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Rich66 · 05-24-2009, 08:23 PM

Tranilast inhibits the growth and metastasis of mammary carcinoma
Chakrabarti, Rabindranath; Subramaniam, Venkateswaran; Abdalla, Salma; Jothy, Serge; Prud'homme, Gérald J.
Anti-Cancer Drugs, June 2009,20(5):334-345
Preclinical Reports

Fig. 6
Fig. 6 Tranilast inhibits metastasis of 4T1 mammary carcinoma to the lungs and liver. Mice were transplanted with 4T1 cells and treated with tranilast by gavage, as in Fig. 5 (eight mice in each group). On day 28 of treatment, the mice were killed and the lungs and liver were collected and examined histologically. (a) Number of tumors per lung section (▪, vehicle; □, tranilast), expressed as the mean±SD (n = 8). (b) Histological sections of lungs showing metastatic tumors. As shown here, the tranilast-treated mice had smaller tumors than the vehicle-treated group (marked areas). (c) The area of each metastatic tumor lesions was measured in each lung section by a morphometric method. The mean area per metastatic tumor was significantly higher in the vehicle group than in the tranilast group (PAnti-Cancer Drugs, June 2009,20(5)

Fig. 3
Fig. 3 Tranilast inhibits TGF-β1-induced epithelial-to-mesenchymal transition (EMT) in 4T1 cells. (a) 4T1 cells were attached to cover slips and cultured for 48 h in the presence and absence of tranilast (800 μmol/l) and TGF-β1 (2 ng/ml). The cells were stained for F-actin with Alexa Fluor 568-conjugated phalloidin. Photographs were taken at a magnification of ×400. Addition of TGF-β1 to the cultures induced EMT as revealed from massive F-actin fiber organization and this was markedly reduced by tranilast treatment. Four experiments yielded similar results. Tranilast also inhibited EMT at a concentration of 200 μmol/l, but the effect was less marked (data not shown). (b) 4T1 cells were deprived of serum for 24 h followed by culture in complete medium in the presence or absence of vehicle (dimethyl sulfoxide) or tranilast (800 μmol/l) for 48 h. The cells were lysed and analyzed for vimentin (mesenchymal marker) by western blot as in Materials and methods. Upper panel: picture of the western blot; lower panel: densities of vimentin bands as percentage of β-actin controls. Tranilast treatment drastically reduced the vimentin level. Two experiments yielded similar results.
Anti-Cancer Drugs, June 2009,20(5)

Fig. 4
Fig. 4 Effect of tranilast on cytokine release/production by spleen cells and 4T1 cell. (a) CD4+25- and CD8+ T cells were isolated from C57BL/6 mice and stimulated with plate-bound anti-CD3 and anti-CD28 mAbs, in the presence or absence of tranilast for 48 h. Then the supernatant was acidified for TGF-β1 activation, and TGF-β1 levels in the culture supernatant were measured by enzyme-linked immunosorbent assay. The control T cells (without tranilast) generated TGF-β1 levels of 83±8.7 pg/ml in CD4+ cells and 39±5.2 pg/ml in CD8+ T cells (mean±SD; n = 3). At all concentrations, tranilast significantly inhibited the production of TGF-β1 by both cell types (PAnti-Cancer Drugs, June 2009,20(5)

Fig. 1
Fig. 1 Effect of tranilast on the growth of breast cancer and other tumor cell lines in vitro. (a) Various tumor cells (LA7, 4T1, MDA-MB-231, LLC, EL4, MCF-7 cells) were allowed to attach to the wells (10×103 cells/well) of a flat-bottom 96-well plate. Various concentrations of tranilast were added to the cells and cultured for 48 h. Cell growth was monitored by MTT assay. Growth or proliferation in all cell lines was suppressed, with LA7 being the most sensitive. The suppression of cell growth was not accompanied by cytotoxicity (data not shown). Results are the mean±SD of three separate experiments. (b) 4T1 cells were cultured for 24 h in serum-free medium, followed by the culture in complete medium in the presence or absence of dimethyl sulfoxide (DMSO) or tranilast for 24 h. Then cell-cycle progression was analyzed by flow cytometry. The percentage of cells in the G1, S, or G2 phases is reported inside each histogram. Tranilast inhibited cells cycle in the S-phase at 200 μmol/l, and primarily at the G1 phase at 800 μmol/l. Two experiments yielded similar results.
Anti-Cancer Drugs, June 2009,20(5)

Fig. 5
Fig. 5 Tranilast inhibits the growth of transplanted 4T1 mammary carcinoma. 4T1 cells were transplanted orthotopically in mammary fat pads of 6-week-old female BALB/c mice (eight mice in each group). Tranilast was administered by gavage at a dose of 300 mg/kg body weight from day 0 (day of cancer cell transplantation) to the end of the treatment. Tumor volume (mean±SD, n = 8) over time is reported (▪, vehicle group; □, tranilast group). At all time points, tumor size in the tranilast group was significantly lower than in the vehicle group (PAnti-Cancer Drugs, June 2009,20(5)

Fig. 2
Fig. 2 Tranilast inhibits TGF-β1-induced Smad2 phosphorylation and serum-induced extracellularly regulated kinase 1 and 2 (ERK1/2) phosphorylation in 4T1 cells. (a) 4T1 cells were attached directly to microscopic slides and incubated for 20 h in the presence of TGF-β1 (2 ng/ml), tranilast (800 μmol/l), or both, and stained for phosphorylated Smad2 (pSmad2) as described in Materials and methods. The cells receiving only vehicle [dimethyl sulfoxide (DMSO)] treatment served as control. TGF-β1 increased nuclear staining for pSmad2, and this was almost completely blocked by tranilast. Photomicrographs were taken at ×400 magnification. Four experiments yielded similar results. (b) 4T1 cells were deprived of serum for 24 h followed by culture in complete medium in the presence or absence of vehicle (DMSO) or tranilast (800 μmol/l) for 24 h. The cells were lysed and analyzed for pSmad2 by western blotting. Upper panel: picture of the western blot; lower panel: densities of pSmad2 bands as percentage of β-actin controls. There is approximately 38% reduction in the level of pSmad2. This is one of the two experiments that yielded similar results. (c) 4T1 cells were grown in flat-bottom 96-well plates overnight, followed by serum deprivation for 24 h. After that, cells were cultured for another 24 h in complete medium in the presence of DMSO (control) or 800 μmol/l tranilast. The level of phosphorylated and total ERK1/2 and JNK were measured by CASE enzyme-linked immunosorbent assay, and the relative extent of phosphorylation was calculated, according to the supplier's protocol. The extent of phosphorylation refers to the proportion (fraction) of the total protein (ERK or JNK) that is phosphorylated. Results are the mean±SD of three separate experiments. Tranilast significantly (PAnti-Cancer Drugs, June 2009,20(5)

05-24-2009, 08:23 PM	#8
Rich66 Senior Member Join Date: Feb 2008 Location: South East Wisconsin Posts: 3,431	Tranilast inhibits the growth and metastasis of mammary carcinoma Chakrabarti, Rabindranath; Subramaniam, Venkateswaran; Abdalla, Salma; Jothy, Serge; Prud'homme, Gérald J. Anti-Cancer Drugs, June 2009,20(5):334-345 Preclinical Reports Fig. 6 Fig. 6 Tranilast inhibits metastasis of 4T1 mammary carcinoma to the lungs and liver. Mice were transplanted with 4T1 cells and treated with tranilast by gavage, as in Fig. 5 (eight mice in each group). On day 28 of treatment, the mice were killed and the lungs and liver were collected and examined histologically. (a) Number of tumors per lung section (▪, vehicle; □, tranilast), expressed as the mean±SD (n = 8). (b) Histological sections of lungs showing metastatic tumors. As shown here, the tranilast-treated mice had smaller tumors than the vehicle-treated group (marked areas). (c) The area of each metastatic tumor lesions was measured in each lung section by a morphometric method. The mean area per metastatic tumor was significantly higher in the vehicle group than in the tranilast group (PAnti-Cancer Drugs, June 2009,20(5) Fig. 3 Fig. 3 Tranilast inhibits TGF-β1-induced epithelial-to-mesenchymal transition (EMT) in 4T1 cells. (a) 4T1 cells were attached to cover slips and cultured for 48 h in the presence and absence of tranilast (800 μmol/l) and TGF-β1 (2 ng/ml). The cells were stained for F-actin with Alexa Fluor 568-conjugated phalloidin. Photographs were taken at a magnification of ×400. Addition of TGF-β1 to the cultures induced EMT as revealed from massive F-actin fiber organization and this was markedly reduced by tranilast treatment. Four experiments yielded similar results. Tranilast also inhibited EMT at a concentration of 200 μmol/l, but the effect was less marked (data not shown). (b) 4T1 cells were deprived of serum for 24 h followed by culture in complete medium in the presence or absence of vehicle (dimethyl sulfoxide) or tranilast (800 μmol/l) for 48 h. The cells were lysed and analyzed for vimentin (mesenchymal marker) by western blot as in Materials and methods. Upper panel: picture of the western blot; lower panel: densities of vimentin bands as percentage of β-actin controls. Tranilast treatment drastically reduced the vimentin level. Two experiments yielded similar results. Anti-Cancer Drugs, June 2009,20(5) Fig. 4 Fig. 4 Effect of tranilast on cytokine release/production by spleen cells and 4T1 cell. (a) CD4+25- and CD8+ T cells were isolated from C57BL/6 mice and stimulated with plate-bound anti-CD3 and anti-CD28 mAbs, in the presence or absence of tranilast for 48 h. Then the supernatant was acidified for TGF-β1 activation, and TGF-β1 levels in the culture supernatant were measured by enzyme-linked immunosorbent assay. The control T cells (without tranilast) generated TGF-β1 levels of 83±8.7 pg/ml in CD4+ cells and 39±5.2 pg/ml in CD8+ T cells (mean±SD; n = 3). At all concentrations, tranilast significantly inhibited the production of TGF-β1 by both cell types (PAnti-Cancer Drugs, June 2009,20(5) Fig. 1 Fig. 1 Effect of tranilast on the growth of breast cancer and other tumor cell lines in vitro. (a) Various tumor cells (LA7, 4T1, MDA-MB-231, LLC, EL4, MCF-7 cells) were allowed to attach to the wells (10×103 cells/well) of a flat-bottom 96-well plate. Various concentrations of tranilast were added to the cells and cultured for 48 h. Cell growth was monitored by MTT assay. Growth or proliferation in all cell lines was suppressed, with LA7 being the most sensitive. The suppression of cell growth was not accompanied by cytotoxicity (data not shown). Results are the mean±SD of three separate experiments. (b) 4T1 cells were cultured for 24 h in serum-free medium, followed by the culture in complete medium in the presence or absence of dimethyl sulfoxide (DMSO) or tranilast for 24 h. Then cell-cycle progression was analyzed by flow cytometry. The percentage of cells in the G1, S, or G2 phases is reported inside each histogram. Tranilast inhibited cells cycle in the S-phase at 200 μmol/l, and primarily at the G1 phase at 800 μmol/l. Two experiments yielded similar results. Anti-Cancer Drugs, June 2009,20(5) Fig. 5 Fig. 5 Tranilast inhibits the growth of transplanted 4T1 mammary carcinoma. 4T1 cells were transplanted orthotopically in mammary fat pads of 6-week-old female BALB/c mice (eight mice in each group). Tranilast was administered by gavage at a dose of 300 mg/kg body weight from day 0 (day of cancer cell transplantation) to the end of the treatment. Tumor volume (mean±SD, n = 8) over time is reported (▪, vehicle group; □, tranilast group). At all time points, tumor size in the tranilast group was significantly lower than in the vehicle group (PAnti-Cancer Drugs, June 2009,20(5) Fig. 2 Fig. 2 Tranilast inhibits TGF-β1-induced Smad2 phosphorylation and serum-induced extracellularly regulated kinase 1 and 2 (ERK1/2) phosphorylation in 4T1 cells. (a) 4T1 cells were attached directly to microscopic slides and incubated for 20 h in the presence of TGF-β1 (2 ng/ml), tranilast (800 μmol/l), or both, and stained for phosphorylated Smad2 (pSmad2) as described in Materials and methods. The cells receiving only vehicle [dimethyl sulfoxide (DMSO)] treatment served as control. TGF-β1 increased nuclear staining for pSmad2, and this was almost completely blocked by tranilast. Photomicrographs were taken at ×400 magnification. Four experiments yielded similar results. (b) 4T1 cells were deprived of serum for 24 h followed by culture in complete medium in the presence or absence of vehicle (DMSO) or tranilast (800 μmol/l) for 24 h. The cells were lysed and analyzed for pSmad2 by western blotting. Upper panel: picture of the western blot; lower panel: densities of pSmad2 bands as percentage of β-actin controls. There is approximately 38% reduction in the level of pSmad2. This is one of the two experiments that yielded similar results. (c) 4T1 cells were grown in flat-bottom 96-well plates overnight, followed by serum deprivation for 24 h. After that, cells were cultured for another 24 h in complete medium in the presence of DMSO (control) or 800 μmol/l tranilast. The level of phosphorylated and total ERK1/2 and JNK were measured by CASE enzyme-linked immunosorbent assay, and the relative extent of phosphorylation was calculated, according to the supplier's protocol. The extent of phosphorylation refers to the proportion (fraction) of the total protein (ERK or JNK) that is phosphorylated. Results are the mean±SD of three separate experiments. Tranilast significantly (PAnti-Cancer Drugs, June 2009,20(5)