Killing Of Pancreatic Cancer Cells Research Articles

CT109-SN-38, a Novel Antibody-drug Conjugate with Dual Specificity for CEACAM5 and 6, Elicits Potent Killing of Pancreatic Cancer Cells.

CEACAM5 and CEACAM6 are glycosylphosphatidylinositol (GPI)- linked members of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family, which are frequently upregulated in epithelial cancers where they contribute to invasion, metastasis, immune evasion, and resistance to anoikis. CT109 is a novel antibody with dual specificity to both CEACAM5 and 6. In this study, we aimed to perform the preclinical characterization of CT109 and antibody- drug conjugate (ADCs) derivatives of CT109, focusing on CT109-SN-38. CT109's cognate epitope was characterized by scanning mutagenesis. CT109 specificity and internalization kinetics were assessed by immunoblot and flow cytometry, respectively. Cognate antigen expression prevalence in colorectal cancer and normal tissue arrays was determined by immunohistochemistry. CT109 conjugations were generated by the reaction of reduced CT109 cysteines with maleimide-functionalized payload linkers. In vitro cytotoxic activity of CT109 ADCs was characterized on antigen-positive and negative pancreatic ductal adenocarcinoma cell (PDAC) lines using a luminometric viability assay. In vivo efficacy of CT109-SN-38 was assessed on a PDAC tumor xenograft model at 10 and 25 mg/kg concentrations. CT109 was shown to bind a glycoepitope centered on N309. CT109 is internalized in the CEACAM5+/CEACAM6+ double-positive PDAC line, BxPC-3, with a t1/2 of 2.3 hours. CT109 ADCs elicit a dose and antigen-dependent cytotoxic effect, with CT109-SN-38 exhibiting an IC50 value of 21 nM in BxPC-3 cells. In a BxPC-3 tumor xenograft model, CT109-SN-38 reduced tumor growth and induced regression in 3/10 mice at a concentration 25 mg/kg. These data suggest that further preclinical and clinical development of CT109-SN-38 is warranted.

Combined chemotherapy based on bioactive black phosphorus for pancreatic cancer therapy

Pancreatic cancer is the most aggressive malignant tumor with difficulty in early diagnosis, very short survival time in advanced stage, and lack of effective treatment options. In this work, a novel combination chemotherapy strategy based on bioactive black phosphorus (BP) and gemcitabine (GEM) is developed for efficient treatment of pancreatic cancer. The combined cell cycle blockage in G2/M phase induced by BP and G0/G1 phase by GEM results in synergistic killing of pancreatic cancer cells with the combination index (CI)<1. The iRGD modified zein nanoparticles co-loaded with BP quantum dots (BPQDs) and GEM are designed and prepared as a targeted nanoplatform (BP-GEM@NPs). After intravenous injection, the in vivo distribution and pharmacokinetics results demonstrate that BP-GEM@NPs shows excellent tumor targeting capability and significantly prolonged blood circulation time. The targeted co-delivery of BPQDs and GEM induces much more pancreatic tumor cell apoptosis and synergistically inhibits tumor growth in both subcutaneous xenograft and orthotopic models. Meanwhile, BP-GEM@NPs exhibit good biocompatibility without bring adverse effects. This work indicates the great potential of BP-GEM@NPs as a combination chemotherapy for pancreatic cancer and provides insights into development of biomedicine by exploring the intrinsic bioactivities of nanomaterials.

Therapeutic efficacy, pharmacokinetic profiles, and toxicological activities of humanized antibody-drug conjugate Zt/g4-MMAE targeting RON receptor tyrosine kinase for cancer therapy

BackgroundAberrant expression of the RON receptor tyrosine kinase is a pathogenic feature and a validated drug target in various types of cancers. Currently, therapeutic antibodies targeting RON for cancer therapy are under intensive evaluation. Here we report the development and validation of a novel humanized anti-RON antibody-drug conjugate for cancer therapy.MethodsAntibody humanization was achieved by grafting sequences of complementarity-determining regions from mouse monoclonal antibody Zt/g4 into human IgG1/κ acceptor frameworks. The selected humanized Zt/g4 subclone H1L3 was conjugated with monomethyl auristatin E using a dipeptide linker to form H-Zt/g4-MMAE. Pharmacokinetic analysis of H-Zt/g4-MMAE was determined using hydrophobic interaction chromatography and a MMAE ADC ELISA kit. Biochemical and biological assays were used for measuring RON expression, internalization, cell viability and death. Therapeutic efficacies of H-Zt/g4-MMAE were validated in vivo using three pancreatic cancer xenograft models. Toxicological activities of H-Zt/g4-MMAE were determined in mouse and cynomolgus monkey.ResultsH-Zt/g4-MMAE had a drug to antibody ratio of 3.77:1 and was highly stable in human plasma with a dissociation rate less than 5% within a 20 day period. H-Zt/g4-MMAE displayed a favorable pharmacokinetic profile in both mouse and cynomolgus monkey. In vitro, H-Zt/g4-MMAE induced RON internalization, which results in killing of pancreatic cancer cells with IC50 values at 10–20 nM. In vivo, H-Zt/g4-MMAE inhibited pancreatic cancer xenograft growth with tumoristatic concentrations at 1~3 mg/kg bodyweight. Significantly, H-Zt/g4-MMAE eradicated tumors across multiple xenograft models regardless their chemoresistant and metastatic statuses. Moreover, H-Zt/g4-MMAE inhibited and eradicated xenografts mediated by pancreatic cancer stem-like cells and by primary cells from patient-derived tumors. Toxicologically, H-Zt/g4-MMAE is well tolerated in mice up to 60 mg/kg. In cynomolgus monkey, H-Zt/g4-MMAE up to 30 mg/kg had a manageable and reversible toxicity profile.ConclusionsH-Zt/g4-MMAE is superior in eradication of pancreatic cancer xenografts with favorable pharmacokinetic profiles and manageable toxicological activities. These findings warrant the transition of H-Zt/g4-MMAE into clinical trials in the future.

Quinazolinedione SIRT6 inhibitors sensitize cancer cells to chemotherapeutics

The NAD+-dependent sirtuin SIRT6 is highly expressed in human breast, prostate, and skin cancer where it mediates resistance to cytotoxic agents and prevents differentiation. Thus, SIRT6 is an attractive target for the development of new anticancer agents to be used alone or in combination with chemo- or radiotherapy. Here we report on the identification of novel quinazolinedione compounds with inhibitory activity on SIRT6. As predicted based on SIRT6's biological functions, the identified new SIRT6 inhibitors increase histone H3 lysine 9 acetylation, reduce TNF-α production and increase glucose uptake in cultured cells. In addition, these compounds exacerbate DNA damage and cell death in response to the PARP inhibitor olaparib in BRCA2-deficient Capan-1 cells and cooperate with gemcitabine to the killing of pancreatic cancer cells. In conclusion, new SIRT6 inhibitors with a quinazolinedione-based structure have been identified which are active in cells and could potentially find applications in cancer treatment.

Abstract 408: ACP-196, an orally bioavailable covalent selective inhibitor of Btk, modulates the innate tumor microenvironment, exhibits antitumor efficacy and enhances gemcitabine activity in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma exists in a complex desmoplastic microenvironment that provides stromal support for tumor growth and conceals the tumor from immune surveillance. Tumor-associated stroma comprises a mix of fibroblasts, immunosuppressive T regulatory cells (Tregs), myeloid suppressive monocytes (MDSCs) and tumor-associated macrophages (TAMs) that promote tumor growth and restrain immune-mediated tumor cell killing. The targeting of immune infiltrates may impair stromal support and enhance immune-mediated killing of pancreatic cancer cells. Bruton's tyrosine kinase (Btk) is a nonreceptor enzyme in the Tec kinase family expressed among cells of hematopoietic origin including B cells, myeloid cells, mast cells and platelets, but not T cells, where it regulates multiple cellular processes. Here we describe an unexpected finding of ACP-196, a potent, novel, second generation Btk inhibitor with improved selectivity and target coverage that binds covalently to a cysteine residue (Cys481) in the front position of the ATP-binding pocket. In an orthotopic mouse model of pancreatic cancer, KPC derived pancreatic cancer cells (KrasG12D; Trp53R172H; Pdx1-Cre) were injected into the pancreases. Vehicle, single agent ACP-196 (15 mg/kg/BID, gavage), single agent gemcitabine (50 mg/kg, IV) and combination ACP-196 with gemcitabine were evaluated for efficacy. By 4 weeks of treatment, mice in the vehicle group showed signs of health deterioration and all mice were euthanized, tumors were collected and measured. Relative to the vehicle treatment, ACP-196 monotherapy resulted in a &amp;gt;2-fold reduction in tumor growth compared with less than a 2-fold reduction with gemcitabine alone. The combination of ACP-196 and gemcitabine resulted in a further reduction in tumor growth when compared to each single agent. Interestingly, analysis of tumor tissues showed that single agent ACP-196 inhibited immunosuppressive populations of TAMs and MDSCs. Surprisingly, Treg populations were also reduced with a robust expansion of CD8+ T cells in the tumors. None of these effects were observed with gemcitabine alone. Although Btk is not expressed in T cells, this finding maybe the result of inhibiting the MDSC and TAM populations within the tumor microenvironment, a mechanism of action which is currently under investigation. Taken together, these data identify Btk as a novel target for modulating tumor immune escape and suggest that pharmacologic targeting of suppressive myeloid cells by ACP-196 induces therapeutic benefit. ACP-196 is currently being evaluated in clinical trials including frontline and salvage pancreatic cancer. Citation Format: Brian J. Lannutti, Michael Gulrajani, Fanny Krantz, Elena Bibikova, Todd Covey, Katti Jessen, Wayne Rothbaum, David M. Johnson, Roger Ulrich. ACP-196, an orally bioavailable covalent selective inhibitor of Btk, modulates the innate tumor microenvironment, exhibits antitumor efficacy and enhances gemcitabine activity in pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 408. doi:10.1158/1538-7445.AM2015-408

3,3′-Diindolylmethane Enhances Chemosensitivity of Multiple Chemotherapeutic Agents in Pancreatic Cancer

Clinical management of pancreatic cancer is a major problem, which is in part due to both de novo and acquired resistance to conventional therapeutics. Here, we present in vitro and in vivo preclinical evidence in support of chemosensitization of pancreatic cancer cells by 3,3-diindolylmethane (DIM), a natural compound that can be easily obtained by consuming cruciferous vegetables. DIM pretreatment of pancreatic cancer cells led to a significantly increased apoptosis (P < 0.01) with suboptimal concentrations of chemotherapeutic agents (cisplatin, gemcitabine, and oxaliplatin) compared with monotherapy. It is known that resistance to chemotherapy in pancreatic cancer is associated with constitutively activated nuclear factor-kappaB (NF-kappaB), which becomes further activated by chemotherapeutic drugs. Our data provide mechanistic evidence for the first time showing that DIM potentiates the killing of pancreatic cancer cells by down-regulation of constitutive as well as drug-induced activation of NF-kappaB and its downstream genes (Bcl-xL, XIAP, cIAP, and survivin). Most importantly, using an orthotopic animal model, we found reduction in tumor size (P < 0.001) when DIM was given in combination with oxaliplatin compared with monotherapy. This was accompanied by loss of phospho-p65 and down-regulation of NF-kappaB activity and its downstream genes (Bcl-xL, survivin, and XIAP), which correlated with reduced cell proliferation (as assessed by Ki-67 immunostaining of tumor specimens) and evidence of apoptosis [as assessed by poly(ADP-ribose) polymerase cleavage and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining]. These results provide strong in vivo evidence in support of our hypothesis that DIM could abrogate chemotherapeutic drug (cisplatin, gemcitabine, and/or oxaliplatin)-induced activation of NF-kappaB, resulting in the chemosensitization of pancreatic tumors to conventional therapeutics.

Antitumor Activity of Gemcitabine and Oxaliplatin Is Augmented by Thymoquinone in Pancreatic Cancer

Previous studies have shown biological activity of thymoquinone, an active compound extracted from Nigella sativa, in pancreatic cancer cells; however, preclinical animal studies are lacking. Here, we report, for the first time, the chemosensitizing effect of thymoquinone to conventional chemotherapeutic agents both in vitro and in vivo using an orthotopic model of pancreatic cancer. In vitro studies revealed that preexposure of cells with thymoquinone (25 mumol/L) for 48 h followed by gemcitabine or oxaliplatin resulted in 60% to 80% growth inhibition compared with 15% to 25% when gemcitabine or oxaliplatin was used alone. Moreover, we found that thymoquinone could potentiate the killing of pancreatic cancer cells induced by chemotherapeutic agents by down-regulation of nuclear factor-kappaB (NF-kappaB), Bcl-2 family, and NF-kappaB-dependent antiapoptotic genes (X-linked inhibitors of apoptosis, survivin, and cyclooxygenase-2). As shown previously by our laboratory, NF-kappaB gets activated on exposure of pancreatic cancer cells to conventional chemotherapeutic agents; interestingly, thymoquinone was able to down-regulate NF-kappaB in vitro, resulting in chemosensitization. In addition to in vitro results, here we show for the first time, that thymoquinone in combination with gemcitabine and/or oxaliplatin is much more effective as an antitumor agent compared with either agent alone. Most importantly, our data also showed that a specific target, such as NF-kappaB, was inactivated in animal tumors pretreated with thymoquinone followed by gemcitabine and/or oxaliplatin. These results provide strong in vivo molecular evidence in support of our hypothesis that thymoquinone could abrogate gemcitabine- or oxaliplatin-induced activation of NF-kappaB, resulting in the chemosensitization of pancreatic tumors to conventional therapeutics.

Retracted: In vitro and in vivo molecular evidence of genistein action in augmenting the efficacy of cisplatin in pancreatic cancer

We recently reported the potential of genistein in augmenting gemcitabine-induced killing of pancreatic cancer (Banerjee, S. et al., Cancer Research 2005;65:9064-72). Since cis-diaminedichloroplatinum (II) (cisplatin) is widely used against solid tumors, we further investigated whether genistein pretreatment could be used as a novel strategy for cisplatin-induced killing of pancreatic cancer cells in vitro and enhanced antitumor activity in vivo. Our in vitro results showed that pretreatment of cells with genistein followed by cisplatin resulted in significant loss of cell viability and potentiated apoptosis irrespective of the metastatic ability of cells. Mechanistically, genistein augmented cisplatin induced killing by down regulating transcription factor-NF-kappaB and anti-apoptotic Akt expression. NF-kappaB was found upregulated when pancreatic cancer cells were exposed to cisplatin, suggesting the potential mechanism of acquired chemo-resistance. In addition, we also showed, for the first time, that genistein in combination with cisplatin is more effective antitumor agent in our orthotopic tumor model. But most importantly, our data also showed that a specific target, such as NF-kappaB, was inactivated in animal tumors treated with genistein and cisplatin. Immunohistochemical data showed reduced staining for phospho-p65, Bcl-xL and MMP-9 in treated tumors compared to control tumors, but the lowest activity was seen in the combination group. These results provide strong molecular in vivo evidence in support of our hypothesis that inactivation of the NF-kappaB signaling pathway by genistein results in the chemo-sensitization of pancreatic tumors to cisplatin, which is likely to be an important and novel strategy for the treatment of pancreatic cancer.

MOLECULAR EVIDENCE OF 3,3???-DIINDOLYLMETHANE INDUCED ENHANCEMENT IN CHEMOSENSITIVITY OF PANCREATIC CANCER CELLS TO MULTIPLE CHEMOTHERAPEUTIC AGENTS

Purpose: Despite recent advances in therapies, the beneficial effect unfortunately remains modest in pancreatic cancer due to a high degree of inherent and acquired chemo-resistance. We previously reported that genistein augments pancreatic tumor sensitivity to Gemcitabine and Cisplatin due to down regulation of NF-κB and Akt (Cancer Res. 2005; 65:9064-72; Cancer 2006; 106:1260-68). Further, as a "proof of concept" in support of our hypothesis, we investigated whether 3,3′-Diindolylmethane (DIM), an inhibitor of NF-κB could elicit similar effects on Panc-1, COLO 357, MDA Panc-28 and stably transfected MDA Panc-28/IκBαM (the MDA Panc-28/ IkBaM cells expressing phosphorylation defective IκBα [S32, 36A] that blocks NF-κB activation) pancreatic cancer cells. Method: Panc-1, COLO 357, MDA Panc-28 and MDA Panc-28/ IκBαM cells were cultured in complete DMEM media with and without DIM (0-100 μM) alone for 72 hrs. The response to multiple chemotherapeutic agents (Oxaliplatin, Gemcitabine and Cisplatin) was assessed in vitro using MTT and quantifying cytoplasmic histone-DNA fragments using Cell Death Detection ELISA kit upon pretreatment with 25-50 μM DIM (48 hrs) followed by chemotherapeutic agents for an additional 48-72 hrs. The molecular mechanism for enhanced chemosensitivity was investigated by performing Western immunobloting for pAkt, Survivin, Bcl-2, Bcl-xL, and PARP by single regimen treatment and under conditions of pretreatment with DIM. Additionally, gel shift assay was performed to elucidate whether activation of the basal NF-κB transcription factor was responsible for suppressing apoptosis and whether DIM mediated abrogation of NF-κB leads to sensitization and killing of pancreatic cancer cells by multiple chemotherapeutic agents. Results: The MDA Panc-28/ IκBαM and COLO 357 cells without NF-κB expression were found relatively more sensitive to DIM compared to parental MDA Panc-28 cells. DIM pretreatment also significantly enhanced the anti-proliferative and apoptotic effect of Oxaliplatin, Gemcitabine and Cisplatin in these cells relative to untreated control and the parental MDA Panc-28 cells. Several downstream regulatory target molecules under influence of NF-κB, such as Bcl-xL, Bcl-2, Cyclin D1, Survivin and MMP-9, were affected to different degrees under DIM pretreatment conditions. Conclusion: Our results strongly suggest that chemosensitization of human pancreatic cancer cells is, at least, due to inactivation of NF-κB and Akt signaling caused by DIM. Thus, our results provide evidence in support of rational design of pre-clinical in vivo and human studies targeting NF-κB and Akt signaling pathway by DIM, a known chemopreventive agent for better outcome of chemotherapy of pancreatic cancer.

Enhanced killing of pancreatic cancer cells by expression of fusogenic membrane glycoproteins in combination with chemotherapy

Pancreatic cancer has a poor prognosis with an annual mortality rate close to the annual incidence rate. We evaluated whether the expression of measles virus fusogenic membrane glycoproteins (FMG) H and F will enhance chemotherapy. Using Chou-Talalay analysis, we showed in vitro in pancreatic cancer cells that the expression of FMG often synergistically enhances clinically relevant chemotherapy. Furthermore, cell fusion in combination with chemotherapy resulted in strongly enhanced Annexin V binding, an early marker for apoptosis, when compared with single treatment. We showed in an i.p. and s.c. pancreatic xenograft model that the administration of a replication-defective adenoviral vector Ad.H/F encoding tumor-restricted FMG in combination with gemcitabine significantly enhanced treatment outcome when compared with treatment with each compound individually. To improve tumor transduction efficiency, the Ad.H/F vector was also transcomplemented with an oncolytic replication-restricted adenovirus (Ad.COX*MK), resulting in significantly improved treatment efficacy. We assessed treatment efficacy by survival analysis or measuring growth, respectively. In the i.p. model, on day 120, three of eight animals treated with this novel triple therapy consisting of Ad.H/F, gemcitabine, and Ad.COX*MK were alive and tumor free. Treatment with Ad.H/F and Ad.COX*MK resulted in one long-term survivor. In all other treatment groups, there were no long-term survivors. The significantly improved therapeutic outcome of animals receiving the triple therapy was attributed to multiple factors, including most likely improved FMG expression throughout the tumor and enhanced sensitivity of the tumor cells to gemcitabine by adenoviral gene products but also FMG expression. Qualitatively similar results were obtained in a s.c. pancreatic xenograft model.

CISPLATIN-INDUCED CYTOTOXICITY IS ENHANCED BY GENISTEIN IN PANCREATIC CANCER CELLS WITH VARIED METASTATIC POTENTIAL.

Purpose: Conventional chemotherapeutic agents including cisplatin are not very effective in pancreatic cancer, which could be due to either de novo or acquired resistance, partly, mediated by activation of NF-κB activity. The purpose of the present study was to test whether inactivation of NF-κB by genistein could be associated with increased cisplatin induced killing of pancreatic cancer cells. Methods: Colo357 FS (non-metastatic) and Colo357 L3.6pl (L3.6pl; meta-static clone of Colo357 FS with higher endogenous NF-κB activity) cells were cultured with and without genistein (0,10,25,50 and 100μM) for 72 hrs to investigate its anti-proliferative and pro-apoptotic effect by MTT and DNA-Histone ELISA, respectively. Chemosensitivity to cisplatin was also assessed by pre-treating these cells with 30μM genistein for 24 hrs followed by cisplatin (1μM) for an additional 72 hrs. EMSA was used for measuring the DNA-binding activity of NF-κB, and Western blotting for assessing the levels of Bcl-xL, Bcl-2, caspase-3, caspase-9 and PARP to assess cisplatin induced killing of pancreatic cancer cells exposed to genistein. Results: Cisplatin (250nM-1μM) alone had no significant effect on cell viability in either of these cell lines. L3.6pl cells were found to be relatively more sensitive to genistein-induced killing, and these results were correlated with higher endogenous activity of NF-κB. We also found that these effects are caspase-mediated as shown by degradation of PARP, caspase-3, and caspase-9. We also found down regulation of Bcl-xL and Bcl-2 in genistein treated cells. Genistein pretreatment significantly enhanced the pro-apoptotic effects of cisplatin in both the cell lines. Moreover, genistein down regulated the constitutively active and/or TNF-α and cisplatin induced NF-κB activation, thereby orchestrating a cascade of events leading to apoptotic cell death. Conclusions: Our results suggest that genistein-induced chemosensitization of pancreatic cancer cells to cisplatin is partly mediated by inactivation of NF-κB activity, supporting that genistein pre-treatment could be a promising strategy in combination with existing arsenals.

Potentiation of apoptosis inducing activity of BL-193, an inhibitor of Bcl-XL protein, by genistein in pancreatic cancer cells

3133 Background: Several important survival molecules such as NF-κB and Bcl-XL are highly activated in pancreas cancer. NF-κB is responsible for transcriptional activation of many tumor-promoting genes including Bcl-XL. We have previously shown that genistein down regulates the activation of NF-κB, and in turn decreases Bcl-XL. We have also shown that a small molecule inhibitor (SMI) BL-193 is a potent inhibitor of Bcl-XL,and to a lesser degree of Bcl-2. Therefore, we hypothesize that inactivation of NF-κB by genistein leads to decreased transcription of Bcl-XL,which will lead to the induction of apoptotic cell death. We also hypothesize that complete inactivation of Bcl-XLmay not be achievable by genistein treatment alone, therefore further inactivation of residual Bcl-XLby BL-193 may lead to complete inactivation of Bcl-XL, thereby enhancing the killing of pancreatic cancer cells. Methods: Results: In support of our hypotheses, we have shown by MTT assay that 30 μM genistein exposure of BxPC-3 pancreatic cancer cells for 24 hours caused inhibition in cell growth. We have also demonstrated that treatment with 3μM BL-193 alone for 72 hours caused significant growth inhibition. Moreover, cells pre-treated for 24 hours with genistein followed by 72 hours of BL-193 demonstrated significantly greater growth inhibition compared to either agent alone. The observed growth inhibition could be due to either cell cycle arrest or induction of apoptosis hence we determined the degree of apoptosis induced by these agents alone or in combination as analyzed by Histone-DNA ELISA. We found that BL-193-induced apoptosis was much more pronounced in cells pre-treated with genistein, and the results were correlated with genistein-induced inactivation of NF-κB and Bcl-XL. Conclusions: These results are consistent with our hypotheses and suggest that pre-treatment of pancreatic cancer cells with genistein down regulates NF-κB and Bcl-XL, which may play important roles in potentiating the apoptosis-inducing effect of BL-193. No significant financial relationships to disclose.

A nitroimidazole derivative, PR-350, enhances the killing of pancreatic cancer cells exposed to high-dose irradiation under hypoxia.

The radiosensitizing effects of PR-350, a nitroimidazole derivative, were examined concerning the cell killing of human pancreatic cancer cell lines exposed to high doses of gamma-ray irradiation in vitro. The percentages of dead cells were analyzed with a multiwell plate reader to measure the fluorescence intensity of propidium iodide before and after a digitonin treatment. The sensitizing effect of PR-350 on cell killing by high-dose irradiation was confirmed by time-course, dose-dependency, and microscopic observations. In five of seven pancreatic cancer cell lines in which the number of dead cells was determined 5 days after 30 Gy irradiation in the presence of PR-350, the number was significantly increased under hypoxic conditions, but not under aerobic conditions. The selective radiosensitive effect of PR-350 on hypoxic cells was also confirmed by flow cytometry. The results indicate that PR-350 can enhance the killing of pancreatic cancer cells by high-dose irradiation under hypoxia, which supports its clinical radiosensitizing effects when administered during intraoperative irradiation to pancreatic cancer.