Antiangiogenic and antihepatocellular carcinoma activities of the Juniperus chinensis extract.

BACKGROUND: To identify a novel therapeutic agent for hepatocellular carcinoma (HCC), for which no promising therapeutic agent exists, we screened a panel of plants and found that Juniperus chinensis exhibited potential antiangiogenic and anti-HCC activities. We further investigated the antiangiogenic and anti-HCC effects of the active ingredient of J. chinensis extract, CBT-143-S-F6F7, both in vitro and in vivo. METHODS: A tube formation assay conducted using human umbilical vein endothelial cells (HUVECs) was first performed to identify the active ingredient of CBT-143-S-F6F7. A series of angiogenesis studies, including HUVEC migration, Matrigel plug, and chorioallantoic membrane (CAM) assays, were then performed to confirm the effects of CBT-143-S-F6F7 on angiogenesis. The effects of CBT-143-S-F6F7 on tumor growth were investigated using a subcutaneous and orthotopic mouse model of HCC. In vitro studies were performed to investigate the effects of CBT-143-S-F6F7 on the cell cycle and apoptosis in HCC cells. Moreover, protein arrays for angiogenesis and apoptosis were used to discover biomarkers that may be influenced by CBT-143-S-F6F7. Finally, nuclear magnetic resonance analysis was conducted to identify the compounds of CBT-143-S-F6F7. RESULTS: CBT-143-S-F6F7 showed significantly antiangiogenic activity in various assays, including HUVEC tube formation and migration, CAM, and Matrigel plug assays. In in vivo studies, gavage with CBT-143-S-F6F7 significantly repressed subcutaneous Huh7 tumor growth in severe combined immunodeficient (SCID) mice, and prolonged the survival of orthotopic Huh7 tumor-bearing SCID mice (a 40 % increase in median survival duration compared with the vehicle-treated mice). Immunohistochemical staining of subcutaneous Huh7 tumors in CBT-143-S-F6F7-treated mice showed a significantly decrease in the cell cycle regulatory protein cyclin D1, cellular proliferation marker Ki-67, and endothelial marker CD31. CBT-143-S-F6F7 caused arrest of the G2/M phase and induced Huh7 cell apoptosis, possibly contributing to the inhibition of HCC tumors. Protein array analysis revealed that several angiogenic and antiapoptotic factors were suppressed in CBT-143-S-F6F7-treated Huh7 cells. Finally, five compounds from CBT-143-S-F6F7 were identified. CONCLUSIONS: According to these results, we report for the first time the antiangiogenic and anti-HCC activities of CBT-143-S-F6F7, the active fractional extract of J. chinensis. We believe that CBT-143-S-F6F7 warrants further evaluation as a new anti-HCC drug.

RFX-1-dependent activation of SHP-1 inhibits STAT3 signaling in hepatocellular carcinoma cells.

Regulatory factor X-1 (RFX-1) is a transcription factor that has been linked to negative regulation of tumor progression; however, its biological function and signaling cascades are unknown. Here, we performed several studies to elucidate the roles of RFX-1 in the regulation of SHP-1 in hepatocellular carcinoma (HCC) cells. Overexpression of RFX-1 resulted in the activation of SHP-1 and repressed colony formation of HCC cells. In addition, by a mouse xenograft model, we demonstrated that RFX-1 overexpression also inhibited the tumor growth of HCC cells in vivo, suggesting that RFX-1 is of potential interest for small-molecule-targeted therapy. We also found that SC-2001, a bipyrrole molecule, induced apoptosis in HCC cells through activating RFX-1 expression. SC-2001 induced RFX-1 translocation from the cytosol to nucleus, bound to the SHP-1 promoter, and activated SHP-1 transcription. In a xenograft model, knockdown of RFX-1 reversed the antitumor effect of SC-2001. Notably, SC-2001 is much more potent than sorafenib, a clinically approved drug for HCC, in in vitro and in vivo assays. Our study confirmed that RFX-1 acts as a tumor suppressor in HCC and might be a new target for HCC therapy. The findings of this study also provide a new lead compound for targeted therapy via the activation of the RFX-1/SHP-1 pathway.

The H3K9 methyltransferase G9a is a marker of aggressive ovarian cancer that promotes peritoneal metastasis.

BACKGROUND: Ovarian cancer (OCa) peritoneal metastasis is the leading cause of cancer-related deaths in women with limited therapeutic options available for treating it and poor prognosis, as the underlying mechanism is not fully understood. METHOD: The clinicopathological correlation of G9a expression was assessed in tumor specimens of ovarian cancer patients. Knockdown or overexpression of G9a in ovarian cancer cell lines was analysed with regard to its effect on adhesion, migration, invasion and anoikis-resistance. In vivo biological functions of G9a were tested by i.p. xenograft ovarian cancer models. Microarray and quantitative RT-PCR were used to analyze G9a-regulated downstream target genes. RESULTS: We found that the expression of histone methyltransferase G9a was highly correlated with late stage, high grade, and serous-type OCa. Higher G9a expression predicted a shorter survival in ovarian cancer patients. Furthermore, G9a expression was higher in metastatic lesions compared with their corresponding ovarian primary tumors. Knockdown of G9a expression suppressed prometastatic cellular activities including adhesion, migration, invasion and anoikis-resistance of ovarian cancer cell lines, while G9a over-expression promoted these cellular properties. G9a depletion significantly attenuated the development of ascites and tumor nodules in a peritoneal dissemination model. Importantly, microarray and quantitative RT-PCR analysis revealed that G9a regulates a cohort of tumor suppressor genes including CDH1, DUSP5, SPRY4, and PPP1R15A in ovarian cancer. Expression of these genes was also inversely correlated with G9a expression in OCa specimens. CONCLUSION: We propose that G9a contributes to multiple steps of ovarian cancer metastasis and represents a novel target to combat this deadly disease.

Differential proteomic analysis of cancer stem cell properties in hepatocellular carcinomas by isobaric tag labeling and mass spectrometry.

Malignant tumors are relatively resistant to treatment due to their heterogeneous nature, drug resistance, and tendency for metastasis. Recent studies suggest that a subpopulation of cancer cells is responsible for the malignant outcomes. These cells are considered as cancer stem cells (CSC). Although a number of molecules have been identified in different cancer cells as markers for cancer stem cells, no promising markers are currently available for hepatocellular carcinoma cells. In this study, two clones of Hep3B cell lines were functionally characterized as control or CSC-like cells, based on properties including spheroid formation, drug resistance, and tumor initiation. Furthermore, their protein expression profiles were investigated by isobaric tags for relative and absolute quantitation (iTRAQ), and a total of 1,127 proteins were identified and quantified from the combined fractions; 50 proteins exhibited at least 2-fold differences between these two clones. These 50 proteins were analyzed by GeneGo and were found to be associated with liver neoplasms, hepatocellular carcinoma (HCC), and liver diseases. They were also components of metabolic pathways, immune responses, and cytoskeleton remodeling. Among these proteins, the expressions of S100P, S100A14, and vimentin were verified in several HCC cell lines, and their expressions were correlated with tumorigenicity in HCC cell lines. The functional significance of vimentin and S100A14 were also investigated and verified.

The role of COX-2/PGE2 in gossypol-induced apoptosis of colorectal carcinoma cells.

Our previous study showed that gossypol (GOS) exhibits potent cytotoxic effects via apoptosis induction against human colorectal carcinoma cells; however, the role of cyclooxygenase (COX)-2/prostaglandin (PG)E(2) on GOS-induced apoptosis is still unknown. In the present study, 12-O-tetradecanoylphorbol-13-acetate (TPA) addition significantly inhibited GOS-induced apoptosis in human colorectal carcinoma HT-29 cells in accordance with inducing COX-2 protein/PGE(2) production. TPA inhibition of GOS-induced apoptosis was blocked by adding protein kinase (PK)C inhibitors including staurosporine (ST), GF109203X (GF), and H7, characterized by the occurrence of cleaved caspase 3 proteins and a decrease in COX-2 protein/PGE(2) production in HT-29 cells. The addition of COX activity inhibitors, including NS398 (NS), aspirin (AS), diclofenac (DI), and indomethacin (IN), suppressed TPA protection of GOS-induced apoptosis with decreased PGE(2) production in HT-29 cells. Application of PGE(2), but not it analogs PGD(2), PGJ(2), or PGF(2α), protected HT-29 cells from GOS-induced DNA ladders, and the E-prostanoid (EP(1)) receptor agonist, 17PT-PGE(2), mimicked the protection induced by PGE(2), whereas the selective EP(2) receptor agonist, butaprostol (BUT), the EP(3) receptor agonist, sulprostol (SUL), and the EP(4) receptor agonist, PGE(1) alcohol (PGE(1)), showed no significant effects on GOS-induced apoptosis in HT-29 cells. PGE(2) 's protection against GOS-induced apoptosis was reversed by adding the selective EP(1) receptor antagonist, SC-19220. Furthermore, GOS had an effective apoptotic effect on COLO205 colorectal carcinoma cells which expressed undetectable level of endogenous COX-2 protein than HT-29 cells, and the decreased COX-2 protein level via COX-2 siRNA or addition of COX-2 activity inhibitor NS significantly elevated GOS-induced cell death in HT-29 cells. COLO205-T cells were established through sustained TPA incubation of COLO205 cells, and COLO205-T cells showed a lower sensitivity to GOS-induced cell death with increased COX-2 (not Bcl-2 and Mcl-1) protein than parental COLO-205 cells. A decrease in COX-2 protein expression in COLO205-T cells by COX-2 siRNA transfection or enhanced GOS-induced cell death according to MTT assay and DNA integrity assay. The notion of COX-2/PGE(2) activation against GOS-induced apoptosis in colon carcinoma cells was demonstrated, and the combination of GOS and COX-2 inhibitors to treat colon carcinoma possesses clinical potential worthy of further investigation.

Chlorella sorokiniana-Induced Activation and Maturation of Human Monocyte-Derived Dendritic Cells through NF-κB and PI3K/MAPK Pathways.

Chlorella sorokiniana (CS) is a unicellular green alga. The extracts of Chlorella have been used as treatments for relieving hypertension and modulating immune response. The detailed mechanisms are not clear yet. In this study, we sought to study the molecular mechanisms for the polysaccharide fraction of CS-induced immune response. We pulsed dendritic cells (DCs) with CS and found that CS could maturate DCs. CS-maturated DC could activate naïve T cells and stimulate T-cell proliferation and IFN-γ secretion. Furthermore, CS activated PI3K and MAPKs signaling pathways in DCs by interacting with TLR4 receptor. These CS-activated signaling pathways could further activate NF-κB and induce IL-12 production in DCs. This study provides molecular mechanisms for CS-induced DCs activation and immune response.

Pharmacophore modeling and virtual screening to identify potential RET kinase inhibitors.

Chemical features based 3D pharmacophore model for REarranged during Transfection (RET) tyrosine kinase were developed by using a training set of 26 structurally diverse known RET inhibitors. The best pharmacophore hypothesis, which identified inhibitors with an associated correlation coefficient of 0.90 between their experimental and estimated anti-RET values, contained one hydrogen-bond acceptor, one hydrogen-bond donor, one hydrophobic, and one ring aromatic features. The model was further validated by a testing set, Fischer's randomization test, and goodness of hit (GH) test. We applied this pharmacophore model to screen NCI database for potential RET inhibitors. The hits were docked to RET with GOLD and CDOCKER after filtering by Lipinski's rules. Ultimately, 24 molecules were selected as potential RET inhibitors for further investigation.

Reciprocal activation of macrophages and breast carcinoma cells by nitric oxide and colony-stimulating factor-1.

Induction of inducible nitric oxide synthase (iNOS) gene expression, nitric oxide (NO) production and migration of RAW264.7 macrophages by coculture with breast cancer MDA-MB-231 cells or the addition of conditioned medium derived from MDA-MB-231 cells (MDA-CM) was identified. Increased iNOS/NO induction and migration of macrophages by MDA-CM were significantly blocked by adding the c-Jun-N-terminal protein kinase (JNK) inhibitor, SP600125, the nuclear factor-kappa B (NF-κB) inhibitor, BAY117082 and pyrrolidine dithiocarbamic acid and a dominant-negative JNK. The addition of an NO donor, Diethylenetriamine-NONOate, significantly activated expressions of MMP-9 and VEGF-A genes in breast carcinoma MDA-MD-231 cells and invasion of MDA-MB-231 cells in coculture with RAW264.7 macrophages as determined using Transwell systems, but that was inhibited by adding SP600125, BAY117082 and the nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester. Induction of heme oxygenase-1 in macrophages reduced MDA-CM-induced iNOS/NO, JNK and NF-κB activations in accordance with inhibiting VEGF-A and MMP-9 gene expressions by MDA-MB-231 cells via Transwell assays. Furthermore, VEGF, sRANKL, TNF-α, IL-1α, TGF-ß, CSF-1 and MCP-1 were applied, and CSF-1 showed the most potent stimulation of iNOS/NO production and migration of macrophages. MCF-7 cells with lower CSF-1 expression than MDA-MB-231 cells showed a poor stimulatory effect on iNOS/NO production and migration of macrophages. Neutralization of CSF-1 in MDA-CM using CSF-1 antibody inhibited MDA-CM-induced iNOS protein expression and migration of macrophages, and CSF-1-induced iNOS protein and migration was blocked by adding JNK inhibitor SP and NF-κB inhibitor BAY. The reciprocal activation of breast cancer and macrophages via NO-CSF-1 is first elucidated herein.

Novel azulene-based derivatives as potent multi-receptor tyrosine kinase inhibitors.

A series of azulene-based derivatives were synthesized as potent inhibitors for receptor tyrosine kinases such as FMS-like tyrosine kinase 3 (FLT-3). Systematic side chain modification of prototype 1a was carried out through SAR studies. Analogue 22 was identified from this series and found to be one of the most potent FLT-3 inhibitors, with good pharmaceutical properties, superior efficacy, and tolerability in a tumor xenograft model.

Synthesis and structure-activity relationship of 6-arylureido-3-pyrrol-2-ylmethylideneindolin-2-one derivatives as potent receptor tyrosine kinase inhibitors.

A series of new ureidoindolin-2-one derivatives were synthesized and evaluated as inhibitors of receptor tyrosine kinases. Investigation of structure-activity relationships at positions 5, 6, and 7 of the oxindole skeleton led to the identification of 6-ureido-substituted 3-pyrrolemethylidene-2-oxindole derivatives that potently inhibited both the vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR) families of receptor tyrosine kinases. Several derivatives showed potency against the PDGFR inhibiting both its enzymatic and cellular functions in the single-digit nanomolar range. Among them, compound 35 was a potent inhibitor against tyrosine kinases, including VEGFR and PDGFR families, as well as Aurora kinases. Inhibitor 36 (non-substituted on the pyrrole or phenyl ring) had a moderate pharmacokinetic profile and completely inhibited tumor growth initiated with the myeloid leukemia cell line, MV4-11, in a subcutaneous xenograft model in BALB/c nude mice.

Quercetin inhibition of tumor invasion via suppressing PKC delta/ERK/AP-1-dependent matrix metalloproteinase-9 activation in breast carcinoma cells.

Quercetin (QUE; 3,5,7,3',4'-tetrahydroxyflavone) has been shown to possess several beneficial biological activities including antitumor, anti-inflammation and antioxidant properties; however, the effects of QUE in preventing invasion by breast carcinoma cells are still undefined. Increases in the protein, messenger RNA and enzyme activity levels of matrix metalloproteinase (MMP)-9 were observed in 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated MCF-7 cells, and these were blocked by QUE, but not by quercitrin or rutin. A translocation of protein kinase C (PKC)delta from the cytosol to the membrane followed by activation of extracellular signal-regulated kinase (ERK) and c-Jun/activator protein-1 (AP-1) by TPA was demonstrated, and TPA-induced MMP-9 activation and migration were inhibited by the pan PKC inhibitor, GF109203X, the specific PKCdelta inhibitor, rottlerin, an ERK inhibitor (PD98059) and an AP-1 inhibitor (curcumin). Application of QUE significantly suppressed TPA-induced activation of the PKCdelta/ERK/AP-1-signaling cascade. To elucidate the importance of hydroxyl (OH) substitutions to QUE's inhibition of tumor migration, several structurally related flavones of QUE including 3',4'-diOH, 3',4'-diOCH(3), 3,5,7-triOH, 3,4',4'-triOH, 3,3',4'-triOCH(3), luteolin and fisetin were used. Results suggested that OH groups at both C3' and C4' play central roles in QUE's inhibition of TPA-induced MMP-9 activation and migration, and an additional OH at C3, C5 or C7 may increase the inhibitory potency of the 3',4'-diOH flavone against TPA-induced MMP-9 activity and migration. The antitumor invasion and migration effects of breast carcinoma cells induced by QUE with the structure-activity relationship analysis were identified.

Myricetin inhibits matrix metalloproteinase 2 protein expression and enzyme activity in colorectal carcinoma cells.

Colorectal carcinoma is a leading cause of human mortality due to its high metastatic ability. Because the activation of matrix metalloproteinases (MMP) is a key factor in the metastatic process, agents with the ability to inhibit MMP activity have potential in the treatment of colorectal carcinoma. In the present study, among 36 flavonoids examined, myricetin was found to be the most potent inhibitor of MMP-2 enzyme activity in COLO 205 cells (IC50 = 7.82 micromol/L). Myricetin inhibition of MMP-2 enzyme activity was also found in the human colorectal carcinoma cell lines COLO 320HSR, COLO 320DM, HT 29, and COLO 205-X (IC50 = 11.18, 11.56, 13.25, and 23.51 micromol/L, respectively). In contrast, no inhibitory effect of MMP-2 protein expression or enzyme activity was observed in myricitrin (myricetin-3-rhamnoside)-treated cells. In 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated COLO 205 cells, an increase in MMP-2 protein expression and enzyme activity, as well as of protein kinase C (PKC) alpha protein translocation, extracellular signal-regulated kinase (ERK) 1/2 protein phosphorylation, and c-Jun protein expression was observed. ERK inhibitor (PD98059) and PKC inhibitors (GF-109203X and H-7), but not p38 inhibitor (SB203580) or c-jun-NH2-kinase inhibitor (SP600125), significantly inhibited TPA-induced MMP-2 protein expression, with reduced ERK phosphorylation and c-Jun protein expression. Addition of myricetin but not myricitrin suppressed TPA-induced MMP-2 protein expression in COLO 205 cells by blocking the TPA-induced events, including translocation of PKCalpha from cytosol to membrane, phosphorylation of ERK1/2 protein, and induction of c-Jun protein expression. Addition of PD98059 or GF-109203X significantly enhanced the inhibitory effect of myricetin on MMP-2 enzyme activity induced by TPA. Furthermore, myricetin, but not myricitrin, suppressed TPA-induced invasion of COLO 205 cells in an in vitro invasion assay using Engelbreth-Holm-Swarm sarcoma tumor extract Matrigel-coated Transwells. Results of the present study indicate that myricetin significantly blocked both endogenous and TPA-induced MMP-2 enzyme activity by inhibiting its protein expression and enzyme activity. The blockade involved suppression of PKC translocation, ERK phosphorylation, and c-Jun protein expression.

Mitochondrial-dependent, reactive oxygen species-independent apoptosis by myricetin: roles of protein kinase C, cytochrome c, and caspase cascade.

Abrogation of mitochondrial permeability and induction of reactive oxygen species (ROS) production have been observed in chemical-induced apoptosis; however, the relationship between the mitochondria and intracellular ROS levels in apoptosis is still unclear. In the present study, myricetin (ME) but not its respective glycoside, myricitrin (MI; myricetin-3-O-rhamnose) reduced the viability of human leukemia HL-60 cells via apoptosis, characterized by the occurrence of DNA ladders and hypodiploid cells. Results of Western blotting and caspase activity assays showed that activation of caspases 3 and 9 but not caspases 1, 6 or 8 with cleavage of PARP and D4-GDI proteins is involved in ME-induced apoptosis. A reduction in mitochondrial functions characterized by a decrease in the Bcl-2/Bax protein ratio and translocation of cytochrome c (cyt c) from the mitochondria to the cytosol in accordance with a decrease in mitochondrial membrane potential were observed in ME-treated HL-60 cells. No significant induction of intracellular ROS levels by ME was observed by the DCHF-DA assay, DPPH assay or plasmid digestion assay, and antioxidants including N-acetyl-cysteine (NAC), catalase (CAT), superoxide dismutase (SOD), and tiron (TIR) showed no protective effects on ME-induced apoptosis. A PKC activator, 12-O-tetradecaoylphorbol-13-acetate (TPA) significantly attenuated ME-induced apoptosis via preventing cytochrome c release to the cytosol and maintaining the mitochondrial membrane potential by inhibiting the decrease in the Bcl-2/Bax protein ratio; these effects were blocked by protein kinase C (PKC) inhibitors including GF-109203X, H7, and staurosporin. Removing mitochondria by ethidium bromide (EtBr) treatment reduced the apoptotic effect of ME. Results of SAR studies showed that the presence of OH at C3', C4', and C5' is important for the apoptosis-inducing activities of ME, and that ME induces apoptosis in another leukemia cell line, Jurkat cells, but not in primary human polymorphonuclear (PMN) cells or in murine peritoneal macrophages (PMs). The results of the present study suggest that apoptosis induced by ME occurs through a novel mitochondrion-dependent, ROS-independent pathway; TPA protects cells from ME-induced apoptosis via PKC activation which prevents the occurrence of mitochondrial destruction during apoptosis.

Hydroxylation at C4' or C6 is essential for apoptosis-inducing activity of flavanone through activation of the caspase-3 cascade and production of reactive oxygen species.

Previous studies demonstrated that hydroxyl groups play important roles in the antioxidative activities of flavonoids; however, the importance of structurally related hydroxylation in their apoptosis-inducing activities is still undefined. In the present study, flavanone with hydroxylation at C4' and C6 had a significant cytotoxic effect in human leukemia HL-60 cells accompanied by the occurrence of DNA ladders, apoptotic bodies, and hypodiploid cells, characteristics of apoptosis. The replacement of a hydroxyl group (OH) by a methoxyl (OCH3) group at C4' or C6 attenuated the apoptotic effect in cells, and there was no significant cytotocity of flavanone or flavanone with OH or OCH3 in C7-treated HL-60 cells. Induction of enzyme activity of caspase-3 and -9, but not caspase-1 and -8, accompanied by release of cytocrome C from mitochondria to cytosol and the appearance of cleaved of PARP (85 kDa), D4-GDI (23 kDa), and caspase-3 (p17/p15) fragments, was identified in 4'-OH- or 6-OH- flavanone-treated HL-60 cells. Caspase-3 and -9 inhibitors Ac-DEVD-FMK and Ac-LEHD-FMK, but not caspase-1 and -8 inhibitors Ac-YVAD-FMK and Ac-LETD-FMK, attenuated 4'-OH- or 6-OH-flavanone-induced cell death. And, inhibition of capsase-9 activity by Ac-LEHD-FMK suppresses caspase-3 protein procession induced by 4'-OH- and 6-OH-flavanone, indicative of caspase-9 activation locating upstream of caspase-3. A decrease in the antiapoptotic protein Mcl-1 and increases in the pro-apoptotic proteins Bax and Bad were found in 4'-OH- or 6-OH-flavanone-treated HL-60 cells. Induction of endogenous ROS production was detected in 4'-OH- or 6-OH-flavanone-treated HL-60 cells by the DCHF-DA assay. Antioxidants such as N-acetylcysteine (NAC), catalase (CAT), superoxide dismutase (SOD), and allopurinol (ALL), but not pyrrolidine dithiocarbamate (PDTC) or diphenylene iodonium (DPI), significantly inhibited 4'-OH- or 6-OH-flavanone-induced ROS production, with blocking of the apoptosis induced by 4'-OH- or 6-OH-flavanone. The apoptosis-inducing activity of 4'-OH- or 6-OH-flavanone was also observed in another leukemia cell line (Jurkat), but was not found in mature monocytic cells (THP-1) and normal human polymorphonuclear neutrophils (PMNs). This suggests that hydroxylation at C4' or C6 is important to the apoptosis-inducing activities of flavanone through ROS production, and that activation of the caspase-3 cascade, downstream of caspase-9 activation, is involved.

Emodin induces apoptosis in human promyeloleukemic HL-60 cells accompanied by activation of caspase 3 cascade but independent of reactive oxygen species production.

Emodin (1,3,8-trihydroxy-6-methylanthraquinone) is an active constituent of Rheum palmatum, and showed inhibitory activity on lipopolysaccharide-induced NO production in our previous study. However, the apoptosis-inducing activity of emodin has remained undefined. Among three structurally related anthraquinones, including emodin, physcion, and chrysophanol, emodin showed the most potent cytotoxic effects on HL-60 cells, accompanied by the dose- and time-dependent appearance of characteristics of apoptosis including an increase in DNA ladder intensity, morphological changes, appearance of apoptotic bodies, and an increase in hypodiploid cells. Emodin at apoptosis-inducing concentrations causes rapid and transient induction of caspase 3/CPP32 activity, but not caspase 1 activity, according to cleavage of caspase 3 substrates poly(ADP-ribose) polymerase and D4-GDI proteins, the appearance of cleaved caspase 3 fragments being detected in emodin- but not physcion- or chrysophanol-treated HL-60 cells. A decrease in the anti-apoptotic protein, Mcl-1, was detected in emodin-treated HL-60 cells, whereas other Bcl-2 family proteins including Bax, Bcl-2, Bcl-XL, and Bad remained unchanged. The caspase 3 inhibitor, Ac-DEVD-CHO, but not the caspase 1 inhibitor, Ac-YVAD-CHO, attenuated emodin-induced DNA ladders, associated with the blockage of PARP and D4-GDI cleavage. Free radical scavenging agents including NAC, catalase, SOD, ALL, DPI, L-NAME and PDTC showed no preventive effect on emodin-induced apoptotic responses, whereas NAC, CAT and PDTC prevented HL-60 cells from ROS (H(2)O(2))-induced apoptosis through inhibition of caspase 3 cascades. Induction of catalase, but not SOD, activity was detected in emodin-treated HL-60 cells by in gel activity assays, and H(2)O(2)-induced intracellular peroxide level was significantly reduced by prior treatment of emodin in HL-60 cells. Our experiments provide evidence that emodin is an effective apoptosis inducer in HL-60 cells through activation of the caspase 3 cascade, but that it is independent of ROS production.

Flavone inhibition of tumor growth via apoptosis in vitro and in vivo.

Colorectal carcinoma is a human malignant tumor, which is very resistant to currently available methods of treatment. Therefore, developing an effective agent with anti-colorectal carcinoma activity is important. In the present study, 8 structurally related flavones including flavone, 3-OH flavone, 5-OH flavone, 7-OH flavone, quercetin, kaempferol, quercetin, and morin were used to study their effects on colorectal carcinoma cells (HT29, COLO205, COLO320-HSR). Results of MTT assay indicated that flavone shows the most potent cytoxic effect among them on these three cell types. The cytotoxicity induced by flavone is mediated by inducing the occurrence of apoptosis characterized by the appearance of DNA ladders, apoptotic bodies and hypodiploid cells. Activation of caspase 3 protein procession and enzyme activity with inducing cleavage of caspase 3 substrates PARP was identified in flavone-treated cells, and an inhibitory peptide Ac-DEVD-FMK for caspase 3, but not Ac-YVAD-FMK for caspase 1, attenuates the cytotoxic effect of flavone in COLO205 and HT29 cells. Elevation of p21 but no p53 protein was observed in flavone-treated cells. Increasing intracellular peroxide level was detected in flavone-treated cells by DCHF-DA assay, and antioxidants such as tiron, catalase, SOD, PDTC, but not DPI, suppress flavone-induced cytotoxic effect. In vivo anti-tumor study indicates that flavone exhibits ability to inhibit tumor formation elicited by s.c. injection of COLO205 cells in nude mice, and apoptotic cells and an increase in p21, but not p53, protein were observed in tumor tissues derived from flavone-treated group. Additionally, flavone induced apoptosis in primary colon carcinoma cells COLO205-X with appearance of DNA ladders, caspase 3 protein procession, PARP protein cleavage, and an increase in p21 (not p53) protein. These data provide evidence to suggest that flavone is an effective agent to induce apoptosis in colorectal carcinoma cells in vitro and in vivo; activation of caspase 3, ROS production, and increasing p21 protein are involved.

In vitro and in vivo inhibitory activities of rutin, wogonin, and quercetin on lipopolysaccharide-induced nitric oxide and prostaglandin E(2) production.

Flavonoids are widely distributed in plants, but their biological functions are still unclear. In the present study, in vitro and in vivo experiments were performed to demonstrate the inhibitory activities of rutin, wogonin, and quercetin on lipopolysaccharide-induced nitric oxide (NO) and prostaglandin E(2) production in RAW 264.7 macrophages, primary peritoneal macrophages, and Balb/c mice, respectively. In vitro results showed that wogonin and quercetin dose-dependently suppressed lipopolysaccharide-induced NO production in RAW 264.7 macrophages and primary peritoneal macrophages without a notable cytotoxic effect on either cell types associated with a decrease in inducible nitric oxide synthase (iNOS) protein expression in both cells. Rutin, at 80 microM only, had a slight but obvious inhibitory effect on lipopolysaccharide-induced NO production in primary peritoneal macrophages. Both wogonin and quercetin attenuated lipopolysaccharide-induced prostaglandin E(2) production in vitro. Intravenous injection of lipopolysaccharide (10 mg/kg, i.v.) resulted in a time-dependent induction of NO production in serum, and pretreatment with the L-arginine analog N-nitro-L-arginine methyl ester (L-NAME) blocked this induction. Intravenous pretreatment of Balb/c mice with rutin, wogonin or quercetin for 1 h followed by lipopolysaccharide treatment significantly inhibited lipopolysaccharide-induced NO production, but no inhibition of prostaglandin E(2) production was found. A decrease in iNOS protein, but not cyclooxygenase-2 protein, was detected in liver and lung specimens of lipopolysaccharide-treated Balb/c mice in the presence of rutin, wogonin or quercetin. In conclusion, data obtained both in vitro and in vivo suggest that wogonin and quercetin exert inhibitory activity on lipopolysaccharide-induced NO production through suppression of iNOS expression.

Activation of JNK contributes to evodiamine-induced apoptosis and G2/M arrest in human colorectal carcinoma cells: a structure-activity study of evodiamine.

Evodiamine (EVO; 8,13,13b,14-tetrahydro-14-methylindolo[2'3'-3,4]pyrido[2,1-b]quinazolin-5-[7H]-one derived from the traditional herbal medicine Evodia rutaecarpa was reported to possess anticancer activity; however, the anticancer mechanism is still unclear. In this study, we investigated the anticancer effects of EVO on human colon COLO205 and HT-29 cells and their potential mechanisms. MTT and lactate dehydrogenase (LDH) release assays showed that the viability of COLOL205 and HT-29 cells was inhibited by EVO at various concentrations in accordance with increases in the percentage of apoptotic cells and cleavage of caspase-3 and poly(ADP ribose) polymerase (PARP) proteins. Disruption of the mitochondrial membrane potential by EVO was accompanied by increased Bax, caspase-9 protein cleavage, and cytochrome (Cyt) c protein translocation in COLO205 and HT-29 cells. Application of the antioxidant N-acetyl-L-cysteine (NAC) inhibited H2O2-induced reactive oxygen species (ROS) production and apoptosis, but did not affect EVO-induced apoptosis of COLO205 or HT-29 cells. Significant increases in the G2/M ratio and cyclinB1/cdc25c protein expression by EVO were respectively identified in colon carcinoma cells via a flow cytometric analysis and Western blotting. Induction of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) protein phosphorylation was detected in EVO-treated cells, and the JNK inhibitor, SP600125, but not the ERK inhibitor, U0126, inhibited EVO-induced phosphorylated JNK protein expression, apoptosis, and G2/M arrest of colon carcinoma cells. Data of the structure-activity analysis showed that EVO-related chemicals containing an alkyl group at position 14 were able to induce apoptosis, G2/M arrest associated with increased DNA ladder formation, cleavage of caspase-3 and PARP, and elevated cycB1 and cdc25c protein expressions in COLO205 and HT-29 cells. Evidence supporting JNK activation leading to EVO-induced apoptosis and G2/M arrest in colon carcinoma cells is provided, and alkylation at position 14 of EVO is a critical substitution for treatment of colonic cancer.

RFX1-dependent activation of SHP-1 induces autophagy by a novel obatoclax derivative in hepatocellular carcinoma cells.

Obatoclax is a small molecule which targets the Bcl-2 family, and is to treat leukemia, lymphoma and lung carcinoma. Previously, an obatoclax analogue, SC-2001, was found to disrupt the protein-protein interactions of the Bcl-2 family and also repress Bcl-XL and Mcl-1 expression via STAT3 inactivation. Here, we report a novel mechanism of autophagy induction by SC-2001 in liver cancer cells. The findings indicate that SC-2001 induced the autophagy marker LC3-II in four hepatocellular carcinoma (HCC) cells. Autophagosomes induced by SC-2001-treated cells were confirmed by electron microscopy. SC-2001 activated SHP-1, dephosphorylated STAT3 and Mcl-1, and subsequently released free beclin 1. Overexpression of STAT3 and Mcl-1 in PLC5 cells attenuated the induction of SC-2001 on autophagy. Abolishment of SHP-1 by a specific inhibitor aboragated the autophagic effects induced by SC-2001. In addition, it was further revealed that RFX-1, a transcription factor of SHP-1, is a critical regulator in SC-2001-mediated autophagy. Downregulation of RFX-1 by si-RNA protected cells from SC-2001-induced autophagy. Importantly, Huh7 tumor-bearing nude mice treated with SC-2001 showed downregulation of Mcl-1 and p-STAT3 protein expression and upregulation of SHP-1, LC3II, and RFX-1 protein expression. In summary, our data suggest that SC-2001 induces autophagic cell death in a RFX1/SHP-1/STAT3/Mcl-1 signaling cascade.

High-purity separation of cancer cells by optically induced dielectrophoresis.

Detecting and concentrating cancer cells in peripheral blood is of great importance for cancer diagnosis and prognosis. Optically induced dielectrophoresis (ODEP) can achieve high resolution and low optical intensities, and the electrode pattern can be dynamically changed by varied light patterns. By changing the projected light pattern, it is demonstrated to separate high-purity gastric cancer cell lines. Traditionally, the purity of cancer cell isolation by negative selection is 0.9% to 10%; by positive selection it is 50% to 62%. An ODEP technology is proposed to enhance the purity of cancer cell isolation to about 77%.