Figure 1.
BMP type I receptor antagonists decrease Smad 1/5/8 activity in H1299 cells.
(A) H1299 cells were transfected with BRE-luciferace reporter. After 48 hours the cells were treated with DMSO, 10 µM Dorsomorphin, or 1 µM DMH1, 1 µM DMH2, or 1 µM LDN for 48 hours. BRE-luciferace activity is reported as the percent of the DMSO control treated cells. Data represents the mean of 3 experiments in triplicate. The mean of the control cells was compared to the mean of the treated cells. * p<0.05. (B) Immunoblot analysis for phosphorylated Smad 1/5/8 of H1299 cells treated with 1 µM DMSO, DMH1, or DMH2 for 12, 24, and 48 hours.
Figure 2.
BMP antagonists decrease the expression of Id family members in A549 and H1299 cells.
(A–B) Quantitative RT-PCR for Id1, Id2, and Id3 on (A) A549 and (B) H1299 cells treated with DMSO, 10 µM Dorsomorphin, 1 µM DMH1, or 1 µM DMH2 for 48 hours. Data represents the mean of at least 3 experiments performed in duplicate and presented as the percent of control treated cells. The mean of the control cells was compared to the mean of the treated cells. * p<0.05. (C–D) Western blot analysis for Id1 and Id3 on (C) A549 and (D) H1299 cells treated with 1 µM DMSO or 1 µM of selective BMP type I receptor antagonist for 12, 24, and 48 hours. These studies were performed at least 3 times.
Figure 3.
Multiple BMP type I receptors mediate BMP signaling in lung cancer cell lines.
(A) Quantitative RT-PCR of the A549 and H1299 cells showing expression of alk2, alk3, and alk6 but not alk1 (n = 3). (B) H1299 cells were transfected with siRNA targeting each type I BMP receptor and quantitative RT-PCR was performed for that BMP receptor. (C) Knockdown of each BMP type I receptor in H1299 cells was performed and quantitative RT-PCR performed for all 3 type I receptors. (B–C) Data represents the mean of 2 experiments performed in duplicate. (D) Knockdown in H1299 cells of a single BMP type I receptor or both alk2 and alk3. After 48 hours the expression of Id1 was examined by quantitative RT-PCR. Data represents the mean of 3 experiments performed in duplicate. (E) Western blot analysis for Id1 in H1299 cells with knockdown of a single type I BMP receptor or combination knockdown of alk2 and alk3, or all 3 BMP type I receptors. Studies were done at least 2 times. Studies show silencing more than one receptor is required to decrease Id1 expression. (F) Quantitative RT-PCR of the type I BMP receptors after knockdown of alk2 and alk3, or all 3 BMP type I receptors. Studies done 3 times in duplicate reported as percent of control. (G) H1299 cells were co-transfected with insertless vector, constitutively active alk3 (ca-alk3), or constitutively active alk6 (ca-alk6) expression vectors and the BRE-luciferase reporter. Cells were then treated with 1 µM DMSO or 1 µM BMP receptor antagonist for 24 hours and luciferace activity measured. Data demonstrates the mean of at least 3 independent experiments shown as the percent of control.
Figure 4.
Antagonizing BMP type I receptors decreases cell growth, proliferation, and clonogenicity of lung cancer cell lines.
(A) A549 and H1299 cells cultured in DMEM 5% FCS were treated with DMSO, 10 µM Dorsomorphin, 1 µM DMH1, 1 µM DMH2, or 1 µM LDN for 7 days and cell counts performed. (B) H1299 cells cultured in SFM were treated with 1 µM DMSO or 1 µM DMH2 for 7 days and cell counts performed. (C) BrdU incorporation of H1299 cells treated with DMSO or 1 µM or 5 µM DMH2 for 24 and 48 hours. (D) BrdU incorporation of H1299 cells transfected with siRNA targeting of all type I BMP receptors or siRNA control. (C–D) Data is the mean of 3 experiments in triplicate reported as the percent of control treated cells. (E–G) Colony growth of A549 and H1299 cells treated with 1 µM DMSO or 1 µM of selective BMP receptor antagonist. (E) Photograph of a representative experiment. (F–G) The data shows the mean of at least 3 independent experiments reported as the percent of control. (G) DMH1 decreases anchorage independent growth of lung cancer cell lines. A549 and H1299 cells in soft agar were treated with 1 µM DMS0 or 1 µM DMH1 for 2 weeks and the number of colonies counted. The data shown is the mean of 3 independent experiments reported as the percent of control.
Figure 5.
Inhibition of type I BMP receptors in lung cancer cell lines induces cell death.
(A–B) H1299 and A549 cultured in DMEM 5% FCS were treated with DMSO or a BMP receptor antagonist (10 µM Dorsomorphin or 1 µM of selective antagonist). The percentage of cells that take up ethidium bromide was then determined. The data is reported as the mean of at least 3 independent experiments. (C) H1299 cells cultured in DMEM 5% FCS were treated with DMSO, 1 µM and μ5 M of DMH2 for 7 days, stained with Typan Blue, and cell counts performed. Data represents the mean of 4 experiments reported as the percent dead cells. (D) H1299 cells cultured in SFM were treated with DMSO or 1 µM of DMH2 for 7 days, stained with Typan Blue, and cell counts performed. Data represents the mean of 5 experiments reported as percent dead cells. (E) Cell death was determined using flow cytometry detecting uptake of an amine-reactive fluorescent dye in H1299 cells treated with DMS0 or DMH2 for 4 days. Data represent the mean of 3 independent experiments. (F) The H1299 cells were transfected with control siRNA and siRNA targeting alk2, alk3, and alk6. After 2 days the percentage of cells staining for ethidium bromide was determined. The data is reported as the mean of 6 independent experiments.
Figure 6.
Lung cancer cell lines secrete BMP2 and knockdown of BMP2 decreases BMP signaling and induces cell death.
(A) A BMP2 Elisa was performed on the DMEM 5% FCS and SFM in the absence of cells (medium). A BMP2 Elisa was performed on the DMEM 5% FCS and SFM cell culture medium containing A549 and H1299 cells for 48 hours. Experiments represent the mean of at least 5 experiments performed in triplicate. (B) BMP antagonists decrease BMP signaling of lung cancer cells cultured in SFM. H1299 cells cultured in SFM were treated with 1 µM DMSO, 1 µM of DMH2 or 1 µM of LDN for 48 hours and Western blot analysis performed. (C) Quantitative RT-PCR for BMP2 expression 48 hours after transfecting H1299 cells with control siRNA or siRNA targeting BMP2. Data represents the percent of control of the average of 4 independent experiments. (D) Western blot analysis of H1299 cells in SFM 48 hours after being transfected with control siRNA or siRNA targeting BMP2. BMP2 knockdown causes a decrease in expression of pSmad 1/5 and Id1. (E) Knockdown of BMP2 induces cell death. H1299 cells in SFM were transfected with control siRNA or siRNA targeting BMP2. After 48 hours the percentage of dead cells was determined by ethidium bromide staining. The data represents the mean of 4 independent experiments performed in triplicate.
Figure 7.
Id1 and Id3 regulate cell growth and survival of lung cancer cells.
(A) Western blot analysis for Id1 and Id3 48 hours after H1299 cells were transfected with control siRNA and siRNA targeting Id1 or Id3. (B) Quantitative RT-PCR for Id1 and Id3 after H1299 cells were transfected with control siRNA and siRNA targeting Id1 or Id3. Data represents the percent control of the mean of 2 experiments. (C) H1299 cells were transfected with control siRNA or siRNA targeting Id1 or Id3. After 48 hours the percentage of cells staining for ethidium bromide was determined. The data is reported as the mean of 4 independent experiments. (D–F) H1299 cells were transfected with control siRNA and siRNA targeting Id1 or Id3. After 7 days the cells were stained with Trypan Blue and the number of alive and dead cells was determined. The data represents the percent change from siRNA control of (D) alive or (F) dead cells. (E) Represents the percentage of cells that were dead. The data represents the mean of 4 independent experiments.
Figure 8.
Forced expression of Id3 prevents growth suppression and cell death induced by DMH2.
H1299 cells were stably transfected with Id1 and Id3 expression vectors or the insertless vector. (A–B) Western blot analysis showing increased expression of (A) Id1 or (B) Id3 in the transfected cell line. (C–D) H1299/Id1 and H1299/Id3 cells were treated with 1 µM DMSO or 1 µM DMH2 for 7 days and the percent alive and dead cells determined. (C) DMH2 caused growth suppression of vector control and H1299/Id1 cells but not H1299/Id3 cells. (D) DMH2 induced cell death in the vector control cells (H/con) but not in the H1299/Id3 cells (H/Id3). The data represents the mean of at least 3 experiments reported as the percent of control treated cells. (E) DMH2 deceases cell growth of immortalized normal human bronchial epithelial cells (HBEC) but not human aortic endothelial cells (HAEC). Cell lines growing in SFM were treated with 1 µM DMSO or 1 µM DMH2 for 7 day and cell counts performed. Data represents the mean of at least 3 experiments reported as the percent of control treated cells. (F) Western blot analysis showing higher expression of pSmad 1/5 and Id1 in HBEC compared to HAEC. 1 µM of DMH2 for 48 hours decreased expression of pSmad 1/5 and ID1 in HBEC but not HAEC.