Biomedical Microdevices

, 21:23 | Cite as

Enhancement of gemcitabine cytotoxicity in pancreatic adenocarcinoma through controlled release of nitric oxide

  • R. Araujo-Gutierrez
  • J. L. Van Eps
  • D. Kirui
  • N. S. Bryan
  • Y. Kang
  • J. B. Fleming
  • J. S. Fernandez-MoureEmail author


Gemcitabine (GEM) is the first-line treatment for pancreatic adenocarcinoma (PAC) yet chemoresistance is common. Nitric oxide (NO) is the predominant species responsible for the cytotoxic action of macrophages against cancer cells yet localized delivery is difficult given the short half-life. We sought to study the effect of locally delivered NO on GEM mediated PAC cytotoxicity and the potential role of SMAD4 in this effect. We hypothesized that NO would enhance the cytotoxicity of GEM in a SMAD4 dependent manner. NO-Silica nanoparticles (NO-Si) were synthesized via a co-condensation of tetraethoxysilane with aminoalkoxysilane under high-pressure nitrous oxide. NO release was measured using chemiluminescence. A SMAD4 negative PAC cell line (SMAD4-) was made using retroviral knockdown of Panc1 PAC cells. Panc1 and SMAD4- cells were treated with gemcitabine (100 nm (hi) to 30 μm (lo)), 30 mg NOSi particles, or both (NOSihi or NOSilo) and cell viability assessed. NoSi reduced cell viability by 25.99% in Panc1 and 24.38% in SMAD4-. When combined with gemcitabine, further reductions were seen in a dose dependent manner for both cell lines. We have demonstrated the in-vitro dose dependent cytotoxic effects of NOSi. When combined with GEM there is a synergistic effect resulting in improved cytotoxicity seen in both Panc1 and SMAD4- PAC cells with a differential pattern of cell death seen at high concentrations of NO. These findings suggest not only that NO is useful chemosensitizing agent but that SMAD4- may play a role in its synergism with GEM.


Pancreatic adenocarcinoma Gemcitabine Nitric oxide SMAD4 PANC1 


Financial Support

The authors acknowledge financial support from the following source: Physical Sciences in Oncology - National Cancer Institute of Health grant (U54CA143837).

Compliance with ethical standards

Conflict of interest statement

There are no financial disclosures or conflicts of interest to report for any of the authors.


  1. R. Altieri, M. Fontanella, A. Agnoletti, P.P. Panciani, G. Spena, E. Crobeddu, et al., Role of nitric oxide in glioblastoma therapy: Another step to resolve the terrible puzzle? Transl Med UniSa 12, 54–59 (2015)Google Scholar
  2. D. Ansari, A. Gustafsson, R. Andersson, Update on the management of pancreatic cancer: Surgery is not enough. World J. Gastroenterol. 21(11), 3157–3165 (2015). CrossRefGoogle Scholar
  3. N. Bardeesy, K.H. Cheng, J.H. Berger, G.C. Chu, J. Pahler, P. Olson, et al., Smad4 is dispensable for normal pancreas development yet critical in progression and tumor biology of pancreas cancer. Genes Dev. 20(22), 3130–3146 (2006). CrossRefGoogle Scholar
  4. K. Bian, F. Murad, What is next in nitric oxide research? From cardiovascular system to cancer biology. Nitric Oxide 43, 3–7 (2014). CrossRefGoogle Scholar
  5. Y. Binenbaum, S. Na'ara, Z. Gil, Gemcitabine resistance in pancreatic ductal adenocarcinoma. Drug Resist. Updat. 23, 55–68 (2015). CrossRefGoogle Scholar
  6. A. Blackford, O.K. Serrano, C.L. Wolfgang, G. Parmigiani, S. Jones, X. Zhang, et al., SMAD4 gene mutations are associated with poor prognosis in pancreatic cancer. Clin. Cancer Res. 15(14), 4674–4679 (2009). CrossRefGoogle Scholar
  7. M. Brunori, A. Giuffre, P. Sarti, G. Stubauer, M.T. Wilson, Nitric oxide and cellular respiration. Cell. Mol. Life Sci. 56(7–8), 549–557 (1999)CrossRefGoogle Scholar
  8. N.S. Bryan, M.B. Grisham, Methods to detect nitric oxide and its metabolites in biological samples. Free Radic. Biol. Med. 43(5), 645–657 (2007). CrossRefGoogle Scholar
  9. A.W. Carpenter, M.H. Schoenfisch, Nitric oxide release: Part II. Therapeutic applications. Chem. Soc. Rev. 41(10), 3742–3752 (2012). CrossRefGoogle Scholar
  10. Y.W. Chen, P.J. Hsiao, C.C. Weng, K.K. Kuo, T.L. Kuo, D.C. Wu, et al., SMAD4 loss triggers the phenotypic changes of pancreatic ductal adenocarcinoma cells. BMC Cancer 14, 181 (2014). CrossRefGoogle Scholar
  11. H. Cheng, L. Wang, M. Mollica, A.T. Re, S. Wu, L. Zuo, Nitric oxide in cancer metastasis. Cancer Lett. 353(1), 1–7 (2014). CrossRefGoogle Scholar
  12. A. Cid-Arregui, V. Juarez, Perspectives in the treatment of pancreatic adenocarcinoma. World J. Gastroenterol. 21(31), 9297–9316 (2015). CrossRefGoogle Scholar
  13. L. de Sousa Cavalcante, G. Monteiro, Gemcitabine: Metabolism and molecular mechanisms of action, sensitivity and chemoresistance in pancreatic cancer. Eur. J. Pharmacol. 741, 8–16 (2014). CrossRefGoogle Scholar
  14. R. Farias-Eisner, F. Teng, M. Oliveira, R. Leuchter, B. Karlan, L.D. Lagasse, J.S. Berek, The influence of tumor grade, distribution, and extent of carcinomatosis in minimal residual stage III epithelial ovarian cancer after optimal primary cytoreductive surgery. Gynecol. Oncol. 55(1), 108–110 (1994). CrossRefGoogle Scholar
  15. E. Fokas, E. O'Neill, A. Gordon-Weeks, S. Mukherjee, W.G. McKenna, R.J. Muschel, Pancreatic ductal adenocarcinoma: From genetics to biology to radiobiology to oncoimmunology and all the way back to the clinic. Biochim. Biophys. Acta 1855(1), 61–82 (2015). CrossRefGoogle Scholar
  16. L.K. Folkes, P. O'Neill, DNA damage induced by nitric oxide during ionizing radiation is enhanced at replication. Nitric Oxide 34, 47–55 (2013). CrossRefGoogle Scholar
  17. L.J. Frederiksen, R. Sullivan, L.R. Maxwell, S.K. Macdonald-Goodfellow, M.A. Adams, B.M. Bennett, et al., Chemosensitization of cancer in vitro and in vivo by nitric oxide signaling. Clin. Cancer Res. 13(7), 2199–2206 (2007). CrossRefGoogle Scholar
  18. D. Fukumura, S. Kashiwagi, R.K. Jain, The role of nitric oxide in tumour progression. Nat. Rev. Cancer 6(7), 521–534 (2006). CrossRefGoogle Scholar
  19. A.F. Hezel, A.C. Kimmelman, B.Z. Stanger, N. Bardeesy, R.A. Depinho, Genetics and biology of pancreatic ductal adenocarcinoma. Genes Dev. 20(10), 1218–1249 (2006). CrossRefGoogle Scholar
  20. S.W. Hung, H.R. Mody, R. Govindarajan, Overcoming nucleoside analog chemoresistance of pancreatic cancer: A therapeutic challenge. Cancer Lett. 320(2), 138–149 (2012). CrossRefGoogle Scholar
  21. A. Jemal, R. Siegel, E. Ward, T. Murray, J. Xu, M.J. Thun, Cancer statistics, 2007. CA Cancer J. Clin. 57(1), 43–66 (2007)CrossRefGoogle Scholar
  22. Y. Kang, J. Ling, R. Suzuki, D. Roife, X. Chopin-Laly, M.J. Truty, et al., SMAD4 regulates cell motility through transcription of N-cadherin in human pancreatic ductal epithelium. PLoS One 9(9), e107948 (2014). CrossRefGoogle Scholar
  23. L.K. Keefer, Fifty years of diazeniumdiolate research. From laboratory curiosity to broad-spectrum biomedical advances. ACS Chem. Biol. 6(11), 1147–1155 (2011). CrossRefGoogle Scholar
  24. E. Kogias, N. Osterberg, B. Baumer, N. Psarras, C. Koentges, A. Papazoglou, et al., Growth-inhibitory and chemosensitizing effects of the glutathione-S-transferase-pi-activated nitric oxide donor PABA/NO in malignant gliomas. Int. J. Cancer 130(5), 1184–1194 (2012). CrossRefGoogle Scholar
  25. R. Leone, P. Giussani, S. De Palma, C. Fania, D. Capitanio, M. Vasso, et al., Proteomic analysis of human glioblastoma cell lines differently resistant to a nitric oxide releasing agent. Mol. BioSyst. 11(6), 1612–1621 (2015). CrossRefGoogle Scholar
  26. K. Luberice, D. Downs, B. Sadowitz, S. Ross, A. Rosemurgy, Has survival improved following resection for pancreatic adenocarcinoma? Am. J. Surg. 214(2), 341–346 (2017). CrossRefGoogle Scholar
  27. J.F. Quinn, M.R. Whittaker, T.P. Davis, Delivering nitric oxide with nanoparticles. J. Control. Release 205, 190–205 (2015). CrossRefGoogle Scholar
  28. D.P. Ryan, T.S. Hong, N. Bardeesy, Pancreatic adenocarcinoma. N. Engl. J. Med. 371(11), 1039–1049 (2014). CrossRefGoogle Scholar
  29. J.H. Shin, S.K. Metzger, M.H. Schoenfisch, Synthesis of nitric oxide-releasing silica nanoparticles. J. Am. Chem. Soc. 129(15), 4612–4619 (2007). CrossRefGoogle Scholar
  30. D.J. Stuehr, M.A. Marletta, Mammalian nitrate biosynthesis: Mouse macrophages produce nitrite and nitrate in response to Escherichia coli lipopolysaccharide. Proc. Natl. Acad. Sci. U. S. A. 82(22), 7738–7742 (1985)CrossRefGoogle Scholar
  31. F. Wang, X. Xia, C. Yang, J. Shen, J. Mai, H.C. Kim, et al., SMAD4 gene mutation renders pancreatic cancer resistance to radiotherapy through promotion of autophagy. Clin. Cancer Res. 24, 3176 (2018). CrossRefGoogle Scholar
  32. J.M. Winter, M.F. Brennan, L.H. Tang, M.I. D'Angelica, R.P. Dematteo, Y. Fong, et al., Survival after resection of pancreatic adenocarcinoma: Results from a single institution over three decades. Ann. Surg. Oncol. 19(1), 169–175 (2012). CrossRefGoogle Scholar
  33. H. Ying, P. Dey, W. Yao, A.C. Kimmelman, G.F. Draetta, A. Maitra, R.A. DePinho, Genetics and biology of pancreatic ductal adenocarcinoma. Genes Dev. 30(4), 355–385 (2016). CrossRefGoogle Scholar
  34. M. Zijlstra, N. Bernards, I.H. de Hingh, A.J. van de Wouw, S.H. Goey, E.M. Jacobs, et al., Does long-term survival exist in pancreatic adenocarcinoma? Acta Oncol. 55(3), 259–264 (2016). CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Heart Failure & Transplant CardiologyHouston Methodist Research InstituteHoustonUSA
  2. 2.Department of SurgeryHouston Methodist HospitalHoustonUSA
  3. 3.Department of Maxillofacial Injury and Disease US Navy Medical Research CenterSan AntonioUSA
  4. 4.Department of Molecular and Human GeneticsBaylor College of MedicineHoustonUSA
  5. 5.Department of Gastrointestinal OncologyH Lee Moffitt Cancer CenterTampaUSA
  6. 6.Department of Surgery, Division of Traumatology, Critical Care, and Emergency SurgeryUniversity of PennsylvaniaPhiladelphiaUSA

Personalised recommendations