Neocarzinostatin as a probe for DNA protection activity--molecular interaction with caffeine.

Neocarzinostatin (NCS), a potent mutagen and carcinogen, consists of an enediyne prodrug and a protein carrier. It has a unique double role in that it intercalates into DNA and imposes radical-mediated damage after thiol activation. Here we employed NCS as a probe to examine the DNA-protection capability of caffeine, one of common dietary phytochemicals with potential cancer-chemopreventive activity. NCS at the nanomolar concentration range could induce significant single- and double-strand lesions in DNA, but up to 75 ± 5% of such lesions were found to be efficiently inhibited by caffeine. The percentage of inhibition was caffeine-concentration dependent, but was not sensitive to the DNA-lesion types. The well-characterized activation reactions of NCS allowed us to explore the effect of caffeine on the enediyne-generated radicals. Postactivation analyses by chromatographic and mass spectroscopic methods identified a caffeine-quenched enediyne-radical adduct, but the yield was too small to fully account for the large inhibition effect on DNA lesions. The affinity between NCS chromophore and DNA was characterized by a fluorescence-based kinetic method. The drug-DNA intercalation was hampered by caffeine, and the caffeine-induced increases in DNA-drug dissociation constant was caffeine-concentration dependent, suggesting importance of binding affinity in the protection mechanism. Caffeine has been shown to be both an effective free radical scavenger and an intercalation inhibitor. Our results demonstrated that caffeine ingeniously protected DNA against the enediyne-induced damages mainly by inhibiting DNA intercalation beforehand. The direct scavenging of the DNA-bound NCS free radicals by caffeine played only a minor role.

Danthron, an anthraquinone derivative, induces DNA damage and caspase cascades-mediated apoptosis in SNU-1 human gastric cancer cells through mitochondrial permeability transition pores and Bax-triggered pathways.

Anthraquinones have been shown to induce apoptosis in different types of tumor cells, but the mechanisms of danthron-induced cytotoxicity and apoptosis in human gastric cancer cells have not been adequately explored. This study investigated the roles of caspase cascades, ROS, DNA damage, mitochondrial disruption, and Bax and Bcl-2 proteins in danthron-induced apoptosis of SNU-1 human gastric cancer cells, a commonly used cell culture system for in vitro studies. Cells were incubated with different concentrations of danthron in a time- and/or dose-dependent manner. Cell morphological changes (shrinkage and rounding) were examined by a phase-contrast microscope, whereas cell viability and apoptotic populations were determined by flow cytometric analysis using propidium iodide (PI) and annexin V-FITC staining. The fluorescent DAPI nucleic acid stain and Comet assay were applied to detect danthron-induced chromatin condensation (an apoptotic characteristic) and DNA damage. Increasing the levels of caspase-3, -8, and -9 activities was involved in danthron-induced apoptosis, and they could be attenuated by inhibitors of specific caspases, indicating that danthron triggered the caspase-dependent apoptotic pathway. Further studies with flow cytometric analyses indicated that cellular levels of ROS, cytosolic Ca(2+), and mitochondrial permeability transition (MPT) pore opening were increased, but the level of mitochondrial membrane potential (ΔΨ(m)) was decreased. Also, the ratio of Bax/Bcl-2 levels and other proapoptotic proteins associated with modulating the ΔΨ(m) were up-regulated. Apoptotic signaling was also stimulated after exposure to danthron and determined by Western blotting and real-time PCR analyses. In summary, it is suggested that danthron-induced apoptotic cell death was involved in mitochondrial depolarization, which led to release of cytochrome c, apoptosis-inducing factor (AIF), and endonuclease G (Endo G) and caused the activation of caspase-9 and -3 in SNU-1 human gastric cancer cells.

Bee venom induced cell cycle arrest and apoptosis in human cervical epidermoid carcinoma Ca Ski cells.

Although it has been previously reported that bee venom (BV) can induce apoptosis in many cancer cell lines, there is no information on the effect of BV on human cervical cancer cells and its molecular mechanisms of action are not fully elucidated. In this study, the possible mechanisms of apoptosis by which BV acts on human cervical cancer Ca Ski cells were investigated. BV induced morphological changes and decreased the percentage of viable Ca Ski cells in a dose- and time-dependent manner. Flow cytometric analysis demonstrated that BV induced the production of reactive oxygen species, increased the level of cytoplasmic Ca2+, reduced mitochondrial membrane potential which led to cytochrome c release, and promoted the activation of caspase-3 which then led to apoptosis. BV also induced an increase in the levels of Fas, p53, p21 and Bax, but a decrease in the level of Bcl-2. The activities of both caspase-8 and caspase-9 were enhanced by BV, promoting caspase-3 activation, leading to DNA fragmentation. Based on the DNA fragmentation and DAPI staining, BV-induced apoptosis was mitochondrial-dependent and caspase-dependent. BV also promoted the expression of AIF and Endo G in the Ca Ski cells. Both AIF and Endo G proteins were released from the mitochondria, and then induced apoptosis which was not through activation of caspase. In conclusion, our data demonstrated that BV-induced apoptosis occurs via a Fas receptor pathway involving mitochondrial-dependent pathways and is closely related to the level of cytoplasmic Ca2+ in Ca Ski cells.

NK3 and NK4 of HGF enhance filamin production via STAT pathway, but not NK1 and NK2 in human breast cancer cells.

AIM: The purpose of this study was to reveal the effects of hepatocyte growth factor (HGF) variants on human breast cancer cells and the differential signaling pathways of the variants in controlling cell proliferation and invasion. METHODS: Four HGF variants (NK1, NK2, NK3, and NK4) were created by gene engineering, and the variant DNA fragments were cloned into pGEM-T for DNA sequencing and then transferred to a pTrcHis-A plasmid for expression. Recombinant proteins were purified from Escherichia coli, and a series of assays, including cell proliferation and invasion were carried out. Phosphorylated components in the HGF-c-Met and STAT (signal transducers and activators of transcription) pathways were detected by immunoprecipitation-Western blots. RESULTS: All the HGF variants inhibited the vigorous growth of the cancer cells differently and dose-dependently, but the effect of NK3 or NK4 was 7.5-fold higher than NK1 or NK2. In addition, the assays for the phosphorylation of the components in the HGF-c-Met pathway showed that NK3 and NK4 inhibited invasion via the STAT pathway, whereas NK1 and NK2 were via the HGF-c-Met pathway. CONCLUSION: The engineered HGF variants inhibited the proliferation of human breast cancer cells via different signaling pathways, NK1 and NK2 via the HGF-c-Met pathways, and NK3 and NK4 via the STAT pathway, the latter being a possible key route for the inhibition of cell invasion. All of the HGF variants have the potential to become pharmaceutical drugs in the treatment of human cancer.

Morin inhibits the growth of human leukemia HL-60 cells via cell cycle arrest and induction of apoptosis through mitochondria dependent pathway.

The effects of morin (3,5,7,20,40-pentahydroxyflavone) on human leukemia HL-60 cells in vitro were investigated and the molecular mechanisms of morin-induced G2/M arrest and apoptosis in HL-60 cells were examined. Morin induced morphological changes and decreased the percentage of viable cells via induction of G2/M-phase arrest and apoptosis. Morin-induced G2/M-phase arrest was accompanied by the promotion of p21 and Wee1, and decreased levels of Cdc25c and cyclins A and B1 complex. Morin-induced apoptosis in HL-60 cells was also confirmed by flow cytometric assay, DNA gel electrophoresis for DNA fragmentation and DAPI staining. Morin induced apoptosis in time- and dose-dependent manners. Morin-induced apoptosis was associated with elevated intracellular reactive oxygen species (ROS) increased and Ca2+ production; decreased the levels of mitochondria membrane potential (deltapsi(m)) and increased caspase-3 activation. Collectively, these results suggest that the morin-induced apoptosis in HL-60 cells may result from the activation of caspase-3 and intracellular Ca2+ release, as well as the mitochondria membrane potential pathway.

Shikonin-induced apoptosis involves caspase-3 activity in a human bladder cancer cell line (T24).

Apoptosis is a process that leads to programmed cell death and also a therapeutic target of cancer. In this study, potential apoptotic effects of shikonin on human bladder cancer cells (T24) in vitro were evaluated. Apoptosis induction, cell viability and morphological changes were investigated and caspase-3 and -9 activity was determined by flow cytometric assay and reverse transcription-polymerase chain reaction. The results showed marked differences in G0/G1 cell cycle arrest and cell death of the T24 cells between shikonin treated and untreated groups. Within 72 hours of treatment, shikonin influenced the cyclin dependent kinase (CDK) and cyclin activity by increasing p21 and decreasing cyclin E, CDK2 and CDK4 protein levels. A marked increase was found in apoptosis induction when the T24 cells were treated with shikonin compared to the untreated group, also confirmed by flow cytometry assay. Shikonin also promoted caspase-3 activity, which led to the induction of caspase-activated DNase (CAD) and cleavage poly(ADP-ribose)polymerase. Furthermore, the shikonin-induced apoptosis of the T24 cells was markedly blocked by the broad-spectrum caspase inhibitor, z-VAD-fmk. Shikonin may be a potential agent for the treatment of bladder transitional cell carcinoma since it induces apoptosis through the activation of caspase-3 activity in T24 human bladder cancer cells.

Sodium ascorbate inhibits growth via the induction of cell cycle arrest and apoptosis in human malignant melanoma A375.S2 cells.

Vitamin C has been reported to be useful in the treatment and prevention of cancer. Inconsistent effects from growth stimulation to induction of apoptosis of malignant tumor cells, however, have been reported. Melanoma is an increasingly common and potentially lethal malignancy. It was reported that melanoma cells were more susceptible to ascorbate toxicity than any other tumor cells. The mechanisms accounting for ascorbate-induced apoptosis in human melanoma cells, however, have remained unclear. This study was undertaken to investigate the effect of sodium ascorbate on cytotoxicity and apoptosis in human malignant melanoma A375.S2 cells. A375.S2 cells were incubated with a certain range of concentrations of sodium ascorbate for various time periods. In order to examine the effects of sodium ascorbate on cell proliferation, cell cycle, apoptosis and necrosis, we performed 4,6-diamidino-2-phenylindole dihydrochloride assays and flow cytometry analysis. Polymerase chain reaction was used to examine the mRNA levels of p53, p21, p27, cyclin A, cyclin E, CDK2 and CDK4, which are associated with cell cycle S-phase arrest and apoptosis. Flow cytometric analysis showed that sodium ascorbate significantly induced cell cycle arrest and apoptosis in the A375.S2 cell line in a dose-dependent manner. The increased expressions of p53 and p21, and the decreased expressions of cyclin A, cyclin E, CDK2 and CDK4, indicated the cell cycle arrest at G1/S phase after the cells had been treated with sodium ascorbate. Induction of apoptosis involved an increase in the levels of p53, p21 and cellular Ca, and a decrease in mitochondrial membrane potential and activation of caspase 3 before culminating in apoptosis in sodium ascorbate-treated A375.S2 cells.

Involvement of Bax, Bcl-2, Ca2+ and caspase-3 in capsaicin-induced apoptosis of human leukemia HL-60 cells.

The role of Ca2+ on the effects of capsaicin on human leukemia HL-60 cells in vitro and the molecular mechanisms of capsaicin-induced apoptosis were investigated. The flow cytometric analysis indicated that capsaicin decreased the percentage of viable HL-60 cells, via the induction of G0/G1-phase cell cycle arrest and apoptosis. Capsaicin-induced G0/G1-phase arrest involved the suppression of CDK2 and the cyclin E complex, which are check-point enzymes for cells moving from G0/G1- to S-phase. Capsaicin-induced apoptosis was associated with the elevation of intracellular reactive oxygen species and Ca2+ production, decreased the levels of mitochondrial membrane potential, promoted cytochrome c release and increased the activation of caspase-3. An intracellular Ca2+ chelator (BAPTA) significantly inhibited capsaicin-induced apoptosis. Capsaicin-induced apoptosis was time-and dose-dependent. These results suggest that the capsaicin-induced apoptosis of HL-60 cells may result from the activation of caspase-3 and the intracellular Ca2+ release pathway.

The effects of emodin on the expression of cytokines and functions of leukocytes from Sprague-Dawley rats.

Emodin has been reported to induce apoptosis in many human cancer cell lines, although its effects on leukocyte functions in vitro have not been demonstrated Therefore, the purpose of this study was to assess the effect of emodin on the phagocytosis of macrophages, the activity of natural killer cells and the expression of cytokines in leukocytes from Sprague-Dawley rats. Leukocytes, isolated from rats, were placed into culture plates for incubation with or without various concentrations of emodin for 1-6 hours and the functions of macrophages and natural killer cells were evaluated by flow cytometric analysis. The results indicated that emodin caused a decrease in phagocytosis of macrophages after treatment for up to 4 hours but 6-hour treatments led to an increase in the phagocytosis of macrophages. Further, emodin increased the activity of natural killer cells, both effects being dose-dependent. The levels of cytokines from the examined leukocytes were evaluated by ELISA and the results indicated that emodin increased the levels of IL-1beta and TNF-alpha, results which were confirmed by PCR assay for the mRNA expressions of the examined cytokines.

N-acetyltransferase is involved in baicalein-induced N-acetylation of 2-aminofluorene and DNA-2-aminofluorene adduct formation in human leukemia HL-60 cells.

Many arylamine and hydrazine drugs are acetylated by cytosolic N-acetyltransferase (NAT). The human promyelocytic leukemia cell line (HL-60) has been shown to acetylate arylamine and contain NAT activity. The purpose of this study was to determine whether or not baicalein could affect N-acetylation of 2-aminofluorene (AF) in HL-60 cells. Acetylated and nonacetylated AF were determined by using high performance liquid chromatography. Baicalein displayed a dose-dependent inhibition of cytosolic and intact cells' NAT activity and reduced the number of viable cells. Time-course experiments showed that N-acetylation of AF, measured from intact HL-60 cells, was inhibited by baicalein for up to 48 h. Baicalein also decreased AF-DNA adduct formation in the examined cells. The effects of baicalein on NAT were examined by flow cytometry and NAT gene expression was examined by polymerase chain reaction. The results demonstrated that baicalein inhibited NAT1 mRNA gene expression and reduced the level of NAT in HL-60 cells. These results show that baicalein can affect the NAT activity of human leukemia cells in vitro.

Shikonin inhibits the growth and N-acetylation of 2-aminofluorene in Helicobacter pylori from ulcer patients.

N-acetyltransferase (NAT) plays an important role in the N-acetylation of arylamine carcinogens and drugs. Shikonin has been shown to induce apoptosis and inhibit angiogenesis in vivo and in vitro. In this study, shikonin elicited dose-dependent bacteriostatic activity in Helicobacter pylori cultures. Arylamine N-acetyltransferase (NAT) activity (N-acetylation of 2-aminofluorene) was determined in the Helicobacter pylori isolated from peptic ulcer patients. Bacterial cytosols or intact cells with or without specific concentrations of shikonin co-treatment showed different percentages of 2-aminofluorene acetylation. Shikonin also inhibited N-acetylation of 2-aminofluorene in these examined Helicobacter pylori cytosols and intact cells in a dose-dependent manner. The apparent values of Km and Vmax were decreased after co-treatment with 4 microM shikonin in the cytosol of Helicobacter pylori.

Evidence for arylamine N-acetyltransferase activity in Klebsiella pneumoniae.

Arylamine N-acetyltransferase (NAT) enzymes have been found in laboratory animals, humans, microorganisms (fungi, bacteria and parasites), and in plants. But the characteristics of NAT from Klebsiella pneumoniae are not clear. NAT activities with p-aminobenzoic acid (PABA) and 2-aminofluorene (AF) as substrates were examined in the cytosol of K. pneumoniae. NAT activity (N-acetylation of substrates) was determined using an acetyl coenzyme A recycling assay and high performance liquid chromatography for determining the amounts of acetylated or non-acetylated PABA or AF. NAT activities from a number of K. pneumoniae isolates were found to be 0.72 +/- 0.08 nmol/min/mg protein for AF, and 0.49 +/- 0.04 nmol/min/mg protein for PABA. The kinetic parameters of apparent Michaelis constant (Km) and maximum velocity (Vmax) obtained were 2.92 +/- 0.48 mM and 7.89 +/- 0.82 nmol/min/mg protein, respectively, for AF and 2.42 +/- 0.28 mM and 9.87 +/- 0.64 nmol/min/mg protein, respectively, for PABA. The optimal pH value for the NAT activity was 7.0 for AF and PABA. The optimal temperature for NAT activity was 37 degrees C for both substrates. The NAT activity was inhibited by 50% with 0.25 mM iodoacetamide, and by more than 90% at 1.0 mM. Among a series of divalent cations and salts, Cu2+ and Zn2+ were the most potent inhibitors of NAT activity.

Bufalin induces G0/G1 phase arrest through inhibiting the levels of cyclin D, cyclin E, CDK2 and CDK4, and triggers apoptosis via mitochondrial signaling pathway in T24 human bladder cancer cells.

Most of the chemotherapy treatments for bladder cancer aim to kill the cancer cells, but a high recurrence rate after medical treatments is still occurred. Bufalin from the skin and parotid venom glands of toad has been shown to induce apoptotic cell death in many types of cancer cell lines. However, there is no report addressing that bufalin induced cell death in human bladder cancer cells. The purpose of this study was investigated the mechanisms of bufalin-induced apoptosis in a human bladder cancer cell line (T24). We demonstrated the effects of bufalin on the cell growth and apoptosis in T24 cells by using DAPI/TUNEL double staining, a PI exclusion and flow cytometric analysis. The effects of bufalin on the production of reactive oxygen species (ROS), the level of mitochondrial membrane potential (ΔΨ(m)), and DNA content including sub-G1 (apoptosis) in T24 cells were also determined by flow cytometry. Western blot analysis was used to examine the expression of G(0)/G(1) phase-regulated and apoptosis-associated protein levels in bufalin-treated T24 cells. The results indicated that bufalin significantly decreased the percentage of viability, induced the G(0)/G(1) phase arrest and triggered apoptosis in T24 cells. The down-regulation of the protein levels for cyclin D, CDK4, cyclin E, CDK2, phospho-Rb, phospho-AKT and Bcl-2 with the simultaneous up-regulation of the cytochrome c, Apaf-1, AIF, caspase-3, -7 and -9 and Bax protein expressions and caspase activities were observed in T24 cells after bufalin treatment. Based on our results, bufalin induces apoptotic cell death in T24 cells through suppressing AKT activity and anti-apoptotic Bcl-2 protein as well as inducing pro-apoptotic Bax protein. The levels of caspase-3, -7 and -9 are also mediated apoptosis in bufalin-treated T24 cells. Therefore, bufalin might be used as a therapeutic agent for the treatment of human bladder cancer in the future.

Glutathione-S-transferase enhances proliferation-migration and protects against shikonin-induced cell death in breast cancer cells.

Glutathione-S-transferase (GST) is a cytoplasmic protein responsible for detoxification, but the effect of the enzyme on cell biological events, including proliferation and migration, has never been reported. Thus, we evaluated the detoxification effect of in vitro-applied GST on cancer cell proliferation and migration. Assays for proliferation and migration of human breast cancer cells in the presence of GST were carried out. Binding of GST on the surface of the cancer cells was studied by flow cytometry. Detoxification through GST pathway was studied in the presence of shikonin. The effective dosage of GST in enhancement of cell proliferation was 10-50 nM, and the cell migration could be significantly enhanced after 6 hours in the presence of 2-50 nM GST. Therefore, overall cell proliferation and migration could be enhanced in the presence of 10nM or greater concentration of GST, and 15 µM shikonin-induced toxification of the cancer cells could be neutralized by 1.0 µM GST. Flow cytometry showed that GST directly bound to the surface of the cancer cells, and this was confirmed by fluorescence confocal microscopic observation. It is concluded that human class π-GST enhances proliferation and migration of human breast cancer cells by means of direct binding to the cell surface and maintaining cell viability by detoxification.

Cantharidin induces apoptosis in human bladder cancer TSGH 8301 cells through mitochondria-dependent signal pathways.

Cantharidin has shown potent anticancer activities on many types of human cancer cells. This study was performed to elucidate whether mitochondria and caspases are involved in the modulation of apoptosis and cell cycle arrest by cantharidin in human bladder cancer cells. The effect of cantharidin on cell cycle arrest, apoptosis, caspases, reactive oxygen species (ROS) and mitochondrial membrane potential (ΔΨ(m)) were measured by flow cytometry, and the levels of apoptosis-associated proteins and its regulatory molecules were studied by Western blotting. Cantharidin-induced apoptosis and DNA damage was determined by flow cytometric analysis, DAPI staining and Comet assay. After cantharidin treatment, the active forms of caspase-3, -8 and -9 were promoted. Cantharidin-induced apoptosis was associated with enhanced ROS and Ca(2+) generations, caused DNA damage, decreased the levels of ΔΨ(m) and promoted Endo G and AIF released from mitochondria. Cantharidin-induced G0/G1 arrest was associated with a marked decrease in the protein expressions of cyclin E and Cdc25c but promoted the levels of p21 and p-p53. Cantharidin-induced apoptosis was accompanied with up-regulation of the protein expression of Bax and PARP, but down-regulation of the protein levels of Bcl-2, resulting in dysfunction of mitochondria then led to Endo G and AIF release for causing induction of apoptosis.

Phenethyl isothiocyanate inhibits migration and invasion of human gastric cancer AGS cells through suppressing MAPK and NF-kappaB signal pathways.

Cell motility involves metastasis suppressors and other regulators that play an important role in tumor invasion and metastasis. Phenethyl isothiocyanate (PEITC), found in dietary cruciferous vegetables, has been found to exhibit antitumor properties and therefore is of special interest for the development of chemopreventive and chemotherapeutic agent for human cancers. Here, we report that in addition to its function as an anticancer agent, and PEITC can inhibit migration and invasion through the extracellular signal-regulated kinases 1/2 (ERK1/2), protein kinase C (PKC) and nuclear factor-kappaB (NF-kappaB) signaling pathways in human gastric cells. The results from wound healing and Boyden chamber assays (migration and invasion) assay indicated that PEITC exhibited an inhibitory effect on the migration and invasion of AGS cells. Results from Western blotting examination demonstrated that PEITC exerted an inhibitory effect on the ERK1/2, mitogen-activated protein kinase kinase 7 (MKK7), MAP kinase kinase kinase 3 (MEKK3), son of sevenless 1 (SOS1), PKC, Ras homolog gene family, member A (Rho A) and urokinase-type plasminogen activator (uPA), causing the inhibition of matrix metallopeptidase-2 (MMP-2) and -9 then followed by the inhibition of invasion and migration of GAS cells in vitro. PEITC also inhibited Ras, growth factor receptor-bound protein 2 (GRB2), vascular endothelial growth factor (VEGF), focal adhesion kinase (FAK), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), causing inhibition of cell proliferation of AGS cells. Results from real-time PCR showed that PEITC inhibited the gene expressions of MMP-2, -7 and -9, FAK and RhoA after PEITC treatment for 24 and 48 h of AGS cells. Taken together, these findings may provide insight into a new mechanisms and functions of PEITC in migration and invasion of human gastric cancer AGS cells. Our data imply that molecular targeting of PKC leading to the inhibition of MMP-2 and -9 might be a useful strategy for the inhibition of migration and invasion of human gastric cancer.

Mitochondrial-dependent caspase activation pathway is involved in baicalein-induced apoptosis in human hepatoma J5 cells.

Baicalein has been reported to induce growth-inhibitory activity in vitro in human cancer cells; however, the molecular mechanism of action is not completely understood. A pharmacological dose (10-100 microM) of baicalein exerted a cytotoxic effect on human hepatoma J5 cells resulting in G2/M arrest and apoptosis. In addition to cytotoxicity in J5 cells, several apoptotic events including mitochondrial cytochrome c release, activation of caspase-9 and -3 occurred. Baicalein induced AIF and Endo G release from mitochondria indicating that baicalein stimulates apoptosis through the caspase-independent pathway, while undergoing apoptosis, there was a remarkable accumulation of G2/M cells. Also, the ratio of Bax/Bcl-2 was increased leading to changes in mitochondria membrane potential (DeltaPsim) and release of cytochrome c, whereas the baicalein-induced apoptosis was partially abrogated by pretreatment with the pan-caspase inhibitor z-VAD-fmk, the accumulation of G2/M cells remained. These results demonstrate that the cytotoxicity of baicalein in J5 cells is attributable to apoptosis mainly involving G2/M-arrest in an ER-dependent manner, via a mitochondria-dependent caspase pathway and as well as contributions of AIF and Endo G pathways.

Novel quinolone CHM-1 induces apoptosis and inhibits metastasis in a human osterogenic sarcoma cell line.

Novel 2-phenyl-4-quinolone compounds have potent cytotoxic effects on different human cancer cell lines. In this study, we examined anticancer activity and mechanisms of 20-fluoro-6,7-methylenedioxy-2-phenyl-4-quinolone (CHM-1) in human osterogenic sarcoma U-2 OS cells. CHM-1-induced apoptosis was determined by flow cytometric analysis, DAPI staining, Comet assay, and caspase inhibitors. CHM-1-inhibited cell migration and invasion was assessed by a wound healing assay, gelatin zymography, and a Transwell assay. The mechanisms of CHM-1 effects on apoptosis and metastasis signaling pathways were studied using Western blotting and gene expression. CHM-1 induced G2/M arrest and apoptosis at an IC(50) (3 microM) in U-2 OS cells and caspase-3, -8, and -9 were activated. Caspase inhibitors increased cell viability after exposure to CHM-1. CHM-1-induced apoptosis was associated with enhanced ROS generation, DNA damage, decreased DeltaPsi(m) levels, and promotion of mitochondrial cytochrome c release. CHM-1 stimulated mRNA expression of caspase-3, -8, and -9, AIF, and Endo G. In addition, CHM-1 inhibited cell metastasis at a low concentration (<3 microM). CHM-1 inhibited the cell metastasis through the inhibition of MMP-2, -7, and -9. CHM-1 also decreased the levels of MAPK signaling pathways before leading to the inhibition of MMPs. In summary, CHM-1 is a potent inducer of apoptosis, which plays a role in the anticancer activity of CHM-1.

Seroprevalence and sources of Toxoplasma infection among indigenous and immigrant pregnant women in Taiwan.

Investigation on seroprevalence and risk factors of Toxoplasma gondii infections among indigenous and immigrant pregnant women in Mid-Taiwan showed that anti Taxoplasma-specific IgG antibody counts were significantly higher in indigenes (40.6%) than in immigrants (18.2%), with an odds ratio of OR=3.34 (95% CI: 1.93-4.80). The titre of Taxoplasma-specific IgG was also significantly higher in indigenes than in immigrants (P<0.001). Differences of living styles for Toxoplasma infection between the two groups were drinking untreated water (OR=2.34, 95% CI: 1.36-4.02), consumption of raw/undercooked meats (OR=10.11 95% CI: 4.92-20.78), especially raw/undercooked pork (P=0.000), or raw/undercooked viscera (OR=9.16, 95% CI: 2.97-27.94), contact with cats (OR=5.69, 95% CI: 2.83-11.47), or soil (OR=2.55 95% CI: 1.72-3.80). Differences of risk factors for Toxoplasma infection in terms of positive IgG in the two groups were consumption of raw/undercooked meats (P=0.005) especially raw/undercooked pork (P=0.004), and contact with cats (P=0.013) or soil (P=0.028). It is concluded that seroprevalence of Toxoplasma infection is higher in indigenous pregnant women and related to their living styles. To prevent congenital toxoplasmosis, health education seems required.

Inhibition of lung cancer cell growth by quercetin glucuronides via G2/M arrest and induction of apoptosis.

Lung cancer is the leading cause of cancer death in many developed countries, including Taiwan. Quercetin, a widely distributed bioflavonoid, is well known to induce growth inhibition in a variety of human cancer cells. Quercetin glucuronides are the main circulating metabolites after dietary supplements with quercetin in humans. However, there is little information available as to how quercetin glucuronides affect human cancer cells. We investigated the effects of quercetin glucuronides in a human lung cancer cell line NCI-H209. We checked the cell viability, cell cycle checkpoint proteins, pro- and antiapoptotic proteins, caspase-3 activity, and gene expression by flow cytometry and Western blot. The viability of cells decreased in a dose- and time-dependent manner. Cell cycle analysis revealed a significant increase of the proportion of cells in G2/M phase and subG0/G1 phase (corresponding to apoptotic cells). Moreover, quercetin glucuronides increased the expressions of cyclin B, Cdc25c-ser-216-p, and Wee1 proteins, indicating the G2/M arrest. We also demonstrated a concurrent decrease of the mitochondrial membrane potential, release of cytochrome c, up-regulation of Bax, down-regulation of Bcl-2, and activation of caspase-3, and subsequently, cleavage of poly(ADP-ribose) polymerase. In addition, quercetin glucuronide-induced apoptosis was totally blocked by the broad-spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone. Taken together, we demonstrated that quercetin glucuronides inhibited proliferation through G2/M arrest of the cell cycle and induced apoptosis via caspase-3 cascade in the human lung cancer cell line NCI-H209. Delineation of the biological effects of specific major quercetin metabolites on chemotherapeutic potential or chemoprevention of human cancers warrants further investigation.