Cell Biology and Toxicology

, Volume 30, Issue 5, pp 269–288 | Cite as

SRJ23, a new semisynthetic andrographolide derivative: in vitro growth inhibition and mechanisms of cell cycle arrest and apoptosis in prostate cancer cells

  • Hui Chyn Wong
  • Charng Choon Wong
  • Sreenivasa Rao Sagineedu
  • Seng Cheong Loke
  • Nordin Haji Lajis
  • Johnson Stanslas
Original Research



3,19-(3-Chloro-4-fluorobenzylidene)andrographolide (SRJ23), a new semisynthetic derivative of andrographolide (AGP), exhibited selectivity against prostate cancer cells in the US National Cancer Institute (NCI) in vitro anti-cancer screen. Herein, we report the in vitro growth inhibition and mechanisms of cell cycle arrest and apoptosis induced by SRJ23.


3-(4,5-Dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used in assessing in vitro growth inhibition of compounds against prostate cancer (PC-3, DU-145 and LNCaP) and mouse macrophage (RAW 264.7) cell lines. Flow cytometry was utilised to analyse cell cycle distribution, whereas fluorescence microscopy was performed to determine morphological cell death. DNA fragmentation and annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) flow cytometry were done to confirm apoptosis induced by SRJ23. Quantitation of cell cycle and apoptotic regulatory proteins were determined by immunoblotting.


AGP and SRJ23 selectively inhibited the growth of prostate cancer cells compared with RAW 264.7 cells at low micromolar concentrations; however, SRJ23 was more potent. Mechanistically, SRJ23-treated PC-3 cells displayed down-regulation of cyclin-dependent kinase (CDK) 1 without affecting levels of CDK4 and cyclin D1. However, SRJ23 induced down-regulation of CDK4 and cyclin D1 but without affecting CDK1 in DU145 and LNCaP cell lines. DNA histogram analysis revealed that the SRJ23 induced G2/M in PC-3 cells but G1 arrest in DU-145 and LNCaP cells. Morphologically, both compounds induced predominantly apoptosis, which was further confirmed by DNA fragmentation and annexin V-FITC staining. The DNA fragmentation was inhibited in the presence of caspase 8 inhibitor (Z-IETD-FMK). Apoptosis was associated with an increase in caspase 8 expression and activation. This thought to have induced cleavage of Bid into t-Bid. Additionally, increased expression and activation of caspase 9 and Bax proteins were apparent, with a concomitant down-regulation of Bcl-2 protein. Similar apoptosis cascade of events was observed in SRJ23-treated DU145 and LNCaP cell lines.


SRJ23 inhibited the growth of prostate cancer cells by inducing G2/M and G1 arrest via down-regulation of CDK1, and CDK4 and cyclin, respectively, and initiated caspase-8-mediated mitochondrial apoptosis. Taken together, these data support the potential of this compound as a new anti-prostate cancer agent.


Andrographolide derivative Apoptosis Caspases Cyclin-dependent kinases G2/M arrest G1 arrest Prostate cancer 





Acridine orange


Cyclin-dependent kinase


Enzyme chemiluminescence


Extracellular signal-regulated kinase


Fluorescein isothiocyanate


Polyacrylamide gel electrophoresis


Propidium iodide

RNase A

Ribonuclease A


Sodium dodecyl sulphate


Self-organising map







The Ministry of Higher Education is thanked for funding this project through the Research University Grant Scheme (RUGS) (Grant 04-02-12-2017RU). Hui Chyn Wong is a recipient of the National Science Fellowship (NSF) awarded by the Ministry of Science, Technology and Innovation of Malaysia (MOSTI). We are grateful to Mr. Lim Siang Hui from Cancer Research Initiatives Foundation (CARIF), Malaysia, for assistance in the flow cytometry work. We are thankful to Mr. Teh Yuan Han (Pharmacotherapeutics Unit, Department of Medicine, Faculty of Medicine and Health Sciences, UPM) for his editorial assistance.

Conflict of interest



  1. Chiou WF, Lin JJ, Chen CF. Andrographolide suppresses the expression of inducible nitric oxide synthase in macrophage and restores the vasoconstriction in rat aorta treated with lipopolysaccharide. Br J Pharmacol. 1998;125:327–34.PubMedCrossRefPubMedCentralGoogle Scholar
  2. Danial NN, Korsmeyer SJ. Cell death: critical control points. Cell. 2004;116:205–19.PubMedCrossRefGoogle Scholar
  3. Gunn EJ, Williams JT, Huynh DT, Iannotti MJ, Han C, Barrios FJ, et al. The natural products parthenolide and andrographolide exhibit anti-cancer stem cell activity in multiple myeloma. Leuk Lymphoma. 2011;52(6):1085–97.PubMedCrossRefGoogle Scholar
  4. Harbour JW, Luo RX, Dei Santi A, Postigo AA, Dean DC. CDK phosphorylation triggers sequential intramolecular interactions that progressively block Rb functions as cell move through G1. Cell. 1998;98:859–69.CrossRefGoogle Scholar
  5. Hocker HJ, Cho KJ, Chen CYK, Rambahal N, Sagineedu SR, Shaari K, et al. Andrographolide derivatives inhibit guanine nucleotide exchange and abrogate oncogenic Ras function. Proc Natl Acad Sci U S A. 2013;110(25):10201–6.PubMedCrossRefPubMedCentralGoogle Scholar
  6. Inaba M, Tashiro T, Sato S, Ohnishi Y, Tanisaka K, Koabayashi H, et al. In vitro-in vivo correlation in anticancer drug sensitivity test using AUC-based concentrations and collagen gel droplet-embedded culture. Oncology. 1996;53:250–7.PubMedCrossRefGoogle Scholar
  7. Jada SR, Hamzah AS, Lajis NH, Saad MS, Stevens MFG, Stanslas J. Semisynthesis and cytotoxic activities of andrographolide analogues. J Enzym Inhib Med Chem. 2006;21:145–55.CrossRefGoogle Scholar
  8. Jada SR, Subur GS, Matthews C, Hamzah AS, Lajis NH, Saad MS, et al. Semisynthesis and in vitro anticancer activities of andrographolide analogues. Phytochem. 2007;68:904–12.CrossRefGoogle Scholar
  9. Jada SR, Matthews C, Saad MS, Hamzah AS, Lajis NH, Stevens MFG, et al. Benzylidene derivatives of andrographolide inhibit growth of breast and colon cancer cell in vitro by inducing G1 cell cycle arrest and apoptosis. Br J Pharmacol. 2008;155:641–54.PubMedCrossRefPubMedCentralGoogle Scholar
  10. Jemal A, Siegel R, Xu J, Ward E. Cancer statistics. CA Cancer J Clin. 2010;60:277–300.PubMedCrossRefGoogle Scholar
  11. Kapril A, Koul IB, Banerjee SK, Gupta BD. Antihepatotoxic effects of major diterpenoid constituents of Andrographis paniculata. Biochem Pharmacol. 1993;46(1):182–5.CrossRefGoogle Scholar
  12. Kim TG, Hwi KK, Hung CS. Morphological and biochemical changes of andrographolide-induced cell death in human prostatic adenocarcinoma PC-3 cells. In Vivo. 2005;19:551–7.PubMedGoogle Scholar
  13. Kuwana T, Bouchier-Hayes L, Chipuk JE, Bonzon C, Sullivan BA, Green DR. BH3 domains of BH3-only proteins differentially regulate Bax-mediated mitochondrial membrane permeabilization both directly and indirectly. Mol Cell. 2005;17:525–35.PubMedCrossRefGoogle Scholar
  14. Li H, Zhu H, Xu CJ, Yuan J. Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell. 1998;94:491–501.PubMedCrossRefGoogle Scholar
  15. Liang FP, Lin CH, Kuo CD, Chao HP, Fu SL. Suppression of v-Src transformation by andrographolide via degradation of the v-Src protein and attenuation of the Erk signalling pathway. J Biol Chem. 2008;283:5023–33.PubMedCrossRefGoogle Scholar
  16. Lim SH (2007) Mechanisms of antitumour activity of 3,19-(2bromobenzylidene) andrographolide (SRJ09). Master thesis, Universiti Putra Malaysia. (
  17. Luo X, Budihardjo I, Zou H, Slaughter C, Wang X. Bid, a Bcl-2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors. Cell. 1998;94:481–90.PubMedCrossRefGoogle Scholar
  18. Manikam ST, Stanslas J. Andrographolide inhibits growth of promyelocytic leukemia cells by inducing retinoic acid receptor-independent cell differentiation and apoptosis. J Pharm Pharmacol. 2009;61:78–9.CrossRefGoogle Scholar
  19. Nanduri S, Nyavanandi VK, Thunuguntla SSR, Kasu S, Pallerla MK, Ram PS, et al. Synthesis and structure-activity relationships of andrographolide analogues as novel cytotoxic agents. Bioorg Med Chem Lett. 2004;14:4711–7.PubMedCrossRefGoogle Scholar
  20. Ozawa S, Sugiyama Y, Mitsuhashi Y, Kobayashi T, Inaba M. Kinetic analysis of cell killing effect induced by cytosine arabinoside and cisplatin in relation to cell cycle phase specificity in human colon cancer and Chinese hamster cells. Cancer Res. 1989;49:3823–8.PubMedGoogle Scholar
  21. Rajagopal S, Kumar RA, Deevi DS, Satyanarayana C, Rajagopalan R. Andrographolide, a potential cancer therapeutic agent isolated from Andrographis paniculata. J Exp Ther Oncol. 2003;3:147–58.PubMedCrossRefGoogle Scholar
  22. Rupniewska Z, Rojarska-Junak A. Apoptosis: mitochondrial membrane permeabilization and the role played by Bcl-2 family proteins. Postepy Hig Med Dosw. 2004;58:538–47.Google Scholar
  23. Shen YC, Chen CF, Chiou WF. Suppression of rat neutrophil reactive oxygen species production and adhesion by the diterpenoid lactone andrographolide. Planta Med. 2000;66:314–7.PubMedCrossRefGoogle Scholar
  24. Shi MD, Lin HH, Chiang TA, Tsai LY, Lee YC, Chen JH. Andrographolide could inhibit human colorectal carcinoma Lovo cells migration and invasion via down-regulation of MMP-7 expression. Chem Bio Int. 2009;180:344–52.CrossRefGoogle Scholar
  25. Stanslas J, Liew PS, Iftikhar N, Lee CP, Saad S, Lajis N, et al. Potential of AG in the treatment of breast cancer. Eur J Cancer. 2001;37 Suppl 6:614.Google Scholar
  26. Tan Y, Chiow KH, Huang D, Wong SH. Andrographolide regulates epidermal growth factor trafficking in epidermoid carcinoma (A-431) cells. Br J Pharmacol. 2010;159(7):1497–510.PubMedCrossRefPubMedCentralGoogle Scholar
  27. Wang ZB, Liu YQ, Cui YF. Pathways to caspase activation. Cell Biol Int. 2005;29:489–96.PubMedCrossRefGoogle Scholar
  28. Wong HC, Sagineedu SR, Lajis NH, Loke SC, Stanslas J. Andrographolide induces cell cycle arrest and apoptosis in PC-3 prostate cancer cells. Afr J Pharm Pharmacol. 2011;5(2):225–33.CrossRefGoogle Scholar
  29. Yi X, Yin XM, Dong Z. Inhibition of Bid-induced apoptosis by Bcl-2. tBid insertion, Bax translocation, and Bax/Bak oligomerization suppressed. J Biol Chem. 2003;278:16992–9.PubMedCrossRefGoogle Scholar
  30. Zhang JH, Xu M. DNA fragmentation in apoptosis. Cell Res. 2000;10(3):205–11.PubMedCrossRefGoogle Scholar
  31. Zhao F, He EQ, Wang L, Liu K. Anti-tumor activities of andrographolide a diterpene from Andrographis paniculata by inducing apoptosis and inhibiting VEGF level. J Asian Nat Prod Res. 2008;10(5):473–9.CrossRefGoogle Scholar
  32. Zhou J, Zhang S, Ong CN, Shen HM. Critical role of pro-apoptotic Bcl-2 family members in andrographolide-induced apoptosis in human cancer cells. Biochem Pharmacol. 2006;72:132–44.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • Hui Chyn Wong
    • 1
  • Charng Choon Wong
    • 2
  • Sreenivasa Rao Sagineedu
    • 1
  • Seng Cheong Loke
    • 3
  • Nordin Haji Lajis
    • 1
  • Johnson Stanslas
    • 1
    • 2
  1. 1.Laboratory of Natural Products, Institute of BioscienceUniversiti Putra MalaysiaSerdangMalaysia
  2. 2.Pharmacotherapeutics Unit, Department of Medicine, Faculty of Medicine and Health SciencesUniversiti Putra MalaysiaSerdangMalaysia
  3. 3.Endocrinology Unit, Department of Medicine, Faculty of Medicine and Health SciencesUniversiti Putra MalaysiaSerdangMalaysia

Personalised recommendations