In vivo microdialysis and in vitro HPLC analysis of the impact of paeoniflorin on the monoamine levels and their metabolites in the rodent brain.

BACKGROUND: Paeoniflorin (PF) possesses several effects such as analgesic, the anti-spasmodic effect on smooth muscle. It protects the cardiovascular system and reveals the neuroprotective effect on cerebral ischemia. Monoamine system has been identified to have complex regulatory effects in pain signaling. There are no reports regarding the impact of PF on monoamine levels in the rodent brain by microdialysis. In this study, the effects of PF on monoamines and their metabolites in the rodent brain using in vivo microdialysis and in vitro high performance liquid chromatography (HPLC) analysis. METHODS: Male S.D. rats were anesthetized, fixed onto the stereotaxic instrument to identify the positions of corpus striatum and cerebral cortex. Drilled a hole in the skull of anesthetic rats and proceeded microdialysis, and gave PF (100 µg, i.c.v.). Collected the dialysate and the concentration of monoamines and their metabolites in dialysate and analyzed with HPLC-ECD. Male ICR mice were administered with PF (96 µg, i.c.v.) and with Ringer solution as a control. After 20 mins of administration, the mice were cut off the brain immediately and separated into eight regions according to the method of Glowinski. Added extraction solution to each region, homogenized and extracted for further procedure. The extract was centrifuged, sucked the transparent layer and centrifuged once more. The transparent layer was filtered with a 0.22 µm nylon filter and analyzed with HPLC-ECD (electrochemical detection). RESULTS: PF increased the content of DOPAC and NE in the cortex, and increased the content of NE and decreased the content of 5-HT in the medulla of the homogenized mice brain tissue. By microdialysis, PF increased the content of DOPAC and 5-HIAA in anesthetic rat cortex and expanded the content of DOPAC, HVA, and 5-HIAA in anesthetic rat striatum. CONCLUSIONS: It reveals that PF could activate the release of monoamines and increase their metabolites in the rodent brain.

Involvement of matrix metalloproteinase-3 in CCL5/CCR5 pathway of chondrosarcomas metastasis.

CCL5 (previously called RANTES) was originally recognized as a product of activated T cells, and plays a crucial role in the migration and metastasis of human cancer cells. It has been reported that the effect of CCL5 is mediated via CCR receptors. We found that human chondrosarcoma tissues had significant expression of the CCL5 and CCR5, which was higher than that in normal cartilage. We also found CCL5 increased the migration and matrix metalloproteinases-3 (MMP)-3 expression in human chondrosarcoma cells (JJ012 cells). In addition, MMP-3 small interfering RNA and inhibitor inhibited the CCL5-induced cell migration. Activations of phosphatidylinositol 3-kinase (PI3K), Akt and NF-kappaB pathways after CCL5 treatment was demonstrated, and CCL5-induced expression of MMP-3 and migration activity was inhibited by the specific inhibitor of PI3K, Akt and NF-kappaB cascades. Taken together, these results indicate that CCL5 and CCR5 interaction enhanced migration of chondrosarcoma cells through the increase of MMP-3 production.

Danthron inhibits the migration and invasion of human brain glioblastoma multiforme cells through the inhibition of mRNA expression of focal adhesion kinase, Rho kinases-1 and metalloproteinase-9.

In this study, we investigated the effect of danthron on the cell migration and invasion of human brain glioblastoma multiforme GBM 8401 cells in vitro. The changes of migration and invasion of GBM 8401 cells after treatment with danthron were detected by cell migration assay and cell invasion assay. The levels of mRNA gene expression associated with cell migration and invasion were detected by real-time PCR. Results indicated that human brain glioblastoma multiforme GBM 8401 cells treated with danthron in vitro migrated and invaded less than cells treated with phosphate-buffered saline (PBS) (control). Western blotting showed that danthron inhibited the protein levels of FAK, MMP-7, MMP-9 and uPA in GBM 8401 cells. Real-time PCR assay also showed that danthron inhibited the mRNA expression of matrix metalloproteinase-9 (MMP-9), FAK and ROCK-1 of GBM 8401 cells. These results showed that danthron inhibited invasion and migration of GBM 8401 cells by downregulating mRNA expression associated with these processes, resulting in reduced metastasis. Thus, danthron may be considered a therapeutic agent that can inhibit primary tumor growth and prevent metastasis.

A. cantoniensis inhibits the proliferation of murine leukemia WEHI-3 cells in vivo and promotes immunoresponses in vivo.

Ampelopsis cantoniensis (AC) has been used as a folk medicine for reducing pain in the Taiwanese population. Our previous studies have shown that the crude extract of AC induced apoptosis in human promyelocytic leukemia HL-60 cells. In this study, the in vivo effects of AC on leukemia WEHI-3 cells and immune responses such as phagocytosis and natural killer (NK) cell activity were investigated. The weights of the livers and spleens were decreased in the AC-treated groups compared to the control groups. The AC treatment increased the percentage of CD3 and CD19 marker cells in WEHI-3-injected mice, indicating that the precursors of T and B cells were inhibited. The AC treatment promoted the activity of macrophage phagocytosis in the peripheral blood mononuclear cells (PBMC) and peritoneal cells. It was found that the NK cells from mice after treatment with AC can kill the YAC-1 target cells. Therefore, the AC treatment increased NK cell activity. In conclusion, AC can affect WEHI-3 cells in vivo and promote macrophage and NK cell activities.

CCL5/CCR5 axis promotes the motility of human oral cancer cells.

CCL5 (previously called RANTES) is in the CC-chemokine family and plays a crucial role in the migration and metastasis of human cancer cells. On the other hand, the effect of CCL5 is mediated via CCR receptor. RT-PCR and flow cytometry studies demonstrated CCR5 but not CCR1 and CCR3 mRNA in oral cancer cell lines, especially higher in those with high invasiveness (SCC4) as compared with lower levels in HSC3 cells and SCC9 cells. Stimulation of oral cancer cells with CCL5 directly increased the migration and metalloproteinase-9 (MMP-9) production. MMP-9 small interfering RNA inhibited the CCL5-induced MMP-9 expression and thereby significantly inhibited the CCL5-induced cell migration. Activations of phospholipase C (PLC), protein kinase Cdelta (PKCdelta), and NF-kappaB pathways after CCL5 treatment was demonstrated, and CCL5-induced expression of MMP-9 and migration activity was inhibited by the specific inhibitor of PLC, PKCdelta, and NF-kappaB cascades. In addition, migration-prone sublines demonstrate that cells with increasing migration ability had more expression of MMP-9, CCL5, and CCR5. Taken together, these results indicate that CCL5/CCR5 axis enhanced migration of oral cancer cells through the increase of MMP-9 production.

Cyr61 increases migration and MMP-13 expression via alphavbeta3 integrin, FAK, ERK and AP-1-dependent pathway in human chondrosarcoma cells.

Cysteine-rich 61 (Cyr61), from the CCN gene family, is a secreted and matrix-associated protein, which is involved in many cellular activities such as growth and differentiation. However, the effect of Cyr61 on migration activity in human chondrosarcoma cells is mostly unknown. Here, we found that Cyr61 increased the migration and expression of matrix metalloproteinase (MMP)-13 in human chondrosarcoma cells (JJ012 cells). RGD peptide, alphavbeta3 monoclonal antibody and mitogen-activated protein kinase (MEK) inhibitors (PD98059 and U0126) but not RAD peptide inhibited the Cyr61-induced increase of the migration and MMP-13 upregulation of chondrosarcoma cells. Cyr61 stimulation increased the phosphorylation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK). In addition, activator protein-1 (AP-1) decoy oligodeoxynucleotide also suppressed the MMP-13 messenger RNA and enzyme activity enhanced by Cyr61. Moreover, Cyr61 increased the binding of c-Fos and c-Jun to the AP-1 element on the MMP-13 promoter. Taken together, our results indicated that Cyr61 enhances the migration of chondrosarcoma cells by increasing MMP-13 expression through the alphavbeta3 integrin receptor, FAK, ERK, c-Fos/c-Jun and AP-1 signal transduction pathway.

Diallyl disulfide (DADS) induces apoptosis in human cervical cancer Ca Ski cells via reactive oxygen species and Ca2+-dependent mitochondria-dependent pathway.

The mechanisms of apoptosis induced by diallyl disulfide (DADS) were explored in human cervical cancer Ca Ski cells. Flow cytometric analysis, DNA gel electrophoresis and DAPI staining demonstrated that DADS induced apoptosis in Ca Ski cells. DADS induced apoptosis through the production of reactive oxygen species and Ca2+, and induced abrogation of mitochondrial membrane potential (Deltapsim) and cleavage of Bid protein (t-Bid). DADS increased the levels of p53, p21 and Bax, but caused a decrease in the level of Bcl-2. DADS also promoted the activities of caspase-3 leading to DNA fragmentation, thus indicating that DADS-induced apoptosis is caspase-3 dependent. In addition, DADS induced an increase in the level of cytochrome c in the cytoplasm, which was released from mitochondria. BAPTA attenuated the Deltapsim abrogation and significantly diminished the occurrence of DADS-induced apoptosis in Ca Ski cells. In conclusion, DADS-induced apoptosis occurs via production of ROS and caspase-3 and a mitochondria-dependent pathway in Ca Ski cells.

Diallyl disulfide induced signal transducer and activator of transcription 1 expression in human colon cancer colo 205 cells using differential display RT-PCR.

Diallyl disulfide is one of the components of garlic and has been demonstrated to induce apoptosis in many cancer cell lines, though it is not reported to be associated with signal transducer and activator of transcription 1 (STAT1) expression. Moreover the role of STAT1 does not directly affect apoptosis in cancer cells after exposure to chemotherapy agents, though some reports showed that STAT1 is associated with apoptosis. In this study, differential display RT-PCR was used to examine the effects of diallyl disulfide (DADS) on human colon cancer cells (colo 205). The results demonstrated that DADS induced the expression of STAT1 which was also confirmed using Western blotting. STAT1 decoy oligonucleotides were also used to block STAT1 mRNA and led to a decrease in the levels of STAT1 and to subsequence decrease in the percentage of apoptosis induced by DADS in examined colo 205 cells.

Berberine inhibits WEHI-3 leukemia cells in vivo.

Berberine, an isoquinoline alkaloid, has a wide range of pharmacological effects including anticancer activities, yet the exact effects on leukemia in vivo are unknown. Our previous studies have demonstrated that berberine induced cytotoxicity against murine leukemia WEHI-3 cells in vitro in a dose-dependent manner. In order to understand the berberine action against leukemia, the effect of berberine on WEHI-3 leukemia cells in vivo was studied. The results showed that Mac-3 and CD11b markers were reduced, indicating differentiation inhibition of the macrophages and granulocytes precursors. There was no affect on the CD14 marker but the CD19 marker that was indicating the promotion of the differentiation of the B-cells precursors. The weights of spleen samples from mice treated with berberine were found to be lower when compared to these from untreated animals.

Baicalein induces apoptosis in SCC-4 human tongue cancer cells via a Ca2+-dependent mitochondrial pathway.

BACKGROUND: The effects of baicalein on SCC-4 human tongue cancer cells were examined to better understand its effect on apoptosis and associated possible signal pathways in vitro. MATERIALS AND METHODS: Apoptosis induction, reactive oxygen species (ROS), cytoplasmic Ca2+, mitochondrial membrane potential (MMP) and caspase-3 activity were analyzed using the flow cytometric assay. Apoptosis-associated proteins, such as p53, BAX, BCL-2, cytochrome c, caspase-3 and -9, EndoG and AIF were determined by Western blotting. RESULTS: Our results showed that baicalein promoted the levels of p53, BAX, cytochrome c, capase-3 and -9 and reduced the level of BCL-2, which were associated with the induction of apoptotic cell death of SCC-4 cells. A release of cytochrome c from mitochondria into cytosol was demonstrated and an activation of caspase-3, which led to the occurrence of apoptosis in SCC-4 cells treated with baicalein as determined by Western blot. In order to understand the role of Ca2+ in the induction of apoptosis, cells were pre-treated with BAPTA (intracellular calcium chelator) and baicalein. It was shown that the MMP was restored, and the level of cytoplasmic Ca2+ suppressed, the proportion of cells undergoing apoptosis was also markedly diminished. Our data suggest that cellular Ca2+ modulates baicalein-induced cell death via a Ca2+-dependent mitochondrial death pathway in SCC-4 cells.

Capsaicin induced cell cycle arrest and apoptosis in human esophagus epidermoid carcinoma CE 81T/VGH cells through the elevation of intracellular reactive oxygen species and Ca2+ productions and caspase-3 activation.

Capsaicin (N-vanillyl-8-methyl-1-nonenamide) is found in pungent fruits, especially in red pepper. Many studies have focused on the anticarcinogenic, antimutagenic or chemopreventive activities of capsaicin. However, the effects of capsaicin on human esophagus epidermoid carcinoma cells have never been investigated. In this study, we investigated the effects of capsaicin on esophagus epidermoid carcinoma cells in vitro and further examined the molecular mechanisms of capsaicin-induced apoptosis in esophagus epidermoid carcinoma cells. Capsaicin decreased the percentage of viable cells of CE 81T/VGH cells, via induction of G0-G1 phase cell cycle arrest and apoptosis. Capsaicin induced G0-G1 phase arrest underwent the promotion of p53 and p21, which is an inhibitor of Cdk2 and cyclin E complex before leading to the inhibitions of both compounds. Capsaicin induced apoptosis in time-dependent manners. Capsaicin-induced apoptosis was in association with the elevation of intracellular reactive oxygen species and Ca2+ productions and BAPTA, an intracellular Ca2+ chelator, which significantly inhibited capsaicin-induced apoptosis. Collectively, these results suggest that the capsaicin-induced apoptosis in the CE 81T/VGH cells may result from the activation of caspase-3 and intracellular Ca2+ release pathway, and it is further suggested that capsaicin has potential as a novel therapeutic agent for the treatment of esophagus epidermoid carcinoma cells.

Curcumin-induced cell cycle arrest and apoptosis in human acute promyelocytic leukemia HL-60 cells via MMP changes and caspase-3 activation.

Curcumin (diferuloylmethane), is a natural product derived from the root of the plant Curcuma longa. For centuries, it has been used as a spice and as a herbal medicine in Chinese populations. Curcumin has been shown to inhibit cell proliferation, cell cycle arrest, cyclooxygenase (COX)-1 and -2 expression and apoptosis in several human cancer cell lines. The aim of this investigation was to clarify the mechanisms by which curcumin induced cytotoxicity and apoptosis in human leukemia HL-60 cells. The effects of curcumin on the levels of reactive oxygen species (ROS), Ca+2 production, cyclin E, cdc25c, wee1, Bcl-2, Bax, the changes of mitochondrial membrane potential (MMP), cytochrome c release and the activation of caspase-3 were also investigated in the HL-60 cells. Results of flow cytometry and DAPI staining assays indicated that curcumin induced cytotoxicity and apoptosis in the examined cells. The results from flow cytometry assay indicated that curcumin induced ROS and Ca+2 productions, decreased the levels of MMP and increased the activity of caspase-3, leading to cell apoptosis. Western blot assay also revealed that curcumin increased the levels of Bax and the release of cytochrome c, and decreased the levels of Bcl-2 in the examined cells. The inhibition of caspase-3 activation by z-VAD-fmk (broad-spectrum caspase inhibitor) completely blocked curcumin-induced apoptosis in HL-60 cells.