Plumbagin from Plumbago Zeylanica L Induces Apoptosis in Human Non-small Cell Lung Cancer Cell Lines through NF-κB Inactivation

  • Xu, Tong-Peng ;
  • Shen, Hua ;
  • Liu, Ling-Xiang ;
  • Shu, Yong-Qian
  • Published : 2013.04.30


Objective: To detect effects of plumbagin on proliferation and apoptosis in non-small cell lung cancer cell lines, and investigate the underlying mechanisms. Materials and Methods: Human non-small cell lung cancer cell lines A549, H292 and H460 were treated with various concentrations of plumbagin. Cell proliferation rates was determined using both cell counting kit-8 (CCK-8) and clonogenic assays. Apoptosis was detected by annexin V/propidium iodide double-labeled flow cytometry and TUNEL assay. The levels of reactive oxygen species (ROS) were detected by flow cytometry. Activity of NF-${\kappa}B$ was examined by electrophoretic mobility shift assay (EMSA) and luciferase reporter assay. Western blotting was used to assess the expression of both NF-${\kappa}B$ regulated apoptotic-related gene and activation of p65 and $I{\kappa}B{\kappa}$. Results: Plumbagin dose-dependently inhibited proliferation of the lung cancer cells. The IC50 values of plumbagin in A549, H292, and H460 cells were 10.3 ${\mu}mol/L$, 7.3 ${\mu}mol/L$, and 6.1 ${\mu}mol/L$ for 12 hours, respectively. The compound concentration-dependently induced apoptosis of the three cell lines. Treatment with plumbagin increased the intracellular level of ROS, and inhibited the activation of NK-${\kappa}B$. In addition to inhibition of NF-${\kappa}B$/p65 nuclear translocation, the compound also suppressed the degradation of $I{\kappa}B{\kappa}$. ROS scavenger NAC highly reversed the effect of plumbagin on apoptosis and inactivation of NK-${\kappa}B$ in H460 cell line. Treatment with plumbagin also increased the activity of caspase-9 and caspase-3, downregulated the expression of Bcl-2, upregulated the expression of Bax, Bak, and CytC. Conclusions: Plumbagin inhibits cell growth and induces apoptosis in human lung cancer cells through an NF-${\kappa}B$-regulated mitochondrial-mediated pathway, involving activation of ROS.


Plumbagin;NSCLC;apoptosis;NF-${\kappa}B$;reactive oxygen species;mitochondrial pathway;caspase


  1. Barchowsky A, Dudek EJ, Treadwell MD, et al (1996). Arsenic induces oxidant stress and NF-KB activation in cultured aortic endothelial cells. Free Radic Biol Med, 21, 783-90.
  2. Ahmad A, Banerjee S, Wang Z, et al (2008). Plumbagin-induced apoptosis of human breast cancer cells is mediated by inactivation of NF-kappaB and Bcl-2. J Cell Biochem, 105, 1461-71.
  3. Aziz MH, Dreckschmidt NE, Verma AK (2008). Plumbagin, a medicinal plant-derived naphthoquinone, is a novel inhibitor of the growth and invasion of hormone-refractory prostate cancer. Cancer Res, 68, 9024-32.
  4. Castro FA, Mariani D, Panek AD, et al (2008). Cytotoxicity Mechanism of Two Naphthoquinones (Menadione and Plumbagin) in Saccharomyces cerevisiae. PLoS One, 3, e3999.
  5. Deng L, Adachi T, Kitayama K, et al (2008). Hepatitis C virus infection induces apoptosis through a Bax-triggered, mitochondrion-mediated, caspase 3-dependent pathway. J Virol, 82, 10375-85.
  6. Ding YX, Chen ZJ, Liu S, et al (2005). Inhibition of Nox-4 activity by plumbagin, a plant-derived bioactive naphthoquinone. J Pharm Pharmacol, 57, 111-6.
  7. Freedman VH, Shin SI (1974). Cellular tumorigenicity in nude mice: correlation with cell growth in semi-solid medium. Cell, 3, 355-9.
  8. Hengartner MO (2000).The biochemistry of apoptosis. Nature, 407, 770-6.
  9. Hsu YL, Cho CY, Kuo PL, et al (2006). Plumbagin (5-Hydroxy-2 -methyl-1, 4-naphthoquinone) induces apoptosis and cell cycle arrest in A549 cells through p53 accumulation via c-Jun NH2-terminal kinase-mediated phosphorylation at serine 15 in vitro and in vivo. J Pharmacol Exp Ther, 318, 484-94.
  10. Imlay J, Fridovich I (1992). Exogenous quinones directly inhibit the respiratory NADH dehydrogenase in Escherichia coli. Arch Biochem Biophys, 296, 337-46.
  11. Jemal A, Siegel R, Ward E, et al (2009). Cancer statistics, 2009. CA Cancer J Clin, 59, 225-49.
  12. Kawiak A, Zawacka-Pankau J, Lojkowska E (2012). Plumbagin induces apoptosis in Her2-overexpressing breast cancer cells through the mitochondrial-mediated pathway. J Nat Prod, 75, 747-51.
  13. Karin M (2006). Nuclear factor-kappaB in cancer development and progression. Nature, 441, 431-6.
  14. Kuo PL, Hsu YL, Cho CY (2006). Plumbagin induces G2-M arrest and autophagy by inhibiting the AKT/mammalian target of rapamycin pathway in breast cancer cells. Mol Cancer Ther, 3, 209-21.
  15. Krysko DV, Vanden Berghe T, D'Herde K, et al (2008). Apoptosis and necrosis: detection, discrimination and phagocytosis. Methods, 44, 205-21.
  16. Li J, Shen L, Lu FR, et al (2012). Plumbagin inhibits cell growth and potentiates apoptosis in human gastric cancer cells in vitro through the NF-$\kappa B$ signaling pathway. Acta Pharmacol Sin, 33, 242-9.
  17. Manu KA, Shanmugam MK, Rajendran P, et al (2011). Plumbagin inhibits invasion and migration of breast and gastric cancer cells by downregulating the expression of chemokine receptor CXCR4. Mol Cancer, 10, 107.
  18. Mocellin S, Rossi CR, Pilati P, et al (2005). Tumor necrosis factor, cancer and anticancer therapy. Cytokine Growth Factor Rev, 16, 35-53.
  19. Mossa JS, El-Feraly FS, Muhammad I (2004). Antimycobacterial constituents from juniperus procera, ferula communis and plumbago zeylanica and their in vitro synergistic activity with isonicotinic acid hydrazide. Phytother Res, 18, 934-7.
  20. Nair S, Nair RR, Srinivas P, et al (2008). Radiosensitizing effects of plumbagin in cervical cancer cells is through modulation of apoptotic pathway. Mol Carcino, 47, 22-33.
  21. Nasuhara Y, Adcock IM, Catley M, et al (1999). Differential IkappaB kinase activation and IkappaBalpha degradation by interleukin-1beta and tumor necrosis factor-alpha in human U937 monocytic cells. Evidence for additional regulatory steps in kappaB-dependent transcription. J Biol Chem, 274, 19965-72.
  22. Ngo EO, Sun TP, Chang JY, et al (1991). Menadione-induced DNA damage in a human tumor cell line. Biochem Pharmacol, 42, 1961-8.
  23. Noto V, Taper HS, Jiang YH, et al (1989). Effects of sodium ascorbate (vitamin C) and 2-methyl-1, 4-naphthoquinone (vitamin K3) treatment on human tumor cell growth in vitro. I. Synergism of combined vitamin C and K3 action. Cancer, 63, 901-6.<901::AID-CNCR2820630518>3.0.CO;2-G
  24. Ohe Y, Ohashi Y, Kubota K, et al (2007).Randomized phase III study of cisplatin plus irinotecan versus carboplatin plus paclitaxel, cisplatin plus gemcitabine, and cisplatin plus vinorelbine for advanced non-small-cell lung cancer: Four-Arm Cooperative Study in Japan. Ann Oncol, 18, 317-23.
  25. Powolny AA, Singh SV (2008). Plumbagin-induced apoptosis in human prostate cancer cells is associated with modulation of cellular redox status and generation of reactive oxygen species. Pharm Res, 25, 2171-80.
  26. Pujol JL, Barlesi F, and Daures JP (2006). Should chemotherapy combinations for advanced non-small cell lung cancer be platinum-based? A meta-analysis of phase III randomized trials. Lung Cancer, 51, 335-45.
  27. Qian Y, Guan T, Huang M, et al (2012). Neuroprotection by the soy isoflavone, genistein, via inhibition of mitochondriadependent apoptosis pathways and reactive oxygen induced-NF-$\kappa B$ activation in a cerebral ischemia mouse model. Neurochem Int, 60, 759-67.
  28. Sandur SK, Ichikawa H, Sethi G, et al (2006). Plumbagin (5-Hydroxy-2-methyl-1, 4-naphthoquinone) suppresses NF-$\kappa B$ activation and NF-$\kappa B$-regulated gene products through modulation of p65 and $I\kappa B$ kinase activation, leading to potentiation of apoptosis induced by cytokine and chemotherapeutic agents. J Biol Chem, 281, 17023-33.
  29. Shieh JM, Chiang TA, Chang WT, et al (2010). Plumbagin inhibits TPA-induced MMP-2 and u-PA expressions by reducing binding activities of NF$\kappa B$ and AP-1 via ERK signaling pathway in A549 human lung cancer cells. Mol Cell Biochem, 335, 181-93.
  30. Srinivas P, Gopinath G, Banerji A, et al (2004). Plumbagin induces reactive oxygen species, which mediate apoptosis in human cervical cancer cells. Mol Carcinog, 40, 201-11.
  31. Thasni KA, Rakesh S, Rojini G, et al (2008). Estrogen-dependent cell signaling and apoptosis in BRCA1-blocked BG1 ovarian cancer cells in response to plumbagin and other chemotherapeutic agents. Ann Oncol, 19, 696-705.
  32. Wang CC, Chiang YM, Sung SC, et al (2008). Plumbagin induces cell cycle arrest and apoptosis through reactive oxygen species/c-Jun N-terminal kinase pathways in human melanoma A375.S2 cells. Cancer Lett, 259, 82-98.
  33. Xu KH, Lu DP (2010). Plumbagin induces ROS-mediated apoptosis in human promyelocytic leukemia cells in vivo. Leuk Res, 34, 658-65.

Cited by

  1. Mechanism of Fatty Acid Synthase in Drug Tolerance Related to Epithelial-mesenchymal Transition of Breast Cancer vol.15, pp.18, 2014,
  2. Plumbagin Modulates Leukemia Cell Redox Status vol.19, pp.7, 2014,
  3. Plumbagin reduces human colon cancer cell survival by inducing cell cycle arrest and mitochondria-mediated apoptosis vol.45, pp.5, 2014,
  4. Plumbagin Downregulates Wnt Signaling Independent of p53 in Human Colorectal Cancer Cells vol.77, pp.5, 2014,
  5. Plumbagin induces growth inhibition of human glioma cells by downregulating the expression and activity of FOXM1 vol.121, pp.3, 2015,
  6. Overexpression of interleukin-18 protein reduces viability and induces apoptosis of tongue squamous cell carcinoma cells by activation of glycogen synthase kinase-3β signaling vol.33, pp.3, 2015,
  7. 99mTc labeled plumbagin: estrogen receptor dependent examination against breast cancer cells and comparison with PLGA encapsulated form vol.308, pp.1, 2016,
  8. Anticancer Properties and Pharmaceutical Applications of Plumbagin: A Review vol.45, pp.03, 2017,
  9. Plumbagin exhibits an anti-proliferative effect in human osteosarcoma cells by downregulating FHL2 and interfering with Wnt/β-catenin signalling vol.12, pp.2, 2016,
  10. Current development in novel drug delivery systems of bioactive molecule plumbagin pp.2169-141X, 2018,
  11. Plumbagin-mediating GLUT1 suppresses the growth of human tongue squamous cell carcinoma vol.24, pp.6, 2018,