Dose-response formation of N7-(3-benzo[1,3]dioxol-5-yl-2-hydroxypropyl)guanine in liver and urine correlates with micronucleated reticulocyte frequencies in mice administered safrole oxide.

Safrole oxide (SAFO), a metabolite of naturally occurring hepatocarcinogen safrole, is implicated in causing DNA adduct formation. Our previous study first detected the most abundant SAFO-induced DNA adduct, N7-(3-benzo[1,3] dioxol-5-yl-2-hydroxypropyl)guanine (N7γ-SAFO-G), in mouse urine using a well-developed isotope-dilution high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (ID-HPLC-ESI-MS/MS) method. This study further elucidated the genotoxic mode of action of SAFO in mice treated with SAFO 30, 60, 90, or 120 mg/kg for 28 days. The ID-HPLC-ESI-MS/MS method detected N7γ-SAFO-G with excellent sensitivity and specificity in mouse liver and urine of SAFO-treated mice. Our data provide the first direct evidence of SAFO-DNA adduct formation in rodent tissues. N7γ-SAFO-G levels in liver were significantly increased by SAFO 120 mg/kg compared with SAFO 30 mg/kg, suggesting rapid spontaneous or enzymatic depurination of N7γ-SAFO-G in tissue DNA. Urinary N7γ-SAFO-G exhibited a sublinear dose response. Moreover, the micronucleated peripheral reticulocyte frequencies increased dose-dependently and significantly correlated with N7γ-SAFO-G levels in liver (r = 0.8647; p < 0.0001) and urine (r = 0.846; p < 0.0001). Our study suggests that safrole-mediated genotoxicity may be caused partly by its metabolic activation to SAFO and that urinary N7γ-SAFO-G may serve as a chemically-specific cancer risk biomarker for safrole exposure.

Anticancer effects of cantharidin in A431 human skin cancer (Epidermoid carcinoma) cells in vitro and in vivo.

Cantharidin (CTD), a potential anticancer agent of Traditional Chinese Medicine has cytotxic effects in different human cancer cell lines. The cytotoxic effects of CTD on A431 human skin cancer (epidermoid carcinoma) cells in vitro and in A431 cell xenograft mouse model were examined. In vitro, A431 human skin cell were treated with CTD for 24 and 48 h. Cell phase distribution, ROS production, Ca2+ release, Caspase activity and the level of apoptosis associated proteins were measured. In vivo, A431 cell xenograft mouse model were examined. CTD-induced cell morphological changes and decreased percentage of viable A431 cells via G0/G1 phase arrest and induced apoptosis. CTD-induced G0/G1 phase arrest through the reduction of protein levels of cyclin E, CDK6, and cyclin D in A431 cells. CTD-induced cell apoptosis of A431 cells also was confirm by DNA gel electrophoresis showed CTD-induced DNA fragmentation. CTD reduced the mitochondrial membrane potential and stimulated release of cytochrome c, AIF and Endo G in A431 cells. Flow cytometry demonstrated that CTD increased activity of caspase-8, -9 and -3. However, when cells were pretreated with specific caspase inhibitors activity was reduced and cell viability increased. CTD increased protein levels of death receptors such as DR4, DR5, TRAIL and levels of the active form of caspase-8, -9 and -3 in A431 cells. AIF and Endo G proteins levels were also enhanced by CTD. In vivo studies showed that CTD significantly inhibited A431 cell xenograft tumors in mice. Taken together, these in vitro and in vivo results provide insight into the mechanisms of CTD on cell growth and tumor production. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 723-738, 2017.

Triptolide induced cell death through apoptosis and autophagy in murine leukemia WEHI-3 cells in vitro and promoting immune responses in WEHI-3 generated leukemia mice in vivo.

Triptolide, a traditional Chinese medicine, obtained from Tripterygium wilfordii Hook F, has anti-inflammatory, antiproliferative, and proapoptotic properties. We investigated the potential efficacy of triptolide on murine leukemia by measuring the triptolide-induced cytotoxicity in murine leukemia WEHI-3 cells in vitro. Results indicated that triptolide induced cell morphological changes and induced cytotoxic effects through G0/G1 phase arrest, induction of apoptosis. Flow cytometric assays showed that triptolide increased the production of reactive oxygen species, Ca2+ release and mitochondrial membrane potential (ΔΨm ), and activations of caspase-8, -9, and -3. Triptolide increased protein levels of Fas, Fas-L, Bax, cytochrome c, caspase-9, Endo G, Apaf-1, PARP, caspase-3 but reduced levels of AIF, ATF6α, ATF6ß, and GRP78 in WEHI-3 cells. Triptolide stimulated autophagy based on an increase in acidic vacuoles, monodansylcadaverine staining for LC-3 expression and increased protein levels of ATG 5, ATG 7, and ATG 12. The in vitro data suggest that the cytotoxic effects of triptolide may involve cross-talk between cross-interaction of apoptosis and autophagy. Normal BALB/c mice were i.p. injected with WEHI-3 cells to generate leukemia and were oral treatment with triptolide at 0, 0.02, and 0.2 mg/kg for 3 weeks then animals were weighted and blood, liver, spleen samples were collected. Results indicated that triptolide did not significantly affect the weights of animal body, spleen and liver of leukemia mice, however, triptolide significant increased the cell populations of T cells (CD3), B cells (CD19), monocytes (CD11b), and macrophage (Mac-3). Furthermore, triptolide increased the phagocytosis of macrophage from peripheral blood mononuclear cells (PBMC) but not effects from peritoneum. Triptolide promoted T and B cell proliferation at 0.02 and 0.2 mg/kg treatment when cells were pretreated with Con A and LPS stimulation, respectively; however, triptolide did not significant affect NK cell activities in vivo. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 550-568, 2017.

Allyl Isothiocyanate Induces Cell Toxicity by Multiple Pathways in Human Breast Cancer Cells.

Isothiocyanates (ITCs) occur in many cruciferous vegetables. These compounds, which have significant anticancer actions, can induce apoptosis in different human cancer cell lines. In the present study, we investigated if allyl isothiocyanate (AITC) would induce toxicity in human breast cancer MCF-7 (estrogen receptor positive) and MDA-MB-231 (estrogen receptor negative) cells. We found that AITC stimulated reactive oxygen species and Ca[Formula: see text] production, and decreased the mitochondrial membrane potential. Activity of caspase-8, -9 and -3 was increased by AITC in both cell lines. AITC also induced mitochondrial-mediated apoptosis, as shown by cytochrome c, AIF and Endo G release from mitochondria, activation of caspase-9 and caspase-3, and formation of DAPI-positive cells. There was a significant reduction in the levels of the anti-apoptotic protein Bcl-2 along with a marked increase in the pro-apoptotic protein Bax in both cell lines. AITC induced apoptosis in human breast cancer MCF-7 cells via AIF and Endo G signaling pathways, but in MDA-MB-231 cells apoptosis occurred via the GADD153 pathway. This study has revealed novel anti-cancer mechanisms of AITC, a compound that is ordinarily present in human diets and may have potential therapeutic effects in various cancers.

Crude extract of Euphorbia formosana induces apoptosis of DU145 human prostate cancer cells acts through the caspase-dependent and independent signaling pathway.

Prostate cancer is the most frequently diagnosed malignancy in men and the second highest contributor of male cancer mortality. The crude extract of Euphorbia formosana (CEEF) has been used for treatment of different diseases but the cytotoxic effects of CEEF on human cancer cells have not been reported. The purpose of the present experiments was to determine effects of CEEF on cell cycle distribution and induction of apoptosis in DU145 human prostate cancer cells in vitro. Contrast-phase microscope was used for examining cell morphological changes. Flow cytometric assays were used for cell viability, cell cycle, apoptosis, reactive oxygen species, and Ca2+ production and mitochondria membrane potential (ΔΨm ). Western blotting was used for examining protein expression of cell cycle and apoptosis associated proteins. Real-time PCR was used for examining mRNA levels of caspase-3, -8, and -9, AIF, and Endo G. Confocal laser microscope was used to examine the translocation of AIF, Endo G, and cytochrome in DU145 cells after CEEF exposure. CEEF-induced cell morphological changes, decreased the percentage of viable cells, and induced S phase arrest and apoptosis in DU145 cells. Furthermore, CEEF promoted RAS and Ca2+ production and reduced ΔΨm levels. Real-time QPCR confirmed that CEEF promoted the mRNA expression of caspase-3 and -9, AIF and Endo G and we found that AIF and Endo G and cytochrome c were released from mitochondria. Taken together, CEEF-induced cytotoxic effects via ROS production, induced S phase arrest and induction of apoptosis through caspase-dependent and independent and mitochondria-dependent pathways in DU245 cancer cells. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1600-1611, 2016.

Bufalin inhibits migration and invasion in human hepatocellular carcinoma SK-Hep1 cells through the inhibitions of NF-kB and matrix metalloproteinase-2/-9-signaling pathways.

Metastasis plays an important role in mortality of cancer patients. Migration and invasion are the major characteristics of tumor metastasis. The induction of matrix metalloproteinases (MMPs) such as MMP-2 and -9 are particularly important for the invasiveness of various cancer cells. Bufalin, a class of toxic steroids, was purified from the skin glands of Bufo gargarizans or Bufo melanostictus; it is known to inhibit proliferation of human cancer cells. In this study, we investigated the antiinvasive mechanisms of bufalin in the human hepatocellular cancer cell line SK-Hep1. Bufalin significantly reduced serum-induced cell invasion and migration. Furthermore, bufalin markedly inhibited MMP-2 and -9 activity, mRNA expression and protein levels in SK-Hep1 cells. Bufalin attenuated phosphoinisitide-3-kinase (PI3K) and phosphorylation of AKT which was associated with reduced levels of nuclear factor kappa B (NF-κB). Bufalin also suppressed protein levels of FAK and Rho A, VEGF, MEKK3, MKK7, and uPA and it diminished NF-κB translocation. Based on these observations, we propose that bufalin is acts as an antiinvasive agent by inhibiting MMP-2 and -9 and involving PI3K/AKT and NF-κB pathways. Bufalin is a potential therapeutic agent that may have efficacy in preventing the invasion and metastasis of malignant liver tumors.

Increased risk of urinary tract cancer in ESRD patients associated with usage of Chinese herbal products suspected of containing aristolochic acid.

INTRODUCTION: Both end-stage renal disease (ESRD) and urothelial cancer (UC) are associated with the consumption of Chinese herbal products containing aristolochic acid (AA) by the general population. The objective of this study was to determine the risk of UC associated with AA-related Chinese herbal products among ESRD patients. METHODS: We conducted a cohort study using the National Health Insurance reimbursement database to enroll all ESRD patients in Taiwan from 1998-2002. Cox regression models were constructed and hazard ratios and confidence intervals were estimated after controlling for potential confounders, including age, sex, residence in region with endemic black foot disease, urinary tract infection, and use of non-steroidal anti-inflammatory drugs and acetaminophen. RESULTS: A total of 38,995 ESRD patients were included in the final analysis, and 320 patients developed UC after ESRD. Having been prescribed Mu Tong that was adulterated with Guan Mu Tong (Aristolochia manshuriensis) before 2004, or an estimated consumption of more than 1-100 mg of aristolochic acid, were both associated with an increased risk of UC in the multivariable analyses. Analgesic consumption of more than 150 pills was also associated with an increased risk of UC, although there was little correlation between the two risk factors. CONCLUSION: Consumption of aristolochic acid-related Chinese herbal products was associated with an increased risk of developing UC in ESRD patients. Regular follow-up screening for UC in ESRD patients who have consumed Chinese herbal products is thus necessary.

Bufalin-inhibited migration and invasion in human osteosarcoma U-2 OS cells is carried out by suppression of the matrix metalloproteinase-2, ERK, and JNK signaling pathways.

Bufalin has been shown to exhibit multiple pharmacological activities, including induction of apoptosis in many types of cancer cell lines. Osteosarcoma is a type of cancer which is difficult to treat and the purpose of this study was to investigate the effects of bufalin on the migration and invasion of human osteosarcoma U-2 OS cells. The wound healing assay and Boyden chamber transwell assay were used for examining the migration of U-2 OS cells. Western blotting and gelatin zymography assays were used for theexpression and activities of metalloproteinase (MMP)-2, MMP-7 or MMP-9 levels. Western blotting analysis also was used for measuring the levels of growth factor receptor-bound protein 2 (GRB2), son of sevenless homolog 1 (SOS1), c-Jun N-terminal kinases 1/2 (JNK1/2), extracellular signal-regulated kinase 1/2 (ERK1/2), and p38 in bufalin-treated U-2 OS cells. Bufalin inhibited the cell migration and invasion of U-2 OS cells in vitro. Moreover, bufalin reduced MMP-2 and MMP-9 enzyme activities of U-2 OS cells. Bufalin also suppressed the protein level of MMP-2 and reduced the levels of mitogen-activated protein kinases (MAPKs) such as JNK1/2 and ERK1/2 signals in U-2 OS cells. Our results suggest that signaling pathways for bufalin-inhibited migration and invasion of U-2 OS cells might be mediated through blocking MAPK signaling and resulting in the inhibition of MMP-2. Bufalin could be a useful agent to develop as a novel antitumor agent by virtue of its ability to inhibit tumor cell migration and invasion.

Quercetin inhibits migration and invasion of SAS human oral cancer cells through inhibition of NF-κB and matrix metalloproteinase-2/-9 signaling pathways.

Quercetin, a principal flavanoid compound in onions, has been shown to possess a wide spectrum of pharmacological properties, including anticancer activities. Our earlier study showed that quercetin induced cytotoxic effects on SAS human oral cancer cells. In this study, we found that quercetin significantly reduced wound closure of SAS cells in culture plates after 12- and 24-h treatments. Results indicated that quercetin inhibited the expression and activity of matrix metalloproteinase (MMP)-2 and MMP-9, as measured by western blotting and gelatin zymography. The results from western blotting also showed that quercetin reduced the protein levels of MMP-2, -7, -9 and -10, vascular endothelial growth factor (VEGF), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65, inductible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), urokinase-type plasminogen activator (uPA), phosphatidylinositide-3 kinases (PI3K), nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IKBα), IKB-α/ß, phosphorylated nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor kinase, alpha/beta (p-IKKα/ß), focal adhesion kinase (FAK), son of sevenless homolog-1 (SOS1), growth factor receptor-bound protein-2 (GRB2), mitogen-activated protein kinase kinase kinase-3 (MEKK3), MEKK7, extracellular-signal-regulated kinase 1/2 (ERK1/2), p-ERK1/2, c-Jun N-terminal kinase 1/2 (JNK1/2), p38, p-p38, Jun proto-oncogene (c-JUN) and p-c-JUN but it did not affect Ras homolog gene family, member A (RhoA), Protein kinase C (PKC) and rat sarcoma viral oncogene homolog (RAS) in SAS cells. Confocal laser microscopy also showed that quercetin promoted the expressions of RhoA and Rho-associated, coiled-coil containing protein kinase-1 (ROCK1), but inhibited the expression of NF-κB p65 in SAS cells. It is concluded from these data that inhibition of migration and invasion of SAS cells by quercetin is associated with the down-regulation of PKC and RhoA by blocking MAPK and PI3K/AKT signaling pathways and NF-κB and uPA, resulting in inhibition of MMP-2 and MMP-9 signaling.

Capsaicin induces apoptosis in SCC-4 human tongue cancer cells through mitochondria-dependent and -independent pathways.

Although there have been advances in the fields of surgery, radiotherapy, and chemotherapy of tongue cancer, the cure rates are still not substantially satisfactory. Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide) is the major pungent ingredient of hot chili pepper and has been reported to have an antitumor effect on many human cancer cell types. The molecular mechanisms of the antitumor effect of capsaicin are not yet completely understood. Herein, we investigated whether capsaicin induces apoptosis in human tongue cancer cells. Capsaicin decreased the percentage of viable cells in a dose-dependent manner in human tongue cancer SCC-4 cells. In addition, capsaicin produced DNA fragmentation, decreased the DNA contents (sub-G1 phase), and induced G0/G1 phase arrest in SCC-4 cells. We demonstrated that capsaicin-induced apoptosis is associated with an increase in reactive oxygen species and Ca²âº generations and a disruption of the mitochondrial transmenbrane potential (ΔΨ(m)). Treatment with capsaicin induced a dramatic increase in caspase-3 and -9 activities, as assessed by flow cytometric methods. A possible mechanism of capsaicin-induced apoptosis is involved in the activation of caspase-3 (one of the apoptosis-executing enzyme). Confocal laser microscope examination also showed that capsaicin induced the releases of AIF, ATF-4, and GADD153 from mitochondria of SCC-4 cells.

Phenethyl isothiocyanate (PEITC) promotes G2/M phase arrest via p53 expression and induces apoptosis through caspase- and mitochondria-dependent signaling pathways in human prostate cancer DU 145 cells.

Phenethyl isothiocyanate (PEITC), one of many compounds found in cruciferous vegetables, has been reported as a potential anticancer agent. In earlier studies, PEITC was shown to inhibit cell growth and induction of apoptosis in many cancer cell lines. However, no report has shown whether PEITC can induce apoptosis in human prostate cancer cells. Herein, we aimed to determine whether PEITC has anticancer activity in DU 145 human prostate cancer cells. As a result, we found that PEITC induced a dose-dependent decrease in cell viability through induction of cell apoptosis and cell cycle arrest in the G(2)/M phase of DU 145 cells. PEITC induced morphological changes and DNA damage in DU 145 cells. The induction of G(2)/M phase arrest was mediated by the increase of p53 and WEE1 and it reduced the level of CDC25C protein. The induction of apoptosis was mediated by the activation of caspase-8-, caspase-9- and caspase-3-depedent pathways. Results also showed that PEITC caused mitochondrial dysfunction, increasing the release of cytochrome c and Endo G from mitochondria, and led cell apoptosis through a mitochondria-dependent signaling pathway. This study showed that PEITC might exhibit anticancer activity and become a potent agent for human prostate cancer cells in the future.

An experimental study on the antileukemia effects of gypenosides in vitro and in vivo.

PURPOSE: Gypenosides (Gyp), found in Gynostemma pentaphyllum Makino, have been used as folk medicine for centuries and have exhibited diverse pharmacological effects, including antileukemia effects in vitro and in vivo. In the present study, Gyp were used to examine effects on cell viability, cell cycle, and induction of apoptosis in vitro. They were administered in the diet to mice injected with WEHI-3 cells in vivo. EXPERIMENTAL DESIGN: Effects of Gyp on WEHI-3 cells were determined by flow cytometric assay and Western blotting. RESULTS: Gyp inhibited the growth of WEHI-3 cells. These effects were associated with the induction of G0/G1 arrest, morphological changes, DNA fragmentation, and increased sub-G1 phase. Gyp promoted the production of reactive oxygen species, increased Ca(2+) levels, and induced the depolarization of the mitochondrial membrane potential. The effects of Gyp were dose and time dependent. Moreover, Gyp increased levels of the proapoptotic protein Bax, reduced levels of the antiapoptotic proteins Bcl-2, and stimulated release of cytochrome c, AIF (apoptosis-inducing factor), and Endo G (endonuclease G) from mitochondria. The levels of GADD153, GRP78, ATF6-α, and ATF4-α were increased by Gyp, resulting in ER (endoplasmic reticular) stress in WEHI-3 cells. Oral consumption of Gyp increased the survival rate of mice injected with WEHI-3 cells used as a mouse model of leukemia. CONCLUSIONS: Results of these experiments provide new information on understanding mechanisms of Gyp-induced effects on cell cycle arrest and apoptosis in vitro and in an in vivo animal model.

Gallic acid induces apoptosis in A375.S2 human melanoma cells through caspase-dependent and -independent pathways.

The natural antioxidant gallic acid (GA) has demonstrated a significant inhibition of cell proliferation and induction of apoptosis in a series of cancer cell lines. However, there is no available information to show whether GA induces apoptosis in human skin cancer cells. In the present study, we report GA-induced apoptosis in A375.S2 human melanoma cells. GA affected morphological changes, decreased the percentage of viable cells and induced apoptosis in A375.S2 cells in a dose- and time-dependent manner. Observation of the molecular mechanism of apoptosis in A375.S2 cells showed that GA up-regulated the proapoptotic proteins such as Bax, and induced caspase cascade activity, but down-regulated antiapoptotic proteins such as Bcl-2. GA induced reactive oxygen species (ROS) and intracellular Ca2+ productions and decreased the level of mitochondrial membrane potential (DeltaPsim) in A375.S2 cells in a time-dependent manner. GA triggered cytosolic release of apoptotic molecules, cytochrome c, promoted activation of caspase-9 and caspase-3, and ultimately apoptotic cell death. In addition, GA also promoted cytosolic release of apoptosis-inducing factor (AIF) and endonuclease G (Endo G). Therefore, GA may also induce apoptosis through a caspase-independent pathway. Our results suggest that GA might be a potential anticancer compound; however, in depth in vivo studies are needed to elucidate the exact mechanism.

Aristolochic acid suppresses DNA repair and triggers oxidative DNA damage in human kidney proximal tubular cells.

Aristolochic acid (AA), derived from plants of the Aristolochia genus, has been proven to be associated with aristolochic acid nephropathy (AAN) and urothelial cancer in AAN patients. In this study, we used toxicogenomic analysis to clarify the molecular mechanism of AA-induced cytotoxicity in normal human kidney proximal tubular (HK-2) cells, the target cells of AA. AA induced cytotoxic effects in a dose-dependent (10, 30, 90 microM for 24 h) and time-dependent manner (30 microM for 1, 3, 6, 12 and 24 h). The cells from those experiments were then used for microarray experiments in triplicate. Among the differentially expressed genes analyzed by Limma and Ingenuity Pathway Analysis software, we found that genes in DNA repair processes were the most significantly regulated by all AA treatments. Furthermore, response to DNA damage stimulus, apoptosis, and regulation of cell cycle, were also significantly regulated by AA treatment. Among the differentially expressed genes found in the dose-response and time-course studies that were involved in these biological processes, two up-regulated (GADD45B, NAIP), and six down-regulated genes (TP53, PARP1, OGG1, ERCC1, ERCC2, and MGMT) were con-firmed by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). AA exposure also caused a down-regulation of the gene expression of anti-oxidant enzymes, such as superoxide dismutase, glutathione reductase, and glutathione peroxidase. Moreover, AA treatment led to increased frequency of DNA strand breaks, 8-hydroxydeoxyguanosine-positive nuclei, and micronuclei in a dose-dependent manner in HK-2 cells, possibly as a result of the inhibition of DNA repair. These data suggest that oxidative stress plays a role in the cytotoxicity of AA. In addition, our results provide insight into the involvement of down-regulation of DNA repair gene expression as a possible mechanism for AA-induced genotoxicity.

Emodin, aloe-emodin and rhein induced DNA damage and inhibited DNA repair gene expression in SCC-4 human tongue cancer cells.

In our primary studies, we have shown that emodin, aloe-emodin and rhein induced cytotoxic effects, including cell cycle arrest and apoptosis in SCC-4 human tongue cancer cells. However, details regarding their effects on DNA damage and repair gene expression in SCC-4 cells are not clear. We investigated whether or not emodin, aloe-emodin and rhein induced DNA damage and inhibited DNA repair gene expression in SCC-4 cells. Comet assay (single cell electrophoresis) indicated that incubation of SCC-4 cells with 0, 20, 30 and 40 microM of emodin, 0, 25, 50 and 100 microM of aloe-emodin or rhein led to a longer DNA migration smear (comet tail). This means that all examined agents induced DNA damage in SCC-4 cells and these effects are dose-dependent but emodin is stronger than that of aloe-emodin or rhein. The results from real-time PCR assay demonstrated that 30 microM of emodin or aloe-emodin used for 24 and 48 h treatment in SCC-4 cells significantly inhibited expression of genes associated with DNA damage and repair [ataxia telangiectasia mutated (ATM); ataxia-telangiectasia and Rad3-related (ATR); 14-3-3sigma (14-3-3sigma); breast cancer 1, early onset (BRCA1); and DNA-dependent serine/threonine protein kinase (DNA-PK)]; only rhein suppressed the expression of O(6)-methylguanine-DNA methyltransferase (MGMT) mRNA with 48 h treatment, but had no effect on ATM expression. On 24 h treatment, only aloe-emodin significantly affected ATM expression. These effects may be the vital factors for emodin, aloe-emodin and rhein induction of DNA damage in vitro. In conclusion, these agents induced DNA damage followed by the inhibition of DNA repair-associated gene expressions, including ATM, ATR, 14-3-3sigma, BRCA1, DNA-PK and MGMT in SCC-4 human tongue cancer cells.

Emodin, aloe-emodin and rhein inhibit migration and invasion in human tongue cancer SCC-4 cells through the inhibition of gene expression of matrix metalloproteinase-9.

Emodin, aloe-emodin and rhein are major compounds in rhubarb (Rheum palmatum L.), used in Chinese herbal medicine, and found to have antitumor properties including cell cycle arrest and apoptosis in many human cancer cells. Our previous studies also showed that emodin, aloe-emodin and rhein induced apoptosis in human tongue cancer SCC-4 cells. However, the detail regarding emodin, aloe-emodin and rhein affecting migration and invasion in SCC-4 cells are not clear. In the present study, we investigated whether or not emodin, aloe-emodin and rhein inhibited migration and invasion of SCC-4 cells. Herein, we demonstrate that emodin, aloe-emodin and rhein inhibit the protein levels and activities of matrix metalloproteinase-2 (MMP-2) but did not affect gene expression of MMP-2, however, they inhibited the gene expression of MMP-9 and all also inhibited the migration and invasion of human tongue cancer SCC-4 cells. MMP-9 (gelatinase-B) plays an important role and is the most associated with tumor migration, invasion and metastasis in various human cancers. Results from zymography and Western blotting showed that emodin, aloe-emodin and rhein treatment decrease the levels of MMP-2, urokinase plasminogen activator (u-PA) in a concentration-dependent manner. The order of inhibition of associated protein levels and gene expression of migration and invasion in SCC-4 cells are emodin >aloe-emodin >rhein. Our results provide new insight into the mechanisms by which emodin, aloe-emodin and rhein inhibit tongue cancers. In conclusion, these findings suggest that molecular targeting of MMP-9 mRNA expression by emodin, aloe-emodin and rhein might be a useful strategy for chemo-prevention and/or chemo-therapeutics of tongue cancers.

Microarray analysis reveals the inhibition of nuclear factor-kappa B signaling by aristolochic acid in normal human kidney (HK-2) cells.

AIM: To study the molecular mechanism underlying the effect of aristolochic acid (AA), a major active component of plants from the Aristolochiaceae family using microarray analysis. METHODS: Human kidney (HK-2) cells were treated with AA (0, 10, 30, and 90 micromol/L) for 24 h, and the cell viability was measured by a 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay. Complementary DNA microarrays were used to investigate the gene expression pattern of HK-2 cells exposed to AA in triplicate. A quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) assay was used to verify the microarray data for selected nuclear factor kappa B (NF-kappaB)-regulated genes. Furthermore, the subcellular localization of NF-kappaB p65 was visualized by immunofluorescence confocal microscopy in HK-2 cells. The NF-kappaB activity was examined by a luciferase reporter assay in HK-2/NF-kappaB transgenic cells. RESULTS: AA exhibited a dose-dependent cytotoxic effect in HK-2 cells and induced alterations in the gene expression profiles related to the DNA damage response, DNA repair, macromolecule metabolic process, carbohydrate metabolic process, DNA metabolic process, apoptosis, cell cycle, and transcription. In addition, 9 biological pathways associated with immunomodulatory functions were down-regulated in AA-treated HK-2 cells. A network analysis revealed that NF-kappaB played a central role in the network topology. Among NF-kappaB-regulated genes, 8 differentially expressed genes were verified by qRT-PCR. The inhibition of NF-kappaB activity by AA was further confirmed by immunofluorescence confocal microscopy and by NF-kappaB luciferase reporter assay. CONCLUSION: Our data revealed that AA could suppress NF-kappaB activity in normal human cells, perhaps partially accounting for the reported anti-inflammatory effects of some plants from the genus Aristolochia.

Population-based case-control study of Chinese herbal products containing aristolochic acid and urinary tract cancer risk.

BACKGROUND: Consumption of Chinese herbs that contain aristolochic acid (eg, Mu Tong) has been associated with an increased risk of urinary tract cancer. METHODS: We conducted a population-based case-control study in Taiwan to examine the association between prescribed Chinese herbal products that contain aristolochic acid and urinary tract cancer. All patients newly diagnosed with urinary tract cancer (case subjects) from January 1, 2001, to December 31, 2002, and a random sample of the entire insured population from January 1, 1997, to December 31, 2002 (control subjects), were selected from the National Health Insurance reimbursement database. Subjects who were ever prescribed more than 500 pills of nonsteroidal anti-inflammatory drugs and/or acetaminophen were excluded, leaving 4594 case patients and 174,701 control subjects in the final analysis. Adjusted odds ratios (ORs) and 95% confidence intervals (CIs) were estimated by using multivariable logistic regression models for the association between prescribed Chinese herbs containing aristolochic acid and the occurrence of urinary tract cancer. Models were adjusted for age, sex, residence in a township where black foot disease was endemic (an indicator of chronic arsenic exposure from drinking water [a risk factor for urinary tract cancer]), and history of chronic urinary tract infection. Statistical tests were two-sided. RESULTS: Having been prescribed more than 60 g of Mu Tong and an estimated consumption of more than 150 mg of aristolochic acid were independently associated with an increased risk for urinary tract cancer in multivariable analyses (Mu Tong: at 61-100 g, OR = 1.6, 95% CI = 1.3 to 2.1, and at >200 g, OR = 2.1, 95% CI = 1.3 to 3.4; aristolochic acid: at 151-250 mg, OR = 1.4, 95% CI = 1.1 to 1.8, and at >500 mg, OR = 2.0, 95% CI = 1.4 to 2.9). A statistically significant linear dose-response relationship was observed between the prescribed dose of Mu Tong or the estimated cumulative dose of aristolochic acid and the risk of urinary tract cancer (P < .001 for both). CONCLUSIONS: Consumption of aristolochic acid-containing Chinese herbal products is associated with an increased risk of cancer of the urinary tract in a dose-dependent manner that is independent of arsenic exposure.

Increased risks of chronic kidney disease associated with prescribed Chinese herbal products suspected to contain aristolochic acid.

AIM: Nephropathy associated with aristolochic acid (AA) has been documented by human and animal studies. Ancient Chinese herbology claimed to reduce toxicity in their mixtures. It was the objective of this study to determine the risk of chronic kidney disease (CKD) associated with AA-related Chinese herbal products (CHP) or mixtures of herbs in a national cohort. METHODS: A retrospective follow-up study was conducted, using a systematic random sample (200 000 people) in the National Health Insurance reimbursement database during 1997-2002. The incidence rates of CKD and end-stage renal disease (ESRD) were calculated for the whole sample and those that had used CHP suspected to contain AA. Cox regression models were constructed to control potential confounders, including age, sex, hypertension, diabetes mellitus, and use of non-steroidal anti-inflammatory drugs and acetaminophen. RESULTS: A total of 199 843 persons were included in the final analysis, 102 464 (51.3%) men and 97 379 (48.7%) women, with an average incidence rate of 1964/10(6) person-years for CKD and 279/10(6) person-years for ESRD. After controlling other risk factors, the hazard ratios for development of CKD seemed to increase for patients that had consumed more than 30 g Mu-Tong, and more than 60 g Fangchi. CONCLUSION: Prescription of more than 30 g Mu-Tong or more than 60 g Fangchi CHP was associated with an increased risk of developing CKD. In addition to prohibiting the use of Guan-Mu-Tong and Guang-Fangchi, patients who have used these CHP should continue to be followed up.