In vivo anti-tumour activity of corilagin on Hep3B hepatocellular carcinoma.

We have investigated the potential in vivo anti-tumour activity of corilagin using the Hep3B hepatocellular carcinoma cell line and an athymic nude mice xenograft model. The purity of corilagin was confirmed by high performance liquid chromatographic analysis. Corilagin was administrated intraperitoneally for a continuous period of 7 days at a concentration of 15 mg/kg of body weight per day. A significant inhibition of tumour growth was observed when treated mice are compared with control groups. Furthermore, analysis of enzymes markers of liver function, including alanine aminotransferase and asparate aminotransferase, suggested that current therapeutic dosage of corilagin did not exert adverse effect on liver. Our observations support the view that corilagin is considerably effective to retard the in vivo growth of xenografted Hep3B hepatocellular carcinoma.

Synthesis of 9,9-dialkyl-4,5-diazafluorene derivatives and their structure-activity relationships toward human carcinoma cell lines.

A homologous set of 9,9-dialkyl-4,5-diazafluorene compounds were prepared by alkylation of 4,5-diazafluorene with the appropriate alkyl bromide and under basic conditions. The structures of these simple organic compounds were confirmed by spectroscopic techniques (FTIR, NMR, and FABMS). Their biological effects toward a panel of human carcinoma cells, including Hep3B hepatocellular carcinoma, MDAMB-231 breast carcinoma, and SKHep-1 hepatoma cells, were investigated; a structure-activity correlation was established with respect to the length of the alkyl chain and the fluorene ring structure. The relationship between the mean potency [log(1/IC(50))] and alkyl chain length was systematically studied. The results show that compounds with butyl, hexyl, and octyl chains exhibit good growth inhibitory effects toward these three human carcinoma cell lines, and the 9,9-dihexyl-4,5-diazafluorene further exhibits antitumor activity in athymic nude mice Hep3B xenograft models. For the structurally related dialkylfluorenes that lack the diaza functionality, in vitro cytotoxicity was not observed at clinically relevant concentrations.

In vitro cytotoxicity of (-)-EGCG octaacetate on MDAMB-231 and SKHep-1 human carcinoma cells: a pharmacological consideration on prodrug design.

Esterification of acetate with generic pharmaceutical compound has been commonly employed to produce ester prodrug for improving its potency when compared with the mother compound. Acetate, on the other hand, has been recognized to have inhibitory effect on the respiratory biochemistry. Here we demonstrate that acetate at a concentration of 400 microM exhibited significant growth inhibitory activity on two human cancer cell lines, the MDAMB-231 breast cancer and the SKHep-1 hepatoma cell lines. To establish the ester prodrug with multi-acetate ester conjugates as our experimental model, one molecule of (-)-epigallocatechin gallate was required to conjugate with eight molecules of acetate forming the corresponding (-)-epigallocatechin gallate octaacetate prodrug. Chemical structure of this epigallocatechin gallate octaacetate ester prodrug was confirmed by both 13C and 1H nuclear magnetic resonance spectra and mass spectrometry. Further cytotoxic assay using both MDAMB-231 and SKHep-1 human carcinoma cell lines showed that acetate at a concentration of 400 microM exhibits an additional cytotoxic effect with (-)-epigallocatechin gallate at a concentration of 50 microM, although the additional effect was not as high as (-)-epigallocatechin gallate octaacetate ester prodrug alone at a concentration of 50 microM. Our results thus raise a pharmacological consideration of using multi-acetate conjugate as the ester prodrug where the release of free acetate by esterase could be part of the explanation for the improved in vitro cytotoxicity.

Synthesis and anti-cancer activity of benzothiazole containing phthalimide on human carcinoma cell lines.

Phthalic anhydride is a highly toxic substance, facing, however, the problem of hydrolysis. In fact, it is rapidly hydrolyzed in aqueous medium, generating phthalic acid as the final product, which is almost harmless to viable cells. Here we describe the 'one pot' condensation reaction for the synthesis of phthalic imide derivative (benzothiazole containing phthalimide), exhibiting in vitro cytotoxic potential on human cancer cell lines. We further demonstrated that both caspase-dependent and -independent pathways are involved in our novel benzothiazole containing phthalimide induced apoptosis on cancer cells.

Paradoxical proliferative potential of iron (II) sulphate on cancer cells after the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay.

There are several scientific approaches for the determination of cellular growth influences of known or novel substances under in vitro conditions, among which colourimetric absorption measurement is considered to be one of the convenient methods. [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] (MTS) assay is one of the commonly used colourimetric absorption assays based on the ability of dehydrogenase from viable cells to produce the brown soluble formazan detectable at 490 nm. Here we have tested the possible growth influence of iron (II) sulphate on two human cancer cell lines, the K562 chronic myelogenous leukaemia and T47D breast carcinoma cells, based on the MTS assay. We found that iron (II) sulphate possessed an inhibitory effect when added at 16- to 125-microM concentrations, but iron (II) sulphate became growth stimulatory when its concentration was further increased to 1000 microM. In addition, a dose-dependent increase in absorbance at the same wavelength was observed when we repeated the experiments without the addition of MTS and phenazine methosulfate. When we further repeated the cell growth determinations using adenosine triphosphate content assay for K562 and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for T47D, iron (II) sulphate showed a consistent dose-dependent growth inhibitory effect. Morphological investigation after methylene blue staining clearly demonstrated that iron (II) sulphate, at a concentration of 1000 microM, is cytotoxic to T47D cells. Interestingly, a consistent increment for the absorbance at 490 nm was further observed with increased iron (II) sulphate concentration either in the presence or absence of MTS even in a cell-free environment. Thus we conclude that iron (II) sulphate is actually growth inhibitory and even cytotoxic at high concentrations towards the K562 and T47D cancer cells and the paradoxical proliferative activity of iron (II) sulphate on these two cancer cell lines using the MTS assay was solely due to the oxidation of initial pale green iron (II) to brownish iron (III) during incubation in the aqueous condition.

Apoptogenic activity of a synthetic cantharimide in leukaemia: implication on its structural activity relationship.

Cantharidin isolated from Mylabris caraganae and other insects has been used as an anti-cancer drug in China for many years. However, its toxicity on the renal system and suppression effect on bone marrow limits its usage clinically. A synthetic analogue of cantharidin (CAN 037) has been shown to have cytotoxic effect on the SK-Hep 1 hepatoma cell line but its underlying working principle remains undefined. Here we further report the action of CAN 037 on an acute myelogenous leukaemia (AML) cell line, KG1a. [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] (MTS) assay was used to demonstrate the cytotoxicity of CAN 037 on KG1a cells. Morphological changes of CAN 037-treated leukaemia cells were recorded under an inverted microscope. Possible activation of caspase 3, 8 and 9 from KG1a cells was also investigated. KG1a AML cells were sensitive to CAN 037. Morphological changes including cell shrinkage and loss of colony formation ability were observed. Caspase 3, 8 and 9 activity was elevated, whereas pre-incubating the KG1a cells with the generic caspase inhibitor z-VAD-fmk could only partially reverse the CAN 037-induced cell death. In addition to the SK-Hep-1 hepatoma cell line, CAN 037 is also effective in inducing the death of KG1a AML cells in vitro. Apoptosis is involved in the action of CAN 037 including the activation of the caspase family. Caspase-dependent cell death pathway may be necessary but not essential in CAN 037-induced apoptosis of KG1a cells. Further consideration of the structural activity relationship of CAN 037 may provide opportunities to improve its therapeutic value.

Antiproliferative ability of a combination regimen of crocodile egg extract, wild radix ginseng and natural Ganoderma lucidum on acute myelogenous leukemia.

Chinese practitioners have employed the use of traditional Chinese medicine as an anti-cancer agent since the ancient period. Different combinations have been formulated for various purposes. Some have been claimed for post-chemotherapy use but their direct actions on cancer cells may not be significantly reported. In the present study, we have tested the possible anti-leukemia potential of a combination regimen including crocodile egg extract, wild radix ginseng and natural Ganoderma lucidum (CGG extract) on acute myelogenous leukemia (AML) in vitro. A water soluble CGG extract was prepared and its antiproliferative activity was tested on the KG1a AML cell line and two freshly prepared bone marrow aspirate samples isolated from patients with de novo AML during presentation by a MTS/PMS assay. Furthermore, the possible activity of the CGG extract on the regeneration potential of KG1a cells was also investigated using a semi-solid methyl-cellulose colony formation assay. Lastly, the acute toxicity of CGG extract was further examined by a single high-dose oral feeding to rats. We found that the CGG extract could possess significant antiproliferative activity on AML cells. A strong colony formation inhibition was further demonstrated on KG1a cells. After feeding the rats with an excessive dose of CGG extract, we observed no development of acute toxicity. We concluded that the CGG extract has growth inhibitory potential on KG1a cells and AML bone marrow samples in vitro. An in vivo toxicity test revealed that no acute toxicity was observed after feeding the rats a high dosage of the CGG extract. Further animal model tests are necessary to investigate the possible chronic toxicity of the CGG extract.

In vitro anti-cancer activity of a novel microbial fermentation product on human carcinomas.

The possible anti-proliferation and cell death induction potential of a novel microbial fermentation extract named as oncogen XP-180 (or simply as XP-180) was tested on three human solid tumour carcinoma cell lines (non-small cell lung cancer A549, breast cancer MDA-MB231, liver adenocarcinoma SK-Hep1) and on the acute myelogenous leukaemia KG1a cell line. Anti-proliferative activity of XP-180 was observed on all of these cancer cell lines with comparable efficiency and in a dose-dependent manner. Morphological investigation further suggested that common features of apoptosis, including cell shrinkage and rounding, are present in XP-180 treated cells. Loss of adhesion properties of these solid tumour cell lines was observed upon XP-180 incubation. Anchorage-dependent clonogenicity assay on solid tumour cell lines and semi-solid methylcellulose colony formation assay on leukaemia cell line further revealed that XP-180 strongly inhibited the regeneration potential of these cancer cells. Using KG1a as an experimental model system, XP-180 was shown to stimulate the activity of caspase 3, 8 and 9 without significant change in caspase 6 activity. Furthermore, XP-180 readily induced collapse of mitochondrial membrane potential after 2 h of incubation. However, the use of the generic caspase specific inhibitor Z-VAD-FMK does not significantly reverse XP-180 mediated cell death. The results obtained suggest that XP-180-mediated cancer cell death could involve mitochondria and both caspase-dependent and -independent pathways. Therefore, XP-180 is an efficient anti-cancer regimen in vitro.

Transforming capacity of two novel genes JS-1 and JS-2 located in chromosome 5p and their overexpression in human esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) has a high mortality rate and geographic differences in incidence. Previous studies of comparative genomic hybridization (CGH) showed that chromosomal 5p is frequently amplified in cell lines and primary ESCC of Hong Kong Chinese origin. In this report, attempt was made to study two novel genes, named as JS-1 and JS-2, which are located in chromosome 5p15.2 and are 5' upstream to delta catenin for their roles in molecular pathogenesis of ESCC. Eleven cell lines, 27 primary ESCC cases and multiple human tissue cDNA panels (MTC) of digestive system were studied for the expression level of JS-1 and JS-2 by RT-PCR. The full-length cDNA sequences of JS-1 and JS-2 were determined from a non-tumor esophageal epithelial cell line by 3' and 5' rapid amplification of cDNA ends (RACE). The transforming capacity of JS-1 and JS-2 was also investigated by transfecting NIH 3T3 cells with the expression vector pcDNA3.1(-) cloned with the full coding sequences and it was followed by the study of foci formation of the transfected cells under confluence growth and the anchorage-independent growth in soft agar. Forty-five percent (5/11) and 18% (2/11) of the ESCC cell lines showed overexpression of JS-1 and JS-2 respectively, while 55% (15/27) and 14% (3/22) primary ESCC cases showed overexpression of JS-1 and JS-2 respectively. JS-1 overexpression was most common in patients with stage II ESCC (6/27; 22%) whereas JS-2 was only overexpressed in a dysplastic lesion (1/22; 4%) and stage III tumors (2/22; 9%). The expression levels of JS-1 and JS-2 are both low in normal esophageal tissues. Overexpression of JS-1 in NIH 3T3 cells caused foci formation in confluence growth and colony formation in soft agar but not for JS-2. A high grade sarcoma was formed in the athymic nude mice when NIH 3T3 cells overexpressing JS-1 were injected subcutaneously. Our results thus indicate that the frequent overexpression of JS-1 in ESCC and its transforming capacity in normal cells may play a critical role in the molecular pathogenesis of ESCC. The present study also forms the ground work for further identification of novel mechanisms of molecular carcinogenesis in ESCC and other cancers.

Inhibition of proteasome activity in Gleditsia sinensis fruit extract-mediated apoptosis on human carcinoma cells.

The anomalous fruit extract of Gleditsia sinensis (GSE) was shown to possess anticancer potential on various solid tumour and leukaemia cell lines in vitro. We have recently demonstrated that the mitochondrial-dependent apoptotic pathway including mitochondrial membrane potential depolarization, changes in the level of reactive oxygen species and activation of caspase 3 were recruited in GSE-induced apoptosis. Whether receptor-dependent APO-1/Fas apoptotic pathway is also involved remains uncertain. Using two solid tumour cell lines, the HepG2 hepatoblastoma carcinoma cells and MDA-MB231 breast cancer cells, we demonstrated that the Fas ligand and Fas receptor protein levels did not have significant variation after GSE incubation. Caspase 8 activity increased only weakly when compared with that of caspase 3. The chrymotrypsin-like activity of proteasome was partially inhibited up to 30-40% when compared with the untreated control. Taken together, we believe that GSE- mediated apoptosis on HepG2 and MDA-MB231 carcinoma cells is mainly dictated by the mitochondrial-dependent pathway while inhibition of proteasome activity may also be involved in GSE-induced apoptosis.

Gleditsia sinensis fruit extract-induced apoptosis involves changes of reactive oxygen species level, mitochondrial membrane depolarization and caspase 3 activation.

Recently, we have shown that the anomalous fruit extract of Gleditsia sinensis (GSE) processes apoptotic activity on numerous solid tumour and leukaemia cell lines as well as primary cultured leukaemia cells obtained from bone marrow aspirate of patients. GSE treated cancer cells exhibited apoptotic features as readily illustrated by morphological investigation, DNA fragmentation analysis and TUNEL labelling methods. Elevation of intracellular superoxide dismutase activity was observed. However, the detailed mechanism still remains undefined. Here we further demonstrated that cell cycle arrest, increment of hydrogen peroxide production, changes of intracellular acid-base equilibrium and mitochondrial membrane potential depolarization (DeltaPsi(m)) were induced from cancer cells after GSE incubation. Caspase 3 protease activity was significantly enhanced upon GSE treatment. Taken together, a defined signaling pathway for the mechanistic action of GSE on cancer cells was worked out.

Growth inhibitory potential of effective microorganism fermentation extract (EM-X) on cancer cells.

The effective microorganism (EM-X) fermentation extract is derived from rice bran and seaweed extract. It has been shown to possess anti-oxidation activity both in vitro and in vivo. To our knowledge, the possible in vitro anti-cancer potential of EM-X has not been demonstrated. Here we showed that the double concentrate of EM-X (EM-X2) at concentrations of 20-30% by volume, had growth inhibitory activity on MDA-MB231 breast cancer cell line and K-562 chronic myelogenous leukaemia cell lines by [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2-H-tetrazolium, inner salt] (MTS) assay. No characteristic features of apoptosis could be observed morphologically. Colony formation assay illustrated that both MDA-MB231 breast cancer and K-562 CML cells lost part of their regeneration potential after treatment with EM-X2 at 30% concentration by volume for 24 h. At these concentrations, only slight growth inhibitory effect was observed in 293 human kidney fibroblast cells and in three non-malignant bone marrows. Intracellular nitro blue tetrazolium (NBT) reduction assay showed that both MDA-MB231 breast cancer and K-562 CML cells had about 30% reduction of intracellular NBT after incubation with 30% of EM-X2. Increased activity of superoxide dismutase (SOD) could be detected from both MDA-MB231 and K-562 cell lines after incubating with 30% of EM-X2. Taken together, our data suggested that EM-X could inhibit growth and reduce the regeneration potential of cancer cells, possibly through its antioxidation activity.

Superoxide anion is involved in the early apoptosis mediated by Gleditsia sinensis fruit extract.

Changes in the intracellular level of reactive oxygen species (ROS) including superoxide anion, hydroxyl radical, hydrogen peroxide and finally cellular acid-base equilibrium are reported to play an important role in the early step of apoptosis. All of which would precede the loss of mitochondrial membrane potential and releasing of those apoptotic inducing factors such as cytochrome c as well as caspases activation. Any potential chemotherapeutic agent that could drive such changes in ROS would be particularly attractive. Recently we have reported the potential use of Gleditsia sinensis extract (GSE) in cancer therapy including solid tumour and leukaemia cell lines as well as primary cultured leukaemia cells in vitro. We demonstrated that apoptotic activity is involved. Here we further showed that the mechanism of GSE induced apoptosis, including an early decreasing of intracellular superoxide anion as measured by nitroblue tetrazolium (NBT) reduction assay. This phenomenon readily occurred before any shrinkage of cancer cells including MDA-MB231 breast cancer, CNE-2 nasopharyngeal carcinoma, K-562 chronic myelogenous leukaemia and KG1-a, acute myelogenous leukaemia. Cell viability was determined by morphological investigation and the [3-(4,5-dimethyl-thiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] (MTS) assay. Furthermore, the superoxide dismutase activity from those cellular extracts after GSE treatment seemed to be increased. Taken together, we speculate that the GSE-induced apoptosis, via ROS pathway, involves an early decrease of intracellular superoxide anion.

Gleditsia sinensis fruit extract induced growth inhibition involves basic fibroblast growth factor and nitric oxide.

Recently we have shown the antiproliferative activity of Gleditsia sinensis fruit extract (GSE) on various solid tumour and leukaemia cell lines as well as primary cultured bone marrow cells isolated from patients with acute and chronic myelogenous leukaemia. We further studied whether the growth inhibitory effect of GSE involves basic fibroblast growth factor (bFGF) in cancer cell lines including breast cancer MDA-MB231, nasopharyngeal cancer CNE-2 and prostate cancer LNCaP. We also investigated whether GSE could alter the production of nitric oxide (NO) pattern from these cancer cell lines. Growth inhibition assay was quantitated by sulforhodamine B protein staining method. Enzyme linked immunosorbent assay (ELISA) was used to quantitate the total bFGF protein. The amount of NO secreted into culture medium in terms of nitrite ion concentration was measured by the Greiss method. ELISA showed that GSE could stimulate total bFGF protein level which was dose- dependent. NO production was also stimulated from these cancer cell lines after treating with GSE. Both of the increment in total bFGF and NO levels were correlated with the degree of growth inhibition. Changes involving cell shrinkage and detachment of cancer cells could readily be observed. Taken together, our results here suggest that growth inhibition induced by GSE in these solid tumour cell lines may involve both bFGF and NO regulations.

Anti-angiogenic potential of Gleditsia sinensis fruit extract.

Blood supply plays a crucial role in solid tumour development and leukaemogenesis. It has been suggested that blocking of angiogenesis could be possible in cancer therapy. We have demonstrated the antiproliferative activity of Gleditsia sinensis fruit extract (GSE) on various human solid tumour cancer cell lines as well as leukaemia cell lines and primary cultured leukaemia cells obtained from leukaemia patients. However, the antiangiogenic potential of GSE has not been demonstrated. Here we demonstrated that GSE could reduce vascular endothelial growth factor (VEGF) mRNA expression in dose- and time course-dependently in MDA-MB231 breast cancer and HepG2 hepatoblastoma cell lines as measured by reverse transcriptase polymerase chain reaction. Enzyme-linked immunosorbent assay further showed that GSE could reduce the VEGF secretion from various cancer cell lines including MDA-MB231, HepG2, HL-60 (acute promyelocytic leukaemia) and eleven primary cultured leukaemia cells obtained from acute myelogenous leukaemia patients. In vivo chick chorioallantoic membrane assay illustrated that GSE could reduce the angiogenic activity of basic fibroblast growth factor. Taken together, the information suggested that GSE could be potentially used as an angiogenic inhibitor in both solid tumour and leukaemia therapy.

Homocysteine stimulates inducible nitric oxide synthase expression in macrophages: antagonizing effect of ginkgolides and bilobalide.

Hyperhomocysteinemia is an independent risk factor for atherosclerotic diseases. Inducible nitric oxide synthase (iNOS) is mainly expressed in macrophages upon stimulation. Overproduction of nitric oxide (NO) by iNOS can exacerbate the development of atherosclerosis. Our previous studies demonstrated that the extract of ginkgo biloba leaves (EGb) inhibited the iNOS-mediated NO production in monocyte-derived macrophage. We also reported that homocysteine could stimulate monocyte chemoattractant protein-1 (MCP-1) expression in vascular cells causing enhanced monocyte chemotaxis. The objective of the present study was to investigate the effect of homocysteine on iNOS-mediated NO production in macrophages and the antagonizing effect of EGb. Human monocytic cell (THP-1)-derived macrophages were incubated with homocysteine for various time periods. Homocysteine at concentrations of 0.05-0.1 mM significantly stimulated NO production and iNOS activity in macrophages via increased expression of iNOS mRNA and protein. The increased iNOS expression was associated with activation of nuclear factor-kappa B (NF-kappaB) arising from reduced expression of inhibitor protein (IkappaB alpha) mRNA as well as increased phosphorylation of IkappaB alpha protein in homocysteine-treated cells. EGb and its terpenoids (ginkgolide A, ginkgolide B and bilobalide) could antagonize the homocysteine effect on iNOS expression in macrophages via their antioxidant effect resulting in attenuation of NF-kappaB activation. Taken together, our results have demonstrated that homocysteine, at pathophysiological concentrations, stimulates iNOS-mediated NO production in macrophages. EGb and its terpenoids can antagonize such stimulatory effect via antioxidation and attenuation of NF-kappaB activation.

Cholestin inhibits cholesterol synthesis and secretion in hepatic cells (HepG2).

Hyperlipidemia is a well-known risk factor for atherosclerosis and statins are widely used to treat patients with elevated levels of lipids in their plasma. Notwithstanding the proven benefits of statin drugs on both primary and secondary prevention of heart disease, the high cost of statin treatment, in addition to possible side effects such as liver function abnormalities, may limit their widespread use. We conducted a study on a natural product as an alternative to statin treatment. Cholestin, a dietary supplement, is prepared from rice fermented with red yeast (Monascus purpureus), which has been shown to significantly decrease total cholesterol levels in hyperlipidemic subjects. Our objective was to determine the cellular effect of Cholestin on cholesterol synthesis in human hepatic cells (HepG2) and the mechanism by which it caused a change in lipid metabolism. Cholestin had a direct inhibitory effect on HMG-CoA reductase activity (78-69% of control). Cholesterol levels in HepG2 cells treated with Cholestin (25-100 microg/mL) were significantly reduced in a dose-dependent manner (81-45% of control, respectively). This reduction was associated with decreased synthesis and secretion of both unesterified cholesterol (54-31 and 33-14% of control, respectively) and cholesteryl ester (18-6 and 37-19% of control, respectively). These results indicate that one of the anti-hyperlipidemic actions of Cholestin is a consequence of an inhibitory effect on cholesterol biosynthesis in hepatic cells and provide the first documentation of a biomolecular action of red yeast rice.