Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Control of cyclooxygenase-2 expression and tumorigenesis by endogenous 5-methoxytryptophan

doi: 10.1073/pnas.1209919109

Figure Lengend Snippet: Suppression of cancer cell COX-2 expression by soluble factors from fibroblasts. (A) A549 lung cancer cells (106 cells) were cocultured with HsFb at various cell densities in a Boyden chamber for 24 h. A549 cells were removed and treated with PMA (100 nM) for 4 h. COX-2 proteins in the cell lysates were analyzed by Western blotting. (B) Conditioned medium (CM) was collected from Hs68 fibroblasts (HsFb) or A549 cells cultured in medium containing 2.5% FBS for 24 h. (Left) HsFb CM was added to washed A549 cells. (Right) A549 CM was added to washed serum-starved Hs68 fibroblasts. Cells were treated with PMA for 4 h, and COX-2 proteins were analyzed by Western blotting. Error bars denote mean ± SEM (n = 3). (C) CMF2 fractions prepared from HsFb or A549 CM were added to washed A549, HT29, or serum-starved HsFb, and PMA-induced COX-2 was analyzed. CMF2 of HsFb inhibited COX-2 expression whereas CMF2 of A549 had no effect. Two independent experiments were performed with similar results. (D) Representative mass spectra (m/z 0–300) of CMF2 prepared from HsFb or A549. Control denotes F2 fraction of the basal DMEM containing 2.5% FBS. Metabolomic analysis of CMF2 was performed using UPLC–QTof mass spectrometry. (E) Comparison of relative intensity of m/z 276.1 and m/z 262.1 between HsFb CMF2 and A549 CMF2. Error bars denote mean ± SEM (n = 3). (F) Effect of cancer-associated fibroblasts on PMA-induced COX-2 expression in A549 cells. CM and CMF2 prepared from prostate cancer-associated fibroblasts (PcFb) inhibited COX-2 expression whereas neither CM nor CMF2 of breast cancer-associated fibroblasts (bCAF) did. (G and H) The metabolomic profile of (G) PcFb-CMF2 was similar to that of HsFb-CMF2, whereas the profile of (H) bCAF-CMF2 reveals diminished m/z 276.1, m/z 262.1, and other m/z peaks.

Article Snippet: Human cancer cell lines A549, HT-29, Hep3B, and MCF7 and MCF10A were from the Bioresource Collection and Research Center.

Techniques: Expressing, Western Blot, Cell Culture, Control, Mass Spectrometry, Comparison

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Control of cyclooxygenase-2 expression and tumorigenesis by endogenous 5-methoxytryptophan

doi: 10.1073/pnas.1209919109

Figure Lengend Snippet: Tryptophan hydroxylase (TPH-1)-derived 5-HTP suppresses COX-2 expression. (A) TPH-1 proteins in HsFb vs. A549 cells were analyzed by Western blotting. The error bars refer to mean ± SEM (n = 3). (B) TPH-1 protein levels in siRNA-transfected HsFb vs. control scRNA-transfected HsFb. “Basal” denotes untransfected cells. (C) Analysis of COX-2 proteins in HsFb transfected with TPH-1 siRNA or scRNA. COX-2 was increased in siRNA-treated cells, and COX-2 suppression was restored by 5-HTP (10 μM) or 5-MTP (10 μM) supplement. 5-MTP supplement completely inhibited COX-2 expression whereas 5-HTP partially suppressed COX-2 proteins. (D) HsFb were pretreated with 5-HTP followed by PMA for 4 h. COX-2 proteins were analyzed by Western blotting. (E) Metabolomic analysis of CMF2 prepared from CM of HsFb with or without siRNA or scRNA transfection.

Article Snippet: Human cancer cell lines A549, HT-29, Hep3B, and MCF7 and MCF10A were from the Bioresource Collection and Research Center.

Techniques: Derivative Assay, Expressing, Western Blot, Transfection, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Control of cyclooxygenase-2 expression and tumorigenesis by endogenous 5-methoxytryptophan

doi: 10.1073/pnas.1209919109

Figure Lengend Snippet: Endogenous and exogenously added 5-MTP inhibits COX-2 expression. (A) Pure 5-MTP at increasing concentrations was added to A549 treated with or without PMA for 4 h. COX-2 proteins were analyzed by Western blotting. (B) 5-MTP restored COX-2 suppression in TPH-1 siRNA-transfected HsFb in a concentration-dependent manner. (C) Addition of 5-MTP to A549 cells enhances m/z 276.1 and 262.1 peaks on mass spectrometry. (D) HsFbs were transfected with TPH-1 siRNA or scRNA. l-Tryptophan (Try) or 5-HTP was added to the transfected cells. Conditioned medium was collected, and 5-MTP concentration was measured by EIA. Each bar denotes mean ± SEM (n = 3). ns, not statistically significant. (E) Analysis of COX-2 proteins by Western blotting in HsFb transfected with HIOMT siRNA or a control scRNA. (Upper) Representative blot. (Lower) Quantitative analysis by densitometry. Error bars denote mean ± SEM (n = 3). Concentration of 5-MTP was 10 μM. (F) Measurement by EIA of 5-MTP in the CM of HsFb under the indicated treatment; 10 μM of 5-HTP or 5-MTP was added. Each error bar denotes mean ± SEM of three independent experiments done in triplicate. ns, not statistically significant.

Article Snippet: Human cancer cell lines A549, HT-29, Hep3B, and MCF7 and MCF10A were from the Bioresource Collection and Research Center.

Techniques: Expressing, Western Blot, Transfection, Concentration Assay, Mass Spectrometry, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Control of cyclooxygenase-2 expression and tumorigenesis by endogenous 5-methoxytryptophan

doi: 10.1073/pnas.1209919109

Figure Lengend Snippet: 5-MTP rescues A549 defects in 5-MTP synthesis and cell proliferation and migration. (A) Analysis of HIOMT proteins in A549 vs. HsFb. (Upper) Representative blot. (Lower) Densitometric analysis of three independent experiments. The error bars are mean ± SEM (*P < 0.01). (B) Measurement of 5-MTP level in A549 vs. HsFb CM. Error bars are mean ± SEM (n = 3). (C) Neither Try nor 5-HTP increased A549 5-MTP production, whereas addition of exogenous 5-MTP increased the 5-MTP level measured by EIA. The error bars are mean ± SEM (n = 3). (D) A549 cells labeled with MTT were treated with 5-MTP for 4 or 24 h. Each bar denotes mean ± SEM (n = 3). (E) A549 cells were pretreated with 5-MTP (10 μM) for 30 min followed by PMA (100 nM) for 6 or 24 h. (Upper panels) Representative cell migration. (Lower panels) Migrated cell counts expressed as percentage of basal controls. Error bars denote mean ± SEM (n = 3). (F) A549 cells were seeded on regular or matrigel-coated membrane and pretreated with 5-MTP for 30 min followed by PMA. (Upper) Representative cell migration and invasion. (Lower panels) Quantitative analysis of migration and invasion.

Article Snippet: Human cancer cell lines A549, HT-29, Hep3B, and MCF7 and MCF10A were from the Bioresource Collection and Research Center.

Techniques: Migration, Labeling, Membrane

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Control of cyclooxygenase-2 expression and tumorigenesis by endogenous 5-methoxytryptophan

doi: 10.1073/pnas.1209919109

Figure Lengend Snippet: 5-MTP reduces cancer growth and metastasis in a murine tumor xenograft model. A549 (5 × 106 cells) were transfected with luciferase (A549-luc-c8) and injected s.c. into the flank of SCID-Beige mice. Ten mice each received i.p. injection of 5-MTP (100 mg/kg) or vehicle twice weekly. (A) Caliper measurement of the s.c. tumor volume periodically for 7 wk. *P < 0.05 and **P < 0.01. (B) Tumor growth was monitored by in vivo bioluminescent imaging (IVIS). The time courses of IVIS of two representative vehicle-treated and two representative 5-MTP–treated mice are illustrated. (C) 5-MTP reduces lung metastasis. Mice were euthanized and both lungs were removed. Nodules were counted. Each nodule was sectioned and stained with hematoxylin-eosine and examined under the microscope. 5-MTP pretreatment significantly attenuated lung metastasis. (D) Metabolic scheme illustrating 5-MTP biosynthesis in HsFb vs. A549 cancer cells. The dotted line denotes possible defects in the synthetic pathway.

Article Snippet: Human cancer cell lines A549, HT-29, Hep3B, and MCF7 and MCF10A were from the Bioresource Collection and Research Center.

Techniques: Transfection, Luciferase, Injection, In Vivo, Imaging, Staining, Microscopy