Multifunctional role of Bcl-2 in malignant transformation and tumorigenesis of Cr(VI)-transformed lung cells

Abstract

B-cell lymphoma-2 (Bcl-2) is an antiapoptotic protein known to be important in the regulation of apoptosis in various cell types. However, its role in malignant transformation and tumorigenesis of human lung cells is not well understood. We previously reported that chronic exposure of human lung epithelial cells to the carcinogenic hexavalent chromium Cr(VI) caused malignant transformation and Bcl-2 upregulation; however, the role of Bcl-2 in the transformation is unclear. Using a gene silencing approach, we showed that Bcl-2 plays an important role in the malignant properties of Cr(VI)-transformed cells. Downregulation of Bcl-2 inhibited the invasive and proliferative properties of the cells as well as their colony forming and angiogenic activities, which are upregulated in the transformed cells as compared to control cells. Furthermore, animal studies showed the inhibitory effect of Bcl-2 knockdown on the tumorigenesis of Cr(VI)-transformed cells. The role of Bcl-2 in malignant transformation and tumorigenesis was confirmed by gene silencing experiments using human lung carcinoma NCI-H460 cells. These cells exhibited aggressive malignant phenotypes similar to those of Cr(VI)-transformed cells. Knockdown of Bcl-2 in the H460 cells inhibited malignant and tumorigenic properties of the cells, indicating the general role of Bcl-2 in human lung tumorigenesis. Ingenuity Pathways Analysis (IPA) revealed potential effectors of Bcl-2 in tumorigenesis regulation. Additionally, using IPA together with ectopic expression of p53, we show p53 as an upstream regulator of Bcl-2 in Cr(VI)-transformed cells. Together, our results indicate the novel and multifunctional role of Bcl-2 in malignant transformation and tumorigenesis of human lung epithelial cells chronically exposed to Cr(VI).

Figure 1. Bcl-2 expression and effect of Bcl-2 knockdown on cell growth.

(A) Endogenous Bcl-2 levels in BEAS-2B, BEAS-Cr and H460 cells determined by Western blot analysis. Densitometry was performed to determine the relative Bcl-2 levels after reprobing the membrane with β-actin antibody. (B) Bcl-2 knockdown experiments were performed in BEAS-Cr and H460 cells by infecting the cells with Bcl-2 shRNA (shBcl-2) viral particles or control shRNA (shCon) particles as described under Materials and Methods. Clonal selection and Bcl-2 expression were performed to identify mutants with downregulated Bcl-2. (C) BEAS-2B, BEAS-Cr and H460 cells (1×10⁵ cells) were seeded on 60-mm cell culture dishes and were incubated at 37°C in a 5% CO₂ incubator. At the indicated times, cells were trypsinized and analyzed for cell number using an electronic cell counter. (D) Effect of Bcl-2 on cell growth of the respective cell lines and their mutants. Cells (1×10⁵ cells) were seeded and incubated at 37°C for 96 h, after which they were analyzed for cell number. Values are means (± SD) (n = 4). *P<0.05 versus passage-control BEAS-2B cells. ^# P<0.05 versus the respective BEAS-Cr and H460 cells.

Figure 2. Effect of Bcl-2 knockdown on colony formation of BEAS-Cr and H460 cells.

(A) BEAS-2B, BEAS-Cr and H460 cells (3×10⁴ cells) were seeded on 0.5% agar plates and incubated at 37°C in a 5% CO₂ incubator. After 2 weeks, colony formation capacity of the cells was scored under a light microscope. (B) Effect of Bcl-2 on colony formation capacity of the respective cell lines and their mutants. Cells (3×10⁴ cells) were seeded on soft agar plates and the colonies were scored after 2 weeks. (C) Representative micrographs of colonies formed by the cell lines and mutants on soft agar are shown. Values are means (± SD) (n = 4). *P<0.05 versus passage-control BEAS-2B cells. ^# P<0.05 versus the respective BEAS-Cr and H460 cells.

Figure 3. Effect of Bcl-2 knockdown on cell invasion and migration of BEAS-Cr and H460 cells.

(A) BEAS-2B, BEAS-Cr and H460 cells (1×10⁵ cells) were added to Transwell® inserts coated with Matrigel® and incubated for 24 h. Invading cells were stained and counted under a light microscope. Plots show relative invasion of BEAS-2B, BEAS-Cr and H460 cells. (B) Effect of Bcl-2 on cell invasion of the respective cell lines and their mutants. Experiments were repeated with the indicated cell lines and analyzed for cell invasion. (C) Confluent monolayers of BEAS-2B, BEAS-Cr and H460 cells were wounded, and the cells were allowed to migrate for 24 h. Wound space was visualized by light microscopy and analyzed by comparing the relative change in wound space as compared to control cell monolayers. (D) Effect of Bcl-2 on cell migration of the respective cell lines and their mutants. Cells were wounded and analyzed for cell migration over a 24 h period. (E) Representative micrographs of cells stained for invasion are shown. Values are means (± SD) (n = 4). *P<0.05 versus passage-control BEAS-2B cells. ^# P<0.05 versus the respective BEAS-Cr and H460 cells.

Figure 4. Effect of Bcl-2 knockdown on angiogenic activity of BEAS-Cr and H460 cells.

(A) BEAS-2B, BEAS-Cr, and H460 cell supernatants were incubated with HUVEC cells, and endothelial capillary tube formation was detected under a light microscope. The number of nodes formed by the tubes were scored and plotted. (B) Effect of Bcl-2 knockdown on angiogenic activity of the respective cell lines. Experiments were performed with the indicated cell lines and analyzed for endothelial tube formation as described. (C) Representative micrographs of tube formation are shown. Values are means (± SD) (n = 4). *P<0.05 versus passage-control BEAS-2B cells. ^# P<0.05 versus the respective BEAS-Cr and H460 cell supernatants.

Figure 5. Apoptosis response to Cr(VI) treatment in BEAS-2B, BEAS-Cr, and H460 cells.

(A) BEAS-2B, BEAS-Cr and H460 cells were treated with or without Cr(VI) (20 µM) for 12 h and apoptosis was determined by Hoechst 33342 assay. (B) and (C) Effect of Bcl-2 knockdown on Cr(VI)-induced apoptosis of the respective cell lines and their mutants. Values are means (± SD) (n = 4). *P<0.05 versus non-treated control. ^# P<0.05 versus Cr(VI)-treated passage-control BEAS-2B cells. **P<0.05 versus Cr(VI)-treated respective BEAS-Cr and H460 cells.

Figure 6. Effect of Bcl-2 knockdown on tumor-associated properties in vivo.

(A) Mice were injected subcutaneously with 1×10⁶ passage-control BEAS-2B, BEAS-Cr, or shBcl-2 BEAS-Cr cells. Tumor formation was determined at 14 d post-injection. Representative photographs are shown. (B) Mice were similarly injected with control BEAS-2B, H460, or shBcl-2 H460 cells. Tumor formation and representative micrographs at 14 d post-injection are shown. Data are means (± SD) (n = 4). *P<0.05 versus passage-control BEAS-2B cells. ^# P<0.05 versus the respective BEAS-Cr and H460 cells.

Figure 7. Ingenuity Pathways Analysis software output for the Bcl-2-interactome.

IPA query for the Bcl-2-interactome. The interactome was filtered for interactions reported in humans only and presented by cellular localization. 56 molecules were reported to interact either directly (solid lines) or indirectly (dash lines). Arrow direction indicates direction of functionality.

Figure 8. Ingenuity Pathways Analysis software output for the downstream components of human Bcl-2-interactome.

Twenty molecules of downstream components were reported to date and were labeled in orange, while upstream molecules were labeled in green.

Figure 9. Bcl-2 and p53 expression in BEAS-2B, BEAS-Cr and H460 cells.

(A) Cells were either left untreated or treated with Cr(VI) (20 µM) for 12 h. Cell lysates were prepared and analyzed for Bcl-2 and p53 by Western blotting. β-actin was used as a loading control. Densitometry was performed to determine the relative levels of Bcl-2 and p53 compared to β-actin. Representative blots are shown. (B) BEAS-Cr cells were transiently transfected with pcDNA (vector) or p53 plasmid, and Bcl-2 and p53 expression levels were determined by Western blotting. Values are means (± SD) (n = 4). *P<0.05 versus BEAS-2B controls. ^# P<0.05 versus vector-transfected control.