Discovery of WS-157 as a highly potent,selective and orally active EGFR inhibitor

EGFR tyrosine kinase inhibitor (EGFR-TKI) has been used successfully in clinic for the treatment of solid tumors. In the present study, we reported the discovery of WS-157 from our in-house diverse compound library, which was validated to be a potent and selective EGFR-TKI. WS-157 showed excellent inhibitory activities against EGFR (IC₅₀ = 0.81 nmol/L), EGFR^[d746−750] (IC₅₀ = 1.2 nmol/L) and EGFR^[L858R] (IC₅₀ = 1.1 nmol/L), but was less effective or even inactive against other nine kinases. WS-157 also displayed excellent antiproliferative activities against a panel of human cancer cell lines, and exhibited the ability to reduce colony formation and wound healing the same as gefitinib. We found that WS-157 upon oral administration showed better anti-tumor activity in A431 bearing xenograft mouse models compared to gefitinib. In addition, WS-157 showed better intestinal absorption than gefitinib and had favorable pharmacokinetic properties and microsomal metabolic stability in different species. These studies indicate that WS-157 has strong antitumor activity in vitro and in vivo, and could be used for the development of anti-lung cancer agent targeting EGFR.     

Yhhu3813 is a novel selective inhibitor of c-Met kinase that inhibits c-Met-dependent neoplastic phenotypes of human cancer cells

Aim： c-Met kinase deregulation is strongly associated with the formation, progression and dissemination of human cancers. In this study we identified Yhhu3813 as a small-molecule inhibitor of c-Met kinase and characterized its antitumor properties both in vitro and in vivo.
Methods： The activities of different kinases were measured using ELISA assays and signaling proteins in the cells were detected with Western blotting. Cell proliferation was assessed using SRB or MTT assay in twenty human cell lines and cell cycle distribution was determined with flow cytometry. Transwell-based assay was used to evaluate cell migration and invasion. Cell invasive growth was detected by a morphogenesis assay. c-Met overactivated human NSCLC cell line EBC-1 xenografts were used to evaluate the in vivo anti-tumor efficacy.

Results： Yhhu3813 potently inhibited c-Met kinase activity in vitro with an IC50 value of 2.4±0.3 nmol/L, 〉400-fold higher than that for a panel of 15 different tyrosine kinases, suggesting a high selectivity of Yhhu3813. The compound （20, 100 and 500 nmol/L） dose-dependently inhibited the phosphorylation of c-Met and its key downstream Akt and Erk signal cascades in multiple c-Met aberrant human cancer cell lines, regardless of the mechanistic complexity in c-Met activation across different cellular contexts. In 20 human cancer cell lines harboring different backgrounds of c-Met expression/activation, Yhhu3813 potently inhibited c-Met-driven cell proliferation via arresting cells at G1/S phase. Furthermore, Yhhu3813 substantially impaired c-Met-mediated cell migration, invasion, scattering, and invasive growth. Oral administration of EBC-1 xenograft mice with Yhhu3813 （50 or 100 mg·kg-1·d-1, qd, for 2 weeks） dose-dependently suppressed the tumor growth, which was correlated with a reduction in the intratumoral proliferation index and c-Met signaling.

Conclusion： Yhhu3813 is a potent selective inhibitor of c-Met that inhibits c-Met-dependent neoplastic phenotypes of human cancer cells in vitro and in vivo.     

Correlation of Tissue-Plasma Partition Coefficients Between Normal Tissues and Subcutaneous Xenografts of Human Tumor Cell Lines in Mouse as a Prediction Tool of Drug Penetration in Tumors

Understanding drug distribution and accumulation in tumors would be informative in the assessment of efficacy in targeted therapy; however, existing methods for predicting tissue drug distribution focus on normal tissues and do not incorporate tumors. The main objective of this study was to describe the relationships between tissue-plasma concentration ratios (K_p) of normal tissues and those of subcutaneous xenograft tumors under nonsteady-state conditions, and establish regression equations that could potentially be used for the prediction of drug levels in several human tumor xenografts in mouse, based solely on a K_p value determined in a normal tissue (e.g., muscle). A dataset of 17 compounds was collected from the literature and from Genentech. Tissue and plasma concentration data in mouse were obtained following oral gavage or intraperitoneal administration. Linear regression analyses were performed between K_p values in several normal tissues (muscle, lung, liver, or brain) and those in human tumor xenografts (CL6, EBC-1, HT-29, PC3, U-87, MCF-7-neo-Her2, or BT474M1.1). The tissue-plasma ratios in normal tissues reasonably correlated with the tumor-plasma ratios in CL6, EBC-1, HT-29, U-87, BT474M1.1, and MCF-7-neo-Her2 xenografts (r² in the range 0.62-1) but not with the PC3 xenograft. In general, muscle and lung exhibited the strongest correlation with tumor xenografts, followed by liver. Regression coefficients from brain were low, except between brain and the glioblastoma U-87 xenograft (r² in the range 0.62–0.94). Furthermore, reasonably strong correlations were observed between muscle and lung and between muscle and liver (r² in the range 0.67–0.96). The slopes of the regressions differed depending on the class of drug (strong vs. weak base) and type of tissue (brain vs. other tissues and tumors). Overall, this study will contribute to our understanding of tissue-plasma partition coefficients for tumors and facilitate the use of physiologically based pharmacokinetics (PBPK) modeling for chemotherapy in oncology studies.     

Molecular pharmacology and antitumor activity of Zalypsis in several human cancer cell lines

Zalypsis^® is a new synthetic alkaloid tetrahydroisoquinoline antibiotic that has a reactive carbinolamine group. This functionality can lead to the formation of a covalent bond with the amino group of selected guanines in the DNA double helix, both in the absence and in the presence of methylated cytosines. The resulting complex is additionally stabilized by the establishment of one or more hydrogen bonds with adjacent nucleotides in the opposite strand as well as by van der Waals interactions within the minor groove. Fluorescence-based thermal denaturation experiments demonstrated that the most favorable DNA triplets for covalent adduct formation are AGG, GGC, AGC, CGG and TGG, and these preferences could be rationalized on the basis of molecular modeling results. Zalypsis^®-DNA adducts eventually give rise to double-strand breaks, triggering S-phase accumulation and apoptotic cell death. The potent cytotoxic activity of Zalypsis^® was ascertained in a 24 cell line panel. The mean IC₅₀ value was 7 nM and leukemia and stomach tumor cell lines were amongst the most sensitive. Zalypsis^® administration in four murine xenograft models of human cancer demonstrates significant tumor growth inhibition that is highest in the Hs746t gastric cancer cell line with no weight loss of treated animals. Taken together, these results indicate that the potent antitumor activity of Zalypsis^® supports its current development in the clinic as an anticancer agent.     

Preclinical studies of the triazolo[1,5-a]pyrimidine derivative WS-716 as a highly potent,specific and orally active P-glycoprotein (P-gp) inhibitor

Multidrug resistance (MDR) is the main cause of clinical treatment failure and poor prognosis in cancer. Targeting P-glycoprotein (P-gp) has been regarded as an effective strategy to overcome MDR. In this work, we reported our preclinical studies of the triazolo[1,5-a]pyrimidine-based compound WS-716 as a highly potent, specific, and orally active P-gp inhibitor. Through direct binding to P-gp, WS-716 inhibited efflux function of P-gp and specifically reversed P-gp-mediated MDR to paclitaxel (PTX) in multiple resistant cell lines, without changing its expression or subcellular localization. WS-716 and PTX synergistically inhibited formation of colony and 3D spheroid, induced apoptosis and cell cycle arrest at G2/M phase in resistant SW620/Ad300 cells. In addition, WS-716 displayed minimal effect on the drug-metabolizing enzyme cytochrome P4503A4 (CYP3A4). Importantly, WS-716 increased sensitivity of both pre-clinically and clinically derived MDR tumors to PTX in vivo with the T/C value of 29.7% in patient-derived xenograft (PDX) models. Relative to PTX treatment alone, combination of WS-716 and PTX caused no obvious adverse reactions. Taken together, our preclinical studies revealed therapeutic promise of WS-716 against MDR cancer, the promising data warrant its further development for cancer therapy.     

Optimized functional and structural design of dual-target LMRAP,a bifunctional fusion protein with a 25-amino-acid antitumor peptide and GnRH Fc fragment

To develop fusion protein of a GnRH Fc fragment and the integrin targeting AP25 antitumor peptide for GnRH receptor-expressing cancer therapy. The LMRAP fusion protein was constructed. A transwell invasion assay was performed. The gene mRNA and protein levels of GnRHR-I, α5β1, and αvβ3 in different cancer cell lines were assessed. Cell proliferation was measured using a cell counting kit-8. An antagonist assay was performed on GnRH receptors. Anti-tumor activity was evaluated with a mouse xenograft tumor model. Immunohistochemistry (IHC) was applied to detect CD31 and CD34 expressions. Pharmacokinetic characteristics were determined with an indirect competition ELISA. The developed bifunctional fusion protein LMRAP not only inhibited HUVEC invasion, but also inhibited proliferation of GnRHR-I, α5β1, and αvβ3 high expression cancer cells. The IC₅₀ for LMRAP in the GnRH receptor was 6.235 × 10⁻⁴ mol/L. LMRAP significantly inhibited human prostate cancer cell line 22RV1 proliferation in vivo and in vitro. LMRAP significantly inhibited CD31 and CD34 expressions. The elimination half-life of the fusion protein LMRAP was 33 h in rats. The fusion protein made of a GnRH Fc fragment and the integrin targeting AP25 peptide retained the bifunctional biological activity of GnRHR blocking, angiogenesis inhibition, prolonged half-life and good tolerance.     

Inhibition of tumor growth in vitro and in vivo by fucoxanthin against melanoma B16F10 cells

The present study was designed to evaluate the molecular mechanisms of fucoxanthin against melanoma cell lines (B16F10 cells). Fucoxanthin reduced the proliferation of B16F10 cells in a dose-dependent manner accompanied by the induction of cell cycle arrest during the G₀/G₁ phase and apoptosis. Fucoxanthin-induced G₀/G₁ arrest was associated with a marked decrease in the protein expressions of phosphorylated-Rb (retinoblastoma protein), cyclin D (1 and 2) and cyclin-dependent kinase (CDK) 4 and up-regulation of the protein levels of p15^INK4B and p27^Kip1. Fucoxanthin-induced apoptosis was accompanied with the down-regulation of the protein levels of Bcl-xL, an inhibitor of apoptosis proteins (IAPs), resulting in a sequential activation of caspase-9, caspase-3, and PARP. Furthermore, the anti-tumor effect of fucoxanthin was assessed in vivo in Balb/c mice. Intraperitoneal administration of fucoxanthin significantly inhibited the growth of tumor mass in B16F10 cells implanted mice.     

Synthesis of 8-Hydroxyquinoline Derivatives as Novel Antitumor Agents

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A new rearranged eudesmane sesquiterpene and bioactive sesquiterpenes from the twigs of <Emphasis Type="Italic">Lindera glauca</Emphasis> (Sieb. et Zucc.) Blume

A new rearranged eudesmane sesquiterpene, named eudeglaucone (1), and five known sesquiterpenes including (+)-faurinone (2) and four eudesmane-type sesquiterpenes (3–6), were isolated from the twigs of Lindera glauca (Sieb. et Zucc.) Blume. The structure of 1 was elucidated by a combination of extensive spectroscopic analyses, including extensive 2D NMR (¹H-¹H COSY, HMQC, HMBC, and NOESY) and HR-MS. Compound 1 was a relatively rare rearranged eudesmane sesquiterpene in terpenoids. All isolates were evaluated for their antiproliferative activities against four human tumor cell lines (A549, SK-OV-3, SK-MEL-2, and HCT-15). Compounds 3 and 6 showed significant cytotoxicity against SK-MEL-2 and HCT-15 cell lines with IC₅₀ values ranging from 9.98 to 12.20 μM. We also investigated the anti-neuroinflammatory activities of the isolates (1–6) in the lipopolysaccharide (LPS)-stimulated murine microglia BV-2 cell line by measuring nitric oxide (NO) levels. All isolates significantly inhibited NO production with IC₅₀ values of 3.67–26.48 μM without inducing cell toxicity.     

VCP746, a novel A1 adenosine receptor biased agonist,reduces hypertrophy in a rat neonatal cardiac myocyte model

VCP746 is a novel A₁ adenosine receptor (A₁AR) biased agonist previously shown to be cytoprotective with no effect on heart rate. The aim of this study was to investigate the potential anti‐hypertrophic effect of VCP746 in neonatal rat cardiac myocytes (NCM). NCM hypertrophy was stimulated with interleukin (IL)‐1β (10 ng/mL), tumour necrosis factor (TNF)‐α (10 ng/mL) or Ang II (100 nmol/L) and was assessed by ³H‐leucine incorporation assay. VCP746 significantly inhibited IL‐1β‐, TNF‐α‐ and Ang II‐stimulated NCM hypertrophy as determined by ³H‐leucine incorporation. The anti‐hypertrophic effect of VCP746 was also more potent than that of the prototypical A₁AR agonist, N⁶‐cyclopentyladenosine (CPA). Further investigation with the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) cell viability assay showed that neither CPA nor VCP746 had any effect on cell viability, confirming that the reduction in ³H‐leucine incorporation mediated by CPA and VCP746 was not due to a reduction in cell viability. IL‐1β, TNF‐α and Ang II were also shown to increase the mRNA expression of hypertrophy biomarkers, ANP, β‐MHC and α‐SKA in NCM. Treatment with VCP746 at concentrations as low as 1 nmol/L suppressed mRNA expression of ANP, β‐MHC and α‐SKA stimulated by IL‐1β, TNF‐α or Ang II, demonstrating the broad mechanistic basis of the potent anti‐hypertrophic effect of VCP746. This study has shown that the novel A₁AR agonist, VCP746, is able to attenuate cardiac myocyte hypertrophy. As such, VCP746 is potentially useful as a pharmacological agent in attenuating cardiac remodelling, especially in the post‐myocardial infarction setting, given its previously established cytoprotective properties.     

MET inhibitor tepotinib antagonizes multidrug resistance mediated by ABCG2 transporter: In vitro and in vivo study

Overexpression of ABCG2 transporter in cancer cells has been linked to the development of multidrug resistance (MDR), an obstacle to cancer therapy. Our recent study uncovered that the MET inhibitor, tepotinib, is a potent reversal agent for ABCB1-mediated MDR. In the present study, we reported for the first time that the MET inhibitor tepotinib can also reverse ABCG2-mediated MDR in vitro and in vivo by directly binding to the drug-binding site of ABCG2 and reversibly inhibiting ABCG2 drug efflux activity, therefore enhancing the cytotoxicity of substrate drugs in drug-resistant cancer cells. Furthermore, the ABCB1/ABCG2 double-transfected cell model and ABCG2 gene knockout cell model demonstrated that tepotinib specifically inhibits the two MDR transporters. In mice bearing drug-resistant tumors, tepotinib increased the intratumoral accumulation of ABCG2 substrate drug topotecan and enhanced its antitumor effect. Therefore, our study provides a new potential of repositioning tepotinib as an ABCG2 inhibitor and combining tepotinib with substrate drugs to antagonize ABCG2-mediated MDR.     

Rapid depression of rat liver microsomal calcium pump activity after administration of carbon tetrachloride or bromotrichloromethane and lack of effect after ethanol

Calcium sequestration was studied in microsomes prepared from the livers of rats given acute doses of carbon tetrachloride (CCl₄), bromotrichloromethane (BrCCl₃), or ethanol. Hepatic mitochondrial calcium uptake was also studied in ethanol-treated animals. The effects of dose and time are reported. It was found that a dose of 6 g ethanol/kg had no effect on either microsomal or mitochondrial calcium uptake during the first 20 hr after administration. After administration of 1.0–5.0 ml CCl₄/kg, calcium uptake was reduced 85% from controls in 10 min, and 3 min after administration of 2.5 ml BrCCl₃/kg calcium uptake was reduced 90% from controls. The inhibition of microsomal calcium uptake occurred at the same time as the appearance of microsomal lipid diene conjugates and slightly after the maximal incorporation of ¹⁴C from ¹⁴CCl₄ into rat liver microsomal lipids, as reported by Rao and Recknagel, 1968, Rao and Recknagel, 1969. Exp. Mol. Pathol.10, 219–228]. Decreased microsomal calcium pumping is one of the earliest signs of CCl₄ or BrCCl₃ hepatotoxicity. This finding supports the proposal that an early disturbance of calcium uptake may contribute to the chain of events leading from localized toxigenic haloalkane metabolism to the metabolic disorganization eventuating in cell death. The complete absence of any effects of acute ethanol administration on microsomal or mitochondrial calcium pump activity suggests that liver injury by ethanol does not involve lipid peroxidation in these organelles.     

Alkaloids from Lycoris caldwellii and their particular cytotoxicities against the astrocytoma and glioma cell lines

Phytochemical investigation of the ethanol extract of the bulbs of Lycoris caldwellii afforded four new alkaloids, (+)-N-methoxylcarbonyl-nandigerine (1), (+)-N-methoxycarbonyl-lindcarpine (2), (+)-10-O-methylhernovine N-oxide (3), and (+)-3-hydroxy-anhydrolycorine N-oxide (4). Structural elucidation of all the compounds were performed by spectral methods such as 1D and 2D (¹H-¹H COSY, HMQC, and HMBC) NMR spectroscopy, in addition to high resolution mass spectrometry. All the alkaloids were in vitro evaluated for their cytotoxic activities against eight tumor cell lines (BEN-MEN-1, CCF-STTG1, CHG-5, SHG-44, U251, BGC-823, HepG2, and SK-OV-3). Alkaloids 1 and 2 exhibited particular cytotoxic activities against astrocytoma and glioma cell lines with IC₅₀ of 9.2–11.3 μM and 10.4–12.2 μM respectively.     

Apoptosis-inducing activity of synthetic hydrocarbon-stapled peptides in H358 cancer cells expressing KRASG12C

Lung cancers are the leading cause of cancer deaths worldwide and pose a grave threat to human life and health. Non-small cell lung cancer (NSCLC) is the most frequent malignancy occupying 80% of all lung cancer subtypes. Except for other mutations (e.g., KRAS^G12V/D) that are also vital for the occurrence, KRAS^G12C gene mutation is a significant driving force of NSCLC, with a prevalence of approximately 14% of all NSCLC patients. However, there are only a few therapeutic drugs targeting KRAS^G12C mutations currently. Here, we synthesized hydrocarbon-stapled peptide 3 that was much shorter and more stable with modest KRAS^G12C binding affinity and the same anti-tumor effect based on the α-helical peptide mimic SAH-SOS1_A. The stapled peptide 3 effectively induced G2/M arrest and apoptosis, inhibiting cell growth in KRAS-mutated lung cancer cells via disrupting the KRAS-mediated RAF/MEK/ERK signaling, which was verified from the perspective of genomics and proteomics. Peptide 3 also exhibited strong anti-trypsin and anti-chymotrypsin abilities, as well as good plasma stability and human liver microsomal metabolic stability. Overall, peptide 3 retains the equivalent anti-tumor activity of SAH-SOS1_A but with improved stability and affinity, superior to SAH-SOS1_A. Our work offers a structural optimization approach of KRAS^G12C peptide inhibitors for cancer therapy.     

Lignan constituents of Tilia amurensis and their biological evaluation on antitumor and anti-inflammatory activities

In the recent decade, numerous lignan derivatives isolated from plants have been proven to have the potential as an anti-cancer substance. On the search for anti-cancer compounds from Korean medicinal plants, the methanolic extract from the trunk of Tilia amurensis Rupr. (Tiliaceae) was found to have significant cytotoxicity against A549 (lung carcinoma), SK-OV-3 (ovary malignant ascites), SK-MEL-2 (skin melanoma), and HCT-15 (colon adenocarcinoma) in our screening test. Hence, a bioassay-guided fractionation and chemical investigation of the methanolic extract resulted in the isolation and identification of 10 lignan derivatives (1–10) including two new lignan glycosides named tiliamurosides A (1) and B (2). The structures of these new compounds were determined by spectroscopic methods, namely 1D and 2D nuclear magnetic resonance (NMR) techniques, high resolution mass spectrometry (HRMS), circular dichroism (CD) data, and chemical methods. Tiliamuroside B (2) and schizandriside (3) showed significant cytotoxicity against A549, SK-OV-3, SK-MEL-2, and HCT-15 cell lines with inhibitory concentration (IC₅₀) values of 3.26–8.89 μM. Moreover, (−)-syringaresinol (8) and (−)-pinoresinol 4-O-β-d-glucopyranoside (10) significantly inhibited nitric oxide (NO) production in murine microglia BV-2 with IC₅₀ values of 15.05 and 34.35 μM, respectively.     

Overcoming the stromal barrier for targeted delivery of HPMA copolymers to pancreatic tumors

Delivery of macromolecules to pancreatic cancer is inhibited by a dense extracellular matrix composed of hyaluronic acid, smooth muscle actin and collagen fibers. Hyaluronic acid causes a high intratumoral fluidic pressure which prevents diffusion and penetration into the pancreatic tumor. This study involves the breaking down of hyaluronic acid by treating CAPAN-1 xenograft tumors in athymic nu/nu mice with targeted N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers radiolabeled with ¹¹¹In for single photon emission computerized tomography (SPECT) imaging. Two targeting strategies were investigated including α_vβ₃ integrin and HER2 receptors. HPMA copolymers were targeted to these receptors by conjugating short peptide ligands cRGDfK and KCCYSL to the side chains of the copolymer. Results demonstrate that tumor targeting can be achieved in vivo after treatment with hyaluronidase. This approach shows promise for enhanced delivery of polymer–peptide conjugates to solid tumors.     

Permeability characteristics of novel mydriatic agents using an in vitro cell culture model that utilizes sirc rabbit corneal cells

The purpose of this study was to evaluate the permeability characteristics of a previously reported in vitro corneal model that utilizes SIRC rabbbit corneal cells and to investigate the permeability of three novel esters of phenylephrone chemical delivery systems (CDS) under different pH conditions using this in vitro model. The SIRC rabbit corneal cell line was grown on transwell polycarbonate membranes, and the barrier properties were assessed by measuring transepithelial electrical resistance (TEER) using a voltohmmeter. The permeabilities of esters of phenylephrone CDS across the SIRC cell layers were measured over a pH range 4.0–7.4. The esters tested include phenylacetyl (1), isovaleryl (2), and pivalyl (3). The SIRC rabbit corneal cell line, when grown on permeable filters, formed tight monolayers of high electrical resistance with TEER values increasing from 71.6 ±20.8 Ω·cm² at day 3 in culture to 2233.42 ±15.2 Ω·cm² at day 8 in culture and remained constant through day 14 in culture. The transepithelial permeability coefficients (P_app) at pH 7.4 ranged from 0.58 × 10⁻⁶ cm/s for the hydrophilic marker, mannitol, to 43.5 × 10⁻⁶ cm/s for the most lipophilic molecule, testosterone. The Papp at pH 7.4 for phenylephrine was 4.21 × 10⁻⁶ cm/s. The P_app values and the lag times of the three esters of phenylephrone were pH dependent. The P_app for 1, 2, and 3 at pH 7.4 were 14.76 × 10⁻⁶, 13.19 × 10⁻⁶, and 12.86 × 10⁻⁶ cm/s, respectively and the permeabilities decreased at conditions below pH 7.4. The lag times at pH 7.4 were 0.10, 0.17, and 0.12 h for 1, 2, and 3, respectively, and the values increased at lower pH conditions. The TEER values of SIRC cell line observed at day 8 to day 14 in the present investigation are similar to the resistance value reported for rabbit cornea (2 kΩ·cm²). All the esters showed significantly (p < 0.05) higher permeabilities than phenylephrine at pH 7.4. The rate and extent of transport of the drugs across the cell layers were influenced by the fraction of ionized and un‐ionized species and the intrinsic partition coefficient of the drug. The results indicate that the permeability of ophthalmic drugs through ocular membranes may be predicted by measuring the permeability through the new in vitro cell culture model.     

Tanshinones from Chinese Medicinal Herb Danshen (Salvia miltiorrhiza Bunge) Suppress Prostate Cancer Growth and Androgen Receptor Signaling

Purpose

To test whether tanshinones inhibit prostate cancer (PCa) growth at least in part through inhibiting androgen receptor (AR) signaling.

Methods

We evaluated cell growth, survival and AR signaling parameters of PCa cells after exposure to tanshinones in in vitro models. We also tested the in vivo inhibitory efficacy of tanshinone IIA (TIIA) against LNCaP xenograft model in athymic nude mice.

Results

For androgen-dependent LNCaP cells, a colony growth assay showed strong inhibitory potency following the order of TIIA??cryptotanshinone>tanshinone I, being 10?C30 folds higher than Casodex (racemic). TIIA inhibited growth of LNCaP cells more than several androgen-independent PCa cell lines. All 3 tested tanshinones were devoid of AR agonist activity under castrate condition. Mechanistically, tanshinones inhibited AR nuclear translocation within 2?h, decreased protein and mRNA abundance of AR and its target prostate-specific antigen within 12?h, and stimulated proteosomal degradation of AR. Oral administration of TIIA (25?mg/kg, once daily) retarded LNCaP xenograft growth and down-regulated tumor AR abundance in athymic nude mice.

Conclusion

AR targeting action of tanshinones was distinct from Casodex and contributed to prostate cancer growth suppression in vitro and in vivo.     

In vitro antiproliferative and antiangiogenic effects of synthetic chalcone analogues

As flavonoids, chalcones possess a wide variety of biological activities including anticancer properties. In the present study we have investigated the in vitro antiproliferative and antiangiogenic effects of four synthetic chalcones.E-2-(4′-methoxybenzylidene)-1-benzosuberone (3) was the most active compound with IC₅₀ = 10⁻⁷ mol l⁻¹ in Jurkat cells. In both Jurkat and HeLa chalcone 3-treated cells we found a significant increase in the proportion of cancer cells in the G₂/M phase of the cell cycle as well as an increase in cells having sub-G₀/G₁ DNA content which is considered to be a marker of apoptotic cell death. Apoptosis was also confirmed by annexin V staining and DNA fragmentation. These effects were associated with reduced expression of the anti-apoptotic gene, Bcl-2, and increased expression of the pro-apoptotic gene, Bax.Furthermore, chalcone 3 was selected to evaluate its effect on some angiogenic events. In non-toxic concentrations, chalcone 3 inhibited VEGF-induced migration of human umbilical vein endothelial cells. Moreover, it also decreased secretion of matrix metalloproteinase (mainly MMP-9) and vascular endothelial growth factor (VEGF).In conclusion, the present study has assessed the in vitro antiproliferative/antiangiogenic potential of chalcone 3. This results generate a rationale for in vivo efficacy studies with this compound in preclinical cancer models.     

Interactions of dopamine and the release of [3H]-taurine and [3H]-glycine from the isolated retina of the rat

1 The dose-related, calcium-dependent, potassium-stimulated release of preloaded [³H]-dopamine from the superfused rat retina has been demonstrated.

2 A high-affinity uptake system for dopamine exists in rat retina in vitro; K_m value was calculated as 1.89 μM, V_max value as 1.4 nmol g^-1 tissue h^-1.

3 Dopamine (0.8 and 4 mM) inhibited the spontaneous release of [³H]-glycine from retina, and in the case of 0.8 mM dopamine this inhibitory effect was antagonized by 10 μM (+)-butaclamol but not by 10 μM (-)-butaclamol.

4 The potassium-evoked (25 mM) release of [³H]-glycine from rat retina was similarly inhibited by dopamine (0.4-4 mM) in a dose-related manner when added to the superfusate with the potassium. The effect of 0.8 mM dopamine was antagonized by 10 μM (+)-butaclamol but not by 10 μM (-)-butaclamol.

5 Dopamine (4 mM) significantly reduced the spontaneous release of [³H]-taurine from rat retina.

6 The potassium-stimulated (25 mM) release of [³H]-taurine occurred after the cessation of the depolarizing stimulus. This delayed release of [³H]-taurine was unaffected if dopamine was applied to the superfusate at the same time as the potassium, but it was significantly reduced if dopamine (0.8 and 4 mM) was applied after the depolarizing stimulus had been removed and during the actual amino acid release phase.

7 The inhibition of K⁺-stimulated (25 mM) delayed release of [³H]-taurine by applying dopamine (0.8 mM) after the depolarizing stimulus was blocked by 10 μM (+)-butaclamol but not by 10 μM (-)-butaclamol.

8 The results are discussed with respect to the possible neurotransmitter role for dopamine within the rat retina, and its possible interaction with glycine and taurine.