Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Toll-like Receptor 4-mediated Apoptotic Cell Death in Primary Isolated Human Cervical Cancers

부인과질환 특이적 종양의 TLR4 매개성 apoptosis 유발에 관한 연구

  • Won, Jinyoung (Department of Rehabilitation Science, Graduate School of Inje University) ;
  • Hong, Yunkyung (u-Healthcare & Anti-aging Research Center (u-HARC), Inje University) ;
  • Park, Sookyoung (u-Healthcare & Anti-aging Research Center (u-HARC), Inje University) ;
  • Kim, Joo-Heon (Institute of Animal Medicine, College of Veterinary Medicine, Gyeongsang National University) ;
  • Hong, Yonggeun (Department of Rehabilitation Science, Graduate School of Inje University)
  • 원진영 (인제대학교 대학원 재활과학과) ;
  • 홍윤경 (인제대학교 u-항노화헬스케어연구소(u-HARC)) ;
  • 박수경 (인제대학교 u-항노화헬스케어연구소(u-HARC)) ;
  • 김주헌 (경상대학교 수의과대학 동물의학연구소) ;
  • 홍용근 (인제대학교 대학원 재활과학과)
  • Received : 2018.01.04
  • Accepted : 2018.03.17
  • Published : 2018.06.30
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Abstract

Toll-like receptor 4 (TLR4) has been implicated in cell proliferation and apoptosis in several types of cancer. In this study, the impact of TLR4 activation on apoptotic cell death in gynecologic cancers induced by lipopolysaccharide (LPS) was investigated. Cervical cancer cell lines were produced from isolated surgical specimens supplied by Paik Hospital. The primary cultures of normal myometrium and gynecologic cancers, including cervical, endometrial, and ovarian cancers, were used to examine the differences in morphological characteristics between normal and cancerous cells. A reverse transcription polymerase chain reaction analysis was used to determine the relative expression levels of TLR4 gene involved in apoptosis-associated signaling in cervical cancer cells. The cancer cell colonies showed a tendency to reach high levels of confluency compared with normal cells. In addition, an enhanced growth rate and loss of contact inhibition were observed in gynecologic cancer cells compared with normal cells (doubling times of 16.6 hr vs. 26 hr, respectively). The expression level of ITGA5, an alpha-5 integrin marker, was upregulated in normal myometrial cells, but this tendency was not exhibited in cervical cancer cells. Furthermore, p53 tumor suppressor gene expression was upregulated, whereas TLR4 and caspase-3 gene expressions were downregulated in cervical cancer cells. Notably, the expression levels of TLR4 and caspase-3 were increased significantly in LPS-treated cancer cells compared with those in non-LPS-treated cells. These results suggest that the TLR4-mediated caspase-dependent apoptotic signaling pathway could be suggested as a therapeutic target for the treatment of gynecologic cancers, including cervical cancers.

Toll 유사수용체의 TLR4는 세포자연사(apoptosis)와 관련하여 세포의 생존과 증식에 영향을 미치는 것으로 알려져 있다. 본 연구에서는 TLR4의 활성이 부인과 질환 특이적 종양세포의 세포사멸기작에 어떠한 영향을 미치는지 살펴보았다. TLR4의 활성에 의한 세포자연사를 확인하기 위하여 부인암 조직(자궁경부암, 자궁내막암, 난소암)에서 종양세포를 분리하여 초대배양시스템을 구축하였고, lipopolysaccharide (LPS)에 의한 TLR4의 활성유도 과정에서 종양세포의 형태학적 변화를 살펴보았다. 또한, TLR4 매개성 세포사멸 기작을 확인하기 위하여 역전사 중합효소 연쇄반응(RT-PCR)을 통해 유전자 분석을 진행하였다. 연구 결과, 부인암의 초대배양세포에서 세포접촉저지(contact inhibition)현상이 감소되었고, 세포의 배가시간(doubling time)이 단축되어, 종양세포의 성장률 변화를 확인하였다(p<0.05). 자궁근육층(정상조직)의 초대배양세포에서는 민무늬근육 확인 인자인 ITGA5 (an alpha5 integrin marker)의 유전자 발현이 나타났으나, 자궁경부조직의 초대배양세포에서는 발현변화를 확인할 수 없었다. 종양세포의 유전자분석 결과에서 p53과 같은 종양억제인자의 발현이 유의적으로 증가한 반면(p<0.05), 세포사멸 신호기작과 관련하여 TLR4와 Caspase-3의 발현은 감소하였다(Caspase-3, p<0.05). LPS를 처리한 종양세포에서는 LPS 비처리군과 비교 시, TLR4의 발현증가와 함께 Caspase-3의 발현변화가 동반되었다. 이러한 결과들은 TLR4 매개성 apoptosis 유도가 종양세포의 증식억제에 중요한 영향을 미치는 것을 의미하며, TLR4 신호기작을 이용한 종양세포의 새로운 치료적 접근법을 제시할 것으로 기대한다.

Keywords

References

  1. Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K. and Walter, P. 2002. The Cell Cycle and Programmed Cell Death. Mol. Biol. Cell. pp. 983-1026, 4th ed., Garland Science, NY, USA.
  2. Aubrey, B. J., Kelly, G. L., Janic, A., Herold, M. J. and Strasser, A. 2018. How does p53 induce apoptosis and how does this relate to p53-mediated tumour suppression? Cell. Death. Differ. 25, 104-113. https://doi.org/10.1038/cdd.2017.169
  3. Biewenga, P., Buist, M. R., Moerland, P. D., Ver Loren van Themaat, E., van Kampen, A. H., ten Kate, F. J. and Baas, F. 2008. Gene expression in early stage cervical cancer. Gynecol. Oncol. 108, 520-526. https://doi.org/10.1016/j.ygyno.2007.11.024
  4. Chhabra, S. and Kutchi, I. 2013. Fertility preservation in gynecologic cancers. Clin. Med. Insights Reprod. Health 7, 49-59.
  5. Fleck, J. L., Pavel, A. B. and Cassandras, C. G. 2016. Integrating mutation and gene expression cross-sectional data to infer cancer progression. BMC. Syst. Biol. 10, 12. https://doi.org/10.1186/s12918-016-0255-6
  6. Fukata, M., Chen, A., Klepper, A., Krishnareddy, S., Vamadevan, A. S., Thomas, L. S., Xu, R., Inoue, H., Arditi, M., Dannenberg, A. J. and Abreu, M. T. 2006. Cox-2 is regulated by Toll-like receptor-4 (TLR4) signaling: Role in proliferation and apoptosis in the intestine. Gastroenterology 131, 862-877. https://doi.org/10.1053/j.gastro.2006.06.017
  7. Fulda, S. 2013. Regulation of cell death in cancer-possible implications for immunotherapy. Front. Oncol. 3, 29.
  8. Goustin, A. S., Leof, E. B., Shipley, G. D. and Moses, H. L. 1986. Growth factors and cancer. Cancer Res. 46, 1015-1029.
  9. He, W., Liu, Q., Wang, L., Chen, W., Li, N. and Cao, X. 2007. TLR4 signaling promotes immune escape of human lung cancer cells by inducing immunosuppressive cytokines and apoptosis resistance. Mol. Immunol. 44, 2850-2859. https://doi.org/10.1016/j.molimm.2007.01.022
  10. Hanahan, D. and Weinberg, R. A. 2011. Hallmarks of cancer: the next generation. Cell 144, 646-674. https://doi.org/10.1016/j.cell.2011.02.013
  11. Hong, Y., Won, J., Lee, Y., Lee, S., Park, K., Chang, K. T. and Hong, Y. 2014. Melatonin treatment induces interplay of apoptosis, autophagy, and senescence in human colorectal cancer cells. J. Pineal. Res. 56, 264-274. https://doi.org/10.1111/jpi.12119
  12. Iguchi, Y. Ito, Y. M., Kataoka, F., Nomura, H., Tanaka, H., Chiyoda, T., Hashimoto, S., Nishimura, S., Takano, M., Yamagami, W., Susumu, N., Aoki, D. and Tsuda, H. 2014. Simultaneous analysis of the gene expression profiles of cancer and stromal cells in endometrial cancer. Genes Chromosomes Cancer 53, 725-737. https://doi.org/10.1002/gcc.22182
  13. Iribarren, K., Bloy, N., Buque, A., Cremer, I., Eggermont, A., Fridman, W. H., Fucikova, J., Galon, J., Spísek, R., Zitvogel, L., Kroemer, G. and Galluzzi, L. 2015. Trial Watch: Immunostimulation with Toll-like receptor agonists in cancer therapy. Oncoimmunology 5, e1088631.
  14. Janeway, C. A. Jr. and Medzhitov, R. 2002. Innate immune recognition. Annu. Rev. Immunol. 20, 197-216. https://doi.org/10.1146/annurev.immunol.20.083001.084359
  15. Kawai, T. and Akira, S. 2007. Signaling to NF-kappaB by Toll-like receptors. Trends Mol. Med. 13, 460-469. https://doi.org/10.1016/j.molmed.2007.09.002
  16. Kiraz, Y., Adan, A., Kartal Yandim, M. and Baran, Y. 2016. Major apoptotic mechanisms and genes involved in apoptosis. Tumour. Biol. 37, 8471-8486. https://doi.org/10.1007/s13277-016-5035-9
  17. Liu, Y., Hu, X., Han, C., Wang, L., Zhang, X., He, X. and Lu, X. 2015. Targeting tumor suppressor genes for cancer therapy. Bioessays 37, 1277-1286. https://doi.org/10.1002/bies.201500093
  18. Maxwell, S. A. and Davis, G. E. 2000. Differential gene expression in p53-mediated apoptosis-resistant vs. apoptosis-sensitive tumor cell lines. Proc. Natl. Acad. Sci. USA. 97, 13009-13014. https://doi.org/10.1073/pnas.230445997
  19. Minig, L., Padilla-Iserte, P. and Zorrero, C. 2016. The relevance of gynecologic oncologists to provide high-quality of care to women with gynecologic cancer. Front. Oncol. 5, 308.
  20. Mitchell, J. A., Shynlova, O., Langille, B. L. and Lye, S. J. 2004. Mechanical stretch and progesterone differentially regulate activator protein-1 transcription factors in primary rat myometrial smooth muscle cells. Am. J. Physiol. Endocrinol. Metab. 287, 439-445. https://doi.org/10.1152/ajpendo.00275.2003
  21. Palmberg, L. and Thyberg, J. 1986. Uterine smooth muscle cells in primary culture. Alterations in fine structure, cytoskeletal organization and growth characteristics. Cell Tissue Res. 246, 253-262. https://doi.org/10.1007/BF00215887
  22. Piccinini, A. M. and Midwood, K. S. 2010. DAMPening inflammation by modulating TLR signalling. Mediators Inflamm. 2010, pii: 672395.
  23. Pistritto, G., Trisciuoglio, D., Ceci, C., Garufi, A. and D'Orazi, G. 2016. Apoptosis as anticancer mechanism: function and dysfunction of its modulators and targeted therapeutic strategies. Aging 8, 603-619. https://doi.org/10.18632/aging.100934
  24. Preedy, V. R. and Watson, R. R. 2010. Handbook of Disease Burdens and Quality of Life Measures, pp. 803-823, Springer, NY, USA.
  25. Radogna, F., Dicato, M. and Diederich, M. 2015. Cancertype-specific crosstalk between autophagy, necroptosis and apoptosis as a pharmacological target. Biochem. Pharmacol. 94, 1-11. https://doi.org/10.1016/j.bcp.2014.12.018
  26. Scott, S. H. and Bahnson, B. J. 2011. Senescence marker protein 30: functional and structural insights to its unknown physiological function. Biomol. Concepts 2, 469-480.
  27. Smits, E. L., Ponsaerts, P., Berneman, Z. N. and Van, Tendeloo, V. F. 2008. The use of TLR7 and TLR8 ligands for the enhancement of cancer immunotherapy. Oncologist 13, 859-875. https://doi.org/10.1634/theoncologist.2008-0097
  28. Soussi, T. 2007. p53 alterations in human cancer: more questions than answers. Oncogene 26, 2145-2156. https://doi.org/10.1038/sj.onc.1210280
  29. Sporn, M. B. and Roberts, A. B. 1985. Autocrine growth factors and cancer. Nature 313, 745-747. https://doi.org/10.1038/313745a0
  30. Suh, D. H., Kim, M., Kim, H. J., Lee, K. H. and Kim, J. W. 2016. Major clinical research advances in gynecologic cancer in 2015. J. Gynecol. Oncol. 27, e53. https://doi.org/10.3802/jgo.2016.27.e53
  31. Tan, R. S., Ho, B., Leung, B. P. and Ding, J. L. 2014. TLR cross-talk confers specificity to innate immunity. Int. Rev. Immunol. 33, 443-453. https://doi.org/10.3109/08830185.2014.921164
  32. Tyson, J. J. and Novak, B. 2014. Control of cell growth, division and death: information processing in living cells. Interface Focus 4, 20130070. https://doi.org/10.1098/rsfs.2013.0070
  33. Wang, Y., Weng, Y., Shi, Y., Xia, X., Wang, S. and Duan, H. 2014. Expression and functional analysis of Toll-like receptor 4 in human cervical carcinoma. J. Membr. Biol. 247, 591-599. https://doi.org/10.1007/s00232-014-9675-7
  34. Witsch, E., Sela, M. and Yarden, Y. 2010. Roles for growth factors in cancer progression. Physiology (Bethesda) 25, 85-101.
  35. Yin, J. G., Liu, X. Y., Wang, B., Wang, D. Y., Wei, M., Fang, H. and Xiang, M. 2016. Gene expression profiling analysis of ovarian cancer. Oncol. Lett. 12, 405-412. https://doi.org/10.3892/ol.2016.4663
  36. Zhang, Y., Luo, F., Li, A., Qian, J., Yao, Z., Feng, X. and Chu, Y. 2014. Systemic injection of TLR1/2 agonist improves adoptive antigen-specific T cell therapy in glioma-bearing mice. Clin. Immunol. 154, 26-36. https://doi.org/10.1016/j.clim.2014.06.004