ABSTRACT
Melanoma is a highly metastatic disease with an increasing rate of incidence worldwide. It is treatment refractory and has poor clinical prognosis; therefore, the development of new therapeutic agents for metastatic melanoma are urgently required. In this study, we created a lung-seeking A375LM5IF4g/Luc BRAFV600E mutant melanoma cell clone and investigated the bioefficacy of a plant sesquiterpene lactone deoxyelephantopin (DET) and its novel semi-synthetic derivative, DETD-35, in suppressing metastatic A375LM5IF4g/Luc melanoma growth in vitro and in a xenograft mouse model. DET and DETD-35 treatment inhibited A375LM5IF4g/Luc cell proliferation, and induced G2/M cell-cycle arrest and apoptosis. Furthermore, A375LM5IF4g/Luc exhibited clonogenic, metastatic and invasive abilities, and several A375LM5IF4g/Luc metastasis markers, N-cadherin, MMP2, vimentin and integrin α4 were significantly suppressed by treatment with either compound. Interestingly, DET- and DETD-35-induced Reactive Oxygen Species (ROS) generation and glutathione (GSH) depletion were found to be upstream events important for the in vitro activities, because exogenous GSH supplementation blunted DET and DETD-35 effects on A375LM5IF4g/Luc cells. DET and DETD-35 also induced mitochondrial DNA mutation, superoxide production, mitochondrial bioenergetics dysfunction, and mitochondrial protein deregulation. Most importantly, DET and DETD-35 inhibited lung metastasis of A375LM5IF4g/Luc in NOD/SCID mice through inhibiting pulmonary vascular permeability and melanoma cell (Mel-A+) proliferation, angiogenesis (VEGF+, CD31+) and EMT (N-cadherin) in the tumor microenvironment in the lungs. These findings indicate that DET and DETD-35 may be useful in the intervention of lung metastatic BRAFV600E mutant melanoma.
Subject(s)
Antineoplastic Agents, Phytogenic/isolation & purification , Antineoplastic Agents, Phytogenic/pharmacology , Asteraceae/chemistry , Lactones/isolation & purification , Lactones/pharmacology , Plant Extracts/isolation & purification , Plant Extracts/pharmacology , Sesquiterpenes/isolation & purification , Sesquiterpenes/pharmacology , Animals , Antineoplastic Agents, Phytogenic/chemistry , Apoptosis/drug effects , Apoptosis/genetics , Cell Cycle Checkpoints/drug effects , Cell Cycle Checkpoints/genetics , Cell Line, Tumor , Cell Proliferation/drug effects , Disease Models, Animal , Humans , Immunohistochemistry , Lactones/chemistry , Lung Neoplasms/drug therapy , Lung Neoplasms/secondary , Melanoma/pathology , Mice , Mitochondria/drug effects , Mitochondria/metabolism , Molecular Structure , Oxidative Stress/drug effects , Plant Extracts/chemistry , Proto-Oncogene Proteins B-raf/genetics , Reactive Oxygen Species/metabolism , Sesquiterpenes/chemistry , Tumor Microenvironment/drug effects , Xenograft Model Antitumor AssaysABSTRACT
Phytochemical investigation of the acetone extract from the roots of Aphanamixis polystachya resulted in isolation of four new tetranortriterpenes (1-4) in addition to one protolimonoid (methyl-1ξ,7R-diacetoxy-23R,25-dihydroxy-20S,24R-21,24-epoxy-3,4-seco-apotirucall-4(28),14(15)-diene-3-oate (5)), five known limonoids (rohituka 3 (6), rohituka 7 (7), nymania 1 (8), rubrin G (9), prieurianin (10)) and a steroid (2,3-dihydroxy-5-pregnan-16-one (11)). Their structures were determined by spectroscopic analyses, including 2D-NMR (COSY, HMQC, HMBC, and NOESY) and high-resolution electrospray ionization mass spectrometry (HRESIMS). Cytotoxic and anti-inflammatory activities of these compounds were evaluated. Compounds 4 and 5 showed significant inhibition against superoxide generation and elastase release by human neutrophils in response to (formyl-l-methionyl-l-leucyl-l-phenylalanine/cytochalasin B) (FMLP/CB).
Subject(s)
Leukocyte Elastase/metabolism , Limonins , Meliaceae/chemistry , Neutrophils/metabolism , Plant Extracts/chemistry , Plant Roots/chemistry , Superoxides/metabolism , Humans , Limonins/chemistry , Limonins/isolation & purification , Limonins/pharmacology , Neutrophils/cytologyABSTRACT
Three novel C19 homolignans, taiwankadsurins D (1), E (2) and F (4), and two new C18 lignans kadsuphilins N (3) and O (5) were isolated from the aerial parts of Taiwanese medicinal plant Kadsura philippinensis. The structures of compounds 1-5 were determined by spectroscopic analyses, especially 2D NMR techniques. The structure of compound 5 was further confirmed by X-ray crystallographic analysis. Compounds 1 and 2 have a 3,4-{1'-[(Z)-2''-methoxy-2''-oxoethylidene]}-pentano(2,3-dihydrobenzo[b]furano)-3-(2'''-methoxycarbonyl-2'''-hydroxy-2''',3'-epoxide) skeleton.
Subject(s)
Kadsura/chemistry , Lignans/chemistry , Plant Leaves/chemistry , Plant Stems/chemistry , Lignans/analysis , Molecular Structure , Nuclear Magnetic Resonance, Biomolecular , Plant Extracts/analysis , Plant Extracts/chemistryABSTRACT
Three new polyoxygenated C(18)-dibenzocyclooctadiene lignans, arisanschinins M and N (1 and 2) and schisphenin A (3), together with eight related metabolites (4-11), were isolated from the fruits of Schisandra arisanensis and Schisandra sphenanthera, respectively. The structures of 1-3 were elucidated on the basis of extensive spectroscopic and 2D NMR (HSQC, HMBC, and NOESY) analyses. The configuration of the biphenyl moiety in the octadiene ring was determined by circular dichroism (CD). Compound 1 possessed an unprecedented 3-(1-hydroxypropan-2-yl)-3-methyl-1,4-dioxo-2-one lactonide ring system attaching at C-6/C-14. Pharmacological studies revealed that compounds 3, 4, 6, 7, and 10 exhibited significant anti-hepatic fibrosis activity, while 9 and 11 showed cytotoxicity against HSC-T6 cells. The biogenetic pathway for compound 1 was also proposed.
Subject(s)
Cyclooctanes/chemistry , Fruit/chemistry , Lignans/chemistry , Plant Extracts/chemistry , Schisandra/chemistry , Cell Line , Cell Survival/drug effects , Circular Dichroism , Cyclooctanes/pharmacology , Humans , Lignans/pharmacology , Liver/drug effects , Liver Cirrhosis/drug therapy , Molecular Structure , Nuclear Magnetic Resonance, Biomolecular , Plant Extracts/pharmacologyABSTRACT
Phytochemical investigation of Hypericum nakamurai (Masamune) Robson has led to the isolation of three phloroglucinol derivatives 1-3. The structures of these compounds were determined by the analysis of their spectroscopic data (IR, mass and UV), and by the application of 1-D and 2-D-NMR techniques. Hyperinakin (1) is a new compound. The anti-inflammatory activities of compounds 1-3 were also tested and evaluated. A biogenetic pathway for compounds 1-3 was also proposed.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/isolation & purification , Hypericum/chemistry , Phloroglucinol/analogs & derivatives , Animals , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Cell Line , Mice , Molecular Structure , Phloroglucinol/chemistry , Phloroglucinol/isolation & purification , Plants, Medicinal/chemistryABSTRACT
Two novel highly oxygenated nortriterpenoids, schisarisanlactones A (1) and B (2), have been isolated from the fruits of Schisandra arisanensis, an endemic plant of Taiwan. Compounds 1 and 2 possess an unprecedented 5/5/7/5/5-fused pentacyclic ring system. The structures of both compounds were determined on the basis of spectroscopic analyses, especially 2D NMR and MS. A plausible biogenetic pathway of 1 was proposed. Schisarisanlactone A (1) showed significant anti-HIV activity.
Subject(s)
Fruit/chemistry , Plant Extracts/chemistry , Schisandra/chemistry , Triterpenes/chemistry , Anti-HIV Agents/chemistry , Anti-HIV Agents/isolation & purification , Anti-HIV Agents/pharmacology , HIV/drug effects , Molecular Structure , Plant Extracts/isolation & purification , Plant Extracts/pharmacology , Taiwan , Triterpenes/isolation & purification , Triterpenes/pharmacologyABSTRACT
Fractionation of an acetone extract from the fruits of Schisandra arisanensis afforded five new nortriterpene lactones, compounds 1-5, together with four known compounds, schindilactones D and E (6 and 7) and preschisanartanins A and B (8 and 9). Compound 1, a wuweiziartane-type nortriterpenoid, possesses a new type of fused ring system with a gamma-lactone ring between C-15 and C-17. Compounds 2, 6, and 7 may be categorized as schisanartane-type nortriterpenoids and compounds 3-5, 8, and 9 as preschisanartane-type nortriterpenoids. The structures of the new compounds were elucidated by 1D and 2D NMR spectroscopic data interpretation. The structure and relative configuration of 3 were supported by single-crystal X-ray diffraction analysis. The antiviral activity against HSV-1 and inhibitory effects on superoxide anion generation and elastase release by human neutrophils in response to FMLP/CB of compounds 1-9 were evaluated.
Subject(s)
Antiviral Agents/isolation & purification , Drugs, Chinese Herbal/isolation & purification , Herpesvirus 1, Human/drug effects , Lactones/isolation & purification , Schisandra/chemistry , Triterpenes/isolation & purification , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Crystallography, X-Ray , Drugs, Chinese Herbal/chemistry , Drugs, Chinese Herbal/pharmacology , Fruit/chemistry , Humans , Lactones/chemistry , Lactones/pharmacology , Molecular Conformation , Molecular Structure , Neutrophils/drug effects , Neutrophils/enzymology , Nuclear Magnetic Resonance, Biomolecular , Triterpenes/chemistry , Triterpenes/pharmacologyABSTRACT
Four new oxygenated dibenzocyclooctadiene lignans, arisanschinins A-D ( 1- 4), and a new 1,4-bis(phenyl)-2,3-dimethylbutane lignan, arisanschinin E ( 5), together with 15 known compounds, were isolated from the EtOAc-soluble fraction of the aerial parts of SCHISANDRA ARISANENSIS Hay. The structures of 1- 5 were elucidated on the basis of extensive spectroscopic analyses, including 2D NMR (HMQC, HMBC, and NOESY) experiments. The configurations of the biphenyl and octadiene moieties were deduced from circular dichroism (CD) and NOESY spectra, respectively. Compound 1 showed significant inhibition of α-glucosidase IN VITRO. The radical-scavenging activities of these compounds were evaluated using DPPH.
Subject(s)
Cyclooctanes/pharmacology , Free Radical Scavengers/pharmacology , Lignans/pharmacology , Schisandra/chemistry , Biphenyl Compounds/chemistry , Cyclooctanes/chemistry , Cyclooctanes/isolation & purification , Free Radical Scavengers/chemistry , Free Radical Scavengers/isolation & purification , Lignans/chemistry , Lignans/isolation & purification , Nuclear Magnetic Resonance, Biomolecular , Picrates/chemistry , Plant Extracts/chemistryABSTRACT
An acetone extract of the fruits of the Taiwanese medicinal plant Schisandra arisanensis has yielded 11 new oxygenated lignans. Four of these, named arisantetralones A-D (1-4), possess the aryltetralone skeleton, while the other seven, named arisanschinins F-L (5-11), are polyoxygenated C(18)-dibenzocyclooctadiene lignans. Structures were determined on the basis of spectroscopic analyses, especially 2D-NMR techniques. The structure of compound 1 was confirmed by X-ray crystallographic analysis. Immunomodulatory activity of the isolated lignans was tested and evaluated.
Subject(s)
Immunologic Factors/isolation & purification , Lignans/isolation & purification , Plants, Medicinal/chemistry , Schisandra/chemistry , Crystallography, X-Ray , Fruit/chemistry , Humans , Immunologic Factors/blood , Immunologic Factors/chemistry , Immunologic Factors/pharmacology , Lignans/blood , Lignans/chemistry , Lignans/pharmacology , Models, Molecular , Molecular Conformation , Molecular Structure , Nuclear Magnetic Resonance, Biomolecular , Oxygen/chemistry , TaiwanABSTRACT
A new dimeric phenylpropanoid namely podonaka A (1), along with the 13 known compounds including diterpenes (2 and 3), norditerpenes (4 and 5), benzenoids (6-10), steroids (11 and 12), chalcone (13), and megastigmane (14), was isolated from the EtOH extract of Podocarpus nakaii Hayata. The structure of 1 was elucidated on the basis of spectroscopic analysis including 1D and 2D NMR and MS techniques. Biological evaluation showed that norditerpenes, inumakilactone B (4), and podolactone E (5) have potent cytotoxic activities against Daoy, WiDr, KB, and HeLa tumor cell lines. Moreover, low dosage of 5 may induce early apoptosis in KB cells before 12 h.
Subject(s)
Antineoplastic Agents, Phytogenic/isolation & purification , Antineoplastic Agents, Phytogenic/pharmacology , Diterpenes/isolation & purification , Diterpenes/pharmacology , Phenylpropionates/isolation & purification , Phenylpropionates/pharmacology , Plants, Medicinal/chemistry , Tracheophyta/chemistry , Antineoplastic Agents, Phytogenic/chemistry , Diterpenes/chemistry , Drug Screening Assays, Antitumor , HeLa Cells , Humans , KB Cells , Molecular Structure , Nuclear Magnetic Resonance, Biomolecular , Phenylpropionates/chemistry , Plant Bark/chemistry , Plant Stems/chemistry , TaiwanABSTRACT
Oleanane-type triterpenoidal saponins, hydrocosisaponins A-F (1-6), along with a known saponin, hydrocotyloside VII (7), were isolated from Hydrocotyle sibthorpioides. Their structures were established on the basis of spectroscopic analyses including NMR spectroscopic techniques ((13)C, (1)H, COSY, HMQC, HMBC, TOCSY and NOESY). Biological evaluation established that saponins possessing four sugar units (three d-glucoses and one l-arabinose) (4-7) exhibited moderate cytotoxicity against KB, Daoy and WiDr human tumor cell lines.
Subject(s)
Magnoliopsida/chemistry , Oleanolic Acid/analogs & derivatives , Saponins/chemistry , Triterpenes/chemistry , Drugs, Chinese Herbal/chemistry , Magnetic Resonance Spectroscopy , Molecular Structure , Oleanolic Acid/chemistryABSTRACT
Phytochemical investigation of the leaves and twigs of Taxus sumatrana afforded six new taxane diterpene esters, tasumatrols U-Z ( 1- 6). Compounds 2 and 5 contained a rare five-membered lactone ring at C-8, C-9, C-10, and C-19. The structures were established on the basis of detailed spectroscopic analyses, particularly HRESIMS and 2D NMR techniques. Compound 5 showed cytotoxicity against a human hepatoma (Hep2) cell line.
Subject(s)
Antineoplastic Agents, Phytogenic/isolation & purification , Bridged-Ring Compounds/isolation & purification , Diterpenes/isolation & purification , Plants, Medicinal/chemistry , Taxoids/isolation & purification , Taxus/chemistry , Antineoplastic Agents, Phytogenic/chemistry , Antineoplastic Agents, Phytogenic/pharmacology , Bridged-Ring Compounds/chemistry , Bridged-Ring Compounds/pharmacology , Diterpenes/chemistry , Diterpenes/pharmacology , Drug Screening Assays, Antitumor , Esters , Humans , Molecular Structure , Nuclear Magnetic Resonance, Biomolecular , Taiwan , Taxoids/chemistry , Taxoids/pharmacologyABSTRACT
Eight new oxygenated lignans, kadsuphilols A-H (1-8), were isolated from the leaves and stems of Kadsura philippinensis. Four of the isolated lignans (1-4) possess the normal C18-dibenzocyclooctadiene skeleton, while the other four lignans (5-8) are C19-homolignans possessing a substituted cyclohexadienone ring with a spiro-benzofuranoid moiety. The structures of the isolated metabolites were elucidated through spectroscopic analyses, including 2D NMR experiments. Compounds 1 and 4 are the first report of an R-biphenyl configuration with a beta-oxygenated substituent at the C-9 position. The in vitro radical-scavenging activities of these compounds using DPPH were tested and evaluated. Compound 3 exhibited more potent activity than vitamins C and E.
Subject(s)
Free Radical Scavengers/isolation & purification , Kadsura/chemistry , Lignans/isolation & purification , Plants, Medicinal/chemistry , Ascorbic Acid/pharmacology , Biphenyl Compounds , Free Radical Scavengers/chemistry , Free Radical Scavengers/pharmacology , Lignans/chemistry , Lignans/pharmacology , Molecular Structure , Picrates/pharmacology , Plant Leaves/chemistry , Plant Stems/chemistry , Taiwan , Vitamin E/pharmacologyABSTRACT
Four new nona-oxygenated C18 dibenzocyclooctadiene lignans, kadsuphilins C-F (1-4), were isolated from the EtOAc soluble portion of the alcoholic extract of the aerial parts of Kadsura philippinensis. The structures of 1-4 were elucidated on the basis of extensive spectroscopic analyses, including 2D NMR (HMQC, HMBC, and NOESY) experiments, comparison of the spectral data with those of the related metabolites. The stereochemistries of the biphenyl and octadiene moieties were deduced from circular dichorism (CD) and the NOESY spectra, respectively. The in vitro antiplatelet aggregation activity of metabolites 1-4 also have been evaluated.
Subject(s)
Antineoplastic Agents, Phytogenic/chemistry , Cyclooctanes/chemistry , Kadsura/chemistry , Lignans/chemistry , Plant Extracts/chemistry , Plants, Medicinal/chemistry , Alcohols/chemistry , Antineoplastic Agents, Phytogenic/isolation & purification , Antineoplastic Agents, Phytogenic/pharmacology , Circular Dichroism , Cyclooctanes/analysis , Cyclooctanes/isolation & purification , Cyclooctanes/pharmacology , Lignans/isolation & purification , Lignans/pharmacology , Magnetic Resonance Spectroscopy , Molecular Structure , Platelet Aggregation Inhibitors/metabolism , StereoisomerismABSTRACT
Four new C18 dibenzocyclooctadiene lignans, kadsuphilins A (1) and B (3), 6-epi-gomisin (2), and 1-demethylkadsuphilin A (4), along with eight known related metabolites, were isolated from an EtOAc fraction of an alcoholic extract of the aerial parts of Kadsura philippinensis growing in Taiwan. The structures of 1-4 were elucidated on the basis of spectroscopic analyses, including 2D NMR (HMQC, HMBC, and NOESY) experiments, and by comparison of their spectroscopic data with those of related metabolites. The configurations of the biphenyl and cyclooctadiene moieties were deduced from circular dichroism (CD) and NOESY NMR spectra, respectively. Some of the compounds showed radical-scavenging activity in a DPPH-HPLC method.
Subject(s)
Cyclooctanes/chemistry , Cyclooctanes/isolation & purification , Free Radical Scavengers/chemistry , Free Radical Scavengers/isolation & purification , Kadsura/chemistry , Lignans/chemistry , Lignans/isolation & purification , Plants, Medicinal/chemistry , Biphenyl Compounds , Cyclooctanes/pharmacology , Free Radical Scavengers/pharmacology , Lignans/pharmacology , Molecular Structure , Nuclear Magnetic Resonance, Biomolecular , Picrates/pharmacology , TaiwanABSTRACT
Bioassay-guided fractionation of the EtOAc extract of Casearia membranacea leaves and twigs afforded three new clerodane diterpenes, caseamembrins M-O (1-3), and the known rel-(2S,5R,6R,8S,9S,10R,18S,19R)-2-(2-methylbutyryloxy)-6-hydroxy-18,19-di-O-acetyl-18,19-epoxycleroda-3,13(16),14-triene (4) and caseamembrin D (5). The structures of 1-3, including the relative configurations, were established by extensive NMR spectroscopic analyses. The cytotoxic activities of the isolated diterpenoids against human oral epidermoid (KB), medulla (Med), and colon (DLD-1) cancer cell lines were evaluated.
Subject(s)
Antineoplastic Agents, Phytogenic/isolation & purification , Casearia/chemistry , Diterpenes, Clerodane/isolation & purification , Plants, Medicinal/chemistry , Antineoplastic Agents, Phytogenic/chemistry , Antineoplastic Agents, Phytogenic/pharmacology , Diterpenes, Clerodane/chemistry , Diterpenes, Clerodane/pharmacology , Drug Screening Assays, Antitumor , Humans , Molecular Structure , Plant Leaves/chemistry , Plant Stems/chemistry , Stereoisomerism , Taiwan , Tumor Cells, CulturedABSTRACT
[structures: see text] Three novel C19 homolignans, designated taiwankadsurins A (1), B (2), and C (3), were isolated from the aerial parts of Taiwanese medicinal plant Kadsura philippinensis. The structures of 1-3, which have a 3,4-{1'-[(Z)-2''-methoxy-2''-oxo-ethylidene]}-pentano(2,3-dihydro-benzo[b]furano)-3-(2'''-methoxycarbonyl-2'''-hydroxy-2''',3'-epoxide) skeleton, were determined by spectroscopic analyses, especially 2D NMR techniques (HMBC and NOESY). Compound 2 exhibited mild cytotoxicity against human KB and Hela tumor cells.
Subject(s)
Antineoplastic Agents, Phytogenic/isolation & purification , Kadsura/chemistry , Lignans/isolation & purification , Plants, Medicinal/chemistry , Antineoplastic Agents, Phytogenic/chemistry , Antineoplastic Agents, Phytogenic/pharmacology , Drug Screening Assays, Antitumor , HeLa Cells , Humans , KB Cells , Lignans/chemistry , Lignans/pharmacology , Molecular Structure , Plant Leaves/chemistry , Plant Stems/chemistry , TaiwanABSTRACT
[structure: see text]. Two novel triterpene dilactones, kadsuphilactones A (1) and B (2), were isolated from the Taiwanese medicinal plant Kadsura philippinensis. The structures of 1 and 2 were elucidated on the basis of extensive spectroscopic methods, including two-dimensional NMR techniques, and confirmed by X-ray crystallographic analysis. Kadsuphilactone B (2) exhibited in vitro anti-HBV activity with IC(50) values of 6 microg/mL by HBsAg enzyme immunoassay.
Subject(s)
Antiviral Agents/isolation & purification , Hepatitis B virus/drug effects , Kadsura/chemistry , Lactones/isolation & purification , Plants, Medicinal/chemistry , Triterpenes/isolation & purification , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Crystallography, X-Ray , Inhibitory Concentration 50 , Lactones/chemistry , Lactones/pharmacology , Molecular Structure , Triterpenes/chemistry , Triterpenes/pharmacologyABSTRACT
Bioassay-directed fractionation of the EtOAc extract of Kadsura japonica has led to the isolation of six new C18 dibenzocyclooctadiene lignans, schizanrins I, J, K, L, M, N, along with four known C19 homolignans, taiwanschirins A, B, C, and heteroclitin F. The elucidations of the new structures were based on spectral analysis. Bioassay evaluation against human type B hepatitis revealed that taiwanschirins A and B showed strong activity for anti-HBsAg and a medium effect for anti-HBeAg at 25 microg/mL (12.9 and 11.9 microM for taiwanschirins A and B, respectively).
Subject(s)
Antiviral Agents/pharmacology , Hepatitis B Surface Antigens/drug effects , Hepatitis B e Antigens/drug effects , Kadsura , Phytotherapy , Plant Extracts/pharmacology , Antiviral Agents/chemistry , Cell Line , Cyclooctanes/chemistry , Cyclooctanes/pharmacology , Hepatitis B/prevention & control , Humans , Lignans/chemistry , Lignans/pharmacology , Magnetic Resonance Spectroscopy , Plant Extracts/chemistry , Structure-Activity RelationshipABSTRACT
Phytochemical investigation of Eupatorium kiirunense has resulted in the isolation of eight new sesquiterpene lactones, constituted by five germacranolides, eupakirunsins A-E (1-5), and three heliangolides, eupaheliangolide A (6), 15-acetoxyheliangin (7), and 3-epi-heliangin (8), in addition to the known heliangin (9) and 8,10-epoxy-9-acetoxythymol angelate (10). The structures of the new compounds were established through detailed analysis of their spectroscopic data. Compounds 6, 8, and 9 exhibited cytotoxicity against human oral epidermoid (KB), cervical epitheloid (Hela), and liver (hepa59T/VGH) carcinoma cells.