Cyclooxygenase-2 (COX-2) expression is induced by mitogenic and proinflammatory factors. Its overexpression plays a causal role in inflammation and tumorigenesis. COX-2 expression is tightly regulated, but the mechanisms are largely unclear. Here we show the control of COX-2 expression by an endogenous tryptophan metabolite, 5-methoxytryptophan (5-MTP). By using comparative metabolomic analysis and enzyme-immunoassay, our results reveal that normal fibroblasts produce and release 5-MTP into the extracellular milieu whereas A549 and other cancer cells were defective in 5-MTP production. 5-MTP was synthesized from L-tryptophan via tryptophan hydroxylase-1 and hydroxyindole O-methyltransferase. 5-MTP blocked cancer cell COX-2 overexpression and suppressed A549 migration and invasion. Furthermore, i.p. infusion of 5-MTP reduced tumor growth and cancer metastasis in a murine xenograft tumor model. We conclude that 5-MTP synthesis represents a mechanism for endogenous control of COX-2 overexpression and is a valuable lead for new anti-cancer and anti-inflammatory drug development.tumor suppression | tryptophan metabolism | inflammation control C yclooxygenase-2 (COX-2) is a rate-limiting enzyme in the production of diverse prostanoids with potent biological activities. It is involved in multiple physiological functions and triggers key pathological processes, such as tumorigenesis and inflammation (1, 2). COX-2 is constitutively overexpressed in a wide variety of human cancers and is enhanced by proinflammatory stimuli (3, 4). There is convincing evidence for a causal role of COX-2 in tumorigenesis. Inhibition of COX-2 activities was reported to control human colorectal cancer (5-8). COX-2 induces tumorigenesis by promoting important cellular functions including cell proliferation, migration, and resistance to apoptosis (9-11). The induced COX-2 expression by proinflammatory and mitogenic factors in normal cells is tightly controlled (12) whereas its overexpression in cancer cells is attributed to dysregulated transcription (13). The endogenous control mechanisms for COX-2 expression in normal cells and the mechanisms underlying the dysregulation in cancer cells are poorly understood. We previously identified in the conditioned medium of human fibroblasts small molecules (named cytoguardins) that suppress COX-2 expression induced by proinflammatory mediators (14). NMR analysis of a semipurified fraction revealed compounds with indole moieties (14). However, the exact chemical structures remain elusive. In this study, we elucidated the structure of cytoguardins by comparing the metabolomic profiles between normal and cancer cells. ResultsCytoguardins Inhibit Cancer Cell COX-2. To determine that fibroblast factors are capable of suppressing cancer cell COX-2 expression, we cocultured human Hs68 foreskin fibroblasts (HsFb) with A549 lung cancer cells in a Boyden chamber for 24 h. A549 cells were removed and treated with phorbol 12-myristate 13-acetate (PMA) for 4 h, and COX-2 proteins were analyzed. HsFb suppressed A549 ...
Tien-Hsien Liquid (THL) is a Chinese herbal mixture that has been used worldwide as complementary treatment for cancer patients in the past decade. Recently, THL has been shown to induce apoptosis in various types of solid tumor cells in vitro. However, the underlying molecular mechanisms have not yet been well elucidated. In this study, we explored the effects of THL on acute promyelocytic leukemia (APL) NB4 cells, which could be effectively treated by some traditional Chinese remedies containing arsenic trioxide. The results showed THL could induce G2/M arrest and apoptosis in NB4 cells. Accordingly, the decrease of cyclin A and B1 were observed in THL-treated cells. The THL-induced apoptosis was accompanied with caspase-3 activation and decrease of PML-RARα fusion protein. Moreover, DNA methyltransferase 1 and oncogenic signaling pathways such as Akt/mTOR, Stat3 and ERK were also down-regulated by THL. By using ethyl acetate extraction and silica gel chromatography, an active fraction of THL named as EAS5 was isolated. At about 0.5–1% of the dose of THL, EAS5 appeared to have most of THL-induced multiple molecular targeting effects in NB4 cells. Based on the findings of these multi-targeting effects, THL might be regarding as a complementary and alternative therapeutic agent for refractory APL.
BackgroundThe non-small cell lung cancer (NSCLC) is the leading cause of cancer death worldwide. In NSCLC, the oncogenic AKT-mTOR, ERK and STAT3 pathways are commonly dysregulated and have emerged as attractive targets for therapeutic developments. In a relatively limited subset of NSCLC, these pathways driven by mutant EGFR can be treated by the tyrosine kinase inhibitors (TKIs)-mediated targeted therapy. However, for the most NSCLC, more novel targeted agents are imperatively needed. Therefore, we investigated the inhibitory effects of the active fraction HS7 from Taiwanofungus camphoratus, a unique medicinal fungus in Taiwan, on these pathways in CL1-0 EGFR wild-type human NSCLC cells.MethodsThe active fraction HS7 was prepared by n-hexane extraction of T. camphoratus followed by silica gel chromatography. Its effects on the cell viabilities were determined by sulforhodamine B colorimetric assay. Flow cytometry was used to analyze cell-cycle regulation and apoptosis induction. The changes in cellular protein levels were examined by Western blot.ResultsThe active fraction HS7 vigorously inhibits AKT-mTOR, ERK and STAT3 signaling pathways in CL1-0 cells. At dose of 25 μg/mL, these signaling pathways were almost completely inhibited by HS7, accompanied with induction of cyclin-dependent kinase inhibitors such as p15, p21 and p27. Accordingly, the AKT-mTOR downstream targets p-p70S6K and HIF-1α were also suppressed as well. At this dose, the cell proliferation was profoundly suppressed to 23.4% of control and apoptosis induction was observed.ConclusionsThe active fraction HS7 from n-hexane extract of T. camphoratus exerts multi-targeting activity on the suppression of AKT-mTOR, ERK and STAT3 pathways and induction of p15, p21 and p27 in EGFR wild-type NSCLC cells. This multi-targeting activity of HS7 suggests its potential as an alternative medicine for the treatment of EGFR TKIs resistant NSCLC.Electronic supplementary materialThe online version of this article (10.1186/s13020-017-0154-9) contains supplementary material, which is available to authorized users.
Leukemia is a serious disease that affects both children and adults. Although all-trans retinoic acid (ATRA) and targeted drug such as imatinib could effectively treat acute promyelocytic (APL) and chronic myelogenous leukemia (CML), respectively, rare therapeutics is available when drug resistance or recurrence occurred. Searching for alternative agent for refractory patient is urgently needed. Our previous studies had found that a human urine extract CDA-2 could induce differentiation of HL60 APL cells as well as potentiate the effects of ATRA and imatinib on HL60 and K562 CML cells, respectively. Purification of active compound possessing these anti-leukemic effects from CDA-2 was thus performed. Recently, an active compound named as P18.9 was isolated. It induced differentiation of K562 CML cells as evidenced by the nitroblue-tetrazolium (NBT) reduction test. At higher dose, flow cytometry analysis showed that P18.9 induced sub-G1 apoptotic fraction of K562 cells and enhanced the sub-G1 fraction induced by imatinib at a scale of about two folds. Although P18.9 did not induce differentiation of HL60 APL cells, it substantially enhanced the ATRA-induced differentiation. This is the first study to isolate an active compound from human urine, which exerts anti-leukemic effects alone or cooperatively with clinical used drugs. The chemical structure of P18.9 had been identified and further subsequent structure modification is ongoing. Continuing the effort to develop P18.9 and its derivatives as novel therapeutics for refractory APL or CML patients is warranted. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C189.
Purpose: Apoptosis induction by Tien-Hsien Liquid (THL) had been reported in various types of human cancer cells. Although our previous report had demonstrated its multiple molecular targeting effects on acute promyelocytic leukemia (APL) cells, whether THL may also exert the similar anticancer effects and play radiosensitizing role in solid tumor have not yet been well elucidated. In this study, we explored the effects of THL on MCF-7 human breast cancer cells in the aspects of cell cycle regulation, signaling pathways inhibition as well as enhancement of radiosensitivity. Materials and Methods: THL was provided by Feida Union Pharmaceutical Manufactory and its active fraction EAS5 was obtained by ethyl acetate extraction followed by silica gel chromatography. The cell viability was determined by sulforhodamine B assay. Cell-cycle distribution was analyzed by flow cytometry. Western blot and antibodies against specific phosphorylated proteins were employed to analyze the activities of affected signaling pathways. Irradiation was performed by a Cesium-137 source. Amido black staining was used to examine the colony formation ability. Results: THL and the active fraction EAS5 could inhibit MCF-7 cells growth dose-dependently, the IC50 were 1.6 mg/ml and 10μg/ml, respectively. The DNA methyltransferase 1 (DNMT1) were down regulated dose-dependently by THL/EAS5. Cell cycle arrest in G2/M phase by THL/EAS5 were shown in flow cytometry, in accompany with the down regulation of cyclin A and cyclin B1 and increase of p15 and p21. The phosphorylatd Akt protein was down-regulated dose-dependently by THL, and the inhibition of MAPK/ERK could be observed at higher dose of THL. On the other hand, THL/EAS5 could down-regulate the level of Rad51 dose-dependently. Compared to other radiation inducible repair enzymes, such as Ku70 and Ku86, Rad51 is more sensitive to THL. The inhibition of colony formation by irradiation was further enhanced in the presence of THL at dose of 0.375 and 0.75 mg/ml. The dose of irradiation required for inhibition of 50% colonies at THL doses of 0, 0.375 and 0.75 mg/ml were 7.5, 4.5 and 3.5 Gy, respectively. The enhanced ratio was 1.7 and 2.1 folds, respectively. In the presence of EAS5 at dose of 1.875μg/ml, the Gy required for 50% inhibition of colonies was down from 5.6 to 2.7; the enhanced ratio was 2.1 folds. Conclusion: Our results indicated the multi-molecular targeting anticancer effects of THL on MCF-7 cells. The EAS5 seems to be one hundred times more potent than THL and conserve most of the anticancer activities of THL, similar to the phenomena we observed in APL cells. In addition to G2/M arrest, THL and its active fraction are able to inhibit the DNA repair and then enhance radiosensitivity in breast cancer cells. Therefore, THL could be considered as a potential radiosensitizers for future clinical use. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3656.
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