Phosphodiesterase 2 inhibitor Hcyb1 reverses corticosterone-induced neurotoxicity and depression-like behavior.

RATIONALE: Currently available PDE2 inhibitors have poor brain penetration that limits their therapeutic utility in the treatment of depression. Hcyb1 is a novel selective PDE2 inhibitor that was introduced more lipophilic groups with polar functionality to the scaffold pyrazolopyrimidinone to improve the blood-brain barrier (BBB) penetration. Our previous study suggested that Hcyb1 increased the neuronal cell viability and exhibited antidepressant-like effects, which were parallel to the currently available PDE2 inhibitor Bay 60-7550. OBJECTIVES: The present study investigated whether Hcyb1 protected HT-22 cells against corticosterone-induced neurotoxicity and produced antidepressant-like effects in behavioral tests in stressed mice. METHODS: The neuroprotective effects of Hcyb1 against corticosterone-induced cell lesion were examined by cell viability (MTS) assay. The enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis were used to determine the levels of cAMP or cGMP and expression of pCREB or BDNF, respectively, in the corticosterone-treated HT-22 cells. The antidepressant-like effects of Hcyb1 were determined in the tail suspension and novelty suppressed feeding tests in stressed mice. RESULTS: In the cell-based assay, Hcyb1 significantly increased cell viability of HT-22 cells against corticosterone-induced neurotoxicity in a time- and dose-dependent manner. Hcyb1 also rescued corticosterone-induced decreases in both cGMP and cAMP levels, pCREB/CREB and BDNF expression. These protective effects of Hcyb1 were prevented by pretreatment with either the PKA inhibitor H89 or the PKG inhibitor KT5823. Moreover, Hcyb1 reversed acute stress-induced increases in immobility time and the latency to feed in the tail suspension and novelty suppressed feeding tests, respectively, which were prevented by pretreatment with H89 or KT5823. CONCLUSION: These findings provide evidence that the neuroprotective effects of Hcyb1 are mediated by PDE2-dependent cAMP/cGMP signaling.

Therapeutic effects of JLX001 on cerebral ischemia through inhibiting platelet activation and thrombus formation in rats.

(3ß,5α,16α,20S)-4,4,14-trimethyl-3,20-bis(methylamino)-9,19-cyclopregnan-16-ol-dihydrochloride (JLX001), a derivative of cyclovirobuxine D (CVB-D), is a novel compound from synthesis. This study aims to confirm the therapeutic effect of JLX001 on cerebral ischemia and researchits antiplatelet and antithrombosis activities via thromboxane (TXA2)/phospholipase C-ß-3(PLCß3)/protein kinase C (PKC) pathway suppression. The therapeutic effects of JLX001 was evaluated by infarct sizes, brain edema and neurological scores in Sprague-Dawley (SD) rats with middle cerebral artery occlusion (MCAO). Brain TXA2 and prostacyclin (PGI2) were measured by enzyme-linked immunosorbentassay (ELISA). P-PLCß3and activated PKC were detected by immunohistochemical method. Adenosine diphosphate (ADP) or 9, 11-dieoxy-11α, 9α-epoxymethanoeprostaglandin F2α (U46619) was used as platelet agonist in the in vivo and in vitro platelet aggregation experiments. Clotting time and bleeding time were determined. Besides, two whole-animal experiments including arteriovenous shunt thrombosis and pulmonary thromboembolism model were conducted. Results showed that JLX001 treatment markedly alleviated cerebral infarcts, edema, and neurological scores in permanent middle cerebral artery occlusion (pMCAO) rats. Brain TXA2 level, p-PLCß3and activated PKC were decreased, while PGI2level had no significant change. Besides, JLX001 inhibited platelet aggregation induced by ADP or U46619 and exhibited anti-coagulation effects with a minor bleeding risk. In the two whole-animal experiments, JLX001 inhibited thrombus formation. In summary, JLX001 attenuates cerebral ischemia injury and the underlying mechanisms relate to inhibiting platelet activation and thrombus formation via TXA2/PLCß3/PKC pathway suppression.

Altered grey matter volume and cortical thickness in patients with schizo-obsessive comorbidity.

Recent findings suggest that schizo-obsessive comorbidity (SOC) may be a unique diagnostic entity. We examined grey matter (GM) volume and cortical thickness in 22 patients with SOC, and compared them with 21 schizophrenia (SCZ) patients, 22 obsessive-compulsive disorder (OCD) patients and 22 healthy controls (HCs). We found that patients with SOC exhibited reduced GM volume in the left thalamus, the left inferior semi-lunar lobule of the cerebellum, the bilateral medial orbitofrontal cortex (medial oFC), the medial superior frontal gyrus (medial sFG), the rectus gyrus and the anterior cingulate cortex (aCC) compared with HCs. Patients with SOC also exhibited reduced cortical thickness in the right superior temporal gyrus (sTG), the right angular gyrus, the right supplementary motor area (SMA), the right middle cingulate cortex (mCC) and the right middle occipital gyrus (mOG) compared with HCs. Together with the differences in GM volume and cortical thickness between patients with SOC and patients with only SCZ or only OCD, these findings highlight the GM changes specific to patients with SOC.

A Promising Microtubule Inhibitor Deoxypodophyllotoxin Exhibits Better Efficacy to Multidrug-Resistant Breast Cancer than Paclitaxel via Avoiding Efflux Transport.

Multidrug resistance (MDR) is a common limitation for the clinical use of microtubule-targeting chemotherapeutic agents, and it is the main factor for poor prognoses in cancer therapy. Here, we report on deoxypodophyllotoxin (DPT), a promising microtubule inhibitor in phase 1, as a promising candidate to circumvent this obstacle. DPT remarkably suppressed tumor growth in xenograft mice bearing either paclitaxel (PTX)-sensitive MCF-7/S or acquired resistance MCF-7/Adr (MCF-7/A) cells. Also, DPT exhibited similar accumulation in both tumors, whereas PTX displayed much a lower accumulation in the resistant tumors. In vitro, DPT exhibited a much lower resistance index (0.552) than those of PTX (754.5) or etoposide (38.94) in both MCF-7/S and MCF-7/A cells. Flow cytometry analysis revealed that DPT (5 and 10 nM) caused arrest of the G2/M phase in the two cell lines, whereas PTX (up to 10 nM) had no effect on cell-cycle progression of the MCF-7/A cells. Microtubule dynamics assays revealed that DPT destabilized microtubule assembly in a different mode. Cellular pharmacokinetic assays indicated comparable intracellular and subcellular accumulations of DPT in the two cell lines but a much lower retention of PTX in the MCF-7/A cells. Additionally, transport assays revealed that DPT was not the substrate of P-glycoprotein, breast cancer resistance protein, or MDR-associated protein 2, indicating a lower occurrence rate of MDR. DPT might be a promising microtubule inhibitor for breast cancer therapy, especially for treatment of drug-resistant tumors.

Antitumor effect of Deoxypodophyllotoxin on human breast cancer xenograft transplanted in BALB/c nude mice model.

Recently, biologically active compounds isolated from plants used in herbal medicine have been the center of interest. Deoxypodophyllotoxin (DPT), structurally closely related to the lignan podophyllotoxin, was found to be a potent antitumor and antiproliferative agent, in several tumor cells, in vitro. However, DPT has not been used clinically yet because of the lack of in vivo studies. This study is the first report demonstrating the antitumor effect of DPT on MDA-MB-231 human breast cancer xenografts in nude mice. DPT, significantly, inhibited the growth of MDA-MB-231 xenograft in BALB/c nude mice. The T/C value (the value of the relative tumor volume of treatment group compared to the control group) of groups treated with 5, 10, and 20 mg/kg of intravenous DPT-HP-ß-CD was 42.87%, 34.04% and 9.63%, respectively, suggesting the positive antitumor activity of DPT. In addition, the antitumor effect of DPT-HP-ß-CD (20 mg/kg) in human breast cancer MDA-MB-231 xenograft was more effective than etoposide (VP-16) (20 mg/kg) and docetaxel (20 mg/kg). These findings suggest that this drug is a promising chemotherapy candidate against human breast carcinoma.

Design and synthesis of novel 4'-demethyl-4-deoxypodophyllotoxin derivatives as potential anticancer agents.

A group of podophyllotoxin (PPT) derivatives (7a-j) were synthesized by conjugating aryloxyacetanilide moieties to the 4'-hydroxyl of 4'-demethyl-4-deoxypodophyllotoxin (DDPT), and their anticancer activity was evaluated. It was found that the most potent compound 7d inhibited the proliferation of three cancer cell lines with sub to low micromolar IC50 values. Furthermore, it was demonstrated that 7d induced cell cycle arrest in G2/M phase in MGC-803 cells, and regulated the expression of cell cycle check point proteins, such as cyclin A, cyclin B, CDK1, cdc25c, and p21. Finally, 4 mg/kg of 7d reduced the weights and volumes of HepG2 xenografts in mice. Our findings suggest that 7d might be a potential anticancer agent.

Deoxypodophyllotoxin suppresses tumor vasculature in HUVECs by promoting cytoskeleton remodeling through LKB1-AMPK dependent Rho A activatio.

Angiogenesis plays a critical role in the growth and metastasis of tumors, which makes it an attractive target for anti-tumor drug development. Deoxypodophyllotoxin (DPT), a natural product isolated from Anthriscus sylvestris, inhibits cell proliferation and migration in various cancer cell types. Our previous studies indicate that DPT possesses both anti-angiogenic and vascular-disrupting activities. Although the RhoA/ RhoA kinase (ROCK) signaling pathway is implicated in DPT-stimulated cytoskeleton remodeling and tumor vasculature suppressing, the detailed mechanisms by which DPT mediates these effects are poorly understood. In the current study, we found that DPT promotes cytoskeleton remodeling in human umbilical vein endothelial cells (HUVECs) via stimulation of AMP-activated protein kinase (AMPK) and that this effect is abolished by either treatment with a selective AMPK inhibitor or knockdown. Moreover, the cellular levels of LKB1, a kinase upstream of AMPK, were enhanced following DPT exposure. DPT-induced activation of AMPK in tumor vasculature effect was also verified by transgenic zebrafish (VEGFR2:GFP), Matrigel plug assay, and xenograft model in nude mice. The present findings may lay the groundwork for a novel therapeutic approach in treating cancer.

The potential utility of acetyltanshinone IIA in the treatment of HER2-overexpressed breast cancer: Induction of cancer cell death by targeting apoptotic and metabolic signaling pathways.

Increased lipogenesis and protein synthesis is a hallmark of cancer cell proliferation, survival, and metastatic progression and is under intense investigation as a potential antineoplastic target. Acetyltanshinone IIA (ATA) is a compound that was obtained from chemical modifications of tanshinone IIA (TIIA), a potent anticancer agent extracted from the dried roots of the Chinese herbal medicine Salvia miltiorrhiza Bunge. A previous investigation indicated that ATA is more effective in inhibiting the growth of breast cancer especially cells with HER2 overexpression. However, the molecular mechanism(s) mediating this cytotoxic effect on HER2-positive breast cancer remained undefined. Studies described here report that ATA induced G1/S phase arrest and apoptosis in the HER2-positive MDA-MB-453, SK-BR-3, and BT-474 breast cancer cell lines. Mechanistic investigations revealed that the ATA-induced apoptosis effect is associated with remarkably down-regulation of receptor tyrosine kinases (RTKs) EGFR/HER2 and inhibition of their downstream pro-survival signaling pathways. Interestingly, ATA was found to trigger oxidative and endoplasmic reticulum (ER) stresses and to activate AMP activated protein kinase (AMPK) leading to inactivation of key enzymes involved in lipid and protein biogenesis. Intraperitoneal administration of ATA significantly inhibited the growth of MDA-MB-453 xenografts in athymic mice without causing weight loss and any other side effects. Additionally, transwell migration, invasion, and wound healing assays revealed that ATA could suppress tumor angiogenesis in vitro. Taken together, our data suggest that ATA may have broad utility in the treatment of HER2-overexpressed breast cancers.

Antineoplastic effects of deoxypodophyllotoxin, a potent cytotoxic agent of plant origin, on glioblastoma U-87 MG and SF126 cells.

BACKGROUND: Deoxypodophyllotoxin (DPT) is a semi-synthetic compound derived from the extract of Dysosma versipellis (Hance) M. Cheng, one of the most popular Chinese herbal medicines. The present study evaluates the in vitro cytotoxicity of DPT on a wide panel of human cancer cell lines and investigates its molecular mechanism of action on high grade glioma U-87 MG and SF126 cells. METHODS: The growth inhibitory effect of DPT on different types of human cancer cells was measured by the Cell Counting Kit-8 (CCK-8) assay. For the elucidation of the nature of the cellular response to DPT-treatment; flow cytometry-based assays, light and fluorescent microscopy, caspase colorimetric and inhibition assays, and Western blot analysis were performed. RESULTS: Our data show that DPT possesses a potent growth-inhibitory action, with IC50 values in nanomolar ranges. Cell cycle analysis revealed G2/M phase arrest in a dose- and time-dependent manner before cell death occurred. Additional studies indicated that DPT induced G2 arrest in U-87 MG cells by decreasing the expression of Cdc2, cyclin B1, and Cdc25C proteins. In contrast, DPT failed to down-regulate these cell cycle regulatory molecules in SF126 glioblastoma cells and stopped the cell cycle at M phase. Interestingly, morphological changes and biochemical markers such as phosphatydylserine externalization, DNA fragmentation, and caspase activation, confirmed that DPT-treatment resulted in an induction of apoptosis in both examined cell lines via caspase-dependent pathways. CONCLUSIONS: Taken together, our data demonstrated that DPT possesses a potent in vitro cytotoxic activity and exerts its effect via G2/M arrest and apoptosis.

Deoxypodophyllotoxin induces G2/M cell cycle arrest and apoptosis in SGC-7901 cells and inhibits tumor growth in vivo.

Deoxypodophyllotoxin (DPT), a natural microtubule destabilizer, was isolated from Anthriscus sylvestris, and a few studies have reported its anti-cancer effect. However, the in vivo antitumor efficacy of DPT is currently indeterminate. In this study, we investigated the anti-gastric cancer effects of DPT both in vitro and in vivo. Our data showed that DPT inhibited cancer cell proliferation and induced G2/M cell cycle arrest accompanied by an increase in apoptotic cell death in SGC-7901 cancer cells. In addition, DPT caused cyclin B1, Cdc2 and Cdc25C to accumulate, decreased the expression of Bcl-2 and activated caspase-3 and PARP, suggesting that caspase-mediated pathways were involved in DPT-induced apoptosis. Animal studies revealed that DPT significantly inhibited tumor growth and decreased microvessel density (MVD) in a xenograft model of gastric cancer. Taken together, our findings provide a framework for further exploration of DPT as a novel chemotherapeutic for human gastric cancer.

Validated LC-MS/MS assay for quantitative determination of deoxypodophyllotoxin in rat plasma and its application in pharmacokinetic study.

A rapid and sensitive liquid chromatography/tandem mass spectrometry method was developed and validated for the quantification of deoxypodophyllotoxin (DPT) concentration in rat plasma with diazepam as internal standard (IS). DPT and IS were extracted with ethyl acetate, and the chromatographic separation was accomplished by using a Waters Symmetry C18 analytical column (2.1mm×150mm, 5µm) with a mobile phase consisting of acetonitrile and deionized water (70:30, v:v) containing 0.1% formic acid at a flow rate of 0.2mL/min. Multiple Reaction Monitoring (MRM), using electrospray ionization in positive ion mode, was employed to quantitatively detect DPT and IS. The monitored transitions were set at m/z 399.05-231.00 and m/z 285.00-154.00 for DPT and IS, respectively. The calibration curve was linear over the concentration range of 7.8-1000ng/mL (R(2)≥0.9999). The intra- and inter-day precision values were less than 7%. Similarly, the mean intra- and inter-day accuracy were found to be within -2.8% to 1.9% of the interval, with all samples locating within general assay acceptability criteria for QC samples according to FDA guidelines. This method was further and successfully applied in the pharmacokinetics study of DPT in rat.

Deoxypodophyllotoxin triggers necroptosis in human non-small cell lung cancer NCI-H460 cells.

Deoxypodophyllotoxin (DPT), a naturally occurring microtubule destabilizer, inhibits tubulin polymerization and causes cell cycle arrest at G2/M phase in tumor cells. However, the anti-tumor effect and specific mechanism of DPT in non-small cell lung cancer (NSCLC) are still poorly understood. In this study, we determined the anti-tumor effect and potential mechanism of DPT in the NSCLC cell line, NCI-H460 (H460). First, we demonstrated that DPT significantly inhibits the proliferation of H460 cells in vitro and the growth of H460 xenografts in vivo. In further studies, DPT triggered necroptosis in H460 cells with the following characteristics: (I) necrotic cell death morphology; (II) autophagy; (III) loss of plasma membrane integrity; (IV) loss of mitochondria membrane potential; (V) elevation of reactive oxygen species levels; and (VI) specific inhibition of necroptosis via a small molecule, necrostatin-1. This study also revealed that DPT has a similar effect towards the drug-sensitive cancer cell line, H460, and the drug-resistant cell line, H460/Bcl-xL. To our knowledge, this is the first report to document the induction of necroptosis by a microtubule-targeting agent to circumvent cancer drug resistance, thereby providing a new potential choice for clinical cancer therapy, especially drug-resistant cancer therapy.

Deoxypodophyllotoxin exerts both anti-angiogenic and vascular disrupting effects.

A functioning vascular supply is essential for solid tumor growth and metastases, which means that blood vessels are an ideal target for antitumor drug discovery. Targeting tumor vasculature involves two main approaches, anti-angiogenesis and vascular disruption. The anti-angiogenic and vascular disrupting activities of deoxypodophyllotoxin (DPT), a natural microtubule destabilizer, were examined with several in vitro, ex vivo and/or in vivo models. First, we demonstrated that DPT significantly inhibits the proliferation, migration and tube formation of endothelial cells and inhibits angiogenesis in rat aortic ring and chick chorioallantoic membrane assays. In further studies, DPT induced cytoskeleton reorganization in endothelial cells, which likely contributed to the anti-angiogenic effect at non-cytotoxic concentrations. DPT treatment at higher concentrations for longer time induced the cell cycle arrest, which may contributes to its anti-proliferation effect and anti-angiogenic activity. And DPT dramatically inducted the expression of cyclin B1 and p21 (WAF1/CIP1). Meanwhile, DPT disrupted capillary-like networks in vitro and newly formed vessels from rat aortic rings. Endothelial cell contraction associated with an increase in F-actin via the Rho/Rho kinase pathway likely contributed to the vascular disrupting activity. Taken together, our results provided the initial evidence that DPT exerts potent anti-angiogenic and vascular disrupting effects. This study also provides important insight into the mechanism of action of promising new anticancer drugs with both anti-angiogenic and vascular disrupting activities.

[A brief history of liver transplantation].

Since 1950s, the idea of liver transplantation (LT) was proposed, the technique of LT had been gradually and widely appliedin the field of clinical surgery, experiencing three stages of animal experiment, clinical probation and clinical application. It is generally acknowledged as the common and effective therapeutic method for terminal stage of liver diseases. Recently, the technique of LT has made rapid progress in Chinese mainland, Hong Kong and Taiwan. China is the second biggest LT country in the world, only next to USA.

[Dynamical examination of auditory event-related potentials P300 and sympathetic skin response in people with insomia of Sweet Dream Capsule therapy].

OBJECTIVE: To observe the change of auditory event-related potentials (P300) and sympathetic skin response (SSR) in people with insomia of Sweet Dream Capsule therapy. METHODS: 30 patients meeting criteria for primary insomnia and 30 healthy volunteers with age matching controls were selected for the study. P300 and SSR were measured before treatment of Sweet Dream Capsule and at week 4 , 8 of the therapeutic course. That the change of P300 and SSR before and after treatment and their relations with PSQI were studied. RESULT: Compared with those of normal controls, both P300 latency and SSR latency were prolonged while amplitude was decreased in patients with insomnia (P < 0.01). P300 amplitude was increased significantly at central (Cz) electrode sites only at week 8 when compared with amplitude before treatment (P < 0.05). With improvement of symptom and PSQI scores, latency and amplitude of SSR were improved at week 4 and week 8 (P < 0.05 and P < 0.01) . CONCLUSION: P300 has a some improvement in people with insomia of Sweet Dream Capsule therapy while SSR im proves significantly, and PSQI scores are ameliorated too.

[Investigation and analysis on diagnostic standard for TCM Syndrome Differentiation in 400 patients with climacteric syndrome].

OBJECTIVE: To explore the thinking and method of studying the diagnostic standard for TCM Syndrome Differentiation by means of clinical epidemiological investigation on climacteric syndrome. METHODS: Adopting DME (design measurement evaluation), the field investigation data of 400 patients with climacteric syndrome were statistically managed and analyzed using blind method. The distribution of syndromes, including information of four-diagnosis of various syndromes as well as the laboratory findings in patients were studied using factor analysis and model of structural equation. RESULTS: The Syndrome types of climacteric syndrome were identified as the Gan-Yang exuberance type, the Shen-Yang deficiency type, the Gan-Shen Yin-deficiency type and the Gan-stagnancy injuring Spirit type. CONCLUSION: Thinking of the diagnostic standard study for TCM Syndrome Differentiation is to identify the supposed diagnostic standard of a disease based on retrospective study, to formulate a new hypothetical standard of Syndrome by way of field investigation, category analysis, factor analysis, and critical theory, followed with identification of Syndrome distribution in the disease, its critical syndrome, typical syndrome, complicated syndrome, and criteria for Syndrome Differentiation.