Luteolin inhibits Cr(VI)-induced malignant cell transformation of human lung epithelial cells by targeting ROS mediated multiple cell signaling pathways

Abstract

Hexavalent chromium [Cr(VI)] is a well-known human carcinogen associated with the incidence of lung cancer. Inhibition of metal induced carcinogenesis by a dietary antioxidant is a novel approach. Luteolin, a natural dietary flavonoid found in fruits and vegetables, possesses potent antioxidant and anti-inflammatory activity. We found that short term exposure of human bronchial epithelial cells (BEAS-2B) to Cr(VI) (5μM) showed a drastic increase in ROS generation, NADPH oxidase (NOX) activation, lipid peroxidation, and glutathione depletion, which were significantly inhibited by the treatment with luteolin in a dose dependent manner. Treatment with luteolin decreased AP-1, HIF-1α, COX-2, and iNOS promoter activity induced by Cr(VI) in BEAS-2B cells. In addition, luteolin protected BEAS-2B cells from malignant transformation induced by chronic Cr(VI) exposure. Moreover, luteolin also inhibited the production of pro-inflammatory cytokines (IL-1β, IL-6, IL-8, TNF-α) and VEGF in chronic Cr(VI) exposed BEAS-2B cells. Western blot analysis showed that luteolin inhibited multiple gene products linked to survival (Akt, Fak, Bcl-2, Bcl-xL), inflammation (MAPK, NF-κB, COX-2, STAT-3, iNOS, TNF-α) and angiogenesis (HIF-1α, VEGF, MMP-9) in chronic Cr(VI) exposed BEAS-2B cells. Nude mice injected with BEAS-2B cells chronically exposed to Cr(VI) in the presence of luteolin showed reduced tumor incidence compared to Cr(VI) alone treated group. Overexpression of catalase (CAT) or SOD2, eliminated Cr(VI)-induced malignant transformation. Overall, our results indicate that luteolin protects BEAS-2B cells from Cr(VI)-induced carcinogenesis by scavenging ROS and modulating multiple cell signaling mechanisms that are linked to ROS. Luteolin, therefore, serves as a potential chemopreventive agent against Cr(VI)-induced carcinogenesis.

Keywords: Angiogenesis; Carcinogenesis; Hexavalent chromium; Inflammation; Luteolin.

Figure 1

Luteolin inhibits Cr (VI)-induced cytotoxicity. (A) Chemical structure of luteolin (B) Cell viability, determined by MTT, was assessed in BEAS-2B cells treated with Cr(VI) (5 μM) for 24 h in the presence of luteolin (0, 5, 10, 20 μM). (C–D) BEAS-2B cells were treated with 2 μM or 4 μM Cr(VI) with or without 20 μM luteolin for 48 h, reseeded and cultured in drug free medium for an additional 7 days and stained with crystal violet. The data are expressed as the mean ± SD of three independent experiments.*(p<0.05).

Figure 2

Luteolin inhibits Cr(VI)-induced ROS generation. (A–B) BEAS-2B cells were exposed to Cr(VI) (0 or 5 μM) with or without luteolin (0, 5, 10, 20 μM) for 12 h and then were labeled with DHE (5 μM) or (D–E) DCFDA (10 μM). Images were taken with fluorescence microscopy and fluorescent intensity determined by flow cytometry. *(p<0.05). BEAS-2B cells were exposed to Cr(VI) (0 or 5 μM) or were pretreated with SOD (500 U/ml), CAT (1000 U/ml), or APO (50 μM) for 2 h followed by Cr(VI) (5 μM) treatment for 6 h and then were labeled with (C) DHE (5 μM) or (F) DCFDA (10 μM) as described previously. LY83853 (10 μM) and H₂O₂ (0.1 mM) were used as positive controls for DHE and DCF measurements, respectively. The data are expressed as mean ± SD of three independent experiments. *p<0.05, statistically significant difference from control cells.

Figure 3

Luteolin inhibits Cr(VI)-induced NOX activation, Lipid Peroxidation, Glutathione depletion in BEAS-2B cells. (A) NOX activity was measured by the lucigenin chemiluminescence assay with Cr(VI) (0 or 5 μM) in the presence of luteolin (0, 5, 10, 20 μM) for indicated times. The data are expressed as the mean ± SD of three independent experiments. *p<0.05, statistically significant difference from control cells. BEAS-2B cells were treated with Cr(VI) (5 μM) for 24 h in the presence of luteolin (0, 5, 10, 20 μM). Cell lysates were prepared by sonication in ice cold PBS and was used for the determination of oxidative stress in terms of (B) lipid peroxidation and (C) glutathione depletion. The data are expressed as the mean ± SD of three independent experiments. *p<0.05, statistically significant difference from control cells.

Figure 4

Luteolin inhibits Cr(VI)-induced AP-1, Cox-2, iNOS, HIF-1α, dependent transactivation in BEAS-2B cells. BEAS-2B cells transfected with the luciferase reporter constructs were treated with Cr(VI) (5 μM) for 24 h in the presence of luteolin (0, 5, 10, 20 μM). Cellular lysates were assessed for luciferase reporter activity as described previously. The results are expressed as relative activity normalized to the luciferase activity in the control cells without treatment. The data are expressed as the mean ± SD of three independent experiments. *p<0.05, statistically significant difference from control cells.

Figure 5

Luteolin inhibits chronic Cr(VI)-induced malignant transformation. BEAS-2B cells were maintained in a medium containing Cr(VI) (0 or 0.5 μM) with or without luteolin (1 and 2 μM) for 6 months. (A) Cells were cultured in 0.35% soft agar for 5 weeks. Colony numbers in the entire dish were counted. (B) Cells cultured in drug free medium for an additional 7 days and stained with crystal violet. Colony numbers in the entire dish were counted. The data are expressed as the mean ± SD of three independent experiments. *(p<0.05).

Figure 6

Luteolin inhibits mediators of inflammation induced by chronic Cr(VI) exposure in BEAS-2B cells. BEAS-2B cells were treated as described in Fig. 5. Culture medium was collected to estimate the (A) TNF-α, (B) IL-6, (C) IL-1β and (D) IL-8 levels using commercially available ELISA kits according to manufacturer’s recommendation.(E) Total cell lysates were prepared for Western blot analysis using specific antibodies against COX-2, STAT3, iNOS, TNF-α and β-actin. (F) STAT3 activity was measured in the nuclear fraction of cell lysates by ELISA following the manufacturer’s protocol. The data are expressed as the mean ± SD of three independent experiments. Significant difference compared to Cr(VI) alone, *p<0.05.

Figure 7

Luteolin inhibits MAPK, AKT, FAK, Bcl-2, Bcl-xL and NF-κB induced by chronic Cr(VI) exposure in BEAS-2B cells. (A) Luteolin inhibited Cr(VI)-induced MAPK signaling in in BEAS-2B cells. Cells were treated as described in Fig. 5 and lysates were prepared to determine the phosphorylated and total protein levels of ERK1/2, p38, and JNK by Western blot analysis, as described previously. (B) Western blot analysis demonstrating that luteolin inhibited Cr(VI)-induced AKT, FAK, Bcl-2, and Bcl-xL in BEAS-2B cells. (C–E) Western blot analysis demonstrating that luteolin inhibited Cr(VI)-induced activation of NF-κB/p65 and IKKα, and degradation of IκBα in in BEAS-2B cells. (C) nuclear translocation of NF-κB/p65 (D) activation of IKKα, or degradation of IκBα in cytosol. The relative intensities of each band after normalization for the levels of lamin/β-actin are shown under each blot. A representative blots from three independent experiments with identical observations, and equivalent protein loading was confirmed by probing stripped blots for β-actin as shown. (E) The activity of NF-κB in nuclear fraction of cell lysates was measured using ELISA following the manufacturer’s protocol. The data are expressed as the mean ± SD of three independent experiments. Significant difference compared to Cr alone, *p<0.05.

Figure 8

Luteolin inhibits chronic Cr(VI)-induced angiogenic mediators in BEAS-2B cells BEAS-2B cells were treated as described in Fig. 5 and lysates prepared to determine the protein levels of HIF-1α, HIF-1β, VEGF, and MMP-9 by Western blot analysis. (E) Culture medium was collected to estimate VEGF levels using a commercially available ELISA kit according to manufacturer’s recommendation. The data are expressed as the mean ± SD of three independent experiments. Significant difference compared to Cr alone, *p<0.05.

Figure 9

Luteolin inhibits growth of xenograft tumors in mice chronically exposed to Cr(VI). Cells treated as indicated from different treatments were injected into the flanks of 6-week old athymic nude mice (2×10⁶ cells per mouse) and checked daily for tumor appearance; tumor volume was measured every 3 days for 30 days. Tumor volume was determined by Vernier caliper, following the formula of A×B²×0.52, where A is the longest diameter of tumor and B is the shortest diameter. (A) Mice injected with BEAS-2B cells exposed to luteolin along with Cr(VI) showed reduced tumor incidence. (B) Angiogenic (CD31, CD34) and proliferation (Ki67) markers were decreased in tumors treated with both Cr(VI) and luteolin as evident from immunohistochemistry. Frozen tumour sections (5 μM thick) were subjected to immunoperoxidase staining (dark brown) to detect CD31, CD34, and Ki67 expression.

Figure 10

Overexpression of antioxidant enzymes block Cr(VI)-induced malignant cell transformation and tumorigenesis in BEAS-2B cells. Inhibition of in vitro colony formation in BEAS-2B cells with overexpressed antioxidant enzymes is demonstrated in (A) soft agar and (B) clonogenic assay. BEAS-2B cells were stably transfected with CAT (BEAS-2B-CAT), SOD2 (BEAS-2B-SOD2), or their corresponding vectors (BEAS-2B-vectors) as controls. After exposure of above stable cell lines with Cr(VI) (0 or 0.5 μM) for 6 months, soft agar assay and clonogenic assay was performed as previously described. (C) Inhibition of in vivo tumor growth in nude mice with overexpressed antioxidant enzymes. After BEAS-2B-vector controls, BEAS-2B-CAT, and BEAS-2B-SOD2, cells were exposed to Cr(VI) (0 or 0.5 μM) for 6 months, xenograft growth of tumors in nude mice was performed as described previously. The data are expressed as the mean ± SD of three independent experiments. *p < 0.05, statistically significant difference from Cr(VI)-treated cells.

Figure 11