The authors have declared that no competing interests exist.
Conceived and designed the experiments: JH YH. Performed the experiments: RL AC JF CL. Analyzed the data: CP RO BA YH. Contributed reagents/materials/analysis tools: CP RO. Wrote the paper: JH YH AC.
The bone morphogenetic protein (BMP) signaling cascade is aberrantly activated in human non-small cell lung cancer (NSCLC) but not in normal lung epithelial cells, suggesting that blocking BMP signaling may be an effective therapeutic approach for lung cancer. Previous studies demonstrated that some BMP antagonists, which bind to extracellular BMP ligands and prevent their association with BMP receptors, dramatically reduced lung tumor growth. However, clinical application of protein-based BMP antagonists is limited by short half-lives, poor intra-tumor delivery as well as resistance caused by potential gain-of-function mutations in the downstream of the BMP pathway. Small molecule BMP inhibitors which target the intracellular BMP cascades would be ideal for anticancer drug development. In a zebrafish embryo-based structure and activity study, we previously identified a group of highly selective small molecule inhibitors specifically antagonizing the intracellular kinase domain of BMP type I receptors. In the present study, we demonstrated that DMH1, one of such inhibitors, potently reduced lung cell proliferation, promoted cell death, and decreased cell migration and invasion in NSCLC cells by blocking BMP signaling, as indicated by suppression of Smad 1/5/8 phosphorylation and gene expression of Id1, Id2 and Id3. Additionally, DMH1 treatment significantly reduced the tumor growth in human lung cancer xenograft model. In conclusion, our study indicates that small molecule inhibitors of BMP type I receptors may offer a promising novel strategy for lung cancer treatment.
Lung cancer is one of the most common types of cancer and the leading cause of cancer deaths. About 228,190 cases of lung cancer are expected to be newly diagnosed in 2013, accounting for ∼27% of all cancer deaths annually in the US
Bone morphogenetic proteins (BMPs) are members of the TGF-β superfamily and their biological activity is mediated through the formation of heterodimeric complexes of the BMP type I and type II serine/threonine kinases receptors. After the ligand binding, the BMP type I receptors are phosphorylated by the constitutively active type II receptors, leading to phosphorylation of the intracellular Smad 1/5/8 proteins, which then form a complex with Smad4 and translocate into the nucleus to regulate transcriptional response
In an
Chemical structure of DMH1; (B) Western blotting for phosphorylated Smad1/5/8 in A549 cells treated with DMH1 at various concentrations (1, 3, and 5 µM); (C) QPCR results of Id1, 2 and 3 in A549 cells treated with DMH1 and vehicle DMSO. *:
A549 and H460 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA), cultured in RPMI 1640 supplemented with 10% FBS (Gibco) and 1% penicillin-streptomycin (Cellgro) in an atmosphere of 5% CO2 at 37°C.
Cells were lysed in RIPA cell lysis buffer (Santa Cruz, Santa Cruz, CA) supplemented with protease inhibitor cocktail (Santa Cruz) and phosphatase inhibitor cocktail 2 (Santa Cruz). Protein concentrations were determined by using BCA protein kit (Thermo Fisher), and cell lysate was isolated in SDS-PAGE and transferred onto PVDF membrane. Alpha-tubulin and p-Smad1/5/8 were detected by Odyssey system (Li-Cor bioscience) following incubation with the appropriate primary and secondary antibodies. Primary antibodies used were rabbit anti-p-Smad1/5/8 (Cell Signaling Tech, 1∶1000 dilution) and mouse anti-alpha-tubulin (Cell Signaling Tech, 1∶1000 dilution). The secondary antibodies used include IRDye 680-conjugated goat anti-rabbit IgG (Li-Cor Bioscience, 1∶5000 dilution) and IRDye 800CW-conjugated goat anti-mouse IgG (Li-Cor Bioscience, 1∶5000 dilution).
A549 and H460 cells were seeded in 35 mm dishes to create a confluent monolayer. The dishes were allowed to incubate overnight in order to allow the cells to attach to the bottom of the dish. On the following day, wounds were created by a straight scratch from a pipette tip in the center of the culture. The cells were then treated with DMSO or DMH1 at 1 µM and 3 µM concentrations. Photographs were taken when wounds were created and after 24 hour's incubation using phase-contrast microscopy, and gap distances were quantitatively evaluated using software ImageJ (NIH). The gap distances after 24 h incubation were normalized with the gap distance at 0 hr as the migration rates.
About 10,000 A549 cells per well were seeded in 96-well plates and incubated for overnight. The culture medium was then changed to fresh medium containing DMSO or DMH1 at various concentrations. The cells were then incubated for 48 hours and 96 hours before treatment termination by replacing the medium with 100 μL of 10% trichloroacetic acid (Sigma) in 1× PBS, followed by incubation at 4°C for at least 1 hour. Subsequently, the plates were washed with water and air dried. The plates were stained with 50 μL 0.4% sulphorhodamine (SRB, Sigma) assay in 1% acetic acid for 30 minutes at room temperature. Unbound dye was washed off with 1% acetic acid. After air drying and solubilization of the protein-bound dye in 10 mM Tris solution, absorbance was read in a microplate reader at 565 nm.
A549 and H460 cells seeded in 6-well plates at a density of 3×105 cells per well were treated with DMSO or DMH1 for 24 hours. Total RNA was extracted using TRIzol Reagent (Invitrogen) following manufacturer's protocol. cDNA was synthesized using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Real-time PCR was carried out using the Power SYBR Green PCR master mix (Applied Biosystems) and with the Applied Biosystems 7300 PCR System. All amplification controls and samples were performed in triplicate. The primer sequences are available upon request.
Cell invasion was measured using a 24-Multiwell Insert System (8 µM membrane, BD Biosciences) according to the manufacture instruction. The cell culture inserts were coated with matrigel (BD Biosciences). A549 cells were seeded at a concentration of 3×105 cells/chamber, After 24-h incubation with or without DMH1 (3 μM), cells that had not moved to the lower wells were removed from the upper face of the filters using cotton swabs, and the cells that invaded through the matrigel-coated-inserts were counted. Mean values for three randomly selected fields were obtained for each well. Experiments were performed in duplicate. Mean values for three random fields were obtained for each well.
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animal experimental protocol was approved by the Western University of Health Sciences Institutional Animal Care and Use Committees (IACUC). All efforts were made to minimize animal suffering. Sub-confluent A549 cells were trypsinized and then suspended in serum free RPMI 1640 medium. The cell suspension (1×106 cells in 100 µl medium for each injection) was injected subcutaneously into both the right and left flanks of eight-week old NOD SCID mice (Taconic, Hudson, NY) (n = 5 for each group). Mice were given Intraperitoneal (i.p.) injection of the vehicle (12.5% 2-hydroxypropyl-β-cyclodextrin) or 5 mg/kg DMH1 every other day. The tumor sizes were measured with a vernier caliper from the sixth day to the fourth week after tumor implantation. The tumor volume (V) was calculated according to the formulation: Volume = (width)∧2× length/2. The tumor tissues were dissected at the end of study, and were sectioned and stained with H & E, and for immunohistochemical analysis.
Lung tumor tissues from both the vehicle and DMH1 treated mice were excised and fixed immediately in formalin and embedded in paraffin blocks. The embedded tumors were cut into 3 micron thick sections and stained with H&E to determine morphology. Cell proliferation in the tumors was performed at Pathology Inc. Laboratories (Torrance, California) detected by immunostaining with an antibody to Ki67 (Dako MIB-1 Clone, Carpinteria, California), used at 1∶50 on Leica Bond III IHC staining system. The stained sections were visualized and photographed with a Nikon microscope image capture system (Nikon DS-Ri1) and public domain software (NIH Image v1.60).
Data are expressed as mean ± standard error. The data was analyzed using NCSS 2007 (Kaysville, UT) via t-test for comparison of two means, one way ANOVA with Fisher's LSD post-hoc test for comparison of multiple means, and repeated measures ANOVA with Tukey-Kramer post-hoc test for the
We first examined whether DMH1 can block BMP signaling in NSCLC cells using Western blotting and quantitative real-time PCR (qPCR). NSCLC A549 cells were treated with the vehicle (DMSO) or DMH1 at 1, 3, and 5 μM for 24 hours, respectively. Western blotting analysis indicated that A549 cells displayed relatively high basal phospho-Smad 1/5/8 expression, and DMH1 treatment effectively blocked Smad 1/5/8 phosphorylation in a dose dependent manner (
Since cell migration and invasion are known to play an important role in the progression of cancer metastasis, we examined the effects of DMH1 on NSCLC cell migration and invasion
Representative images from scratch-wound assays performed in the cultured A549 cells treated with DMSO, BMP4 and DMH1 (1 µM and 3 µM) for 24 hours. (B) Cell migrations of A549 cells were quantified by the gap distances after 24 hour treatment normalized with the initial gap distances. (C) Cell migrations of H460 cells were quantified in a similar way. (D) The effect of DMH1 (3 µM) on A549 cell invasion was determined using modified Boyden chamber assay in a 24-Multiwell Insert System (8 µM membrane, BD Biosciences) coated with matrigel. *:
We next examined the effects of DMH1 on NSCLC cell proliferation and survival. A549 cells were treated with DMH1 and the vehicle DMSO for 48 hours, and cell proliferation was determined by the sulforhodamine B (SRB) assay. The result showed that 5 µM DMH1 led to about 10% reduction in cell growth after two day treatment, suggesting that inhibition of BMP signaling by DMH1 significantly reduces lung cancer cell proliferation
A549 cells were treated with DMH1 (3 and 5 µM) or DMSO for 48 hours and cell proliferation was determined by the sulforhodamine B (SRB) assay. (B) A549 cells were treated with DMH1 or DMSO for 72 hours, and cells were harvested for cell death assay using Trypan Blue staining. *:
We next examined the effect of DMH1 on lung tumor cell growth
The A549 cells were subcutaneously implanted into the SCID mice followed by intraperitoneal injection of the vehicle (12.5% 2-hydroxypropyl-β-cyclodextrin) or 5 mg/kg DMH1 every other day for 4 weeks. Tumor volumes were measured from the sixth day to four weeks after injection. (B) Representative tumor tissues from mice treated with the vehicle control and DMH1 are compared. (C) Tumor tissues were stained for human specific Ki67 proliferation marker. *:
BMPs are aberrantly expressed in many types of carcinoma cells including prostate, lung, breast, gastric and ovarian
The effects of DMH1 on attenuating lung tumor growth may be mediated by multiple mechanisms, including disrupting the tumor microenvironment or lung cancer stem cells. Previous studies reported that BMP-2 induced the neovascularization of developing tumors, and stimulated blood vessel formation in A549-derived tumors in nude mice
In the in vivo study using the A549 xenograft model, the treatment was initiated on the same day of tumor cell implantation. Therefore, the tumor sizes were relatively small at the end of the in vivo experiments. In future studies, it remains to determine whether treatment by DMH1 following different schedules can result in greater anticancer effects. Although DMH1 treatment significantly attenuated lung cancer growth
In summary, our study demonstrated that antagonizing BMP type I receptors with small molecules is effective on suppressing lung cancer cell proliferation, migration, invasion