Immunohistochemical assessment of possible anticancer effect mechanisms of 2-(6,8-dimethyl-5-nitro-4-chloroquinoline-2-yl)- 5,6,7-trichloro-1,3-tropolone in PDX models of lung cancer

Purpose of the study. Evaluation of the expression of immunohistochemical tumor markers Ki-67, b-catenin, Bcl-2, P53, connexin 32 and connexin 43 when using 2-(6,8-dimethyl-5-nitro-4-chloroquinoline-2-yl)-5,6,7-trichloro-1,3-tropolone in mice with xenographs of squamous cell lung cancer.

Materials and methods. Subcutaneous PDX models of human squamous cell lung cancer were created in immunodeficient BALB/c Nude mice. A fragment of the patient’s tumor (3 × 3 × 3 mm) was implanted subcutaneously in the right thigh of a previously anesthetized mouse. 200 μl of 2-(6,8-dimethyl-5-nitro-4-chloroquinoline-2-yl)-5,6,7-trichloro-1,3-tropolone was administered orally using a probe in 12 doses once every 3 days. All animals were divided into groups depending on the tropolone doses: experimental groups 2–5 with doses of 0.0055, 0.055, 0.55 and 2.75 mg/g, respectively. The control group received 1 % starch gel which was tropolone carrier. The animals were euthanized 36 days after the start of the substance administration, and the tumor tissue was isolated and prepared for the IHC study according to the standard protocol. IHC reactions were performed using antibodies for Ki-67, b-catenin, Bcl-2, P53, connexin 32 and connexin 43.

Results. Higher tropolone doses were associated with decreased expression of Ki-67, b-catenin, and the Bcl-2 protein, but increased expression of the P53 protein. The dosage of tropolone and expression of connexin 43 were directly proportional.

Conclusion. Immunohistochemical analysis of expression of proteins in PDX models of human squamous cell lung cancer when using 2-(6,8-dimethyl-5-nitro-4-chloroquinoline-2-yl)-5,6,7-trichloro-1,3-tropolone showed the changes indicating its antitumor efficacy and suggesting a possible mechanism of action based on the activation of apoptosis.

1. Kit OI, Shaposhnikov AV, Zlatnik EY, Nikipelova EA, Novikova IA. Local cellular immunity in adenocarcinoma and large intes- tine polyps. Siberian Medical Review. 2012;4(76):11–16. (In Russ.). EDN: PBXSWF

2. Kit OI, Frantsiyants EM, Nikipelova EA, Komarova EF, Kozlova LS, Tavaryan IS, et al. Changes in markers of proliferation, neoangiogenesis and plasminogen activation system in rectal cancer tissue. Experimental and Clinical Gastroenterology. 2015;2(114):40–45. (In Russ.). EDN: THKCLP

3. Bang DN, Sayapin YuA, Nguyen HL, Duc D, Komissarov VN. Synthesis and cytotoxic activity of [benzo[b][1,4]oxazepino[7,6,5-de] quinolin-2-yl]-1,3-tropolones. Chemistry of Heterocyclic Compounds. 2015;51(3):291–294.

4. https://doi.org/10.1007/s10593-015-1697-2

5. Tkachev VV, Shilov GV, Aldoshin SM, Sayapin YA, Tupaeva IO, Gusakov EA, et al. Structure of 2-(benzoxazole-2-yl)- 5,7- di(tert-butyl)-4-nitro-1,3-tropolone. Journal of Structural Chemistry. 2018;59(1):197–200. https://doi.org/10.1134/s0022476618010316

6. Burbaeva GSh, Androsova LV, Savushkina OK. Binding of colchicine to tubulin in the brain structuresin normal conditions and in schizophrenia. Нейрохимия. 2020;37(2):183–187. (In Russ.). https://doi.org/10.31857/s1027813320010069 , EDN: UMXPLK

7. Alkadi H, Khubeiz MJ, Jbeily R. Colchicine: A Review on Chemical Structure and Clinical Usage. Infect. Disord. Drug Targets. 2018;18(2):105–121. https://doi.org/10.2174/1871526517666171017114901

8. Florian S, Mitchison TJ. Anti-Microtubule Drugs. Methods Mol Biol. 2016;1413:403–421. https://doi.org/10.1007/978-1-4939-3542-0_25

9. Zhang G, He J, Ye X, Zhu J, Hu X, Minyan Sh, et al. β-Thujaplicin induces autophagic cell death, apoptosis, and cell cycle ar- rest through ROS-mediated Akt and p38/ERK MAPK signaling in human hepatocellular carcinoma. Cell Death Dis. 2019 Mar 15;10(4):255. doi: https://doi.org/10.1038/s41419-019-1492-6

10. Chen SM, Wang BY, Lee CH, Lee HT, Li JJ, Hong GC, et al. Hinokitiol up-regulates miR-494-3p to suppress BMI1 expression and inhibits self-renewal of breast cancer stem/progenitor cells. Oncotarget. 2017 Jun 27;8(44):76057–76068. https://doi.org/10.18632/oncotarget.18648

11. Haney SL, Allen C, Varney ML, Dykstra KM, Falcone ER, Colligan SH, et al. Novel tropolones induce the unfolded protein re- sponse pathway and apoptosis in multiple myeloma cells. Oncotarget. 2017 Jun 16;8(44):76085–76098. https://doi.org/10.18632/oncotarget.18543

12. Lukbanova EA, Mindar MV, Dzhenkova EA, Maksimov AYu, Goncharova AS, Shatova YuS, et al. Experimental approach to obtaining subcutaneous xenograft of non-small cell lung cancer. Research'n Practical Medicine Journal. 2022;9(2):65–76. (In Russ.). https://doi.org/10.17709/2410-1893-2022-9-2-5, EDN: SWTKGU

13. Kolesnikov EN, Lukbanova EA, Vanzha LV, Maksimov AYu, Kit SO, Goncharova AS, et al. The method of anesthesia in BALB/c Nude mice during surgical interventions. Patent RU No. 2712916, publ. 03.02.2020, Bull. No 4. (In Russ.).

14. Minkin VI, Kit OI, Goncharova AS, Lukbanova EA, Sayapin YuA, Gusakov EA, et al. An agent with cytotoxic activity against a cell culture of non-small cell lung cancer A 549. Patent of the Russian Federation. RU2741311 C1. Application No. 2020123736 dated 07/17/20. (In Russ.).

15. Miller I, Min M, Yang C, Tian C, Gookin S, Carter D, et al. Ki67 is a Graded Rather than a Binary Marker of Proliferation versus Quiescence. Cell Rep. 2018 Jul 31;24(5):1105–1112.e5. https://doi.org/10.1016/j.celrep.2018.06.110

16. Albayrak G, Demirtas Korkmaz F. Memantine shifts cancer cell metabolism via AMPK1/2 mediated energetic switch in A549 lung cancer cells. EXCLI J. 2021 Feb 4;20:223–231.

17. Gioacchini FM, Alicandri-Ciufelli M, Rubini C, Magliulo G, Re M. Prognostic value of Bcl-2 expression in squamous cell carci- noma of the larynx: a systematic review. Int J Biol Markers. 2015 May 26;30(2):e155–160. https://doi.org/10.5301/jbm.5000116

18. Lee YS, Choi KM, Kim W, Jeon YS, Lee YM, Hong JT, et al. Hinokitiol inhibits cell growth through induction of S-phase arrest and apoptosis in human colon cancer cells and suppresses tumor growth in a mouse xenograft experiment. J Nat Prod. 2013 Dec 27;76(12):2195–2202. https://doi.org/10.1021/np4005135

19. Aasen T, Johnstone S, Vidal-Brime L, Lynn KS, Koval M. Connexins: Synthesis, Post-Translational Modifications, and Traf- ficking in Health and Disease. Int J Mol Sci. 2018 Apr 26;19(5):1296. https://doi.org/10.3390/ijms19051296

20. Ruch RJ. Connexin43 Suppresses Lung Cancer Stem Cells. Cancers (Basel). 2019 Feb 2;11(2):175. https://doi.org/10.3390/cancers11020175

21. Spagnol G, Trease AJ, Zheng L, Gutierrez M, Basu I, Sarmiento C, et al. Connexin43 Carboxyl-Terminal Domain Directly In- teracts with β-Catenin. Int J Mol Sci. 2018 May 24;19(6):1562. https://doi.org/10.3390/ijms19061562

22. Aasen T, Mesnil M, Naus CC, Lampe PD, Laird DW. Gap junctions and cancer: communicating for 50 years. Nat Rev Cancer. 2016 Dec;16(12):775–788. https://doi.org/10.1038/nrc.2016.105. Erratum in: Nat Rev Cancer. 2017 Jan;17 (1):74.

23. Yang Y, Zhang N, Zhu J, Hong XT, Liu H, Ou YR, et al. Downregulated connexin32 promotes EMT through the Wnt/β-catenin pathway by targeting Snail expression in hepatocellular carcinoma. Int J Oncol. 2017 Jun;50(6):1977–1988. https://doi.org/10.3892/ijo.2017.3985