3 Biotech

, 9:159 | Cite as

Synergistic effect of co-treatment with all-trans retinoic acid and 9-cis retinoic acid on human lung cancer cell line at molecular level

  • Esther Sathya Bama
  • V. M. Berlin GraceEmail author
  • Viswanathan Sundaram
  • Perinba Dansiha Jesubatham
Original Article


The major challenge in treating cancers with ATRA is the limited availability inside the cell and resistance developed in prolonged treatment. We made an attempt for co-treatment of human NSCLC cell lines (A549) with ATRA and its isomeric precursor (9cisRA). In this study, the growth inhibitory effect of ATRA, 9cisRA and combination of both were tested in A549 cells by MTT and Trypan blue assays. As the effects of retinoid are mediated through their receptors, their gene expression levels were analyzed by RT-PCR. The target gene receptor, RAR-β protein expression, was analyzed by immunocytochemistry. The cancer cell (A549) growth inhibitory effect was significantly (p ≤ 0.001) enhanced in combination treatment when compared with the result of individual treatments. The mRNA expression levels of both RAR-β and RXR-β were found to be increased in co-treatment (band density of 0.75 and 0.806, respectively) when compared with 9cisRA treatment (0.25 and 0.112) and ATRA treatment (0.01 and 0.081). A concomitant enhancement in the target RAR-β protein expression was observed in co-treated cells when compared with individual treatments. We thus conclude that the co-treatment had increased the availability of ATRA, by isomerization of the 9cisRA which then resulted in an increased expression of both RAR-β and RXR-β receptors and the target protein RAR-β which in turn inhibited lung cancer cell growth. Our study results have explored the mechanism of synergistic effect of co-treatment with ATRA and 9cisRA and further preclinical studies are necessary to validate the application of co-treatment of retinoid in clinical use.


9-Cis retinoic acid ATRA Retinoic acid receptor Retinoid X receptor RT-PCR Immunocytochemistry 



The authors would like to acknowledge the Karunya Institute of Technology and Sciences for the financial support given through Karunya Short-Term Project Grant. We also acknowledge partial financial support shared from DST-SERB and DBT, Govt. of India. We would also like to acknowledge the valuable technical support of Dr. C. Vani, Assistant Professor and Ms. Jissin Mathew, Research Scholar, Department of Biotechnology, Karunya Institute of Technology and Sciences, Coimbatore.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Altucci L, Leibowitz MD, Ogilvie KM, De Lera AR, Gronemeyer H (2007) RAR and RXR modulation in cancer and metabolic disease. Nat Rev Drug Discovery 6(10):793–810Google Scholar
  2. Bogos K, Renyi-Vamos F, Kovacs G, Tovari J, Dome B (2008) Role of retinoic receptors in lung carcinogenesis. J Exp Clin Cancer Res 27(1):18Google Scholar
  3. Brabender J, Metzger R, Salonga D, Danenberg KD, Danenberg PV, Hölscher AH, Schneider PM (2005) Comprehensive expression analysis of retinoic acid receptors and retinoid X receptors in non-small cell lung cancer: implications for tumor development and prognosis. Carcinogenesis 26(3):525–530Google Scholar
  4. Bushue N, Wan Y-JY (2010) Retinoid pathway and cancer therapeutics. Adv Drug Deliv Rev 62(13):1285–1298Google Scholar
  5. Buskohl PR, Gould RA, Curran S, Archer SD, Butcher JT (2012) Multidisciplinary inquiry-based investigation learning using an ex vivo chicken culture platform: role of vitamin A on embryonic morphogenesis. Am Biol Teacher 74(9):636–643Google Scholar
  6. Cheung B, Yan J, Smith SA, Nguyen T, Lee M, Kavallaris M, Norris MD, Haber M, Marshall GM (2003) Growth inhibitory retinoid effects after recruitment of retinoid X receptor β to the retinoic acid receptor β promoter. Int J Cancer 105(6):856–867Google Scholar
  7. Cras A, Darsin-Bettinger D, Balitrand N, Cassinat B, Soulie A, Toubert M, Delva L, Chomienne C (2007) Epigenetic patterns of the retinoic acid receptor β2 promoter in retinoic acid-resistant thyroid cancer cells. Oncogene 26(27):4018–4024Google Scholar
  8. Di Masi A, Leboffe L, De Marinis E, Pagano F, Cicconi L, Rochette-Egly C, Lo-Coco F, Ascenzi P, Nervi C (2015) Retinoic acid receptors: from molecular mechanisms to cancer therapy. Mol Aspects Med 41:1–115Google Scholar
  9. Dragnev KH, Rigas JR, Dmitrovsky E (2000) The retinoids and cancer prevention mechanisms. Oncologist 5(5):361–368Google Scholar
  10. Han S, Fukazawa T, Yamatsuji T, Matsuoka J, Miyachi H, Maeda Y, Durbin M, Naomoto Y (2010) Anti-tumor effect in human lung cancer by a combination treatment of novel histone deacetylase inhibitors: SL142 or SL325 and retinoic acids. PLoS ONE 5(11):e13834Google Scholar
  11. Hanahan D, Weinberg RA (2000) The hallmarks of cancer. Cell 100(1):57–70Google Scholar
  12. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D (2011) Global cancer statistics. Cancer J Clin 61(2):69–90Google Scholar
  13. Lei M, de Thé H (2003) Retinoids and retinoic acid receptor in cancer. Eur J Cancer Suppl 1(2):13–18Google Scholar
  14. Lotan R (1999) Aberrant expression of retinoid receptors and lung carcinogenesis. Oxford University Press, OxfordGoogle Scholar
  15. Mi L, Wang X, Govind S, Hood BL, Veenstra TD, Conrads TP, Saha DT, Goldman R, Chung F-L (2007) The role of protein binding in induction of apoptosis by phenethyl isothiocyanate and sulforaphane in human non-small lung cancer cells. Can Res 67(13):6409–6416Google Scholar
  16. Minna JD, Roth JA, Gazdar AF (2002) Focus on lung cancer. Cancer Cell 1(1):49–52Google Scholar
  17. Moison C, Senamaud-Beaufort C, Fourrière L, Champion C, Ceccaldi A, Lacomme S, Daunay A, Tost J, Arimondo PB, Guieysse-Peugeot A-L (2013) DNA methylation associated with polycomb repression in retinoic acid receptor β silencing. FASEB J 27(4):1468–1478Google Scholar
  18. Pavan B, Dalpiaz A, Biondi C, Nieddu M, De Luca A, Prasad PD, Paganetto G, Favaloro B (2008) An RPE cell line as a useful in vitro model for studying retinoic acid receptor β: expression and affinity. Biosci Rep 28(6):327–334Google Scholar
  19. Pettersson F, Dalgleish A, Bissonnette R, Colston K (2002) Retinoids cause apoptosis in pancreatic cancer cells via activation of RAR-γ and altered expression of Bcl-2/Bax. Br J Cancer 87(5):555–561Google Scholar
  20. Ramya D, Siddikuzzaman Manjamalai A, Berlin Grace V (2012) Chemoprotective effect of all-trans retinoic acid (ATRA) on oxidative stress and lung metastasis induced by benzo (a) pyrene. Immunopharmacol Immunotoxicol 34(2):317–325Google Scholar
  21. Rhinn M, Dollé P (2012) Retinoic acid signalling during development. Development 139(5):843–858Google Scholar
  22. Siddikuzzaman, Grace VB (2012) Inhibition of metastatic lung cancer in C57BL/6 mice by liposome encapsulated all trans retinoic acid (ATRA). Int Immunopharmacol 14(4):570–579Google Scholar
  23. Soria J-C, Xu X, Liu DD, Lee JJ, Kurie J, Morice RC, Khuri F, Mao L, Hong WK, Lotan R (2003) Retinoic acid receptor β and telomerase catalytic subunit expression in bronchial epithelium of heavy smokers. J Natl Cancer Inst 95(2):165–168Google Scholar
  24. Stewart B, Wild CP (2017) World cancer report 2014. HealthGoogle Scholar
  25. Sun S-Y, Wan H, Yue P, Hong WK, Lotan R (2000) Evidence that retinoic acid receptor β induction by retinoids is important for tumor cell growth inhibition. J Biol Chem 275(22):17149–17153Google Scholar
  26. Szabó DR, Baghy K, Szabó PM, Zsippai A, Marczell I, Nagy Z, Varga V, Éder K, Tóth S, Buzás EI (2014) Antitumoral effects of 9-cis retinoic acid in adrenocortical cancer. Cell Mol Life Sci 71(5):917–932Google Scholar
  27. Wen X, Li Y, Hu K, Dai C, Liu Y (2005) Hepatocyte growth factor receptor signaling mediates the anti-fibrotic action of 9-cis-retinoic acid in glomerular mesangial cells. Am J Pathol 167(4):947–957Google Scholar
  28. Zuccari G, Carosio R, Fini A, Montaldo P, Orienti I (2005) Modified polyvinylalcohol for encapsulation of all-trans-retinoic acid in polymeric micelles. J Control Release 103(2):369–380Google Scholar

Copyright information

© King Abdulaziz City for Science and Technology 2019

Authors and Affiliations

  • Esther Sathya Bama
    • 1
  • V. M. Berlin Grace
    • 1
    Email author
  • Viswanathan Sundaram
    • 1
  • Perinba Dansiha Jesubatham
    • 1
  1. 1.Department of BiotechnologyKarunya Institute of Technology and SciencesCoimbatoreIndia

Personalised recommendations