Background & Aims
The mechanisms and pathways mediating pain and nociception are transcriptionally regulated. Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-?B) is a known regulator of gene expression and contributes to pain/nociception. To understand the relationship between NF-?B and the evolution of pain and hyperalgesia/nociception, it is imperative to unravel the molecular basis of this process. Previously, we demonstrated that OPRM1 (the gene for the µ-opioid receptor, MOR) is methylated and downregulated in oral cancer compared to matched normal tissues in the same patients; these patients complained of oral cancer pain [1]. We reversed methylation and re-expressed OPRM1 with non-viral transfection of OPRM1 DNA in two oral cancer mouse models; this process attenuated nociception (i.e., pain) in both oral cancer mouse models [2]. In this study, we further investigate the mechanism of controlling pain by re-expression of OPRM1 in a cancer via the NF-?B signaling pathway.
Methods
HSC-3 human tongue cancer cells were infected with a lentivirus carrying the gene for NF-?B-luciferase. HSC-3 cells expressing NF-?B-luciferase were transfected with a non-viral hybrid vector that combined cell-permeable peptide and cationic lipids [3] carrying the gene for OPRM1 or green fluorescent protein (GFP). Luminescence expression was detected with immunofluorescence staining and bioluminescence was measured with Bright-Glo Luciferase Assay System. HSC-3 cells expressing NF-?B-luciferase and MOR, or NF-?B-luciferase and GFP were inoculated into the tongue of athymic mice and the bioluminescent intensity of luciferase expression was subsequently measured using the IVIS® In Vivo Imaging System. In addition, localization and expression of MOR were confirmed and quantified using confocal fluorescence microscopy. Oral function and nociception were quantified using the dolognawmeter [4].
Results
Luciferase expression indicating NF-?B activity was attenuated in HSC-3 cells transfected with OPRM1 compared to GFP. The bioluminescence intensity of luciferase expression indicating NF-?B activity in OPRM1 group was significantly lower than GFP group at 11 days following inoculation (p < 0.05, t-test, n = 6). MOR expression was increased in HSC-3 cells transfected with OPRM1 compared to GFP or sham at 1 and 7 days following inoculation. Mice transfected with HSC-3 tumors expressing OPRM1 demonstrated significant antinociception compared to mice transfected with GFP or sham (p < 0.01, OPRM1 vs Sham, Two-way ANOVA Tukey’s multiple comparisons. n = 7).
Conclusions
Nonviral delivery of the OPRM1 gene targeted to the cancer microenvironment produces antinociception in a preclinical cancer model. The mechanism of antinociception involves down regulation of NF-?B. Nonviral gene delivery of OPRM1 is a potential treatment for oral cancer pain.
References
1.Viet CT, Dang D, Aouizerat BE, Miaskowski C, Ye Y, Viet CT, Ono K, Schmidt BL. OPRM1 Methylation Contributes to Opioid Tolerance in Cancer Patients. J Pain. 2017; 18(9):1046-1059.
2.Yamano S, Viet CT, Dang D, Dai J, Hanatani S, Takayama T,Kasai H, Imamura K, Campbell K, Ye Y, Dolan JC, Kwon M, Schneider SD, Schmidt BL. Ex vivo nonviral gene delivery of ?-opioid receptor to attenuate cancer-induced pain. Pain. 2017;158(2):240-251.
3.Yamano S, Dai J, Yuvienco C, Khapli S, Moursi AM, Montclare JK. Modified Tat peptide with cationic lipids enhances gene transfection efficiency via temperature-dependent and caveolae-mediated endocytosis, J Control Release. 2011;152(2):278-85.
4.Dolan JC, Lam DK, Achdjian SH, and Schmidt BL. The Dolognawmeter: A Novel Instrument and Assay to Quantify Nociception in Rodent Models of Orofacial Pain. J Neurosci Methods. 2010;187(2):207-15.
Presenting Author
Kenji Inoue
Poster Authors
Kenji Inoue
PhD
New York University College of Dentistry
Lead Author
Tu Huu Nguyen
NYU
Lead Author
John Dolan
DDS
Transrational Research Center, New York University College of Dentistry
Lead Author
Seiichi Yamano
DDS
Department of Prosthodontics, New York University Dentistry
Lead Author
Brian Schmidt
NYU Dentistry, Translational Research Center
Lead Author
Topics
- Specific Pain Conditions/Pain in Specific Populations: Cancer Pain & Palliative Care