Abstract

To detect effects of plumbagin on proliferation and apoptosis in non-small cell lung cancer cell lines, and investigate the underlying mechanisms. 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-κB was examined by electrophoretic mobility shift assay (EMSA) and luciferase reporter assay. Western blotting was used to assess the expression of both NF-κB regulated apoptotic-related gene and activation of p65 and IκBκ. Plumbagin dose-dependently inhibited proliferation of the lung cancer cells. The IC50 values of plumbagin in A549, H292, and H460 cells were 10.3 μmol/L, 7.3 μmol/L, and 6.1 μmol/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-κB. In addition to inhibition of NF-κB/p65 nuclear translocation, the compound also suppressed the degradation of IκBκ. ROS scavenger NAC highly reversed the effect of plumbagin on apoptosis and inactivation of NK-κB 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. Plumbagin inhibits cell growth and induces apoptosis in human lung cancer cells through an NF-κB-regulated mitochondrial-mediated pathway, involving activation of ROS.

Highlights

  • Lung cancer is currently the commonest malignancy and the leading cause of cancer-related death in the world (Jemal et al, 2009)

  • Cell viability was assayed by treating Non-small cell lung cancer (NSCLC) cell lines, including A549, H292, and H460 cells, with various concentrations of plumbagin followed the cell counting kit-8 (CCK-8) viability assay

  • We observed that cellular proliferation was inhibited by plumbagin for 24 hours in a dose-dependent manner in all three of the NSCLC cell lines (Figure 1B)

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Summary

Introduction

Lung cancer is currently the commonest malignancy and the leading cause of cancer-related death in the world (Jemal et al, 2009). Non-small cell lung cancer (NSCLC) represents about 80% of primary lung cancer cases and approximately two thirds of these patients are diagnosed at an advanced stage (Jemal et al, 2009). Several effective chemotherapeutic agents are administrated, the platinum-based regimen is the standard initial treatment for NSCLC patients (Pujol et al, 2006). The efficacy of the regimen for NSCLC has been reported to be only 30-40% based on NSCLC trials involving unselected patients (Ohe et al, 2007). Plumbagin (5-hydroxy-2methyl-1, 4-naphthoquinone), a Chinese traditional herb extracted from the root of Plumbago zeylanica L has been reported in the literature to have various pharmacological activities, including anti-microbial, hypolipidemic, antiatherosclerotic, and anti-carcinogenic effects (Mossa et al, 2004; Ding et al, 2005; Aziz et al, 2008). It has been shown to exert anti-proliferation influence in diverse cancer cell lines, both in vivo and in vitro, such as leukemia (Xu et al, 2010), prostate (Aziz et al, 2008; Powolny et al, 2008), breast (Kuo et al, 2006; Ahmad et al, 2008), ovarian (Thasni et al, 2008), cervical (Srinivas et al, 2004; Nair et al, 2008) and melanoma (Wang et al, 2008) examples

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