Letter pubs.acs.org/NanoLett
Focal Amplification of HOXD-Harboring Chromosome Region Is Implicated in Multiple-Walled Carbon Nanotubes-Induced Carcinogenicity Ping Wu,†,‡ Shin-Sheng Yuan,§ Chao-Chi Ho,∥ Wan-Yu Hsieh,‡ Qi-Sheng Hong,⊥ Sung-Liang Yu,⊥ Wei Chen,# Hsuan-Yu Chen,§ Chin-Di Wang,§ Ker-Chau Li,§,∇ Pan-Chyr Yang,∥ and Huei-Wen Chen*,‡ †
Department and Institute of Pharmacology, School of Medicine, National Yang-Ming University, Taipei, Taiwan 112 Graduate Institute of Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan 106 § Institute of Statistical Science, Academia Sinica, Taipei, Taiwan 115 ∥ Department of Internal Medicine, National Taiwan University Hospital and National Taiwan University Medical College, Taipei, Taiwan 106 ⊥ Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei, Taiwan 106 # Department of Chemistry, National Taiwan University, Taipei, Taiwan 106 ∇ Department of Statistics, University of California, Los Angeles, Los Angeles, California 90095, United States ‡
S Supporting Information *
ABSTRACT: Multiple-walled carbon nanotubes (MWCNTs) may cause carcinogenesis. We found that long-term exposure to MWCNTs can induce irreversible oncogenic transformation of human bronchial epithelial cells and tumorigenicity in vivo. A genome-wide array-comparative genomic hybridization (aCGH) analysis revealed global chromosomal aberration in MWCNTstreated clones, predominantly at chromosome 2q31−32, where the potential oncogenes HOXD9 and HOXD13 are located. Functional assays confirmed that this variation can modulate oncogenic signaling and plays a part in MWCNTs-induced tumorigenesis, suggesting that MWCNTs are carcinogens that act by altering genomic stability and oncogenic copy numbers. KEYWORDS: Array comparative genomic hybridization analysis (aCGH), carcinogenesis, chromosome aberration, genome-wide, HOXD family, multiple-walled carbon nanotubes (MWCNTs)
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may cause an airway obstructive response, inflammation, and pulmonary granuloma formation via regulating inflammatory signaling and tissue remodeling.7,8 The accumulated evidence suggests that inhalation of CNTs poses a significant occupational hazard. Previous studies have revealed correlations between particulate air pollution levels, respiratory/cardiovascular diseases, various cancers, and mortality.9,10 Exposure of CNTs has been suggested to promote tumorigenesis.11,12 The potential risk of initiating carcinogenesis posed by CNTs, which are similar to some hazardous fibers (e.g., asbestos), may cause mesothelioma in humans and/or experimental animals.13,14 The lungs are the major target organ for airborne CNT exposure, and dozens of previous reports have indicated that CNTs, especially
arbon nanotubes (CNTs) display unique physical and chemical properties that are of interest due to numerous potential novel industrial and biomedical applications.1 Consequently, up to 1000 tons of CNTs are produced in the U.S. per year.2 With the development of CNTs, the residue or pollution generated from CNTs manufacturing or production has raised potential safety issues in regard to both environmental risk and human health.3 CNTs with fibrous structures are manufactured in two main forms: as single-walled CNTs (SWCNTs) and as multiple-walled (MWCNTs). SWCNTs are composed of a single-layer graphene sheet rolled into a cylindrical shape, whereas MWCNTs contain several layers of chrysotile-like structures.4 CNTs, found from both indoors and outdoors in fine particulate matter (PM) size, may cause cytotoxicity in lung epithelial cells.5 In rodent models, a single dose of CNTs can persist in lung for over 60 days and induces inflammatory, fibrotic reactions and granulomas in the bronchial lumen, together with alveolitis in the surrounding tissues.6 Intratracheal administration of SWCNTs to ICR mice © 2013 American Chemical Society
Received: May 7, 2013 Revised: August 9, 2013 Published: August 28, 2013 4632
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Figure 1. MWCNTs induce colony formation under long-term passaging conditions in BEAS-2B cells, leading to irreversible colony formation ability in vitro, as assessed through soft agar assays, in addition to tumorigenicity in NOD/SCID mice in vivo. (A) Exposure to MWCNTs from passages 10 to 40 increased the ability of BEAS2B cells to form colonies. The cells were plated once every 3−4 days. The empty bars indicate the vehicle control group, and black bars indicate MWCNTs-treated BEAS2B cells. * indicates P < 0.05 (compared to the untreated group consisting of cells from different passages). (B) Schematic representation of the strategy applied in MWCNTs-treated cells to test the reversibility of the induction of colony formation. The control group consisted of cells maintained without exposure to MWCNTs, whereas the MWCNTs-P40 cells experienced long-term exposure to MWCNTs. The W-P10 cells were MWCNTs-P40 cells that were maintained for 10 additional passages in the absence of MWCNTs. (C) Cells exposed to MWCNTs showed an irreversible ability to form colonies. Empty bars indicate the control-P40 group; gray bars indicate the colony-forming ability of the MWCNTs-P40 cells; and black bars indicate the significantly increased colony-forming ability observed in W-P10 cells. * indicates P < 0.05 (compared to vehicle controls). (D) Tumors from MWCNTs-treated BEAS2B cells. A549 lung adenocarcinoma cells were used as a positive control for tumor growth. Tumors were observed to form in both the MWCNTs-P40 and W-P10 cells. (E) Hematoxylin and eosin (H&E)-stained images revealed proliferation of the A549, MWCNTs-P40, and W-P40 cells. (F) Tumorigenicity of MWCNTs-treated BEAS2B cells. A549 cells showed 100% tumorigenicity (3/3) in NOD/SCID mice. We performed two vehicle controls to demonstrate the fate of nontumorigenic BEAS2B cells in NOD/SCID mice (the original control, P0, and a long-term control P40). The MWCNTs-P40 cells showed 80% (4/5) tumorigenicity, while the W-P10 cells displayed 100% tumorigenicity.
apoptosis as well as increase the formation of micronuclei (MN) in bone marrow cells or monocytes in mice,25,26 in human lymphocytes,23 and in human lung epithelial cells.27−30 Gonzalez et al. hypothesized that CNTs are capable of disturbing mitotic spindle formation,31 resulting in aneuploidy.32 It has also been suggested that CNTs interact with mitotic spindles, mimicking microtubules or motor proteins, and thus, disturbing the separation of chromosomes during cell division.33 Most interestingly, it has been proposed that CNTs interact with DNA34 and selectively bind to certain sequences according to their chirality and the binding affinity of hydrogen bonds.35 These findings suggest that MWCNTs may directly or indirectly effect genotoxicity and can act as clastogenic and aneugenic agents, thereby giving rise to potential carcinogenesis. In the present study, we discovered that MWCNTsinduced chromosomal instability may occur in certain
MWCNTs, can cause chronic inflammation, pulmonary granuloma, and asbestos-related pathological changes in rodent models.15−18 In a study performed in heterozygous p53 mice,19 Takagi et al. discovered that peritoneal adhesion and granuloma formation occur in a dose-dependent manner following intraperitoneal exposure to MWCNTs. However, the incidence of tumor formation was found to be random in different dosage groups.20 MWCNTs with a length of greater than 5 μm are pathogenic to the pleura in C57Bl/6 mice, suggesting that longscale CNTs may pose an asbestos-like mesothelioma hazard.21 Although the pathogenic effects of MWCNTs are related to tumorigenesis, the mechanism underlying MWCNTs-induced tumorigenesis is unclear at present. MWCNTs-induced cytotoxicity, genotoxicity, and/or regulation of the inflammatory response have been reported in several different cell lines.22−24 MWCNTs can cause DNA fragmentation and 4633
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chromosomal regions. A functional assay showed that the amplification of oncogenic genes (e.g., HOXD9 and HOXD13), located on chromosome 2q31−32, may play a role in MWCNTs-induced carcinogenesis and participate in several lung cancer-related signaling pathways. MWCNTs Induction of Irreversible Malignancies in Human Lung Epithelial Cells. It has been suggested that MWCNTs can induce asbestos-like pathogenicity, mesothelioma, or lung cancer.15,18 Although a previous study showed that MWCNTs can induce cell transformation in mouse fibroblast cell lines,36 we believe that lung epithelial cells are the major target of inhaled nanoparticles. Here, we demonstrated that long-term incubation with MWCNTs could cause malignant cell transformation in human lung epithelial cells (BEAS2B cells). The dosage of MWCNTs to BEAS2B in culture was 1 μg/mL in culture medium. The total exposure volume was 10 mL in 100 mm cell culture dishes, respectively. From this, it may be calculated that an applied particle concentration of 1 μg/mL corresponds to a concentration of 0.16 μg/cm2 on the surface of the culture dishes. It mimics physiologically relevant in vivo doses (30−40 μg/mouse lung), and it was calculated and based on previous studies to induce pathological changes in the lungs, including granuloma, fibrosis, and inflammation.37−39 After subcultures of BEAS2B cells were exposed to manufacture MWCNTs (see Supplementary Figure S1 and Table S1) for 40 passages (P40), we found that MWCNTs could induce anchorage-independent colony formation in the cells (Figure 1A). This result indicated that transformation ability was significantly increased in the MWCNTs-treated cells, whereas the BEAS-2B control culture showed no significant changes in colony-formation ability from P10 to P40. Most importantly, we observed that the MWCNTs-induced oncogenic transformation was irreversible, as demonstrated by the retention of the ability to undergo anchorage-independent colony formation following the removal of MWCNTs for 10 passages (W-P10) (Figure 1B). To demonstrate the tumorigenicity of the transformed cells in a xenograft model, cells from the 40 passage MWCNTs treatment (MWCNTs-P40), from passagematched controls (control-P40), and from the treatment groups in which MWCNTs exposure was withdrawn (WP10) were subcutaneously injected into NOD/SCID mice (N = 5 for each group). The results showed that the MWCNTs-P40 and W-P10 groups both displayed high tumor-generating abilities (80−100%) compared to the control-P40 group, which exhibited no tumorigenicity (0%) in the xenograft model (Figure 1D,E). Histological examination revealed the presence of rapidly proliferating cells with high nucleus/cytoplasmic ratios in the MWCNTs-induced transformed cells forming tumors (Figure 1F). These results indicate that MWCNTs possess the ability to induce irreversible malignant cell transformations in human lung epithelial cells. To further elucidate the effects of MWCNTs in lung epithelial cells and the potential underlying mechanisms. Cells undergoing MWCNTs-induced anchorage-independent growth were collected as highly tumorigenic transformed subclones, in accordance with previous studies40 (Figure 2A), and their ability to form colonies on soft agar was examined (Figure 2B). However, the colonies of control group cannot grow well compared to MWCNTs-transformed BEAS2B cells. Therefore, we choose the parental BEAS2B cells with comparable 40 passages (control-p40) as control group. Ten of the 16 selected clones showed a similar or greater ability to form colonies compared to the mixed clones (Figure 2B). Furthermore, five
Figure 2. Clones harvested from MWCNTs-treated BEAS2B cells inherited diverse colony-forming abilities and tumorigenicity in NOD/ SCID mice. (A) Schematic representation of clones harvested from MWCNTs-treated cells grown in soft agar. We collected approximately 20 clones from soft agar and demonstrated colony formation through a soft agar assay. (B) MWCNTs-selected clones showed diverse colony-forming abilities in a soft agar assay. Clones selected from the MWCNTs-treated BEAS2B cells (clones 1−16) showed different colony-forming abilities than the control group. (C) The incidence of tumorigenicity among parental MWCNTs-treated cells and selected clones. A549 cells showed 100% tumorigenicity (3/3) in NOD/SCID mice. Five different clones, along with controls, were used to demonstrate tumorigenicity in NOD/SCID mice, with the following percentages of tumor formation being obtained: MWCNTsP40 cells showed 80% (4/5) tumorigenicity; the mixed clones, 60% (3/5); clone 2, 80% (4/5); clone 6, 40%; clone 7, 60%; clone 8, 0%; and clone 9, 80%. (D) Histological images of tumors arising from MWCNTs-selected cells, mixed clones, clone 6 and clone 7, showing poorly differentiated cells (H&E staining).
independent clones (MWCNTs-C2, C6, C7, C8, and C9) obtained from the clones that exhibited the greatest colonyforming ability also displayed significant tumorigenicity in vivo, as compared to the nontumorigenic control-P40 group (Figure 2C). The pathohistological evidence indicated that these MWCNTs-selected clones presented the ability to form poorly differentiated carcinomas in the xenograft model (Figure 2D). These findings showed that MWCNTs could induce 4634
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Figure 3. Chromosomal instability in control cells and five different clones observed following the amplification of chromosome 2 from MWCNTsselected clones via aCGH analysis. (A) The aCGH analysis revealed amplification of the 2q31−32 region in mixed clones and in clones 2, 6, 8, and 9. (B) Quantitative PCR analysis of genes expressed in parental MWCNTs-treated cells and selected clones; gene expression was compared to the untreated control group. The genes are listed in order of their location within the 2q31−32 locus. The expression level is indicated by the color bar. Green indicates that expression is down-regulated compared to the control group, and red indicates up-regulation compared to the control group. (C and D) HOXD9 and HOXD13 copy numbers were elevated in the MWCNTs-selected clones. * indicates P < 0.05 (compared to the passage passaging control).
Carcinogenesis is a multistep process and can be associated with irreversible genetic changes. The accumulated evidence and our data support the hypothesis that MWCNTs act as a potential genotoxic nanomaterial (see Supplementary Figure S2).18,27,43,44 Here, we hypothesized that MWCNTs might cause irreversible malignant transformation through modulating chromosomal aberrations, which could affect the amplification of key oncogenic signals. To discover the regions of DNA affected by MWCNTs exposure and the potential carcinogenic mechanisms associated with MWCNTs, a high-resolution aCGH analysis was performed to detect genome-wide alterations in gene copy numbers in MWCNTs-selected clones.
irreversible neoplastic transformation in human lung epithelial cells. Global Chromosomal Aberrations in MWCNT-Selected Clones. Previous studies have analyzed cellular responses to MWCNTs both in vitro and in vivo through gene expression or protein profiling and attempted to elucidate the potential underlying toxicity mechanisms and signaling pathways involved in MWCNTs-induced toxicity.41,42 However, cellular signaling and gene expression can only explain part of the toxicity of MWCNTs, and examination of these processes alone may not reveal the mechanism of MWCNTsinduced carcinogenic transformation of normal epithelial cells. 4635
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Figure 4. Functional validation of HOXD9- or HOXD13-expressing HEK 293 cells using in vitro and in vivo assays. (A) Western blots obtained from stable clones of HOXD9- or HOXD13-expressing HEK293 cells. HOXD9-HA or HOXD13-V5 tagged constructs were introduced into HEK293 cells, which were then selected with G418 for 1 week. (B) Increased anchorage-independent growth was observed in HEK293 cells expressing HOXD9 or HOXD13. Six samples of 2000 cells each were suspended in 0.35% soft agar in 6-well plates; the assay was performed in triplicate. Colonies were further analyzed after 3 weeks of induction. Light gray bars indicate the MOCK control, and dark gray bars indicate cells expressing HOXD9 or HOXD13. * indicates P < 0.05 (compared to the MOCK control). (C) Comparison of the tumorigenicity of HEK293 cells expressing HOXD9 or HOXD13 in NOD/SCID mice. Groups of male NOD/SCID mice (aged 8−10 weeks, weighing approximately 30 g each; n = 4−9 mice per group) were injected subcutaneously in the left dorsum with 2 × 10 to the seventh power cells from each clonal cell line. The resultant tumors were weighed at the termination of the experiment, 21 days after tumor inoculation. * indicates P < 0.05 (compared to the MOCK control). Photographs of representative tumors arising from HEK293 cells expressing HOXD9 or HOXD13 and vector-expressing tumors are presented.
cancer, including lung, breast, and colon cancer.50 Additionally, some of the chromosomal areas that showed recurrent DNA copy number amplifications in the 2q31−32 region have been found in nonsmall cell lung cancers, head and neck squamous cell carcinomas, and endometrium cancers.51−53 HOXDs May Act as Oncogenes and Contribute to MWCNTs-Induced Carcinogenesis. Among the selected 17 putative oncogenic genes in the 2q31−32 region, the top five overexpressed genes in the MWCNTs transformed mix clone were HOXD13, CIR1, HOXD9, ATF2, and SP5. By examining the probe distribution of the aCGH design, we found that HOXD9 and HOXD13 genes were covered with the designed probe, which is highly amplified (corresponding sequence is from chromosome 2, 176934449 to 176996981). To address
As expected, we found that the chromosomal aberrations observed in MWCNTs-selected clones were significantly greater than in untreated cells (Figure 3A). Especially, the region of chromosome 2 from 2q31 to 32 showed significant genome amplification in the mixed clones and single clones 6, 8, and 9 (Figure 3A). There are approximately 200 genes located at this region. Based on surveys of the literature, we selected 17 putative oncogenic genes and conduct real-time PCR to measure gene expression. The results showed that several genes were highly expressed in different MWCNTtreated clones (Figure 3B), such as ITGA6,45 OLA1,46 CIR1/ CROC1,47 ATF2,48 and members of the homeobox D family (HOXD),49 and so forth. Alterations in the somatic copy number have previously been identified in several types of 4636
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Figure 5. Silencing of HOXD9 or/and HOXD13 expression in cells of MWCNTs-selected mixed clones affects cell proliferation and decreases anchorage-independent growth ability and tumorigenicity in vivo. (A) Western blots demonstrating the efficiency of HOXD9 and HOXD13 siRNA treatment 48 h after the transfection of mixed clones. The HOXD9 and HOXD13 protein levels were quantified via densitometry and are expressed relative to actin. Each group was compared to a scrambled control. (B) Cell growth in MWCNTs-selected mixed clones following HOXD9 or/and HOXD13 siRNA transfection. The proliferation of siRNA-transfected cells was measured spectrophotometrically through MTT assays using DMSO to dissolve formazan. * indicates P < 0.05. Error bars represent ± SEM. (C) Decreasing anchorage-independent growth was observed in mixed clone cells in which HOXD9 and/or HOXD13 was silenced. Six samples of 500 cells each were suspended in 6-well plates containing 0.35% soft agar; the assays were performed in triplicate. Colonies were analyzed after 3 weeks of induction. * indicates P < 0.05 (compared to the scrambled control). (D) The tumorigenicity of silencing HOXD9 and HOXD13 in the MWCNTs selected mix clone via xenograft model. Photographs of absent tumors from MWCNTs selected mix clone under silencing of HOXD9 and HOXD13 siRNA. The siRNA transfection protocol was followed by previous experiments. After 48 h transfection, each group (2 × 10 to the sixth power cells in 100 μL of HBSS) was subcutaneously injected into 5-week-old NOD/SCID mice. After 30 days of monitoring, the incidence of tumorigenicity in MWCNTs-mix clone was 83% (5/6); HOXD9 and HOXD13 siRNA treated group were 33% (2/6); Combined siRNA treated group was 16% (1/6). (E) The tumor volume was measured, and the P value was represented by comparing to scramble control. 4637
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Figure 6. Scheme of the effects of MWCNTs regarding transformation in normal bronchial epithelial cells. Chromosomal aberrations and genomic amplification on chromosome 2 (locus 2q31−32) leading to the malignant transformation of cells represents the current suggested mechanism of MWCNTs-induced tumorigenesis. Analysis of KEGG signaling pathways showed lung cancer cell related pathway between vehicle control cells and HOXD9 and D13 expressing cells. Scheme represents reconstructed signaling network that may be related to HOXD9 and HOXD13 regulation. These genes may affect cell proliferation, apoptosis, and EMT. 4638
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colonies and for the tumorigenicity in MWCNTs-selected clones. To shed further light on the potential functions of HOXD9 and HOXD13 in tumorigenicity and the underlying mechanisms, a transcriptomic analysis was performed to examine regulatory signaling in HOXD9- and HOXD13-overexpressing HEK293 cells. Using a comprehensive web server, the Composite Regulatory Signature Database2 (CRSD2, http:// syslab.nchu.edu.tw/crsd2),61 the complex regulatory behaviors associated with the gene expression signatures of HOXD9 and HOXD13 were investigated. According to KEGG pathway analysis, the top 10 major up-regulated pathways are highly correlated with cancer, specifically lung cancer (small cell and nonsmall cell lung cancer) (Figure 6). Several well-known oncogenic genes are implicated in these signaling pathways, including MYC, FOS, and cyclinD1 (CCND1). Other candidate genes, PI3K and protein kinase C is related to epithelial-mesenchymal transition (EMT) and carcinogenesis.62,63 Further, cross-talk pathways (FGFR364 and ITGA865) and downstream signaling may play a role as an inducer for several important oncogenic factors, MYC, FOS, and cyclinD1 to promote cell growth, survival, and transform the cells to malignant66 (Figure 6). Based on the microarray analysis, we suggest that the HOXD’s downstream oncogenic factors, such as CCND1 and c-MYC, may play the roles in MWCNTstransformed lung epithelial cells. Here, we found that the MWCNTs-transformed lung epithelial cells within higher expression levels of these HOXD’s downstream oncogenes (CCND1 and c-MYC)(*, P < 0.05, compared to control-P40) (Supplementary Figure S6). Our data suggest that HOXD9 and D13 may involve in MWCNTs-induced cell transformation and carcinogenesis, whereas CCND1 and c-MYC may contribute on HOXD oncogenic signaling. We conclude that MWCNTs can induce chromosomal aberration, especially the amplification of chromosome 2q31− 32, thereby causing the irreversible cell transformation in lung epithelial cells. We demonstrated both in vitro and in vivo the potential carcinogenicity of genes HOXD9 and HOXD13 that are located in this region. These evidence suggest that HOXD9 and HOXD13 may act as MWCNTs-related oncogenes which trigger the lung cancer−associated signaling pathways. Consequently, our results support the viewpoint that MWCNTs are potentially hazardous to human health and their long-term effects and potential carcinogenic risk of exposure to MWCNTs should be carefully monitored. Guidance on safe usage of CNTs in future industrial applications and more studies on molecular mechanisms of MWCNTs-induced genotoxicity and carcinogenesis are urgently called for.
the importance of HOXD9/D13 on MWCNTs-induced carcinogenesis, we further analyze the relationship between transformation ability and variation of copy number (CNV) in other MWCNTs transformed clones. The copy numbers of HOXD on colonies with lower transformation abilities were measured via copy-number Q-PCR. We found that these colonies (Clone No. 1, 3, 5, 11, 12, and 16) have a much lower CNV than those with higher transformation abilities (Clone No. 2, 6, 7, 8, 9, and mixed clone) (see Supplementary Figure S3). Also, a HOXD genes family, which was concentrated and located at the 2q31−32 chromosomal locus, plays an important role in embryonic development, cell proliferation, and apoptosis and is required for vertebrate limb morphogenesis.54 A previous study showed that HOXD9 play an important role in maintaining tumorigenicity in glioma stem cell.55 Further, HOXD13 also found to fuse with NUP98 and manipulate hematopoietic differentiation into acute leukemia.56,57 According to previous studies and our preliminary results, we further investigate if HOXD9 and HOXD13 play the role in MWCNTs-induced carcinogens. First, we confirmed that HOXD9 and HOXD13 were amplified in the MWCNTsselected clones (the mixed clones and clones 6, 8, and 9) via genomic PCR. This finding is consistent with that the results of the aCGH analysis (Figure 3C,D). To confirm the function of HOXD9 and HOXD13 in MWCNTs-induced carcinogenesis, we further introduced HOXD9 and HOXD13 into HEK293 cells. The results showed that overexpressing either HOXD9 or HOXD13 would display a greater ability to form colonies compared with vehicle-treated controls (Figure 4A,B). Most importantly, HOXD9 and HOXD13 significantly promoted tumor growth in the xenograft model in vivo (Figure 4C). These results suggested that HOXD9 or HOXD13 might contribute to MWCNTs-induced malignant transformation. Aberrations in HOX gene expression have been reported in cases of abnormal development and malignancy, indicating that altered expression of HOX genes could be important factor in oncogenesis.49 A previous comparison of HOX gene expression between adenocarcinoma and squamous cell carcinoma tissues revealed higher expression of the HOXA1, D9, D10, and D11 genes in squamous cell carcinomas.58,59 To explore the clinical significances of the HOXD genes in lung cancer patients, the public available gene expression data sets were downloaded from GEO.60 We carried out the cox regression with proportional hazard and found that HOXD13 has the significant coefficient. The corresponding hazard ratio was estimated to be 1.45 (see Supplementary Figure S4). The results suggest that the increased expression of HOXD13 might be a key genetic event during cancer development. To validate the functions of HOXD9 and HOXD13 in the MWCNTstransformed cells, we also introduced siRNA targeting HOXD9 and HOXD13 into the MWCNTs-selected mixed clones (Figure 5A). The data revealed small or no effects following single-gene knockdown (of only HOXD9 or HOXD13), whereas the application of combined siRNAs targeting HOXD9 and HOXD13 significantly inhibited cell proliferation (Figure 5B). This result was also corroborated through anchorage-independent assays (Figure 5C). Similar results also showed in clone 8 and 9 (see Supplementary Figure S5). Moreover, the incidence of tumorigenicity in MWCNTs selected mix clone was significantly reduced by HOXD siRNA in the xenograft model (Figure 5D, E). Thus, HOXD9 and HOXD13 may be crucial for cells to form
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ASSOCIATED CONTENT
S Supporting Information *
A detailed description of the experimental procedures, TEM and SEM image, gene expression, table for MWCNTs characteristics, and other supporting results. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Graduate Institute of Toxicology, College of Medicine, National Taiwan University, Taipei, 10051, Taiwan. Tel.: +886-2-2312-3456 ext. 88606; Fax: +886-2-2341-0217. Email:
[email protected]. 4639
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Notes
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The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
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REFERENCES
We would like to thank Dr. Yih-Leong Chang from Department of Pathology, National Taiwan University Hospital and National Taiwan University College of Medicine for the technical assistance on pathology. We greatly appreciate the assistance with SEM provided by the Joint Center for Instruments and Researches, College of Bioresources and Agriculture at National Taiwan University. We thank American Journal Experts (certificate verification key: F670-B8F1-B51144FA-2BCA) for English editing. This work was supported by grants from the National Science Council (NSC101-2325-B002-047), the National Taiwan University Cutting-Edge Steering Research Project (NTU CESRP-10R71602C2 and 100R705057), Academia Sinica (AS- 100-TP-AB2), and the National Taiwan University Center of Integrated Core Facilities for Functional Genomics and Microarray Core Facility.
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