Skip to main content
NCBI home page
Toxicology Research logoLink to Toxicology Research
. 2017 Feb 15;6(3):324–332. doi: 10.1039/c6tx00372a

Chromium contributes to human bronchial epithelial cell carcinogenesis by activating Gli2 and inhibiting autophagy

Junpeng Huang a,†, Gang Wu a,†, Rong Zeng a,†, Jinting Wang a, Rui Cai a, James Chung-Man Ho b, Jiren Zhang a,c, Yanfang Zheng a,✉
PMCID: PMC6060711  PMID: 30090501

graphic file with name c6tx00372a-ga.jpgOccupational and environmental inhalation exposure to hexavalent chromium [Cr(vi)] compounds has been confirmed to cause respiratory system injury and cancer.

Abstract

Occupational and environmental inhalation exposure to hexavalent chromium [Cr(vi)] compounds has been confirmed to cause respiratory system injury and cancer. The molecular mechanisms of chromium carcinogenesis still require further study. We established Cr(vi)-transformed cells (BEAS-2B-Cr) after chronic exposure of immortalized normal human bronchial epithelial BEAS-2B cells to low doses of Cr(vi), which obtained the ability of anchorage-independent growth. BEAS-2B-Cr cells not only exhibited stronger proliferation, migration, invasion and tumorigenesis capabilities but also acquired an altered and distinct Gli2 gene expression pattern compared with untreated parental BEAS-2B cells (P-NC) and the control BEAS-2B cells (NC). Interestingly, we found that activation of Gli2 by Cr(vi) treatment prevented the induction of autophagy. Using a gene silencing approach, we showed that Gli2 plays an important role in the malignant properties of BEAS-2B-Cr cells. Downregulation of Gli2 induced autophagy and inhibited cell proliferation and colony forming abilities, which are both upregulated in BEAS-2B-Cr cells compared to NC cells. In addition, inhibition of autophagy by 3-methyladenine (3-MA) partially suppressed the cytotoxicity induced by GANT61-induced inhibition of Gli2. These results demonstrate that hexavalent chromium Cr(vi) activates Gli2 to promote the proliferation of BEAS-2B-Cr cells by inhibition of autophagy, which contributes to human bronchial epithelial cell carcinogenesis. Gli2 may not only play an important role in lung cancer pathogenesis, but also be a promising early indicator in monitoring exposure to chromium.

Introduction

Lung cancer currently has the highest incidence and mortality of malignant tumors worldwide.1 In developing countries, including China, people have experienced serious particulate air pollution in recent years. There is a positive correlation between PM2.5 and lung cancer mortality rate.2 Wu et al. studied the chemical constituents of fine particulate air pollution and showed that this pollution contained chromium.3 Multiple factors, including cigarette smoke, air pollution and heavy metals, have been established as important causes of lung cancer, but the molecular mechanisms of carcinogenesis are still not fully defined. Hexavalent chromium [Cr(vi)] compounds have been recognized as Class I human carcinogens by the IARC based on epidemiological data and a large body of knowledge showing that these compounds are mutagenic and genotoxic.4 Several recent occupational epidemiological studies have shown that inhalation of hexavalent chromium [Cr(vi)] is associated with increased lung cancer risk; this increased risk has been observed among workers in chromate production, plating, pigments and ferrochrome production industries.5 There are few studies suggesting that hexavalent chromium [Cr(vi)] induces microRNA expression, histone modification,6 and DNA methylation,7 which may contribute to its carcinogenicity.8,9 However, the detailed molecular mechanisms of Cr(vi)-induced lung cancer are poorly understood.

Hedgehog (Hh) signaling plays an important role in embryonic development and in the regulation of a variety of cellular functions. Recently, growing evidence has implicated aberrant activation of Hh signaling in several human cancers including lung cancer.10,11 As the downstream targets of Hh signaling are controlled by the dynamics of Gli transcription factors, Gli proteins are essential in Hh signal transduction processes. Gli2 seems to be the primary activator of Hh signaling in cancer, with Gli1 as a transcriptional target of Gli2.12,13 Gli targets and mediates various cellular responses, including notably enhanced cell proliferation and survival, by upregulating Bcl-2.14 Previous publications have shown that the expression of the hedgehog interacting protein (HHIP), a gene that antagonizes hedgehog signaling pathways, was decreased in Cr(vi)-transformed cells.15 Currently, the role of hedgehog signaling pathways in chromium-induced lung carcinogenesis remains unclear.

Autophagy, a highly conserved degradation process, functions to maintain cellular homeostasis.16 Autophagosomes can isolate unnecessary or dysfunctional proteins and organelles; these autophagosomes are subsequently degraded by the lysosomal machinery. Chromium induced DNA damage promotes autophagy.9 Autophagy dysfunction is associated with different human pathologies, including cancer. Autophagy is mainly controlled by three systems: (1) LC3 (MAP1LC3A)-PE conjugation to convert LC3-I to LC3-II, which functions in autophagosome membrane elongation;17 (2) the ULK1 (unc-51-like autophagy activating kinase 1) protein kinase complex, which is regulated by mTOR; and (3) the Beclin-1/Vps34 complex, which facilitates the interaction of Beclin-1 (BECN1) with a class III PI3K (Vps34) and other proteins to initiate autophagosome formation.18,19 Jimenez-Sanchez et al. have shown that hedgehog signaling plays a key role in regulating autophagy20 and confirms that Gli2 is necessary for Hh-induced inhibition of autophagy.

Chromium-induced immortalized normal human bronchial epithelial BEAS-2B cell transformation, a process that converts normal cells into a cancer-like state of unlimited division and proliferation, is a critical step in chromium carcinogenesis. In our study, we performed a set of experiments to elucidate the molecular mechanisms by which hedgehog signaling functions in chromium carcinogenesis. As a result, hexavalent chromium Cr(vi) activates Gli2, which is essential in Hh signal transduction processes, to promote BEAS-2B-Cr cells’ proliferation through inhibition of autophagy to contribute to human bronchial epithelial cell carcinogenesis.

Materials and methods

Chemicals, materials and cell lines

Potassium dichromate (K2Cr2O7) was purchased from Sigma-Aldrich (Shanghai, China), dissolved in double distilled water (ddH2O) to 25 mM as a stock solution and stored at room temperature. DMEM, dimethyl sulfoxide (DMSO) and trypsin-EDTA solution were all purchased from Life Technologies (California, USA). Heat-inactivated fetal bovine serum (FBS) was obtained from Merck Millipore (MA, USA). Human normal bronchial epithelial BEAS-2B cells were purchased from the American Type Culture Collection (Manassas, VA, USA). GANT61 and 3-methyladenine (3-MA) were purchased from Selleck (Houston, USA).

Cell culture and Cr(vi) treatment

BEAS-2B cells were cultured in Dulbecco's modified Eagle medium (Life Technologies, California, USA) containing 10% FBS (Merck Millipore, MA, USA) in an incubator at 37 °C with 5% CO2. 25 mM K2Cr2O7, which is equivalent to 50 mM Cr(vi), was used as the stock solution. Working solution was diluted to 100 μM and may be used for 1 week. In the Cr(vi) exposure group, for every 50 ml of culture medium, 250 μl Cr(vi) working solution was added. The same volume of ddH2O was added in culture medium in the control group. For Cr(vi) exposure, the cells were treated with 0.5 μM Cr(vi) for 4 weeks. The medium contained chromium all the time, even during cell passage before the cells attached. The medium was changed every other day, and the cells were passaged in the presence of Cr(vi) every 3 days.

Cell cytotoxicity assay

When the cultured cells had grown to approximately 80% confluence, the cells were seeded at 1 × 103 cells per well in 96-well plates and treated with different concentrations of Cr(vi) (0.25, 0.5, 1.0, 2.0, 4.0, 8.0 and 16.0 μM) for 24 h, with ddH2O as the solvent control. A viable cell number was determined using the Cell Counting Kit-8 Proliferation Cytotoxicity Assay Kit (Dojindo, Japan) according to the manufacturer's instructions.

Wound-healing assay

The cells were seeded in 6-well plates at a density of 5 × 105 cells per well. When the cultured cells had grown to approximately 80% confluence, they were serum starved in FBS-free medium for 12 h. A wound was produced by scraping across the cell monolayer using a 10 μl sterile polystyrene micropipette tip. The cells were cultured and allowed to migrate into the denuded area for 24 h. The wounds adjacent to the lines were photographed 0 h and 24 h after scratching under a phase contrast microscope using a 20× objective lens. The cells were counted using ImageProPlus 6.0 software in the marked area between the labeled lines and the wound.

Cell migration assay

Cell migration was measured using transwell chambers (8 μm pore size, BD Biosciences) according to the methods described previously.21 The top chambers of the transwells were loaded with 1 × 105 cells in 0.2 ml of medium per chamber, and the bottom chambers contained 0.5 ml of complete medium. After the cells were incubated at 37 °C and 5% CO2 for 24 h, the migratory cells were fixed, stained with crystal violet solution, and counted under a microscope.

Cell invasion assay

The cell invasion assay was performed according to the methods described previously.21 BEAS-2B-Cr cells (5 × 105 cells) were loaded into 24-well invasion chambers (BD Biosciences) that were pre-coated with 40 μl Matrigel solution. Medium containing 10% fetal calf serum was added to the plates (0.5 ml per well). The Matrigel invasion chambers were incubated for 48 h, and the invading cells were stained with crystal violet and observed by microscopy.

Soft agar assays

The cells were plated at 1 × 103 cells per well in 6-well plates with culture medium containing 0.35% low-melting-point agarose over a 0.5% agarose base layer and cultured in a 37 °C incubator with 5% CO2 for 2 weeks. The resulting colonies were stained with INT/BCIP (Roche) and photographed.

Quantitative real-time PCR analysis

The cells were seeded in 6-well plates at a density of 5 × 105 cells and 2 ml fresh medium per well and incubated overnight. Total RNA was extracted from the cells using total RNA kits (OMEGA, United States) and converted to single-stranded cDNA using a PrimeScript™ RT Reagent Kit (Takara Bio Inc., Japan). Quantitative real-time PCR analysis was performed using SYBR Premix Ex Taq (Takara Bio Inc., Japan) on a LightCycler 480 system (Roche, Basel, Switzerland). All PCR reactions were performed in triplicate. Relative gene expression levels were assessed by normalizing to GAPDH and calculated by the 2–△△Ct method. The results are presented as the fold change relative to the levels expressed in normal BEAS-2B cells. Primers for GAPDH forward: 5′-GGCTCATGACCACAGTCCATG-3′; GAPDH reverse: 5′-CAGCTCTGGGATGACCTTG-3′. Gli1 forward: 5′-GAGTCCAGAGGTTCAAGAG-3′; Gli1 reverse: 5′-TGGTGAGTA GACAGAGGTT-3′; Gli2 forward: 5′-TGTGTAGGTGGTGTGGTT-3′; Gli2 reverse: 5′-TGTGTTCAGGAATGATGTCT-3′ (Invitrogen, California, USA).

Western blot analysis

The cells were incubated overnight at a density of 5 × 105 cells per well in 6-well plates. Cultured cells were washed with PBS solution before the cells were lysed. Cell lysates were prepared in RIPA buffer (Beyotime, Shanghai, China) containing protease inhibitors (Beyotime, Shanghai, China) and phosphatase inhibitors (Beyotime, Shanghai, China) for 15 minutes on ice. Lysates were cleared by centrifugation at 12 000g for 15 minutes. Protein concentrations were measured using a BCA protein assay kit (Thermo Scientific, Epsom, UK). Equal amounts of protein were resolved on a 10% SDS-PAGE gel (Beyotime, Shanghai, China) and transferred to a PVDF membrane (Merck Millipore, MA, USA). Western blotting was performed according to the standard protocol with antibodies against Gli2 (Cell Signaling Technology, Boston, USA), Beclin-1 (Cell Signaling Technology, Boston, USA), Bcl-2 (Proteintech, Wuhan, China), and LC3 (Bioworld, Ohio, USA) using standard immunoblotting, and specific protein signals from the respective horseradish peroxidase-linked secondary antibodies were detected by using enhanced chemiluminescence (ECL) western blotting detection reagents (Thermo Scientific, Epsom, UK). The blots were then re-probed with rabbit anti-GAPDH (Proteintech, Wuhan, China) to standardize the results.

Small interfering RNAs (siRNAs) and cell transfection

The cells were seeded in 6-well plates at a density of 1 × 105 cells per well and incubated overnight before transfection. Chemically modified siRNAs were designed and ordered from GenePharma (Shanghai, China). The sequence of siRNA-Gli2 is listed here (sense: GUGACACCAACCAGAACAATT; antisense: UUGUUCUGGUUGGUGUCACTT). Transfection of siRNA-Gli2 was achieved using Lipofectamine 3000 (Invitrogen, California, USA). Nonsense siRNA provided by the manufacturer (GenePharma, Shanghai, China) was used as a negative control. The efficacy of siRNAs targeting Gli2 was tested in BEAS-2B-Cr cells western blotting 72 h after transfection.

Transmission electron microscopy

The cells in the logarithmic growth phase were selected for overnight culturing in 6-well plates (the seeding density was 5 × 105 cells with 2 ml fresh medium per well in 6-well plates). The cells were lifted by cell scrapers during the logarithmic growth phase when the cells grow to 80% confluence. The cells were collected after removing the supernatant by centrifugation and then fixed through adding 2.5% glutaraldehyde (500 μl) at room temperature for 1 h. Glutaraldehyde was eliminated after incubation at 4 °C for 3 h, and PBS was added fully to the cells at 4 °C. The cells were observed and filmed under an electron microscope after the preparing procedure including dehydration, embedding, ultrathin section, and staining based on established procedures.

Statistical analysis

All data are presented as the mean ± standard deviation (mean ± SD). SPSS version 13.0 software was used for the statistical analysis of the data. The differences between two groups were examined by Student's t-test, and differences between 3 or more groups were compared by one-way analysis of variance (ANOVA). If the variance was equal, the F-test was applied directly, and least significant difference (LSD) tests were used for multiple comparisons. If not, the correction F test (Welch) was used with Tamhane's T2 multiple comparison test for variance under misaligned conditions. Differences with P < 0.05 were considered statistically significant.

Results

Establishment of Cr(vi)-transformed cells (BEAS-2B-Cr)

When BEAS-2B cells were acutely exposed to different concentrations (0, 0.25, 0.5, 1.0, 2.0, 4.0, 8.0, and 16.0 μM) of Cr(vi) for 24 h, we found that the half maximal inhibitory concentration (IC50) of Cr(vi) was approximately 8.0 μM based on CCK8 detection, which showed no significant difference (P = 0.0633) at or below 0.5 μM (Fig. 1). This result suggested that 0.5 μM of Cr(vi) has minimal acute toxicity on BEAS-2B cells. To mimic low-dose and long term exposure in vivo, immortalized human bronchial epithelial BEAS-2B cells were continuously cultured in medium containing 0.5 μM of Cr(vi), which was the dose that BEAS-2B cells exhibited minimal toxicity (Fig. 1A).

Fig. 1. Acute toxicity of different concentrations of Cr(vi) on BEAS-2B cells and chronic exposure of Cr(vi) promotes the anchorage-independent growth of BEAS-2B cells. (A) BEAS-2B cells were treated with different concentrations of Cr(vi) for 24 h. CCK8 working solution was added directly to the cultures for proliferation assay (**, p < 0.01). (B & C) BEAS-2B cells were exposed to 0.5 μM Cr(vi) for 4 weeks, and assessed for anchorage-independent growth using a soft agar assay. Cells treated with Cr(vi) formed significantly more colonies compared to untreated parental cells (P-NC group) and passage-matched cells without Cr(vi) treatment (NC group). (B) Representative plates in soft-agar assay are shown. (C) Numbers of colonies per well were counted and presented as the mean ± SD (n = 3).

Fig. 1

Open in a new tab

Apart from hematopoietic cells and lymphoid lineage cells, most normal epithelial cells rely on physical contact with the substratum to maintain normal cell growth. In contrast, many transformed cells lose contact inhibition and can grow and survive without adherence, which is often referred to as anchorage-independent growth. Because of its high correlation with tumor progression in vivo, anchorage-independent growth has been considered a sign of the transformation of normal cells into a cancer-like state of unlimited division and proliferation.22

BEAS-2B-Cr cells were the BEAS-2B cells chronically exposed to a low dose of Cr(vi) (0.5 μM) for 4 weeks. The soft agar assay was used to examine cell transformation. Both BEAS-2B-Cr and untreated control BEAS-2B cells were grown in 0.35% top agar for two weeks. The P-NC group means parental BEAS-2B cells stocked in nitrogen without Cr(vi) treatment. The NC group means the cells passaged the same as BEAS-2B-Cr cells, with ddH2O instead of Cr(vi) adding into DMEM as the control. BEAS-2B-Cr cells formed significantly more colonies in soft agar compared to the P-NC group and NC group (Fig. 1B and C). These results indicated that Cr(vi) exposure was able to significantly enhance anchorage-independent growth of BEAS-2B cells and suggested that chronic Cr(vi) exposure is able to cause malignant cell transformation in BEAS-2B cells, which was not shown in the parent and passage-matched BEAS-2B cells.

Chronic exposure to chromium enhances the migration and invasion of BEAS-2B cells

BEAS-2B-Cr cells increased cell migration up to 3.2 and 2.8 fold compared with the P-NC and NC cells respectively, shown by the wound healing assay (Fig. 2A and B). The result of the Matrigel invasion assay also showed that chronic Cr(vi) stimulation increased the migration and invasion abilities of BEAS-2B-Cr cells (Fig. 2C–E). BEAS-2B-Cr cells increased cell invasiveness approximately 5.9 and 6.2 fold (Fig. 2E) compared with the P-NC and NC cells, respectively. Compared with the P-NC and NC groups, BEAS-2B-Cr cells have enhanced migratory and invasive abilities.

Fig. 2. Chronic exposure of Cr(vi) improved the migration and invasion abilities of BEAS-2B cells. Wound-healing assay showed that BEAS-2B-Cr cells increased cell migration up to 3.2 and 2.8 folds compared with the P-NC and NC cells respectively (A & B). (B) compared the percentage of cells in the scratched area. Transwell chamber experiments showed that BEAS-2B-Cr cells have enhanced migratory and invasive abilities compared with the P-NC and NC groups (C, D, E, **: p < 0.01; ***: p < 0.001). In (C), “Migration cells” means the cells migrated in the bottom chambers, and “Invasion cells” means the cells invaded in bottom chambers. (D & E) compared migratory or invasive cells per field.

Fig. 2

Open in a new tab

Chronic exposure to chromium activates Gli2 in BEAS-2B cells

As the downstream targets of Hh signaling are controlled by the dynamics of Gli transcription factors, Gli proteins are essential in Hh signal transduction processes. Detecting BEAS-2B-Cr cell changes at the genetic level using qPCR, we found that the critical gene Gli2 is up-regulated in BEAS-2B-Cr cells (P < 0.001). However, Gli1 expression did not change (P = 0.08, Fig. 3A). Western blotting confirmed that Gli2 protein expression increased significantly in BEAS-2B-Cr cells (P < 0.001, Fig. 3B). Previous publications reported that Gli2 activators bind to the GACCACCCA motif to regulate the transcription of the key anti-apoptotic factor Bcl-2,14,23 and the Bcl-2 expression is predominantly activated by Gli2 compared with Gli1. Our results show that Bcl-2 protein expression in BEAS-2B-Cr cells increases significantly (Fig. 3C and D).

Fig. 3. Increased expression of Gli2, Bcl-2, LC3-I and decreased expression of LC3-II in BEAS-2B-Cr cells. (A) RT-PCR showed that the Gli2 gene was up-regulated in BEAS-2B-Cr cells while Gli1 gene expression did not change. **, p < 0.01 compared to P-NC and NC groups. (B) BEAS-2B-Cr cells continued to be cultured in normal medium without Cr(vi) and Gli2 protein expression did not decrease with time (***, p < 0.001). (C & D) Western blotting confirmed that Gli2 protein expression in BEAS-2B-Cr cells increased significantly (***, p < 0.001). Bcl-2 protein expression increases significantly, while LC3-II protein expression and LC3-II/I protein ratio were decreased significantly in BEAS-2B-Cr cells (***, p < 0.001 compared to P-NC and NC groups).

Fig. 3

Open in a new tab

Autophagy inhibition in BEAS-2B-Cr cells

Autophagy is a self-degradation process that is important for balancing sources of energy at critical times during development and in response to nutrient stress. Previous research has suggested that autophagic degradation provides an important mechanism to prevent cellular transformation.24 We next sought to determine whether autophagy activity was changed in BEAS-2B-Cr cells. The accumulation of LC3-II is commonly used as a marker of the induction of autophagy.17,25 Two forms of LC3 (microtubule associated protein 1 light chain 3) called LC3-I and LC3-II are produced post-translationally in various cells. LC3-I is cytosolic, whereas LC3-II is membrane bound. We used the LC3-II/I ratio to evaluate the state of autophagy in cells. As shown in Fig. 3C and D, Cr(vi) induced downregulation of LC3-II in BEAS-2B-Cr cells. We found that the LC3-II/I protein expression ratio and autophagosome formation observed under transmission electron microscopy were both reduced, indicating that the ability to initiate autophagy in BEAS-2B-Cr cells decreases significantly.

Gli2 suppresses autophagy promotion in BEAS-2B carcinogenesis

To determine the impact of Gli2 on autophagy, BEAS-2B-Cr cells were treated with small interfering RNA against the Gli2 gene (siRNA-Gli2) via transient transfection to inhibit the Gli2 gene expression. Western blotting confirmed decreasing protein expression of Gli2 after siRNA-Gli2 transfection. The ability to initiate autophagy was restored as shown by an increase in the LC3-II/I protein expression ratio (shown in Fig. 4). The expression of Bcl-2 was decreased after Gli2 silencing, while the expression of Beclin1 did not change.

Fig. 4. Effects of Gli2 silencing with siRNA transfection on expression of Bcl-2, LC3-I, LC3-II and Beclin1 genes in BEAS-2B-Cr cells. Control is the BEAS-2B-Cr cells without siRNA transfection. SiRNA-Gli2 group is the BEAS-2B-Cr cells transfected with SiRNA-Gli2, while SiRNA-control group is the BEAS-2B-Cr cells transfected with non-specific siRNA. Western blotting confirmed decreasing protein expression of Gli2 after siRNA-Gli2 transfection. Decreasing expression of Gli2 caused an increase in the LC3-II/I protein expression ratio, which may suggest higher level of autophagy. The expression of Bcl-2 was decreased after Gli2 silencing, while the expression of Beclin1 did not change (***, p < 0.001 compared to control group).

Fig. 4

Open in a new tab

BEAS-2B-Cr cells were treated with the Gli inhibitor GANT61 (5 μM) alone, and then analyzed for gene expression and autophagy change. Alternatively, the cells were pretreated with 3-methyladenine (3 MA, 60 μM, a classic inhibitor of autophagy at the sequestration stage) prior to GANT61 treatment. As shown in Fig. 5A and B, GANT61 treatment decreased the colony forming ability in the soft-agar assay, while 3 MA treatment rescued the colony forming ability inhibited by GANT61. Western blot analysis showed that GANT61 treatment decreased Bcl-2 expression, and increased LC3-II expression and LC3-II/I ratio. 3 MA together with GANT61 treatment rescued the GANT61's effect on LC3-I and LC3-II, with decreased LC3-II expression and LC3-II/I ratio. 3 MA treatment did not increase Bcl-2 expression (Fig. 5C and D).

Fig. 5. Effects of GANT61 (a Gli inhibitor) and 3 MA (an autophagy inhibitor) on soft agar colony forming ability, gene expression, and autophagy in BEAS-2B-Cr cells. (A, B) GANT61 treatment decreased the colony forming ability in soft-agar assay, while 3 MA treatment rescued the colony forming ability inhibited by GANT61. (C, D) GANT61 treatment decreased Bcl-2 expression, and increased LC3-II expression and LC3-II/I ratio. 3 MA together with GANT61 treatment rescued the GANT61's effect on LC3-I and LC3-II, with decreased LC3-II expression and LC3-II/I ratio. 3 MA treatment did not increase Bcl-2 expression (**, p < 0.01 and ***, p < 0.001). (E) Autophagosome formation was observed by using a transmission electron microscope. BEAS-2B-Cr cells had decreased autophagosome formation compared with NC. GANT61 treatment increased autophagosome formation in BEAS-2B-Cr cells, while 3 MA abolishes GANT61's effects by decreasing autophagosome formation.

Fig. 5

Open in a new tab

Autophagosome formation in cells was observed by using a transmission electron microscope (shown in Fig. 5E). BEAS-2B-Cr cells had decreased autophagosome formation compared with NC. GANT61 treatment increased autophagosome formation in BEAS-2B-Cr cells, while 3 MA abolishes GANT61's effects by decreasing autophagosome formation.

Discussion

Although hexavalent chromium [Cr(vi)] compounds have been classified by the International Agency for Research on Cancer (IRAC, 1990) as carcinogenic in humans, the underlying molecular mechanisms of Cr(vi) in tumorigenesis remain unclear. Bronchial epithelial cells were chosen in this study because they are a key target for Cr(vi)-induced tumorigenesis. BEAS-2B cells have been widely used in the literature to define conditions under which various agents and oncogenes cause neoplastic transformation.26 These cells have also been shown to exhibit characteristics and cellular responses to carcinogens similar to primary or normal lung cells. Although BEAS-2B cells have a mutated p53Ser47 gene, previous studies have shown that this mutation does not affect its growth suppressing and apoptotic functions, which are controlled by p53Ser15 and are normal in BEAS-2B cells.27

In this study, we reported an in vitro model for Cr(vi) tumorigenesis studies using chronically exposed human bronchial epithelial BEAS-2B cells. BEAS-2B cells were chronically exposed to a low dose of Cr(vi) (0.5 μM) for 4 weeks. The soft agar assay was used to examine cell transformation.22,28 Both BEAS-2B-Cr and untreated control BEAS-2B cells were grown in 0.35% top agar for two weeks. The cells treated with 0.5 μM Cr(vi) formed significantly more colonies in soft agar compared to the untreated and passage-matched BEAS-2B cells without treatment (Fig. 1B). These results indicated that Cr(vi) exposure was able to significantly enhance the anchorage-independent growth of BEAS-2B cells and suggested that chronic exposure of BEAS-2B cells to Cr(vi) contributes to malignant cell transformation, which was not observed in the parent and passage-matched BEAS-2B cells. Simultaneously, BEAS-2B-Cr cells have enhanced migration and invasion abilities. These results confirm an altered status of transformed BEAS-2B cells with some cancer cell characters.29

Our results are the first to reveal that continuous exposure to Cr(vi) induces immortalized human normal lung epithelial BEAS-2B cell carcinogenesis by activating the key transcription factor Gli2 but not Gli1 to suppress cell autophagy in the hedgehog pathway. Cr(vi) induces the aberrant activation of Gli2 in BEAS-2B cells, and Gli2 may regulate the expression of the downstream target gene Bcl-2.14 Bcl-2 not only plays an anti-apoptotic role in Cr(vi) carcinogenesis, but also binds to Beclin-1 protein (which stimulates the induction of autophagy) to disable the function of the Beclin-1/Vps34 complex30 and prevent the initiation of autophagy in BEAS-2B-Cr cells. We discovered increasing initiation of autophagy by applying the RNA interference technique to Gli2 gene expression in this study. Then, we confirmed that Gli2 leads to malignant proliferation by inhibiting the autophagy of cells, by using GANT61 to interfere with Gli2 expression and by experiments analyzing cell autophagy and soft agar assay. These findings reveal that chronic exposure to a low dose of Cr(vi) in BEAS-2B cells can induce aberrant expression of Gli2, which plays a critical role in the early carcinogenesis of lung epithelial cells. Gli2 could be a promising early biomarker for monitoring the chronic exposure of Cr(vi) and other environmental carcinogens. Furthermore, the combination treatment of the Gli2-targeted inhibitor GANT61 and autophagy inhibitors/activators in clinical settings to treat lung cancer deserves further studies.

The zinc-finger transcription factor Gli2 has been identified as a critical mediator of Hh signaling at the distal end of the pathway,12 but the molecular mechanisms by which Gli2 regulates cell proliferation or induces epidermal malignancies are still unclear. The detailed molecular mechanism that how chromium upregulates Gli2 and inhibits autophagy has not yet been verified and is the focus of our next study. Beclin-1 (the mammalian ortholog of yeast ATG6) has been well characterized to play a pivotal role in autophagy—a major catabolic pathway in which the cell degrades macromolecules and damaged organelles. The Beclin-1 structure has been identified to contain three identifiable domains, including a short Bcl-2-homology-3 (BH3) motif, a central coiled-coil domain (CCD) and a C-terminal region encompassing an evolutionarily conserved domain (ECD).18 Gli2 up-regulation of Bcl-2 expression may result in an interaction with Beclin-1 and suppression of autophagy, which promotes BEAS-2B cells’ carcinogenesis process.

Conclusion

Hexavalent chromium [Cr(vi)] compounds are human carcinogens related to lung cancer, while the detailed mechanisms are still not clear. Here we report that hexavalent chromium Cr(vi) activates Gli2 to promote the proliferation of BEAS-2B-Cr cells by inhibition of autophagy, which contributes to human bronchial epithelial cell carcinogenesis. Gli2 may not only play an important role in lung cancer pathogenesis, but also be a promising early indicator in monitoring exposure to chromium.

Conflicts of interest

There are no conflicts of interest to declare.

Acknowledgments

This study was supported by a grant from the Natural Science Foundation of Guangdong Province (Grant No. 2015A030313273).

References

  1. Torre L. A., Bray F., Siegel R. L., Ferlay J., Lortet-Tieulent J., Jemal A. CA-Cancer J. Clin. 2015;65:87–108. doi: 10.3322/caac.21262. [DOI] [PubMed] [Google Scholar]
  2. Fu J., Jiang D., Lin G., Liu K., Wang Q. BMJ Open. 2015;5:e009452. doi: 10.1136/bmjopen-2015-009452. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Wu S., Deng F., Hao Y., Shima M., Wang X., Zheng C., Wei H., Lv H., Lu X., Huang J., Qin Y., Guo X. J. Hazard. Mater. 2013;260:183–191. doi: 10.1016/j.jhazmat.2013.05.018. [DOI] [PubMed] [Google Scholar]
  4. Kim D., Dai J., Park Y. H., Fai L. Y., Wang L., Pratheeshkumar P., Son Y. O., Kondo K., Xu M., Luo J., Shi X., Zhang Z. J. Biol. Chem. 2016;291:16271–16281. doi: 10.1074/jbc.M116.715797. [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
  5. Wilbur S., Abadin H., Fay M., Yu D., Tencza B., Ingerman L., Klotzbach J., James S., Abadin H., Fay M., Yu D., Tencza B., Ingerman L., Klotzbach J. and James S., Toxicological Profile for Chromium, Agency for Toxic Substances and Disease Registry (US), Atlanta (GA), 2012. [PubMed] [Google Scholar]
  6. Xia B., Ren X.-H., Zhuang Z.-X., Yang L.-Q., Huang H.-Y., Pang L., Wu D.-S., Luo J., Tan Y.-L., Liu J.-J., Zou F. Toxicol. Lett. 2014;228:241–247. doi: 10.1016/j.toxlet.2014.05.010. [DOI] [PubMed] [Google Scholar]
  7. Lou J., Wang Y., Yao C., Jin L., Wang X., Xiao Y., Wu N., Song P., Song Y., Tan Y., Gao M., Liu K., Zhang X. PLoS One. 2013;8:e71031. doi: 10.1371/journal.pone.0071031. [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
  8. Rodrigues C. F., Urbano A. M., Matoso E., Carreira I., Almeida A., Santos P., Botelho F., Carvalho L., Alves M., Monteiro C., Costa A. N., Moreno V., Alpoim M. C. Mutat. Res. 2009;670:42–52. doi: 10.1016/j.mrfmmm.2009.07.004. [DOI] [PubMed] [Google Scholar]
  9. Zhang S., Shang Z., Zhou P. Toxicol. Res. 2015;4:613–622. [Google Scholar]
  10. Watkins D. N., Berman D. M., Burkholder S. G., Wang B., Beachy P. A., Baylin S. B. Nature. 2003;422:313–317. doi: 10.1038/nature01493. [DOI] [PubMed] [Google Scholar]
  11. Huang L., Walter V., Hayes D. N., Onaitis M. Clin. Cancer Res. 2014;20:1566–1575. doi: 10.1158/1078-0432.CCR-13-2195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Bai C. B., Auerbach W., Lee J. S., Stephen D., Joyner A. L. Development. 2002;129:4753–4761. doi: 10.1242/dev.129.20.4753. [DOI] [PubMed] [Google Scholar]
  13. Ikram M. S., Neill G. W., Regl G., Eichberger T., Frischauf A. M., Aberger F., Quinn A., Philpott M. J. Invest. Dermatol. 2004;122:1503–1509. doi: 10.1111/j.0022-202X.2004.22612.x. [DOI] [PubMed] [Google Scholar]
  14. Regl G., Kasper M., Schnidar H., Eichberger T., Neill G. W., Philpott M. P., Esterbauer H., Hauser-Kronberger C., Frischauf A. M., Aberger F. Cancer Res. 2004;64:7724–7731. doi: 10.1158/0008-5472.CAN-04-1085. [DOI] [PubMed] [Google Scholar]
  15. Sun H., Clancy H. A., Kluz T., Zavadil J., Costa M. PLoS One. 2011;6:e17982. doi: 10.1371/journal.pone.0017982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Mizushima N. Genes Dev. 2007;21:2861–2873. doi: 10.1101/gad.1599207. [DOI] [PubMed] [Google Scholar]
  17. Kabeya Y., Mizushima N., Ueno T., Yamamoto A., Kirisako T., Noda T., Kominami E., Ohsumi Y., Yoshimori T. EMBO J. 2000;19:5720–5728. doi: 10.1093/emboj/19.21.5720. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Fu L. L., Cheng Y., Liu B. Int. J. Biochem. Cell Biol. 2013;45:921–924. doi: 10.1016/j.biocel.2013.02.007. [DOI] [PubMed] [Google Scholar]
  19. Qi Y., Zhang M., Li H., Frank J. A., Dai L., Liu H., Zhang Z., Wang C., Chen G. Cancer Res. 2014;74:3740–3752. doi: 10.1158/0008-5472.CAN-13-3182. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Jimenez-Sanchez M., Menzies F. M., Chang Y.-Y., Simecek N., Neufeld T. P., Rubinsztein D. C. Nat. Commun. 2012;3:1200. doi: 10.1038/ncomms2212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Zeng R., Huang J. P., Li X. F., Xiong W. B., Wu G., Jiang Z. J., Song S. J., Li J. Q., Zheng Y. F., Zhang J. R. Cell Biochem. Funct. 2016;34:133–141. doi: 10.1002/cbf.3170. [DOI] [PubMed] [Google Scholar]
  22. Freedman V. H., Shin S. I. Cell. 1974;3:355–359. doi: 10.1016/0092-8674(74)90050-6. [DOI] [PubMed] [Google Scholar]
  23. Katoh Y., Katoh M. Curr. Mol. Med. 2009;9:873–886. doi: 10.2174/156652409789105570. [DOI] [PubMed] [Google Scholar]
  24. Edinger A. L., Thompson C. B. Cancer Cell. 2003;4:422–424. doi: 10.1016/s1535-6108(03)00306-4. [DOI] [PubMed] [Google Scholar]
  25. Glick D., Barth S., Macleod K. F. J. Pathol. 2010;221:3–12. doi: 10.1002/path.2697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Proctor D. M., Suh M., Campleman S. L., Thompson C. M. Toxicology. 2014;325:160–179. doi: 10.1016/j.tox.2014.08.009. [DOI] [PubMed] [Google Scholar]
  27. Park Y.-H., Kim D., Dai J., Zhang Z. Toxicol. Appl. Pharmacol. 2015;287:240–245. doi: 10.1016/j.taap.2015.06.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Shin S. I., Freedman V. H., Risser R., Pollack R. Proc. Natl. Acad. Sci. U. S. A. 1975;72:4435–4439. doi: 10.1073/pnas.72.11.4435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Hanahan D., Weinberg R. A. Cell. 2011;144:646–674. doi: 10.1016/j.cell.2011.02.013. [DOI] [PubMed] [Google Scholar]
  30. Pattingre S., Tassa A., Qu X., Garuti R., Liang X. H., Mizushima N., Packer M., Schneider M. D., Levine B. Cell. 2005;122:927–939. doi: 10.1016/j.cell.2005.07.002. [DOI] [PubMed] [Google Scholar]

Articles from Toxicology Research are provided here courtesy of Oxford University Press

RESOURCES