Chih-Shiang Chang

Phloroglucinol derivative MCPP induces cell apoptosis in human colon cancer

This study is the first to investigate the anticancer effects of the new phloroglucinol derivative (3,6‐bis(3‐chlorophenylacetyl)phloroglucinol; MCPP) in human colon cancer cells. MCPP induced cell death and antiproliferation in three human colon cancer, HCT‐116, SW480, and Caco‐2 cells, but not in primary human dermal fibroblast cells. MCPP‐induced concentration‐dependent apoptotic cell death in colon cancer cells was measured by fluorescence‐activated cell sorter (FACS) analysis. Treatment of HCT‐116 human colon cancer cells with MCPP was found to induce a number of signature endoplasmic reticulum (ER) stress markers; and up‐regulation of CCAAT/enhancer‐binding protein homologous protein (CHOP) and glucose‐regulated protein (GRP)‐78, phosphorylation of eukaryotic initiation factor‐2α (eIF‐2α), suggesting the induction of ER stress. MCPP also increased GSK3α/β(Tyr270/216) phosphorylation and reduced GSK3α/β(Ser21/9) phosphorylation time‐dependently. Transfection of cells with GRP78 or CHOP siRNA, or treatment of GSK3 inhibitor SB216163 reduced MCPP‐mediated cell apoptosis. Treatment of MCPP also increased caspase‐7, caspase‐9, and caspase‐3 activity. The inhibition of caspase activity by z‐DEVE‐FMK or z‐VAD‐FMK significantly reduced MCPP‐induced apoptosis. Furthermore, treatment of GSK3 inhibitor SB216763 also dramatically reversed MCPP‐induced GRP and CHOP up‐regulation, and pro‐caspase‐3 and pro‐caspase‐9 degradation. Taken together, the present study provides evidences to support that GRP78 and CHOP expression, and GSK3α/β activation in mediating the MCPP‐induced human colon cancer cell apoptosis. J. Cell. Biochem. 112: 643–652, 2011.

Cyclooxygenase-2 enhances α2β1 integrin expression and cell migration via EP1 dependent signaling pathway in human chondrosarcoma cells

BackgroundCyclooxygenase (COX)-2, the inducible isoform of prostaglandin (PG) synthase, has been implicated in tumor metastasis. Interaction of COX-2 with its specific EP receptors on the surface of cancer cells has been reported to induce cancer invasion. However, the effects of COX-2 on migration activity in human chondrosarcoma cells are mostly unknown. In this study, we examined whether COX-2 and EP interaction are involved in metastasis of human chondrosarcoma.ResultsWe found that over-expression of COX-2 or exogenous PGE2 increased the migration of human chondrosarcoma cells. We also found that human chondrosarcoma tissues and chondrosarcoma cell lines had significant expression of the COX-2 which was higher than that in normal cartilage. By using pharmacological inhibitors or activators or genetic inhibition by the EP receptors, we discovered that the EP1 receptor but not other PGE receptors is involved in PGE2-mediated cell migration and α2β1 integrin expression. Furthermore, we found that human chondrosarcoma tissues expressed a higher level of EP1 receptor than normal cartilage. PGE2-mediated migration and integrin up-regulation were attenuated by phospholipase C (PLC), protein kinase C (PKC) and c-Src inhibitor. Activation of the PLCβ, PKCα, c-Src and NF-κB signaling pathway after PGE2 treatment was demonstrated, and PGE2-induced expression of integrin and migration activity were inhibited by the specific inhibitor, siRNA and mutants of PLC, PKC, c-Src and NF-κB cascades.ConclusionsOur results indicated that PGE2 enhances the migration of chondrosarcoma cells by increasing α2β1 integrin expression through the EP1/PLC/PKCα/c-Src/NF-κB signal transduction pathway.

Geraniin-mediated apoptosis by cleavage of focal adhesion kinase through up-regulation of Fas ligand expression in human melanoma cells

Geraniin, a form of tannin separated from geranium, causes cell death through induction of apoptosis; however, cell death characteristics for geraniin have not yet been elucidated. Here, we investigated the mechanism of geraniin-induced apoptosis in human melanoma cells and demonstrated that geraniin was able to induce cell apoptosis in a concentration- and time-dependent manner. We also examined the signaling pathway related to geraniin-induced apoptosis. To clarify the relationship between focal adhesion kinase (FAK) and geraniin-induced apoptosis, we treated human melanoma cells with geraniin and found that this resulted dose- and time-dependent degradation in FAK. However, FAK cleavage was significantly inhibited when cells were pretreated with a selective inhibitor of caspase-3 (Ac-Asp-Glu-Val-Asp-CHO). Here, we demonstrated for the first time that geraniin triggered cell death by caspase-3-mediated cleavage of FAK. There were two possible mechanisms for activating caspase-3, mitochondria-mediated and receptor-mediated apoptosis. To confirm the geraniin-relevant signaling pathway, using immunoblot analysis we found that geraniin-induced apoptosis was associated with the up-regulation of Fas ligand expression, the activation of caspase-8, the cleavage of Bid, and the induction of cytochrome c release from mitochondria to the cytosol. Treatment with geraniin caused induction of caspase-3 activity in a dose- and time-dependent manner followed by proteolytic cleavage of poly-(ADP-ribose) polymerase, and DNA fragmentation factor 45. The geraniin-induced apoptosis may provide a pivotal mechanism for its cancer-chemopreventive action.

The novel phloroglucinol derivative BFP induces apoptosis of glioma cancer through reactive oxygen species and endoplasmic reticulum stress pathways

Prenyl-phloroglucinol derivatives from hop plants have been shown to have anticancer activities. This study is the first to investigate the anticancer effects of the new phloroglucinol derivative (2,4-bis(4-fluorophenylacetyl)phloroglucinol; BFP). BFP induced cell death and anti-proliferation in three glioma, U251, U87 and C6 cells, but not in primary human astrocytes. BFP-induced concentration-dependently cell death in glioma cells was determined by MTT and SRB assay. Moreover, BFP-induced apoptotic cell death in glioma cells was measured by Hochest 33258 staining and fluorescence-activated cell sorter (FACS) of propidine iodine (PI) analysis. Treatment of U251 human glioma cells with BFP was also found to induce reactive oxygen species (ROS) generation, which was detected by a fluorescence dye used FACS analysis. Treatment of BFP also increased a number of signature endoplasmic reticulum (ER) stress markers glucose-regulated protein (GRP)-78, GRP-94, IRE1, phosphorylation of eukaryotic initiation factor-2α (eIF-2α) and up-regulation of CAAT/enhancer-binding protein homologous protein (CHOP). Moreover, treatment of BFP also increased the down-stream caspase activation, such as pro-caspase-7 and pro-caspase-12 degradation, suggesting the induction of ER stress. Furthermore, BFP also induced caspase-9 and caspase-3 activation as well as up-regulation of cleaved PARP expression. Treatment of antioxidants, or pre-transfection of cells with GRP78 or CHOP siRNA reduced BFP-mediated apoptotic-related protein expression. Taken together, the present study provides evidences to support that ROS generation, GRP78 and CHOP activation are mediating the BFP-induced human glioma cell apoptosis.

2-Phenyl-5-(pyrrolidin-1-yl)-1-(3,4,5-trimethoxybenzyl)-1H-benzimidazole, a benzimidazole derivative, inhibits growth of human prostate cancer cells by affecting tubulin and c-Jun N-terminal kinase

BACKGROUND AND PURPOSE The c‐Jun N‐terminal kinase (JNK) and tubulin are, frequently, targets for developing anti‐cancer drugs. A major obstacle to successful development is P‐glycoprotein (P‐gp)‐mediated resistance. Here, we have assessed a compound that inhibited growth of cancer cells, for effects on JNK and tubulin and as a substrate for P‐gp.

Tyrosol and Its Analogues Inhibit Alpha-Melanocyte-Stimulating Hormone Induced Melanogenesis

Melanin is responsible for skin color and plays a major role in defending against harmful external factors such as ultraviolet (UV) irradiation. Tyrosinase is responsible for the critical steps of melanogenesis, including the rate-limiting step of tyrosine hydroxylation. The mechanisms of action of skin hypopigmenting agents are thought to be based on the ability of a given agent to inhibit the activity of tyrosinase and, hence, down regulate melanin synthesis. Tyrosol and its glycoside, salidroside, are active components of Rhodiola rosea, and in our preliminary study we found that Rhodiola rosea extract inhibited melanogenesis. In this study, we examined the effects of tyrosol and its analogues on melanin synthesis. We found that treatment of B16F0 cells to tyrosol (1), 4-hydroxyphenylacetic acid (5), 3-hydroxyphenylacetic acid (6), 2-hydroxyphenylacetic acid (7), or salidroside (11) resulted in a reduction in melanin content and inhibition of tyrosinase activity as well as its expression. Tyrosol (1), 4-hydroxyphenylacetic acid (5) and 2-hydroxyphenylacetic acid (7) suppressed MC1R expression. Tyrosol (1), 4-hydroxyphenylacetic acid (5), 3-hydroxyphenylacetic acid (6), and 2-hydroxyphenylacetic acid (7) inhibited α-MSH induced TRP-1 expression, but salidroside (11) did not. All the compounds did not affect MITF and TRP-2 expression. Furthermore, we found that the cell viability of tyrosol (1), 4-hydroxyphenylacetic acid (5), 3-hydroxyphenylacetic acid (6), and 2-hydroxyphenylacetic acid (7) at concentrations below 4 mM and salidroside (11) at concentrations below 0.5 mM were higher than 90%. The compounds exhibited metal-coordinating interactions with copper ion in molecular docking with tyrosinase. Our results suggest that tyrosol, 4-hydroxyphenylacetic acid, 3-hydroxyphenylacetic acid, 2-hydroxyphenylacetic acid, and salidroside are potential hypopigmenting agents.

Inhibitory effects of Zuo-Jin-Wan and its alkaloidal ingredients on activator protein 1, nuclear factor-κB, and cellular transformation in HepG2 cells.

Zuo-Jin-Wan (ZJW) has been used to treat hepatocellular carcinoma in Asia. This study was to determine whether ZJW and its components blocked activator protein 1 (AP-1) and nuclear factor-κB (NF-κB) activities as well as tumor promotion in hepatoblastoma HepG2 cells. ZJW and its components, Coptis chinensis and Evodia rutaecarpa, inhibited AP-1 and NF-κB activities, and suppressed anchorage-independent growth of HepG2 cells. The major alkaloidal ingredients, berberine and evodiamine, inhibited AP-1 activities and/or NF-κB activation, and further suppressed hepatocellular transformation. In conclusion, ZJW and its constituents, berberine and evodiamine, suppressed tumor promotion primarily through AP-1 and/or NF-κB pathways in HepG2 cells.

Water solution of onion crude powder inhibits RANKL-induced osteoclastogenesis through ERK, p38 and NF-κB pathways

SummaryOnion powder has been reported to decrease the ovariectomy-induced bone resorption of rats. However, the molecular mechanism of onion powder on the bone cells has not been reported. Here, we report that water solution of onion crude powder decreases the osteoclastogenesis from co-cultures of bone marrow stromal cells and macrophage cells. Additionally, water solution of onion crude powder inhibits the RANKL-induced ERK, p38 and NF-κB activation in macrophages. In summary, our data showed that onion powder may benefit bone through an anti-resorption effect on the osteoclasts.IntroductionA nutritional approach is important for both prevention and treatment of osteoporosis. Onion has been reported to decrease the ovariectomy-induced bone resorption. However, the functional effects of onion on the cultured osteoclasts and osteoblasts remain largely unknown. Here, we found that water solution of onion crude powder markedly inhibited the receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclastogenesis through ERK, p38 and NF-κB pathways. Other studies were also designed to investigate the potential signaling pathways involved in onion-induced decrease in osteoclastogenesis.MethodsThe osteoclastogenesis was examined using the TRAP staining method. The MAPKs and NF-κB pathways were measured using Western blot analysis. A transfection protocol was used to examine NF-κB activity.ResultsWater solution of onion crude powder inhibited the RANKL plus M-CSF-induced osteoclastic differentiation from either bone marrow stromal cells or from RAW264.7 macrophage cells. Treatment of RAW264.7 macrophages with RANKL could induce the activation of ERK, p38 and NF-κB that was inhibited by water solution of onion crude powder. On the other hand, it did not affect the cell proliferation and differentiation of human cultured osteoblasts.ConclusionsOur data suggest that water solution of onion crude powder inhibits osteoclastogenesis from co-cultures of bone marrow stromal cells and macrophage cells via attenuation of RANKL-induced ERK, p38 and NF-κB activation.

BFPP, a phloroglucinol derivative, induces cell apoptosis in human chondrosarcoma cells through endoplasmic reticulum stress

Chondrosarcoma is a malignant primary bone tumor that responds poorly to both chemotherapy and radiation therapy. This study is the first to investigate the anticancer effects of the new phloroglucinol derivative (2,4-bis(2-fluorophenylacetyl)phloroglucinol; BFPP) in human chondrosarcoma cells. BFPP induced cell apoptosis in two human chondrosarcoma cell lines, JJ012 and SW1353 but not in primary chondrocytes. BFPP triggered endoplasmic reticulum (ER) stress, as indicated by changes in cytosol calcium levels, and increased glucose-regulated protein 78 (GRP78) expression, but failed to show the same effects on GRP94 expression. BFPP also increased calpain expression and activity. Transfection of cells with GRP78 or calpain siRNA reduced BFPP-mediated cell apoptosis in JJ012 cells. Importantly, animal studies have revealed a dramatic 50% reduction in tumor volume after 21 days of treatment. This study demonstrates novel anticancer activity of BFPP against human chondrosarcoma cells and in murine tumor models.

FPTB, a novel CA-4 derivative, induces cell apoptosis of human chondrosarcoma cells through mitochondrial dysfunction and endoplasmic reticulum stress pathways†

Chondrosarcoma is a malignant primary bone tumor that responds poorly to both chemotherapy and radiation therapy. The aim of this study was to elucidate the mechanism of the novel Combretastatin A‐4 derivative, 2‐(furanyl)‐5‐(pyrrolidinyl)‐1‐(3,4,5‐trimethoxybenzyl)benzoimidazole (FPTB)‐induced human chondrosarcoma cells apoptosis. FPTB induced cell apoptosis in human chondrosarcoma cell line but not primary chondrocytes. FPTB induced up‐regulation of Bax and Bak, down‐regulation of Bcl‐2 and Bcl‐XL and dysfunction of mitochondria in chondrosarcoma. FPTB also triggered endoplasmic reticulum (ER) stress, as indicated by changes in cytosol‐calcium levels. We found that FPTB increased glucose‐regulated proteins (GRP)78 but not GRP94 expression. In addition, treatment of cells with FPTB induced calpain expression and activity. Transfection of cells with GRP78 or calpain siRNA reduced FPTB‐mediated cell apoptosis. Therefore, FPTB‐induced apoptosis in chondrosarcoma cells through the mitochondria dysfunction and involves caspase‐9 and caspase‐3‐mediated mechanism. FPTB also induced cell death mediated by increasing ER stress, GPR78 activation, and Ca2+ release, which subsequently triggers calpain, caspase‐12 and caspase‐3 activity, resulting in apoptosis. J. Cell. Biochem. 112: 453–462, 2011.