Low-dose LPS and calorie restriction combined therapy for pancreatic ductal adenocarcinoma: an in vitro and in vivo study

Abstract

Objective: This study aimed to evaluate the therapeutic effects of lipopolysaccharide (LPS) on Panc02 cells since LPS is an inflammatory agent with the potential to activate the immune system and reorganize the tumor microenvironment. Moreover, calorie restriction (CR) has been investigated in combination with LPS as a prospective adjuvant intervention for cancer therapy. Materials and Methods: Panc02 cells were cultured in two distinct media with low and high-glucose concentrations, and cell viability was investigated after applying varying doses of LPS to culture media. The in vivo effects of LPS and CR were investigated on the mice subcutaneous pancreatic ductal adenocarcinoma (PDAC) tumor model in terms of tumor mass, histological examination, mRNA expression profiles, and biochemical parameters. Results: The lowest cell count was detected in the cells treated with 10 μg/ml LPS in low-glucose environments in vivo. Tumor mass significantly decreased in the P+LPS+CR group in vivo. The mRNA expression analysis of tumor tissues indicated that P+LPS+CR acts through NF-κB, JNK, and IL-6 signaling pathways. Conclusion: Lipopolysaccharide alone is insufficient to show therapeutic effects, but it can inhibit tumor development by acting on NF-κB and JNK pathways when combined with CR. This study gives insight into developing new treatment options for PDAC.

Keywords

• Panc02
• Lipopolysaccharide
• Calorie restriction
• Subcutaneous pancreatic ductal adenocarcinoma model
• Cancer

References

1. Siegel RL, Miller KD, Fuchs HE, Jemal A. Cancer Statistics, 2021. CA Cancer J Clin 2021; 71:7-33. doi: 10.3322/caac.21654.
2. Li C, Cui L, Yang L, et al. Pancreatic stellate cells promote tumor progression by promoting an ımmunosuppressive microenvironment in murine models of pancreatic cancer. Pancreas 2020;49:120-7. doi: 10.1097/ MPA.000.000.0000001464.
3. Chandana SR, Babiker HM, Mahadevan D. Therapeutic trends in pancreatic ductal adenocarcinoma (PDAC). Expert Opin Investig Drugs 2019;28:161-77. doi: 10.1080/13543.784.2019.1557145.
4. Alexander C, Rietschel ET. Bacterial lipopolysaccharides and innate immunity. J Endotoxin Res 2001;7: 167-202. doi: 10.1179/096.805.101101532675.
5. Wu TT, Chen TL, Chen RM. Lipopolysaccharide triggers macrophage activation of inflammatory cytokine expression, chemotaxis, phagocytosis, and oxidative ability via a tolllike receptor 4-dependent pathway: Validated by RNA interference. Toxicol Lett 2009;191:195-202. doi: 10.1016/j. toxlet.2009.08.025.
6. Fang H, Pengal RA, Cao X, et al. Lipopolysaccharide-induced macrophage inflammatory response is regulated by SHIP. J Immunol 2004;173:360-6. doi: 10.4049/jimmunol.173.1.360.
7. Seeley JJ, Ghosh S. Molecular mechanisms of innate memory and tolerance to LPS. J Leukoc Biol 2017;101:107-19. doi: 10.1189/jlb.3mr0316-118rr.
8. Lashinger LM, Malone LM, Brown GW, et al. Rapamycin partially mimics the anticancer effects of calorie restriction in a murine model of pancreatic cancer. Cancer Prev Res 2011;4:1041-51. doi: 10.1158/1940-6207.CAPR-11-0023.

9. O’Flanagan CH, Smith LA, McDonell SB, et al. When less may be more: Calorie restriction and response to cancer therapy. BMC Med 2017;15: 106. doi: 10.1186/s12916.017.0873-x.
10. Vidoni C, Ferraresi A, Esposito A, et al. Calorie restriction for cancer prevention and therapy: mechanisms, expectations, and efficacy. J Cancer Prev 2021;26:224-36. doi: 10.15430/ jcp.2021.26.4.224.
11. Meynet O, Ricci JE. Caloric restriction and cancer: Molecular mechanisms and clinical implications. Trends Mol Med 2014;20:419-27. doi: 10.1016/j.molmed.2014.05.001.
12. Lu Y, Tao F, Zhou MT, Tang KF. The signaling pathways that mediate the anti-cancer effects of caloric restriction. Pharmacol Res 2019;141:512-20. doi: 10.1016/j.phrs.2019.01.021.
13. Lee C, Raffaghello L, Brandhorst S, et al. Fasting cycles retard growth of tumors and sensitize a range of cancer cell types to chemotherapy. Sci Transl Med 2012,4:124ra27. doi: 10.1126/ scitranslmed.3003293.
14. Lanza-Jacoby S, Yan G, Radice G, LePhong C, Baliff J, Hess R. Calorie restriction delays the progression of lesions to pancreatic cancer in the LSL-KrasG12D; Pdx-1/Cre mouse model of pancreatic cancer. Exp Biol Med 2013,238:787-97. doi: 10.1177/153.537.0213493727.
15. Ekiz-Yilmaz T, Isildar B, Gezer A, et al. The role of unfolded protein response in the pathogenesis of endometriosis: contribution of peritoneal fluid. Reprod Biomed Online 2021,42:1-15. doi: 10.1016/j.rbmo.2020.09.012.
16. Sarantis P, Koustas E, Papadimitropoulou A, et al. Pancreatic ductal adenocarcinoma: Treatment hurdles, tumor microenvironment and immunotherapy. World J Gastrointest Oncol 2020,12:173-81. doi: 10.4251/wjgo.v12.i2.173.
17. Beydogan AB, Coskun Yazici ZM, Bolkent S. Influences of calorie restriction and lipopolysaccharide therapy on inflammation, cytokine response, and cell proliferation in pancreatic adenocarcinoma mouse model. J Biochem Mol Toxicol 2023;37:1-10. doi: 10.1002/jbt.23250.
18. Green CL, Mitchell SE, Derous D, et al. The effects of graded levels of calorie restriction: IX. Global metabolomic screen reveals modulation of carnitines, sphingolipids and bile acids in the liver of C57BL/6 mice. Aging Cell 2017;16:529-40. doi: 10.1111/acel.12570.
19. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248- 54. doi: 10.1016/0003-2697(76)90527-3.
20. Aebi H. Catalase in vitro. Methods Enzymol 1984;105:121-6. doi: 10.1016/S0076-6879(84)05016-3.
21. Ledwozyw A, Michalak J, Stȩpień A, Kadziolka A. The relationship between plasma triglycerides, cholesterol, total lipids and lipid peroxidation products during human atherosclerosis. Clin Chim Acta 1986,155:275-83. doı: 10.1016/0009-8981(86)90247-0.
22. Beutler E, Duron O, Kelly BM. Improved method for the determination of blood glutathione. J Lab Clin Med 1963;61:882-8.
23. Sun Y, Oberley LW, Li Y. A simple method for clinical assay of superoxide dismutase. Clin Chem 1988;34:497-500. doi: 10.1093/clinchem/34.3.497.
24. Reznick AZ, Packer L. Oxidative damage to proteins: Spectrophotometric method for carbonyl assay. Methods Enzymol 1994;233:357-63. doi: 10.1016/S0076- 6879(94)33041-7.
25. Honda T, Inagawa H. Molecular response of human monocytes following interaction with colon cancer cells by pre-treatment with low-dose lipopolysaccharide. Anticancer Res 2015;35: 4473-7.
26. Raza H, John A, Shafarin J. Potentiation of LPS-induced apoptotic cell death in human hepatoma HepG2 cells by aspirin via ROS and mitochondrial dysfunction: Protection by N-acetyl cysteine. PLoS One 2016;11:e0157750. doi: 10.1371/ journal.pone.0159750.
27. Honda T, Inagawa H. Usefulness of monocytes/macrophages activated with low-dose lipopolysaccharide in tumor tissue and adipose tissue of obesity. Anticancer Res 2019;39:4475-8. doi: 10.21873/anticanres.13621.
28. Brooks D, Barr LC, Wiscombe S, Mcauley DF, Simpson AJ, Rostrron AJ. Human lipopolysaccharide models provide mechanistic and therapeutic insights into systemic and pulmonary inflammation. Eur Respir J 2020;56:1901298. doi: 10.1183/13993.003.01298-2019.
29. Millischer V, Heinzl M, Faka A, et al. Intravenous administration of LPS activates the kynurenine pathway in healthy male human subjects: a prospective placebocontrolled cross-over trial. J Neuroinflammation 2021;18:158. doi: 10.1186/s12974.021.02196-x.
30. Buters TP, Hameeteman PW, Jansen IME, et al. Intradermal lipopolysaccharide challenge as an acute in vivo inflammatory model in healthy volunteers. Br J Clin Pharmacol 2022;88:680- 90. doi: 10.1111/bcp.14999.
31. Larsson J, Hoppe E, Gautrois M, Cvijovic M, Jirstrand M. Optimizing study design in LPS challenge studies for quantifying drug induced inhibition of TNFα response: Did we miss the prime time? Eur J Pharm Sci 2022;176:106256. doi: 10.1016/j.ejps.2022.106256.
32. Lien EC, Westermark AM, Li Z, Sapp K M, Heiden M G V.Caloric restriction alters lipid metabolism to contribute to tumor growth inhibition. bioRxiv Epub ahead of print 2020. doi: 10.1101/2020.03.09.984302.
33. Lashinger LM, Harrison LM, Rasmussen AJ, et al. Dietary energy balance Heidenmodulation of Kras – and Ink4a/ Arf +/—driven pancreatic cancer: The role of insulin-like growth factor-I. Cancer Prev Res (Phila) 2013;6:1046-55. doi: 10.1158/1940-6207.CAPR-13-0185.
34. Brandhorst S, Longo VD. Fasting and caloric restriction in cancer prevention and treatment. Recent Results Cancer Res 2016;207:241-66. .doi: 10.1007/978-3-319-42118-6_12.
35. Sadeghian M, Rahmani S, Khalesi S, Hejazi E. A review of fasting effects on the response of cancer to chemotherapy. Clin Nutr 2021;40:1669-81. doi: 10.1016/j.clnu.2020.10.037.
36. Jain S, Dash P, Minz AP, et al. Lipopolysaccharide (LPS) enhances prostate cancer metastasis potentially through NF-κB activation and recurrent dexamethasone administration fails to suppress it in vivo. Prostate 2019.79:168-82. doi: 10.1002/ pros.23722.
37. Jiang Y, Ji X, Liu K, et al. Exosomal miR-200c-3p negatively regulates the migraion and invasion of lipopolysaccharide (LPS)-stimulated colorectal cancer (CRC). BMC Mol Cell Biol 2020;21:48. doi: 10.1186/s12860.020.00291-0.
38. Henke E, Nandigama R, Ergün S. Extracellular matrix in the tumor microenvironment and its impact on cancer therapy. Front Mol Biosci 2020;6:160. . doi: 10.3389/fmolb.2019.00160.
39. Salminen A, Huuskonen J, Ojala J, Kauppinen A, Kaarniranta K, Suuronen T. Activation of innate immunity system during aging: NF-kB signaling is the molecular culprit of inflammaging. Ageing Res Rev 2008;7:83-105. doi: 10.1016/j. arr.2007.09.002.
40. Baltimore D. Discovering NF-kappaB. Cold Spring Harb Perspect Biol 2009;1:a000026. doi: 10.1101/cshperspect. a000026.
41. Yarza R, Vela S, Solas M, Ramirez M J. c-Jun N-terminal kinase (JNK) signaling as a therapeutic target for Alzheimer’s disease. Front Pharmacol 2016;6:321. doi: 10.3389/fphar.2015.00321.
42. Nöthlings U, Wilkens LR, Murphy SP, Hankin J H, Henderson M E, Kolonel L N.Meat and fat intake as risk factors for pancreatic cancer: The multiethnic cohort study. J Natl Cancer Inst 2005;97:1458-65. doi: 10.1093/jnci/dji292.
43. Martinez-Useros J, Li W, Cabeza-Morales M, Foncillas J G. Oxidative stress: A new target for pancreatic cancer prognosis and treatment. J Clin Med 2017;6:29. doi: 10.3390/ jcm6030029.
44. Pan L, Yu L, Wang L, et al. Inflammatory stimuli promote oxidative stress in pancreatic acinar cells via Toll-like receptor 4/nuclear factor-κB pathway. Int J Mol Med 2018;42:3582-90. doi: 10.3892/ijmm.2018.3906.