Kanoknetr Suksen

A phytoestrogen diarylheptanoid mediates estrogen receptor/Akt/glycogen synthase kinase 3β protein-dependent activation of the Wnt/β-catenin signaling pathway.

Background: Diarylheptanoid (ASPP 049) isolated from C. comosa exhibits high estrogenic activity. Results: ASPP 049 rapidly induced β-catenin accumulation in the nucleus and activated TCF/LEF-mediated activation of Wnt/β-catenin signaling. Conclusion: ASPP 049 from C. comosa induces preosteoblastic cell proliferation and differentiation through activation of Wnt/β-catenin signaling. Significance: Providing a scientific rationale for using C. comosa as a dietary supplement to prevent bone loss in postmenopausal women. Estrogen promotes growth in many tissues by activating Wnt/β-catenin signaling. Recently, ASPP 049, a diarylheptanoid isolated from Curcuma comosa Roxb., has been identified as a phytoestrogen. This investigation determined the involvement of Wnt/β-catenin signaling in the estrogenic activity of this diarylheptanoid in transfected HEK 293T and in mouse preosteoblastic (MC3T3-E1) cells using a TOPflash luciferase assay and immunofluorescence. ASPP 049 rapidly activated T-cell-specific transcription factor/lymphoid enhancer binding factor-mediated transcription activity and induced β-catenin accumulation in the nucleus. Interestingly, the effects of ASPP 049 on the transcriptional activity and induction and accumulation of β-catenin protein in the nucleus of MC3T3-E1 cells were greater compared with estradiol. Activation of β-catenin in MC3T3-E1 cells was inhibited by ICI 182,780, suggesting that an estrogen receptor is required. In addition, ASPP 049 induced phosphorylations at serine 473 of Akt and serine 9 of GSK-3β. Moreover, ASPP 049 also induced proliferation and expressions of Wnt target genes Axin2 and Runx2 in MC3T3-E1 cells. In addition, ASPP 049 increased alkaline phosphatase expression, and activity that was abolished by DKK-1, a blocker of the Wnt/β-catenin receptor. Taken together, these results suggest that ASPP 049 from C. comosa induced osteoblastic cell proliferation and differentiation through ERα-, Akt-, and GSK-3β-dependent activation of β-catenin signaling. Our findings provide a scientific rationale for using C. comosa as a dietary supplement to prevent bone loss in postmenopausal women.

Estrogenic Activity of Diarylheptanoids from Curcuma comosa Roxb. Requires Metabolic Activation

Curcuma comosa Roxb. has traditionally been used as a dietary supplement for health promotion in peri- and postmenopausal women in Thailand. We investigated the estrogenic activity of 7 naturally occurring diarylheptanoids from the extracts of C. comosa both in vitro and in vivo. A yeast recombinant system containing human estrogen receptor alpha, coactivator TIF2 and a beta-galactosidase reporter gene was used to determine estrogenic activity of diarylheptanoids metabolically activated with rat liver S9-fraction prior to the assay. The most potent compound was (3R)-1,7-diphenyl-(4E,6E)-4,6-heptadien-3-ol, with a relative potency of 4% compared to 17beta-estradiol. The metabolic activation of diarylheptanoids markedly enhanced their efficiency. The chemical structure required for estrogenic activity of diarylheptanoids was the presence of a keto group at C3 and absence of hydroxyl moiety in ring B. Only diarylheptanoids showing full estrogenic efficiency in vitro were able to elicit uterotrophic activity of in immature ovariectomized rat. This is the first evidence for in vivo estrogenic activity of diarylheptanoids from C. comosa. This novel class of natural phytoestrogens has the potential to be developed for use as dietary supplement in the treatment of menopausal symptoms.

New substituted C-19-andrographolide analogues with potent cytotoxic activities.

Andrographolide, the major diterpenoid lactone from Andrographis paniculata, is toxic against cancer cells. In the present study, we investigated the structure-activity relationships (SARs) of 19 andrographolide analogues which were synthesized by modification at the three hydroxyl groups. A number of the andrographolide analogues showed much higher cytotoxic activities than that of the parent compound on cancer cells including P-388, KB, COL-2, MCF-7, LU-1 and ASK cells. SAR studies of the synthetic analogues indicated that the introduction of silyl ether or triphenylmethyl ether group into C-19 of the parent compound led to increase in toxicity against the cancer cells. The 19-O-triphenylmethyl ether analogue 18 showed higher cytotoxic activity than the potent anticancer drug ellipticine, and this analogue may serve as a potential structure lead for the development of new anticancer drugs.

Synthesis and in vitro transfection efficiency of spermine-based cationic lipids with different central core structures and lipophilic tails

Twelve spermine-based cationic lipids with four different central core structures (di(oxyethyl)amino, di(oxyethyl)amino carboxy, 3-amino-1,2-dioxypropyl and 2-amino-1,3-dioxypropyl) and three hydrophobic tails (lauric acid, myristic acid and palmitic acid) were synthesized. The liposomes containing lipids and DOPE showed moderate to good in vitro DNA delivery into HeLa cells. GFP expression experiments revealed that liposomes composed of lipids with 3-amino-1,2-dioxypropyl as a central core structure exhibited highest transfection efficiency under serum-free condition. Whereas, lipid with 2-amino-1,3-dioxypropyl core structure showed highest transfection under 10% serum condition. Moreover, the liposomes and lipoplexes composted of these cationic lipids exhibited low cytotoxicity.

Downregulation of LAT1 expression suppresses cholangiocarcinoma cell invasion and migration.

Currently, there is no effective treatment for cholangiocarcinoma (CCA), which is the most prevalent in the northeastern part of Thailand. A new molecular target for the treatment of CCA is, therefore, urgently needed. Although L-type amino acid transporter 1 (LAT1) is highly expressed in CCA cells, its role in malignant phenotypes of CCA cells remains unclear. This study aimed to investigate the impact of LAT1 on proliferation, migration, and invasion of KKU-M213 cells, the CCA cells derived from Thai patients with intrahepatic cholangiocarcinoma. Results showed that KKU-M213 cells expressed all LAT isoforms (LAT1, LAT2, LAT3 and LAT4). The expressions of LAT1 and its associated protein 4F2hc were highest whereas those of LAT2 and LAT4 were extremely low. Treatment with 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) reduced L-leucine uptake concomitant with an inhibition of cell motility and, to a lesser extent, on cell proliferation. It also induced a time dependent up-regulation of LAT1 and 4F2hc expressions. Similarly, cell migration and invasion, but not proliferation, were reduced in LAT1 knockdown KKU-M213 cells. In addition, silencing of LAT1 inhibited the expressions of 4F2hc mRNA and protein whereas the expression of microRNA-7, the 4F2hc down-regulator, was increased. Furthermore, the phosphorylation levels of ERK1/2 and p70S6K were reduced after LAT1 knockdown. Collectively, these results suggest that suppression of cell invasion and migration in LAT1 knockdown KKU-M213 cells may be partly mediated through the inhibition of the 4F2hc-signaling pathway by the up-regulation of microRNA-7. Based on this finding, LAT1 may be a potential therapeutic target for treating CCA.

12-Amino-andrographolide analogues: synthesis and cytotoxic activity

Andrographolide, a diterpenoid lactone of the plant Andrographis paniculata, has been shown to be cytotoxic against various cancer cells in vitro. In the present study, a series of β-amino-γ-butyrolactone analogues has been synthesized from naturally occurring andrographolide via one pot tandem aza-conjugate addition–elimination reaction. By using economic procedure without any base or catalyst at room temperature, the products obtained were in fair to excellent yields with high stereoselectivity. The cytotoxicity of all new amino analogues were evaluated against six cancer cell lines and revealed their potential for being developed as promising anti-cancer agents.

Cytotoxic alkaloids from stems, leaves and twigs of Dasymaschalon blumei.

Bioassay-guided fractionation of the cytotoxic ethyl acetate extract from the stems of Dasymaschalon blumei (Annonaceae) led to the isolation of four aristololactam alkaloids, including the hitherto unknown 3,5-dihydroxy-2,4-dimethoxyaristolactam (1), as well as the three known compounds, aristolactam BI, goniopedaline, and griffithinam. Additionally, the cytotoxic extract from the combined leaves and twigs of the same plant yielded three known oxoaporphine alkaloids, oxodiscoguattine, dicentrinone, and duguevalline. The structures of aristolactams and oxoaporphine alkaloids were elucidated on the basis of spectroscopic methods. All isolates were evaluated for cytotoxicity against a panel of mammalian cancer cell lines and a noncancerous human embryonic kidney cell Hek 293.

A rhodol-based fluorescent chemosensor for hydrazine and its application in live cell bioimaging

A rhodol cinnamate fluorescent chemosensor (RC) has been developed for selective detection of hydrazine (N2H4). In aqueous medium, the rhodol-based probe exhibited high selectivity for hydrazine among other molecules. The addition of hydrazine triggered a fluorescence emission with 48-fold enhancement based on hydrazinolysis and a subsequent ring-opening process. The chemical probe also displayed a selective colorimetric response toward N2H4 from colorless solution to pink, readily observed by the naked eye. The detection limit of RC for hydrazine was calculated to be 300nM (9.6ppb). RC is membrane permeable and was successfully demonstrated to detect hydrazine in live HepG2 cells by confocal fluorescence microscopy.

Cleistanthoside A tetraacetate-induced DNA damage leading to cell cycle arrest and apoptosis with the involvement of p53 in lung cancer cells.

Lung cancer is the leading cause of cancer-related death worldwide and resistance to chemotherapeutic drugs is the major obstacle for effective treatment. The present study investigated the anticancer potential of cleistanthoside A tetraacetate (CAT), a derivative of cleistanthoside A from Phyllanthus taxodiifolius Beille on human lung cancer cells, LU-1. Multiple molecular approaches were used in this study and include measuring the anti-proliferative effect of CAT in LU-1 cells using flow cytometry; evaluating the induction of apoptosis by monitoring DNA fragmentation, phosphatidylserine externalization and activation of caspase-3 activity; and assaying the expression of regulatory proteins involved in cell cycle arrest and apoptosis using immunoblots. CAT potently inhibited LU-1 proliferation through an early G1 arrest with down-regulation of cdk4/6 and cyclin D1 proteins. CAT also inhibited DNA topoisomerase IIα activity resulting in DNA damage and increased the expression of the p53 protein with the subsequent induction of apoptosis. A decrease in the Bcl-2/Bax ratio, activation of caspase-3 activity and cleavage of PARP accompanied apoptosis. CAT is highly toxic to lung cancer and its primary targets are the inhibition of topoisomerase IIα activity and inducing apoptosis through a G1 arrest. These properties indicate that CAT is a promising anticancer agent for treatment of lung cancer.

Rhodol-based fluorescent probe for Au3+ detection and its application in bioimaging

A turn-on fluorescent probe of propargyl–rhodol conjugate responds selectively to Au3+. In the presence of Au3+, the chemodosimeter undergoes a remarkable change in its absorption spectrum with a 370-fold fluorescence enhancement. This reaction-based Au3+ chemodosimeter is highly sensitive with a detection limit of 7 ppb through an irreversible mechanism. Bioimaging of HeLa cells with this chemical probe has been successfully demonstrated.