Combination treatment strategies with a focus on rosiglitazone and adriamycin for insulin resistant liver cancer.

Insulin resistance promotes the occurrence of liver cancer and decreases its chemosensitivity. Rosiglitazone (ROSI), a thiazolidinedione insulin sensitiser, could be used for diabetes with insulin resistance and has been reported to show anticancer effects on human malignant cells. In this paper, we investigated the combination of ROSI and chemotherapeutics on the growth and metastasis of insulin-resistant hepatoma. In vitro assay, ROSI significantly enhanced the inhibitory effects of adriamycin (ADR) on the proliferation, autophagy and migration of insulin-resistant hepatoma HepG2/IR cells via downregulation of EGFR/ERK and AKT/mTOR signalling pathway. In addition, ROSI promoted the apoptosis of HepG2/IR cells induced by ADR. In vivo assay, high fat and glucose diet and streptozotocin (STZ) induced insulin resistance in mice by increasing the body weight, fasting blood glucose (FBG) level, oral glucose tolerance, fasting insulin level and insulin resistance index. Both the growth of mouse liver cancer hepatoma H22 cells and serum FBG level in insulin resistant mice were significantly inhibited by combination of ROSI and ADR. Thus, ROSI and ADR in combination showed a stronger anti-tumour effect in insulin resistant hepatoma cells accompanying with glucose reduction and might represent an effective therapeutic strategy for liver cancer accompanied with insulin resistant diabetes.

Pantoprazole pretreatment elevates sensitivity to vincristine in drug-resistant oral epidermoid carcinoma in vitro and in vivo.

Resistance to chemotherapeutic agents is a major cause of treatment failure in patients with oral cancer. Proton pump inhibitors (PPIs), essentially H+-K+-ATPase inhibitors which are currently used in the treatment of acid related diseases, have demonstrated promising antitumor and chemo-sensitizing efficacy. The main purpose of the present study was to investigate whether pantoprazole (PPZ, one of PPIs) could increase the sensitivity of chemoresistant oral epidermoid carcinoma cells (KB/V) to vincristine (VCR) and elucidate the underlying action mechanism. Results showed that combination treatment of PPZ and VCR synergistically inhibited the proliferation of KB/V cells in vitro and in vivo. Furthermore, administration of PPZ and VCR not only induce apoptosis and G2/M phase arrest in KB/V cells but also suppress the migration and invasion of KB/V cells. The mechanism underlying synergistic anti-tumor effect of PPZ and VCR was related to the inhibition of the function and expression of P-glycoprotein (P-gp) and the down-regulation of EGFR/MAPK and PI3K/Akt/mTOR signaling pathways in KB/V cells. Additionally, we observed that PPZ treatment induced an increase in lysosomal pH and inhibited the activity of lysosomal enzyme acid phosphatase in KB/V cells, which could functionally reduce the sequestration of VCR in lysosomes and sensitized KB/V cells to VCR. In conclusion, our study demonstrated that PPZ could be included in new combined therapy of human oral cancer (especially on VCR-resistant therapy) together with VCR.

A randomized controlled trial of neuromuscular electrical stimulation for chronic urinary retention following traumatic brain injury.

BACKGROUND: This study aimed to evaluate the effectiveness of neuromuscular electrical stimulation (NMES) therapy for chronic urinary retention (CUR) following traumatic brain injury (TBI). METHODS: This 2-arm randomized controlled trial (RCT) enrolled 86 eligible patients with CUR following TBI. All included patients were randomly allocated to a treatment group (n = 43) or a sham group (n = 43). The administration of NMES or sham NMES, as intervention, was performed for an 8-week period treatment, and 4-week period follow-up. In addition, all subjects were required to undergo indwelling urinary catheter throughout the study period. The primary outcome was assessed by the post-voiding residual urine volume (PV-VRU). The secondary outcomes were evaluated by the voided volume, maximum urinary flow rate (Qmax), and quality of life, as assessed by Barthel Index (BI) scale. In addition, adverse events were also recorded during the study period. All primary and secondary outcomes were measured at baseline, at the end of 8-week treatment, and 4-week follow-up. RESULTS: At the end of 8-week treatment, the patients in the treatment group did not achieve better outcomes in PV-VRU (P = .66), voided volume (P = .59), Qmax (P = .53), and BI scores (P = .67), than patients in the control group. At the end of 4-week follow-up, there were also no significant differences regarding the PV-VRU (P = .42), voided volume (P = .71), Qmax (P = .24), and BI scores (P = .75) between 2 groups. No adverse events occurred in either group. CONCLUSIONS: In summary, the findings of this study showed that NMES therapy may not benefit patients with CUR following TBI.

Discovery and Characterization of 4-Hydroxy-2-pyridone Derivative Sambutoxin as a Potent and Promising Anticancer Drug Candidate: Activity and Molecular Mechanism.

Sambutoxin, a representative derivative of 4-hydroxy-2-pyridone, was isolated from Hericium alpestre for the first time in this study. The possible correlation between the sambutoxin-induced suppression of tumor growth and its influence on cell-cycle arrest and apoptosis was investigated. The effects of sambutoxin on reactive oxygen species (ROS) production, DNA damage, mitochondrial transmembrane potential, cell apoptosis, and the expression of related proteins were evaluated. An in vitro cell viability study demonstrated that sambutoxin could inhibit the proliferation of various cancer cells. Treatment with sambutoxin induced the production of ROS, which caused DNA damage. Furthermore, the subsequent sambutoxin-induced activation of ATM and Chk2 resulted in G2/M arrest, accompanied by decreased expression of cdc25C, cdc2, and cyclin B1. Sambutoxin induced apoptosis by activating the mitochondrial apoptosis pathway through an increased Bax/Bcl-2 ratio, loss of mitochondrial membrane potential (ΔΨm), cytochrome (Cyt) c release, caspase-9 and caspase-3 activation, and poly(ADP-ribose) polymerase (PARP) degradation. The ROS elevation induced the sustained phosphorylation of c-Jun N-terminal kinase (JNK), while SP600125, a JNK inhibitor, nearly completely reversed sambutoxin-induced apoptosis. Accordingly, an in vivo study showed that sambutoxin exhibited potential antitumor activity in a BALB/c nude mouse xenograft model without significant systemic toxicity. Moreover, the expression changes in proteins related to the G2/M phase, DNA damage, and apoptosis in vivo were consistent with those in vitro. Importantly, sambutoxin has remarkable antiproliferative effects and is a promising anticarcinogen candidate for cancer treatment.

Metformin incombination with curcumin inhibits the growth, metastasis, and angiogenesis of hepatocellular carcinoma in vitro and in vivo.

Hepatocellular carcinoma (HCC) has poor prognosis due to the advanced disease stages by the time it is diagnosed, high recurrence rates and metastasis. In the present study, we investigated the effects of metformin (a safe anti-diabetic drug) and curcumin (a turmeric polyphenol extracted from rhizome of Curcuma longa Linn.) on proliferation, apoptosis, invasion, metastasis, and angiogenesis of HCC in vitro and in vivo. It was found that co-treatment of metformin and curcumin could not only induce tumor cells into apoptosis through activating the mitochondria pathways, but also suppress the invasion, metastasis of HCC cells and angiogenesis of HUVECs. These effects were associated with downregulation of the expression of MMP2/9, VEGF, and VEGFR-2, up-regulation of PTEN, P53 and suppression of PI3K/Akt/mTOR/NF-κB and EGFR/STAT3 signaling. Co-administration of metformin and curcumin significantly inhibited HCC tumor growth than administration with metformin or curcumin alone in a xenograft mouse model. Thus, metformin and curcumin in combination showed a better anti-tumor effects in hepatoma cells than either metformin or curcumin presence alone and might represent an effective therapeutic strategy for HCC treatment.

DHPAC, a novel synthetic microtubule destabilizing agent, possess high anti-tumor activity in vincristine-resistant oral epidermoid carcinoma in vitro and in vivo.

Multidrug resistance (MDR) is one of major obstacles to effective chemotherapeutic treatment of cancer. This study showed that DHPAC, 2-(6-ethoxy-3-(3-ethoxyphenylamino) -1-methyl-1,4-dihydroindeno[1,2-c]pyrazol-7-yloxy) acetamide, a novel compound that binds to the same site on microtubules as colchicine, has high anti-tumour activity in vincristine-resistant oral epidermoid carcinoma (KB/V) cells. It found that the presence of DHPAC strongly inhibited KB/V cell growth in vivo and in mice xenograft. The inhibitory effect of DHPAC is much stronger than that by colchicine in these KB/V cells (IC50: 64.4nM and 458.0nM respectively). Treatment of the cells with DHPAC induced cell apoptosis by reducing mitochondrial membrane potential and altered the expression of several apoptosis-related proteins such as Bcl-2, Bax, Caspase-9, Cytochrome c and PARP. DHPAC treatment also caused cell rest in G2/M phase by regulating of the expression of a number of cell cycle-related proteins (e.g. Cyclin B1, Cdc2, Cdc25b, Cdc25c, RSK2). Furthermore, DHPAC presence inhibits PTEN phosphorylation and PTEN/Akt/NF-κB signalling. Thus, DHPAC has potent anti-cancer activity in MDR tumuors and may be a potential therapeutic agent for the treatment of vincristine-resistant human oral epidermoid carcinoma.

The anti-inflammatory effects of Morin hydrate in atherosclerosis is associated with autophagy induction through cAMP signaling.

SCOPE: Although the previous trials of inflammation have indicated that morin hydrate (MO) hold considerable promise, understanding the distinct mechanism of MO against inflammation remains a challenge. METHODS AND RESULTS: This study investigated the effect of MO in atherosclerosis in ApoE-/- mice and underlying cell signaling of MO effect in inflammation in human umbilical vein endothelial cells (HUVECs). Administration of MO significantly reduced serum lipid level, inflammatory cytokines (TNF-α and ICAM-1), and atherosclerotic plaque formation in vivo. MO presence attenuated the expression of TNF-α-induced inflammatory cytokines (ICAM-1, COX-2, and MMP-9), and remarkably enhanced microtubule associated protein 1 light chain 3 beta 2 (MAP1LC3B2) expression and sequestosome 1 (SQSTM1/p62) degradation in HUVECs. These MO effects were significantly prevented by the presence of autophagic inhibitors, 3-methyladenine (3-MA), or chloroquine (CQ), as well as siRNA suppression of ATG5 and BECN1. MO increased intracellular cAMP levels and activated cAMP-PKA-AMPK-SIRT1 signaling in vivo and in vitro. These changes resulted in increased expression of autophagy-related protein MAP1LC3B2 and decreased secretion of inflammatory cytokines (ICAM-1, COX-2, and MMP-9). CONCLUSION: Our results suggest that anti-AS and anti-inflammatory effects of MO are largely associated with its induction of autophagy through stimulation of cAMP-PKA-AMPK-SIRT1 signaling pathway.

Inhibition of PTEN expression and activity by angiotensin II induces proliferation and migration of vascular smooth muscle cells.

PTEN (phosphatase and tensin homolog deleted on chromosome 10) is a tumor suppressor and has been suggested recently to be involved in the regulation of cardiovascular diseases. The molecular mechanisms of this regulation are however poorly understood. This study shows that down regulation of PTEN expression and activity by angiotensin II (Ang II) increased proliferation and migration of vascular smooth muscle cells (VSMCs). The presence of Ang II induced rapid PTEN phosphorylation and oxidation in accordance with increased AKT and FAK phosphorylation. The Ang II-mediated VSMC proliferation and migration was inhibited when cellular PTEN expression was increased by AT1 inhibitor losartan, PPARγ agonist rosiglitazone, NF-κB inhibitor BAY 11-7082. Over expression of PTEN in VSMCs by adenovirus transduction also resulted in inhibition of cell proliferation and migration in response to Ang II. These results suggest that PTEN down-regulation is involved in proliferation and migration of VSMCs induced by Ang II. This provides insight into the molecular regulation of PTEN in vascular smooth muscle cells and suggests that targeting the action of PTEN may represent an effective therapeutic approach for the treatment of cardiovascular diseases.

Riccardin D-26, a synthesized macrocyclic bisbibenzyl compound, inhibits human oral squamous carcinoma cells KB and KB/VCR: In vitro and in vivo studies.

BACKGROUND: Riccardin D-26, a synthesized macrocyclic bisbibenzyl compound, might possess anti-cancer properties. We aimed to evaluate the efficacy of Riccardin D-26 as a candidate compound for treatment of cancers with sensitive or drug resistant cells. METHODS: Experiments were performed on human oral squamous carcinoma KB cells and vincristin-selected MDR KB/VCR cells. The inhibition of cell growth was evaluated by colorimetric and clonogenic assays. The apoptotic cells were determined by the Annexin V-FITC/PI staining assay. JC-1 fluorescence probe was used to examine the mitochondria membrane potential (MMP). Further experiments were performed in nude mice bearing KB or KB/VCR xenografts. Riccardin D-26 was administered by injection for 2weeks. The specimens of KB and KB/VCR xenografts were removed for TUNEL staining and Western blotting analysis. RESULTS: Riccardin D-26 significantly inhibited cancer growth in both KB and KB/VCR cells. Riccardin D-26's activity in cancer cells was greater than that in human normal liver cells. In mice, Riccardin D-26 effectively prevented the growth of KB and KB/VCR xenografts without significant toxicity. Further studies suggested that Riccardin D-26 inhibited cancer growth by inducing apoptosis in the activation of mitochondria-mediated intrinsic apoptosis pathway. Riccardin D-26 also possessed this activity in regulation of mitogen-related protein kinases such as MAPK and PI3K/Akt, which is associated with its inhibitory effect on KB/VCR cells. CONCLUSIONS: Riccardin D-26 possessed an anti-proliferation activity against both sensitive KB and MDR KB/VCR cancer cells. GENERAL SIGNIFICANCE: Riccardin D-26 could be a promising agent for treatment of cancers with sensitive or drug resistant cells.

Retigeric acid B exhibits antitumor activity through suppression of nuclear factor-κB signaling in prostate cancer cells in vitro and in vivo.

Previously, we reported that retigeric acid B (RB), a natural pentacyclic triterpenic acid isolated from lichen, inhibited cell growth and induced apoptosis in androgen-independent prostate cancer (PCa) cells. However, the mechanism of action of RB remains unclear. In this study, we found that using PC3 and DU145 cells as models, RB inhibited phosphorylation levels of IκBα and p65 subunit of NF-κB in a time- and dosage-dependent manner. Detailed study revealed that RB blocked the nuclear translocation of p65 and its DNA binding activity, which correlated with suppression of NF-κB-regulated proteins including Bcl-2, Bcl-x(L), cyclin D1 and survivin. NF-κB reporter assay suggested that RB was able to inhibit both constitutive activated-NF-κB and LPS (lipopolysaccharide)-induced activation of NF-κB. Overexpression of RelA/p65 rescued RB-induced cell death, while knockdown of RelA/p65 significantly promoted RB-mediated inhibitory effect on cell proliferation, suggesting the crucial involvement of NF-κB pathway in this event. We further analyzed antitumor activity of RB in in vivo study. In C57BL/6 mice carrying RM-1 homografts, RB inhibited tumor growth and triggered apoptosis mainly through suppressing NF-κB activity in tumor tissues. Additionally, DNA microarray data revealed global changes in the gene expression associated with cell proliferation, apoptosis, invasion and metastasis in response to RB treatment. Therefore, our findings suggested that RB exerted its anti-tumor effect by targeting the NF-κB pathway in PCa cells, and this could be a general mechanism for the anti-tumor effect of RB in other types of cancers as well.

Site-specific chemical modification of human serum albumin with polyethylene glycol prolongs half-life and improves intravascular retention in mice.

Human serum albumin (HSA) is used as an important plasma volume expander in clinical practice. However, the infused HSA may extravasate into the interstitial space and induce peripheral edema in treating the critical illness related to marked increase in capillary permeability. Such poor intravascular retention also demands a frequent administration of HSA. We hypothesize that increasing the molecular weight of HSA by PEGylation may be a potential approach to decrease capillary permeability of HSA. In the present study, HSA was PEGylated in a site-specific manner and the PEGylated HSA carrying one chain of polyethylene glycol (PEG) (20 kDa) per HSA molecule was obtained. The purity, PEGylated site and secondary structure of the modified protein were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), thiol group blockage method and circular dichroism (CD) measurement, respectively. In addition, the pharmacokinetics in normal mice was investigated, vascular permeability of the PEGylated HSA was evaluated in lipopolysaccharide (LPS)-induced lung injury mouse model and the pharmacodynamics was investigated in LPS-induced sepsis model with systemic capillary leakage. The results showed that the biological half-life of the modified HSA was approximately 2.3 times of that of the native HSA, PEG-HSA had a lower vascular permeability and better recovery in blood pressure and haemodilution was observed in rats treated with PEG-HSA. From the results it can be inferred that the chemically well-defined and molecularly homogeneous PEGylated HSA is superior to HSA in treating capillary permeability increase related illness because of its longer biological half-life and lower vascular permeability.

A112, a tamibarotene dimethylaminoethyl ester, may inhibit human leukemia cell growth more potently than tamibarotene.

A112 is a tamibarotene dimethylaminoethyl ester considered a candidate compound for the treatment of acute promyelocytic leukemia (APL) and acute myeloid leukemia (AML). Our goal in this study was to evaluate the efficacy of anti-cancer activity, beginning by studying its inhibitory effects on leukemia cells and then comparing it to tamibarotene. A112 effectively inhibited the growth of HL-60 and NB4 cells as estimated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The inhibitory effect of A112 was confirmed in mice in which A112 delayed the growth of HL-60 xenografts after 3 weeks' injection. The efficacy of A112 on leukemia cell growth was stronger than that of tamibarotene at the same dosage. The detection of A112 and tamibarotene in plasma of rats showed that A112 might sustain release of its hydrolysate tamibarotene, and the concentration was maintained at a higher level and for a longer time than that of tamibarotene injection. We studied the differentiation morphologies of leukemic cells exposed to A112 or tamibarotene. The number of differentiated NB4 cells was increased, suggesting that A112 possessed differentiation activity in the inhibition of leukemia growth. Further studies showed that the expression of CD11b, a marker of terminal granulocyte differentiation, was increased as estimated by flow cytometry with a direct immunofluorescence assay. A112 was found to induce the activation of CCAAT/enhancer-binding protein ß (C/EBPß) and cyclin-dependent kinase (CDK) inhibitors p21(Waf1/cip1) and p27(Kip1) while cell growth was inhibited. These activities of A112 were greater than those of tamibarotene. The higher efficacy of A112 was also evidenced by induction of apoptosis in leukemia cells. A112 induced a greater number of annexin V-positive cells than did tamibarotene as measured by flow cytometry analysis. Treatment of mice with A112 resulted in stronger terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining in HL-60 xenografts. Western blot analysis revealed that A112 increased the expression of caspase-3, caspase-9 and cleaved poly(ADP-ribose) polymerase (PARP) in leukemia cells both in vitro and in vivo, indicating that induction of apoptosis was involved in the inhibition of leukemia growth. Taken together, these results suggest that A112 is a highly effective derivative of trans retinoic acid and a potential candidate compound for the treatment of leukemia.

Riccardin D, a novel macrocyclic bisbibenzyl, induces apoptosis of human leukemia cells by targeting DNA topoisomerase II.

We studied the effect of riccardin D, a macrocyclic bisbibenzyl, which was isolated from the Chinese liverwort plant, on human leukemia cells and the underlying molecular mechanism. Riccardin D had a significant antiproliferative effect on human leukemia cell lines HL-60, K562 and its multidrug resistant (MDR) counterpart K562/A02 cells, but showed no effect on the topoisomerase-II-deficient HL-60/MX2 cells, as measured by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The pBR322 DNA relaxation assay revealed that riccardin D selectively inhibited the activity of topoisomerase II (topo II). The suppression of topo II activity by riccardin D was stronger than that of etoposide, a known topo II inhibitor. After treatment with riccardin D, nuclear extracts of leukemia K562 and K562/A02 cells left the majority of pBR322 DNA in a supercoiled form. Further examination showed that riccardin D effectively induced HL-60, K562 and K562/A02 apoptosis as evidenced by externalization of phosphatidylserine and formation of DNA ladder fragments. The activation of cytochrome c, caspase-9, caspase-3 and cleaved poly ADP-ribose polymerase (PARP) was also enhanced, as estimated by Western blot analysis. By contrast, riccardin D was unable to induce apoptosis in the topoisomerase-II-deficient HL-60/MX2 cells, indicating that the induction of apoptosis by riccardin D was due to the inhibition of topo II activity. In addition, riccardin D was able to significantly decrease P-glycoprotein (P-gp) expression in K562/A02 cells. Taken together, our data demonstrate that riccardin D is a novel DNA topo II inhibitor which can induce apoptosis of human leukemia cells and that it has therapeutic potential for both regular and MDR strains of leukemia cells.

Inhibition of angiogenesis involves in anticancer activity of riccardin D, a macrocyclic bisbibenzyl, in human lung carcinoma.

Riccardin D is a novel macrocyclic bisbibenzyl compound extracted from Chinese liverwort plant Dumortiera hirsuta. Our previous studies showed that riccardin D is a DNA topo II inhibitor and has therapeutic potential for treatment of cancers. In this combined in vitro and in vivo study, we examined the inhibitory effects of riccardin D on tumor angiogenesis and the subsequent effect of anticancer activity was evaluated. Incubation with riccardin D weakly inhibited the proliferation of human umbilical vascular endothelial cells (HUVEC) as estimated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The scratch wound experiment showed that riccardin D effectively decreased the motility and migration of HUVEC cells. Riccardin D inhibited the formation of capillary tube as demonstrated by decrease of branch points formed by HUVEC cells on 3-D Matrigel. We examined the levels of angiogenic factors including vascular endothelial growth factor (VEGF), VEGF receptor 2, epidermal growth factor receptor (EGF receptor), and matrix metalloproteinase (MMPs) in HUVEC cells. The expressions of VEGF, phospho-VEGF receptor 2, EGF receptor and MMP-2 were significantly reduced by riccardin D as estimated by Western blot assay and real-time quantitative PCR analysis. The decrease of VEGF was also detected in riccardin D-treated human lung cancer H460 cells. The anticancer activity of riccardin D was then evaluated in a mouse model in which riccardin D delayed the growth of H460 xenografts without obvious toxicity to animals after three weeks injection. To evaluate the role of antiangiogenesis of riccardin D in mice, CD34 immunohistochemical staining was employed to analyze the mean vascular density in H460 xenograft tissues. The number of blood vessels was significantly decreased after riccardin D treatment. These results suggest that riccardin D display the inhibitory effect on growth of human lung carcinoma cells and that the inhibition of angiogenesis may involve in anticancer activity of riccardin D.

LYP, a bestatin dimethylaminoethyl ester, inhibited cancer angiogenesis both in vitro and in vivo.

Our previous study revealed that LYP, a bestatin dimethylaminoethyl ester, inhibited the growth of human ovarian carcinoma ES-2 xenografts in mice and suppressed aminopeptidase N (APN/CD13) activity more potently than bestatin. In this study, we examined the inhibitory effect of LYP on migration and formation of capillary tube of human umbilical vascular endothelial cells (HUVECs) in vitro and anti-angiogenesis in ES-2 xenografts in mice. LYP did not possess cytotoxicity to HUVEC proliferation according to the MTT assay and trypan blue exclusion assay. However, APN/CD13 activity on cell surface of HUVECs was suppressed in the presence of LYP as measured by quantifying the enzymatic cleavage of the substrate l-leucine-p-nitroanilide. The assays of scratch and transwell chamber showed that LYP significantly inhibited HUVEC migration and invasion through Matrigel coated polycarbonate filters. Capillary tube formation assay revealed that the number of branch points formed by HUVECs on 3-D Matrigel was reduced after incubation with LYP. The anti-angiogenesis of LYP was verified in ES-2 xenografts in mice. The mean vascular density (MVD) and mean vascular luminal diameter (MVLD) were markedly reduced by LYP after two weeks of intravenous injection as evaluated by CD34 immunohistochemical staining. LYP suppression of cancer angiogenesis was greater than that of bestatin. The inhibition of angiogenic molecules may involve in anti-angiogenesis of LYP. The levels of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and transforming growth factor-alpha (TGF-α) were decreased in HUVECs and ES-2 xenografts after treatment with LYP as determined by Western blot analysis. These results indicated that the high efficacy of LYP may partially relate to the inhibition of angiogenesis.

Ligustrazine derivate DLJ14 reduces multidrug resistance of K562/A02 cells by modulating GSTπ activity.

Multidrug resistance (MDR) of tumor cells is a major obstacle in chemotherapeutic cancer treatment. Over-expression of glutathione S-transferase π (GSTπ) is one of the mechanisms contributing to MDR. In this study, we investigated the reversal of MDR by DLJ14, a ligustrazine derivate, in adriamycin (Adr) resistant human myelogenous leukemia (K562/A02) cells by modulating the expression of GSTπ and the activity of GST-related enzymes. In the MTT test, DLJ14 showed a weak inhibition on proliferation of both K562/A02 and K562 cells, while verapamil at the same concentration showed a much stronger inhibition. The sensitivity of K562/A02 cells to cytotoxic killing by Adr was enhanced by incubation with DLJ14 as a result of the increased intracellular accumulation of Adr. The accumulation of Adr induced by DLJ14 may due to down regulation of GST-related enzyme activity. Western blot analysis and RT-PCR showed that DLJ14 was able to inhibit the protein expression and mRNA expression of GSTπ in K562/A02 cells. Moreover, DLJ14 increased the expression of cellular c-Jun NH(2)-terminal kinase (JNK) in K562/A02 cells exposure to Adr. This is consistent with the inhibition of GSTπ. These results demonstrate that DLJ14 may be an attractive new agent for the chemosensitization of cancer cells.

N1-acetyl substituted pyrrolidine derivative CIP-A5: a novel compound that could ameliorate liver cirrhosis through modulation of hepatic stellate cell activity.

(2S,4R)-methyl 1-acetyl-4-(N-(4-bromophenyl)sulfamoyloxy)pyrrolidine-2-carboxylate (CIP-A5) is the N1-acetyl substituted pyrrolidine derivative which was designed against the structure of matrix metalloproteinase (MMP-2) and MMP-9. CIP-A5 has been considered as a candidate compound for treatment of liver cirrhosis. In this study, we evaluated the efficacy of CIP-A5 on the activity of hepatic stellate cells. CIP-A5 prevented the transforming growth factor ß (TGF-ß)-induced proliferation of hepatic stellate HSC-T6 cells as estimated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. CIP-A5 stimulated MMPs activity as evidenced by an increase of degradation of succinylated gelatin. Gelatin zymography analysis showed that CIP-A5 stimulated the secretion and activity of MMP-2 and MMP-9 in HSC-T6 cells. This stimulatory effect on MMPs was verified by the observation of increased expression of MMP-2 and MMP-9 as evaluated by Western blot assay. At the same time, a significant decrease of the expression of tissue inhibitors of matrix metalloproteinases-1 (TIMP-1) was observed, suggesting a modulation of the balance of MMPs/TIMPs in hepatic stellate cells. CIP-A5 treatment also resulted in suppression of the profibrogenic cytokines, such as TGF-ß, tumor necrosis factor alpha (TNF-α) and connective tissue growth factor (CTGF) in HSC-T6 cells. CIP-A5 did not have cytotoxicity to human normal hepatic cells. These results implied that CIP-A5 could selectively ameliorate the process of liver cirrhosis through modulation of activated hepatic stellate cell activity, which offers hope for prevention and treatment of this devastating end-stage liver disease.

Antagonistic effects of ultra-low-molecular-weight heparin on Aß25-35-induced apoptosis in cultured rat cortical neurons.

Low-molecular-weight heparin (LMWH) and ultra-low-molecular-weight heparin (ULMWH) are heparin's derivatives, having various pharmacological effects. The present study aims to investigate the effect of ULMWH on amyloid ß peptide (Aß25-35)-induced neurotoxicity in cultured rat cortical neurons, and LMWH was employed as a positive control agent. The neurons were incubated with Aß25-35 (35µM), Aß25-35 plus ULMWH (2, 10, 50 µg/ml) or LMWH (10 µg/ml) for 24h. The cell viability was assessed by MTT and LDH release. FITC-Annexin V/PI double staining, Hoechst 33258 staining, TUNEL and Western blotting for bcl-2 and caspase-3 were employed to measure the neuron apoptosis. Furthermore, the intracellular Ca(2+) concentration was measured by a fluorescent dye, Fura-2/AM. The results showed that ULMWH significantly increased cell viability and the protein expression levels of bcl-2 and decreased the LDH release, the number of apoptotic cells, the concentration of intracellular Ca(2+) and the protein expression levels of caspase-3 in cortical neurons, suggesting that ULMWH can obviously reduce Aß25-35-induced neurotoxic effects and might act as a potential agent for Alzheimer's disease.

LYP, a novel bestatin derivative, inhibits cell growth and suppresses APN/CD13 activity in human ovarian carcinoma cells more potently than bestatin.

LYP is a bestatin dimethylaminoethyl ester which inhibits aminopeptidase N (APN/CD13). Our goal in this study was to evaluate LYP as a candidate compound for cancer treatment, beginning by studying its inhibitory effects on tumors and then comparing it to bestatin. Experiments were performed on human ovarian carcinoma (OVCA) ES-2 and SKOV-3 cell lines, which have high and low levels of APN/CD13 respectively. LYP effectively inhibited ES-2 cell growth as estimated by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and the trypan blue dye-exclusion test. LYP significantly suppressed APN/CD13 activity on the surface of ES-2 cells as measured by quantifying the enzymatic cleavage of the substrate L-leucine-p-nitroanilide. The inhibitory effects of LYP were greater than those of bestatin at the same concentrations. In contrast, LYP was a weak inhibitor of SKOV-3 cell growth, suggesting that LYP may inhibit ES-2 cell growth via suppression of APN/CD13. Inhibition of APN/CD13 expression was also demonstrated with immunofluorescent flow cytometry and Western blot analysis. Inhibitory effects of LYP were confirmed by using a mouse model in which LYP delayed the growth of ES-2 xenografts in mice after 2 weeks of LYP injections. Inhibition of APN/CD13 expression was demonstrated in the ES-2 xenografts using Western blot analysis. The inhibitory effects of LYP on the ES-2 xenografts were stronger than those of bestatin. These results suggest that LYP has a powerful inhibitory effect on the growth of OVCA cells and that the mechanism may be via a decrease in the expression of APN/CD13.

TMPDP, a tetramethylpyrazine derivative, protects vascular endothelial cells from oxidation damage by hydrogen peroxide.

A novel ligustrazine derivative, tetramethylpyrazine diphenylmethyl piperazidine (TMPDP), prepared by hybridization and bioisosteric replacement of the molecular structure of TMP, was studied for its protective effects on oxidative damage of human umbilical vein endothelial cells (HUVECs) in response to hydrogen peroxide (H2O2). The antioxidative effect of TMPDP was assessed by the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) test. Cell viability was measured using a 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The activity of lactate dehydrogenase (LDH), superoxide dismutase (SOD) and glutathione peroxidase (GSH) and the content of malondialdehyde (MDA) in cells were determined by commercial kits. The intracellular formation of reactive oxygen species (ROS) and the concentration of free intracellular calcium ([Ca2+]i) were determined using DCFH-DA assay and with fura-2/AM fluorimetry, respectively. Results showed that TMPDP had a moderate antioxidative effect against DPPH. Cell viability was decreased markedly by exposure to H2O2. Introduction of TMPDP, however, significantly increased cell viability, markedly reduced LDH release from cells and decreased lipid peroxidation in response to H2O2 treatment. These effects of TMPDP were accompanied by increased activity of the endogenous antioxidant enzymes, SOD and GSH, reduced production of ROS and reduced intracellular concentration of Ca2+. These results suggest that TMPDP protects HUVECs against oxidative damage by scavenging ROS and regulates intracellular calcium concentration. This might have important implications for the development of new agents for the effective treatment of vascular disease.