, vorinostat. Radiosensitization by vorinostat was analyzed using clonogenic survival assays. Apoptosis was evaluated using flow cytometry and immunoblotting. DNA repair was evaluated using immunofluorescence assessment of histone 2AX phosphorylation and immunoblotting for DNA repair proteins. Prosurvival pathway proteins were measured by immunoblotting and electrophoretic mobility shift assays. Vorinostat significantly sensitized PaCa cells to radiation, but vorinostat-induced apoptosis did not contribute significantly to the observed radiosensitization. However, vorinostat inhibited DNA damage repair by targeting key DNA repair proteins and also abrogated prosurvival pathways responsible for PaCa aggressiveness and radioresistance. Specifically, the constitutively overexpressed epidermal growth factor receptor and nuclear factor κB pathways were shown to be induced by radiation and inhibited by vorinostat. Vorinostat augments the antitumor effects of RT by abrogating radioresistance responses of PaCa cells mediated by prosurvival and DNA repair pathways and promises to be a clinically relevant adjunct to RT for treatment of PaCa.