Microindoline 581, an indole derivative from Microbacterium sp. RP581 as a novel selective antineoplastic agent to combat hepatic cancer cells: Production optimization and structural elucidation

Document Type : Research article


1 Molecular Biotechnology Laboratory, Department of Biology, Faculty of Science, Shiraz. University, Iran. Department of Pharmaceutical Chemistry, Institute of Pharmacy, Center for Molecular Biosciences Innsbruck, University of Innsbruck, CCB–Centrum for Chemistry and Biomedicine, Innrain 80-82, 6020 Innsbruck, Austria.

2 Department of Pharmaceutical Chemistry, Institute of Pharmacy, Center for Molecular Biosciences Innsbruck, University of Innsbruck, CCB–Centrum for Chemistry and Biomedicine, Innrain 80-82, 6020 Innsbruck, Austria.

3 Immunobiology and Stem Cell Laboratory, Department of Internal Medicine V (Hematology and Oncology), Innsbruck Medical University, Anichstrasse 35, 6020 Innsbruck, Austria. Tyrolean Cancer Research Institute, Innrain 66, 6020 Innsbruck, Austria.

4 Molecular Biotechnology Laboratory, Department of Biology, Faculty of Science, Shiraz University, Iran.


Screening of bioactive compounds with potential binding affinity to DNA as one of the target molecules in fighting against cancer cells has gained the attention of many scientists. Finding such compounds in the cellular content of microorganisms, especially marine bacteria as valuable and rich natural resources, is of great importance. Microbacterium sp. RP581, as a member of Actinobacteria phylum, was isolated from the Persian Gulf coastal area and the production of the target compound was optimized using statistical methods in cheap culture ingredients. The purification of the target compound was performed by flash chromatography and preparative HPLC. Both molecular and structural analyses indicated that the compound was an indole derivate which was tentatively named as Microindoline 581. Interaction of Microindoline 581 with genomic and circular DNA revealed that this compound can cause double- strand breaks through binding to the DNA. The analysis of cellular growth and proliferation of various cancer cell lines suggested proper and specific effect Microindoline 581 towards HepG2 cells with an IC50 of 172.2 ± 1.7 µM. Additional studies on cell migration inhibition and cell-death induction indicated a concentration-dependent inhibitory effect on proliferation and induction of death of HepG2 cells. The selective action of Microindoline 581 which was isolated from the Microbacterium sp. RP581 in killing HepG2 cells might be due to its specific metabolism in those cells as a precursor.

Graphical Abstract

Microindoline 581, an indole derivative from  Microbacterium  sp. RP581 as a novel selective antineoplastic agent to combat hepatic cancer cells: Production optimization and structural elucidation


(1) Newman DJ and Cragg GM. Natural products as sources of new drugs over the last 25 years⊥. J. Nat. Prod. (2007) 70: 461-77.
(2) Newman DJ and Cragg GM. Natural products as sources of new drugs over the 30 years from 1981 to 2010. J. Nat. Prod. (2012) 75: 311-35.
(3) Bernardini S, Tiezzi A, Laghezza Masci V and Ovidi E. Natural products for human health: an historical overview of the drug discovery approaches. Nat. Prod. Res. (2018) 32: 1926-50.
(4) Barka EA, Vatsa P, Sanchez L, Gaveau-Vaillant N, Jacquard C, Klenk H-P, Clément C, Ouhdouch Y and van Wezel GP. Taxonomy, physiology, and natural products of Actinobacteria. Microbiol. Mol. Biol. Rev. (2016) 80: 1-43.
(5) Molinski TF, Dalisay DS, Lievens SL and Saludes JP. Drug development from marine natural products. Nature reviews. Drug Discov. (2009) 8: 69. شماره صفحه
(6) Pettit GR, Herald CL, Doubek DL, Herald DL, Arnold E and Clardy J. Isolation and structure of bryostatin 1. J. Am. Chem. Soc. (1982) 104: 6846-8.
(7) Feling RH, Buchanan GO, Mincer TJ, Kauffman CA, Jensen PR and Fenical W. Salinosporamide A: a highly cytotoxic proteasome inhibitor from a novel microbial source, a marine bacterium of the new genus Salinospora. Ang. Chem. Int. Ed. (2003) 42: 355-7.
(8) Yun TY, Feng RJ, Zhou DB, Pan YY, Chen YF, Wang F, Yin LY, Zhang YD and Xie JH. Optimization of fermentation conditions through response surface methodology for enhanced antibacterial metabolite production by Streptomyces sp. 1-14 from cassava rhizosphere. PLOS One (2018) 13: e0206497.
(9) Ahsan T, Chen J, Wu Y and Irfan M. Application of response surface methodology for optimization of medium components for the production of secondary metabolites by Streptomyces diastatochromogenes KX852460. AMB. Exp. (2017) 7: 96.
(10) Torre LA, Siegel RL, Ward EM and Jemal A. Global cancer incidence and mortality rates and trends- an update. Cancer Epidem. Biomar. (2016) 25: 16-27.
(11) Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA and Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA. Cancer J. Clin. (2018) 68: 394-424.
(12) Brown ZJ, Heinrich B and Greten TF. Mouse models of hepatocellular carcinoma: an overview and highlights for immunotherapy research. Nat. Rev. Gastroenterol. Hepatol. (2018) 15: 536-54.
(13) Vahidi H, Barabadi H and Saravanan M. Emerging selenium nanoparticles to combat cancer: a systematic review. J. Cluster Sci. (2020) 31: 301-9.
(14) Zhai B, Hu F, Jiang X, Xu J, Zhao D, Liu B, Pan S, Dong X, Tan G and Wei Z. Inhibition of Akt reverses the acquired resistance to sorafenib by switching protective autophagy to autophagic cell death in hepatocellular carcinoma. Mol. Cancer Ther. (2014) 13: 1589-98.
(15) Chen K-F, Chen HL, Tai WT, Feng WC, Hsu C-H, Chen PJ and Cheng A-L. Activation of phosphatidylinositol 3-kinase/Akt signaling pathway mediates acquired resistance to sorafenib in hepatocellular carcinoma cells. J. Pharm. Exper. Ther. (2011) 337: 155-61.
(16) Liu D, Lin H, Proksch P, Tang X, Shao Z and Lin W. Microbacterins A and B, New Peptaibols from the Deep Sea Actinomycete Microbacterium sediminis sp. nov. YLB-01(T). Org. Let. (2015) 17: 1220-3.
(17) Oh I-K, Yoo S-H, Bae I-Y, Cha JH and Lee HG. Effects of Microbacterium laevaniformans Levans molecular weight on cytotoxicity. J. Microbiol. Biotechnol. (2004) 14: 985-90.
(18) Wicke C, Hüners M, Wray V, Nimtz M, Bilitewski U and Lang S. Production and Structure Elucidation of Glycoglycerolipids from a Marine Sponge-Associated Microbacterium Species. J. Nat. Prod. (2000) 63: 621-6.
(19) Lee L-H, Zainal N, Azman A-S, Eng SK, Goh B-H, Yin W-F, Ab Mutalib NS and Chan KG. Diversity and antimicrobial activities of actinobacteria isolated from tropical mangrove sediments in Malaysia. Sci. World J. (2014) 2014: 698-712.
(20) Mincer TJ, Jensen PR, Kauffman CA and Fenical W. Widespread and persistent populations of a major new marine actinomycete taxon in ocean sediments. Appl. Environ. Microbiol. (2002) 68: 5005-11.
(21) Karbalaei-Heidari HR, Ziaee A-A, Schaller J and Amoozegar MA. Purification and characterization of an extracellular haloalkaline protease produced by the moderately halophilic bacterium, Salinivibrio sp. strain AF-2004. Enz. Microbial. Technol. (2007) 40: 266-72.
(22) Kumar S, Stecher G and Tamura K. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. (2016) 33: 1870-74.
(23) Hwang YL and Hwang KC. Nonlinear Stern-Volmer Fluorescence Quenching of Pyrene by C60/70. Fullerene Sci. Technol. (1999) 7: 437-54.
(24) Fu X-B, Zhang J-J, Liu DD, Gan Q, Gao HW, Mao ZW and Le XY. Cu (II)–dipeptide complexes of 2-(4′-thiazolyl)benzimidazole: Synthesis, DNA oxidative damage, antioxidant and in-vitro antitumor activity. J. Inorg. Biochem. (2015) 143: 77-87.
(25) Pournejati R, Karbalaei Heidari HR and Budisa N. Secretion of recombinant archeal lipase mediated by SVP2 signal peptide in Escherichia coli and its optimization by response surface methodology. Protien Expr. Purif. (2014) 101: 84-90.
(26) Pournejati R and Karbalaei Heidari HR. Searching for new bioactive metabolites from marine bacteria in the Persian Gulf: Antibacterial, cytotoxic and anti-inflammatory agents. Biomacr. J. (2018) 4: 78-92.
(27) Ruiz B, Chávez A, Forero A, García-Huante Y, Romero A, Sánchez M, Rocha D, Sánchez B, Rodríguez-Sanoja R and Sánchez S. Production of microbial secondary metabolites: regulation by the carbon source. Crit. Rev. Microbiol. (2010) 36: 146-67.
(28) Murthy HN, Lee EJ and Paek KY. Production of secondary metabolites from cell and organ cultures: strategies and approaches for biomass improvement and metabolite accumulation. Plant Cell Tiss. Org. (PCTOC) (2014) 118: 1-16.
(29) Szatrowski TP and Nathan CF. Production of large amounts of hydrogen peroxide by human tumor cells. Cancer Res. (1991) 51: 794-8.
(30) Cragg GM and Newman DJ. Natural products: a continuing source of novel drug leads. Biochim. Biophys. Acta (BBA) (2013) 1830: 3670-95.
(31) Niu S, Zhou TT, Xie CL, Zhang GY and Yang XW. Microindolinone A, a Novel 4,5,6,7-Tetrahydroindole, from the Deep-Sea-Derived Actinomycete Microbacterium sp. MCCC 1A11207. Marin Drugs (2017) 15: 230.
(32) de Sa Alves FR, Barreiro EJ and Fraga CA. From nature to drug discovery: the indole scaffold as a 'privileged structure'. Mini Rev. Med. Chem. (2009) 9: 782-93.
(33) Shimazaki Y, Yajima T, Takani M and Yamauchi O. Metal complexes involving indole rings: structures and effects of metal–indole interactions. Coordin. Chem. Rev. (2009) 253: 479-92.
(34) Lafayette EA, de Almeida SMV, Cavalcanti Santos RV, de Oliveira JF, Amorim CAdC, da Silva RMF, Pitta MGdR, Pitta IdR, de Moura RO, de Carvalho Júnior LB, de Melo Rêgo MJB and de Lima MdCA. Synthesis of novel indole derivatives as promising DNA-binding agents and evaluation of antitumor and antitopoisomerase I activities. Eur. J. Med. Chem. (2017) 136: 511-22.
(35) Farahat AA, Ismail MA, Kumar A, Wenzler T, Brun R, Paul A, Wilson WD and Boykin DW. Indole and benzimidazole bichalcophenes: Synthesis, DNA binding and antiparasitic activity. Eur. J. Med. Chem. (2018) 143: 1590-6.
(36) Netz N and Opatz T. Marine indole alkaloids. Marine Drugs (2015) 13: 4814-914.
(37) Chiang DY, Villanueva A, Hoshida Y, Peix J, Newell P, Minguez B, LeBlanc AC, Donovan DJ, Thung SN and Sole M. Focal gains of VEGFA and molecular classification of hepatocellular carcinoma. Cancer Res. (2008) 68: 6779-88.
(38) Calvisi DF, Ladu S, Gorden A, Farina M, Conner EA, Lee JS, Factor VM and Thorgeirsson SS. Ubiquitous activation of Ras and Jak/Stat pathways in human HCC. Gastroenterol. (2006) 130: 1117-28.
(39) Takami T, Kaposi-Novak P, Uchida K, Gomez-Quiroz LE, Conner EA, Factor VM and Thorgeirsson SS. Loss of hepatocyte growth factor/c-Met signaling pathway accelerates early stages of N-nitrosodiethylamine–induced hepatocarcinogenesis. Cancer Res. (2007) 67: 9844-51.
(40) Niu L, Liu L, Yang S, Ren J, Lai PBS and Chen GG. New insights into sorafenib resistance in hepatocellular carcinoma: Responsible mechanisms and promising strategies. Biochim. Biophys. Acta (2017) 1868: 564-70.
(41) Lim SD, Sun C, Lambeth JD, Marshall F, Amin M, Chung L, Petros JA and Arnold RS. Increased Nox1 and hydrogen peroxide in prostate cancer. The Prostate (2005) 62: 200-7.
(42) Yang Y, Karakhanova S, Werner J and V Bazhin A. Reactive oxygen species in cancer biology and anticancer therapy. Cur. Med. Chem. (2013) 20: 3677-92.