[Rich66]

Anticancer Drugs. 2009 Mar 24. [Epub ahead of print]Links

Tranilast inhibits the growth and metastasis of mammary carcinoma.

FULL TEXT

Chakrabarti R, Subramaniam V, Abdalla S, Jothy S, Prudʼhomme GJ.
aDepartment of Laboratory Medicine and Li Ka Shing Knowledge Institute, St Michael's Hospital bDepartment of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada.
Tranilast (N-[3,4-dimethoxycinnamonyl]-anthranilic acid) is a drug of low toxicity that is orally administered, and has been used clinically in Japan as an antiallergic and antifibrotic agent. Its antifibrotic effect is thought to depend on the inhibition of transforming growth factor-beta (TGF-beta). It has also been shown to exert antitumor effects, but its mode of action is unclear. Here, we explored the antitumor effects of tranilast in vitro and in vivo. Tranilast inhibited the proliferation of several tumor cell lines including mouse mammary carcinoma (4T1), rat mammary carcinoma stem cell (LA7), and human breast carcinoma (MDA-MB-231 and MCF-7). Tranilast blocked cell-cycle progression in vitro. In the highly metastatic 4T1 cell line, tranilast inhibited phospho-Smad2 generation, consistent with a blockade of TGF-beta signaling. It also inhibited the activation of MAP kinases (extracellularly regulated kinase 1 and 2 and JNK), which have been linked to TGF-beta-dependent epithelial-to-mesenchymal transition and, indeed, it blocked epithelial-to-mesenchymal transition. Although tranilast only partially inhibited TGF-beta production by 4T1 tumor cells, it potently inhibited the production of TGF-beta, interferon-gamma, IL-6, IL-10, and IL-17 by lymphoid cells, suggesting a general anti-inflammatory activity. In vivo, female BALB/c mice were inoculated with syngeneic 4T1 cells in mammary fat pads and treated with tranilast by gavage. Tranilast reduced (>50%) the growth of the primary tumor. However, its effects on metastasis were more striking, with more than 90% reduction of metastases in the lungs and no metastasis in the liver. Thus, tranilast has potential activity as an antimetastatic agent in breast cancer.
PMID: 19322072 [PubMed - as supplied by publisher

Anticancer Drugs. 2010 Feb 6. [Epub ahead of print]
Tranilast inhibits cell proliferation and migration and promotes apoptosis in murine breast cancer.

Subramaniam V, Chakrabarti R, Prudʼhomme GJ, Jothy S.
aDepartment of Laboratory Medicine and Keenan Research Centre of the Li Ka Shing Knowledge Institute, St Michael's Hospital bDepartment of Laboratory Medicine and Pathobiology, University of Toronto, Ontario, Canada.
The malignant transformation of breast epithelium involves a number of cellular pathways, including those dependent on signaling from TGF beta. Tranilast [N-(3, 4-dimethoxycinnamonyl)-anthranilic acid] is a drug that is used in Japan to control allergic disorders in patients, and its mechanism of action involves TGF beta. In view of the multiple roles of TGF beta in tumor progression, we hypothesized in this study that tranilast impacts cell proliferation, apoptosis, and migration. Using the mouse breast cancer cell line 4T1, our studies showed that tranilast increases AKT1 phosphorylation and decreases ERK1/2 phosphorylation. Alterations in the cell cycle mediators' cyclin D1, p27, cyclin A, pRB, cyclin B, and Cdc2 were observed after exposure to tranilast, favoring cell arrest beyond the G1/S phase. Tranilast reduced tumor cell proliferation even when it was amplified by exogenous TGF beta. TGF beta-neutralizing antibody did not cause a significant decrease in cell proliferation. Tranilast treatment upregulates p53, induces PARP cleavage in vitro, consistent with a promotion of tumor cell apoptosis. TGF beta-neutralizing antibody downregulates endoglin and matrix metalloproteinases (MMP)-9 levels in vitro indicating that the tranilast effect is mediated through TGF beta modulation. Tranilast treatment results in the inhibition of cell migration and invasion. Western blot analysis of tumor lysates from tranilast-treated mice shows decreased levels of TGF beta1, endoglin, and significantly higher levels of p53 and cleaved PARP. Cleaved caspase 3 expression is significantly elevated in tranilast-treated mouse breast tumors. To conclude, tranilast induces cellular and molecular changes in murine breast cancer that can be exploited in preclinical therapeutic trials.






Int J Oncol. 2010 Feb;36(2):341-9.
Tranilast strongly sensitizes pancreatic cancer cells to gemcitabine via decreasing protein expression of ribonucleotide reductase 1.

FULL TEXT


Mitsuno M, Kitajima Y, Ohtaka K, Kai K, Hashiguchi K, Nakamura J, Hiraki M, Noshiro H, Miyazaki K.
Department of Surgery, Saga University Faculty of Medicine, Saga 849-8501, Japan.
Gemcitabine (Gem) is a dFdC analogue with activity against several solid tumors. Gem is intracellularly phosphorylated by dCK, leading to the production of the metabolite dFdCDP. dFdCDP exhibits the cytotoxic effect by inactivating ribonucleotide reductase larger subunit 1 (RRM1), which is a rate limiting enzyme for de novo DNA synthesis. To date, RRM1 expression is believed to determine sensitivity to Gem in pancreatic and non-small cell lung cancer. In the present study, we found that an anti-allergic drug, tranilast strongly enhanced the sensitivity of pancreatic cancer cell line KP4 to Gem. In growth inhibition assay, 100 microM of tranilast plus 1 microM of Gem more strongly suppressed the growth of KP4 at 12.7-fold in IC50 than single Gem treatment, while this compound no longer affected the sensitivity to other drugs such as 5-fluorouracil, irinotecan or paclitaxel. FACS and TUNEL analysis demonstrated the increased apoptotic population in KP4 cells under tranilast plus Gem, compared with single Gem treatment. In Western blot analysis, tranilast treatment decreased RRM1 expression at protein level with dose-dependency in KP4 cells. Proteasome inhibitor MG132 disturbed the reduction of RRM1 expression in tranilast treated KP4 cells, indicating protein degradation by the activated proteasome. Transfection using siRNA against RRM1 increased the sensitivity of KP4 to Gem, suggesting that RRM1 suppression is an important step in increasing Gem efficacy. Finally, we demonstrated that tranilast reduced RRM1 protein and increased Gem efficacy in 4 other pancreatic cell lines. In a future, a novel chemotherapeutic strategy by Gem along with tranilast might improve Gem efficacy against pancreatic cancer.

PMID: 20043067 [PubMed - in process]





PLoS One. 2010 Nov 3;5(11):e13831.
Breast cancer stem-like cells are inhibited by a non-toxic aryl hydrocarbon receptor agonist.

Prud'homme GJ, Glinka Y, Toulina A, Ace O, Subramaniam V, Jothy S.
Source

Department of Laboratory Medicine and Li Ka Shing Knowledge Institute, St Michael's Hospital, Toronto, Canada. prudhommeg@smh.ca

FREE TEXT

Abstract

BACKGROUND:

Cancer stem cells (CSCs) have increased resistance to cancer chemotherapy. They can be enriched as drug-surviving CSCs (D-CSCs) by growth with chemotherapeutic drugs, and/or by sorting of cells expressing CSC markers such as aldehyde dehydrogenase-1 (ALDH). CSCs form colonies in agar, mammospheres in low-adherence cultures, and tumors following xenotransplantation in Scid mice. We hypothesized that tranilast, a non-toxic orally active drug with anti-cancer activities, would inhibit breast CSCs.
METHODOLOGY/FINDINGS:
We examined breast cancer cell lines or D-CSCs generated by growth of these cells with mitoxantrone. Tranilast inhibited colony formation, mammosphere formation and stem cell marker expression. Mitoxantrone-selected cells were enriched for CSCs expressing stem cell markers ALDH, c-kit, Oct-4, and ABCG2, and efficient at forming mammospheres. Tranilast markedly inhibited mammosphere formation by D-CSCs and dissociated formed mammospheres, at pharmacologically relevant concentrations. It was effective against D-CSCs of both HER-2+ and triple-negative cell lines. Tranilast was also effective in vivo, since it prevented lung metastasis in mice injected i.v. with triple-negative (MDA-MB-231) mitoxantrone-selected cells. The molecular targets of tranilast in cancer have been unknown, but here we demonstrate it is an aryl hydrocarbon receptor (AHR) agonist and this plays a key role. AHR is a transcription factor activated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), polycyclic aromatic hydrocarbons and other ligands. Tranilast induced translocation of the AHR to the nucleus and stimulated CYP1A1 expression (a marker of AHR activation). It inhibited binding of the AHR to CDK4, which has been linked to cell-cycle arrest. D-CSCs expressed higher levels of the AHR than other cells. Knockdown of the AHR with siRNA, or blockade with an AHR antagonist, entirely abrogated the anti-proliferative and anti-mammosphere activity of tranilast. Thus, the anti-cancer effects of tranilast are AHR dependent.

CONCLUSION/SIGNIFICANCE:
We show that tranilast is an AHR agonist with inhibitory effects on breast CSCs. It is effective against CSCs of triple-negative breast cancer cells selected for anti-cancer drug resistance. These results suggest it might find applications in the treatment of breast cancer.

PMID:

21072210

[PubMed - indexed for MEDLINE]

PMCID: PMC2972222

Inhibition occurred at tranilast concentrations ≥100 µM, and at 400 µM no mammospheres were seen. Tranilast reduced cell survival in these cultures, but even at the highest concentration (400 µM) almost 50% of cells survived after 7 d in culture (Fig. S1B). Note that at concentrations that can be achieved pharmacologically (100–200 µM) some spheres still formed (although they were smaller), suggesting survival of sphere-forming cells.




Figure 5Tranilast inhibits mammosphere formation by mitoxantrone-selected MDA-MB-231 cells.

We compared the effects of tranilast with paclitaxel and etoposide at various concentrations. In Fig. 5A,B, the results obtained with the highest concentrations of either paclitaxel (60 µM) or etoposide (40 µM) tested are shown. Lower concentrations were less effective (not shown). As seen in Fig. 5A, pharmacological concentrations of tranilast (100–200 µM) were more effective than these drugs at reducing the number of mammospheres.

Tranilast inhibits the following: cell cycling, TGF-β activity, MAPK signaling, epithelial-to-mesenchymal transition (EMT), cell migration and invasion. In vivo, it has prominent anti-metastatic effects [12]. Here we show that tranilast strongly inhibits CSCs in colony forming assays and mammosphere formation assays, at pharmacologically relevant concentrations. Tranilast was highly active at inhibiting mammosphere formation by D-CSCs, and dissociating formed mammospheres. It was effective in vivo, preventing metastasis to the lungs following i.v. injection of MDA-MB-231 mitoxantrone-selected cells.The molecular target(s) of tranilast in cancer have been unclear, but we demonstrate here that it is an aryl hydrocarbon receptor (AHR) agonist. The AHR is a transcription factor known principally as a receptor for toxins such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) [15], but it has other functions [16] and appears to exert some anti-cancer effects [17], [18]. We show that tranilast binds to the AHR, induces its translocation to the nucleus, and stimulates CYP1A1 expression (a classic marker of AHR activity). D-CSCs expressed increased amounts of AHR. Knockdown of the AHR with siRNA, or addition of an AHR antagonist to cultures, both abolished the activity of tranilast in vitro. Thus, the activity of tranilast appears to be highly AHR dependent.
We also show that tranilast is effective at inhibiting CSCs of triple-negative (ER−/PR−/HER-2−) breast cancer cell lines (MDA-MB-231, SUM149 and SUM159) or a triple-positive cell line (ER+/PR+/HER-2+) (BT474). This is particularly relevant because triple-negative cells have lacked a distinct molecular target and their treatment is problematic. Recent studies suggest that the AHR is an excellent target for breast cancer therapy [17], [18], and tranilast appears to be an exceptional drug for this purpose.

Active ingredient in oats?


J Agric Food Chem. 2009 Nov 25;57(22):10619-24.
In vitro antioxidant activity and antigenotoxic effects of avenanthramides and related compounds.

Lee-Manion AM, Price RK, Strain JJ, Dimberg LH, Sunnerheim K, Welch RW.
Northern Ireland Centre for Food and Health, School of Biomedical Sciences, University of Ulster, Coleraine BT52 1SA, United Kingdom.
Avenanthramides are substituted N-cinnamoylanthranilic acids, with hydroxycinnamic acid and anthranilic acid moieties. These alkaloid phenols, which are unique to oats, may confer health benefits via antioxidant or other mechanisms. Synthetic avenanthramides, hydroxycinnamic acids, Tranilast, and ascorbic acid were evaluated for antioxidant activity using two assays, DPPH (2,2-diphenyl-1-picrylhydrazyl) and FRAP (ferric reducing antioxidant potential), and for antigenotoxicity using the Comet assay with stressed human adenocarcinoma colon cells. Of all the compounds tested, N-(3',4'-dihydroxy-(E)-cinnamoyl)-5-hydroxyanthranilic acid (2c), an abundant oat avenanthramide, generally had the highest activity in all three assays. The drug Tranilast showed antigenotoxic effects, but not antioxidant activity, suggesting that antigenotoxicity is not dependent on antioxidant effects. Overall, results show that avenanthramides exert antioxidant and antigenotoxic activities that are comparable to those of ascorbic acid and which have the potential to exert beneficial physiological effects.

PMID: 19874025 [PubMed - in process]




Br J Pharmacol. 2010 Jan 8. [Epub ahead of print]
The anti-allergic compound tranilast attenuates inflammation and inhibits bone destruction in collagen-induced arthritis in mice.

Shiota N, Kovanen PT, Eklund KK, Shibata N, Shimoura K, Niibayashi T, Shimbori C, Okunishi H.
Department of Pharmacology, Shimane University School of Medicine, Shimane, Japan.
Background and purpose: Recent findings suggest the importance of mast cells in the pathogenesis of rheumatoid arthritis and their potential as a therapeutic target. Tranilast is an anti-allergic compound with a potent membrane-stabilizing effect on mast cells and a wide range of anti-inflammatory effects, thus may be advantageous in the treatment of arthritis. Here, we have evaluated the effects of tranilast on the progression of collagen-induced arthritis in mice. Experimental approach: Tranilast (400 mg.kg(-1).day(-1)) was orally administered for 8 weeks to mice with established collagen-induced arthritis. Arthritis was assessed by clinical signs and X-ray scores. In paw tissue, the numbers of mast cells and osteoclasts were measured by histological analysis, and several inflammatory factrs were assessed by RT-PCR and Western blot analysis. Key results: TNF-alpha-positive mast cells were present extensively throughout the inflamed synovium of vehicle-treated arthritic mice, with some mast cells in close proximity to osteoclasts in areas of marked bone and cartilage destruction. Tranilast significantly reduced clinical and X-ray scores of arthritis and decreased numbers of TNF-alpha-positive mast cells and mRNA levels of TNF-alpha, chymase (mouse mast cell protease 4), tryptase (mouse mast cell protease 6), stem cell factor, interleukin-6, cathepsin-K, receptor activator of nuclear factor-kappaB, and of receptor activator of nuclear factor-kappaB-ligand, but increased interleukin-10 mRNA level in paws of arthritic mice. Osteoclast numbers were decreased by treatment with tranilast. Conclusions and implications: Tranilast possesses significant anti-rheumatic efficacy and, probably, this therapeutic effect is partly mediated by inhibition of mast cell activation and osteoclastogenesis.

PMID: 20067475 [PubMed - as supplied by publisher]