OBJECTIVE: To investigate the effects of rutaecarpine on high glucose-induced Alzheimer's disease-like pathological and cognitive dysfunction and its mechanism in rats. METHODS: Adult male SD rats were randomly divided into three groups (n=20): control group, high glucose group and rutaecarpine group. Rats in the control group were fed with conventional feed and tap water. The rats in the high glucose group were fed with conventional feed and 20% sucrose water. The rutaecarpine group was fed with fodder contain 0.01% rutaecarpine and 20% sucrose water. Morris water maze test was used to detect learning and memory and cognitive function of three groups rats after 24 weeks of feeding. Western blot analysis was used to detect tau protein at Thr205 and Ser214 sites in each group. Phosphorylation levels of GSK-3β in serine 9 site (S9-GSK-3β) and PP2A at cycline 307 site (Y307-PP2AC) were also detected. Immunohistochemistry further confirmed tau protein at Thr205 site in each group both in hippocampus and cortex. RESULTS: Compared with the control group, Morris water maze results showed that the latency of finding the hidden platform of the rats in high glucose group was increased significantly and the number of crossing platforms and the target quadrant residence time were significantly decreased (all P＜0.05). Immunohistochemistry showed that the phosphorylation level of tau protein at Thr205 site was significantly increased in the high glucose group compared with the control group, and the phosphorylation level of tau protein at Thr205 site in the rutaecarpine group was higher than that in the high glucose group. Western blot analysis showed that the phosphorylation level of tau protein in the high glucose group was significantly increased at Thr205 and Ser214 site compared with the control group, but the phosphorylation level of pS9-GSK-3β was significantly decreased (all P ＜0.05). Compared with the high glucose group, the latency of finding the hidden platform of the rats in rutaecarpine group was significantly decreased, and the number of crossing platforms and the target quadrant residence time were significantly increased (both P＜0.05). Compared with the high glucose group, the phosphorylation levels of tau protein at Thr205 and Ser214 sites showed a significant decrease, but the phosphorylation level of pS9-GSK-3β was significantly increased (all P＜0.05). CONCLUSION Rutaecarpine can alleviate AD-like cognitive dysfunction induced by high glucose, possibly by enhancing pS9-GSK-3β phosphorylation, down-regulating GSK-3β activity, and thus reducing hyperphosphorylation of tau-associated sites.
Alzheimer Disease ; chemically induced ; drug therapy ; Animals ; Cognitive Dysfunction ; chemically induced ; drug therapy ; Glucose ; Glycogen Synthase Kinase 3 beta ; chemistry ; Indole Alkaloids ; pharmacology ; Male ; Maze Learning ; Phosphorylation ; Quinazolines ; pharmacology ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; tau Proteins ; chemistry

BACKGROUND: Tumor recurrence and drug resistance are the main causes of death in tumor patients. The family of acetaldehyde dehydrogenase (ALDH) is closely related to the proliferation, migration, invasion and resistance of tumor cells, and different ALDH subtypes are expressed in different tumor cells. The aim of this study is to elucidate the ALDH subtype in human lung adenocarcinoma HCC-827/GR cells, which resistant to the gefitinib. METHODS: The human lung adenocarcinoma HCC-827 cells were used to generate the gefitinib-resistant HCC-827/GR cells; the expression of ALDH subtype in either HCC-827 or HCC-827/GR was detected by flow cytometry; The proliferative capacity and sensitivity to gefitinib of hcc-827/GR cells were analyzed by MTT assay before and after treatment with 100 μmol/L diethyllaminaldehyde (DEAB); Real-time quantitative PCR was used to detect the expression of ALDH subtypes at mRNA levels in hcc-827 cells and hcc-827/GR cells. RESULTS: Compared with HCC-827 cells, the positive rate of ALDH in HCC-827/GR cells increased. The proliferation ability of HCC-827/GR cells decreased after treatment with 100 μmol/L DEAB. Compared with HCC-827 cells, the expression of ALDH1A1 and ALDH1L1 mRNA was increased in hcc-827/GR cells, but the ALDH3B2 expression was decreased. CONCLUSIONS ALDH might be used as a molecular biomarker to test the gefitinib-resistant to lung adenocarcinoma cancer cells, and the ALDH1A1 may play a role in gefitinib resistance in lung cancer.
Adenocarcinoma ; pathology ; Adenocarcinoma of Lung ; Aldehyde Oxidoreductases ; antagonists & inhibitors ; genetics ; Cell Line, Tumor ; Drug Resistance, Neoplasm ; genetics ; Enzyme Inhibitors ; pharmacology ; Gefitinib ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; Lung Neoplasms ; pathology ; Quinazolines ; pharmacology

OBJECTIVETo evaluate the potential pharmacokinetic interactions of the anticancer agent gefitinib (Iressa®) and the oriental medications Guipi Decoction (, GPD, Guibi-tang in Korean) and Bawu Decoction (, BWD, Palmul-tang in Korean).

METHODSMethylcellulose (MC, control), GPD (1,200 mg/kg), or BWD (6,000 mg/kg) was orally administered to rats either as a single dose or multiple doses prior to gefitinib administration. To examine the effects of a single dose of the herbal medicines, gefitinib (10 mg/kg) was orally administered after 5 min or 1 h of MC or the herbal medicine pretreatments. To examine the effects of the multiple doses of the herbal medicines, gefitinib (10 mg/kg) was orally administered following 7 consecutive days of the administration of MC or each herbal medicine. The plasma concentrations of gefitinib were determined with liquid chromatography-tandem mass spectrometry assay. The plasma concentration-time profiles of gefitinib were analyzed with a noncompartmental analysis.

RESULTSGefitinib was rapidly absorbed and showed a monoexponential decline with an elimination half-life of 3.7-4.1 h. The pharmacokinetics of gefitinib was not affected by GPD pretreatment. However, a significantly lower maximum plasma concentration (C, P<0.05) and area under the curve (P<0.05), and a delayed time to reach C (T, P<0.01) were observed in both single- and multipledose BWD-pretreated rats compared with the control rats.

CONCLUSIONSBWD and not GPD might delay and interfere with gefitinib absorption. Further evaluations of the clinical significance of these findings are needed.

Animals ; Chromatography, Liquid ; Dose-Response Relationship, Drug ; Drugs, Chinese Herbal ; pharmacology ; Male ; Quinazolines ; administration & dosage ; blood ; pharmacokinetics ; Rats, Sprague-Dawley ; Tandem Mass Spectrometry ; Time Factors

OBJECTIVETo investigate the effects of evodiamine (Evo), a component of Evodiaminedia rutaecarpa (Juss.) Benth, on cardiomyocyte hypertrophy induced by angiotensin II (Ang II) and further explore the potential mechanisms.

METHODSCardiomyocytes from neonatal Sprague Dawley rats were isolated and characterized, and then the cadiomyocyte cultures were randomly divided into control, model (Ang II 0.1 μmol/L), and Evo (0.03, 0.3, 3 μmol/L) groups. The cardiomyocyte surface area, protein level, intracellular free calcium ([Ca]) concentration, activity of nitric oxide synthase (NOS) and content of nitric oxide (NO) were measured, respectively. The mRNA expressions of atrial natriuretic factor (ANF), calcineurin (CaN), extracellular signal-regulated kinase-2 (ERK-2), and endothelial nitric oxide synthase (eNOS) of cardiomyocytes were analyzed by real-time reverse transcriptionpolymerase chain reaction. The protein expressions of calcineurin catalytic subunit (CnA) and mitogen-activated protein kinase phosphatase-1 (MKP-1) were detected by Western blot analysis.

RESULTSCompared with the control group, Ang II induced cardiomyocytes hypertrophy, as evidenced by increased cardiomyocyte surface area, protein content, and ANF mRNA expression; increased intracellular free calcium ([Ca]) concentration and expressions of CaN mRNA, CnA protein, and ERK-2 mRNA, but decreased MKP-1 protein expression (P<0.05 or P<0.01). Compared with Ang II, Evo (0.3, 3 μmol/L) significantly attenuated Ang II-induced cardiomyocyte hypertrophy, decreased the [Ca] concentration and expressions of CaN mRNA, CnA protein, and ERK-2 mRNA, but increased MKP-1 protein expression (P<0.05 or P<0.01). Most interestingly, Evo increased the NOS activity and NO production, and upregulated the eNOS mRNA expression (P<0.05).

CONCLUSIONEvo signifificantly attenuated Ang II-induced cardiomyocyte hypertrophy, and this effect was partly due to promotion of NO production, reduction of [Ca]i concentration, and inhibition of CaN and ERK-2 signal transduction pathways.

Angiotensin II ; Animals ; Atrial Natriuretic Factor ; metabolism ; Calcineurin ; genetics ; metabolism ; Calcium ; metabolism ; Dual Specificity Phosphatase 1 ; genetics ; metabolism ; Extracellular Signal-Regulated MAP Kinases ; genetics ; metabolism ; Hypertrophy ; Myocytes, Cardiac ; drug effects ; metabolism ; pathology ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type III ; metabolism ; Quinazolines ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Rats, Sprague-Dawley

OBJECTIVETo investigate the effect and potential mechanisms of rutaecarpine (Rut) in a rat artery balloon-injury model.

METHODSThe intimal hyperplasia model was established by rubbing the endothelia with a balloon catheter in the common carotid artery (CCA) of rats. Fifty rats were randomly divided into five groups, ie. sham, model, Rut (25, 50 and 75 mg/kg) with 10 rats of each group. The rats were treated with or without Rut (25, 50, 75 mg/kg) by intragastric administration for 14 consecutive days following injury. The morphological changes of the intima were evaluated by hematoxylin-eosin staining. The expressions of proliferating cell nuclear antigen (PCNA) and smooth muscle (SM) α-actin in the ateries were assayed by immunohistochemical staining. The mRNA expressions of c-myc, extracellular signal-regulated kinase 2 (ERK2), MAPK phosphatase-1 (MKP-1) and endothelial nitric oxide synthase (eNOS) were determined by real-time reverse transcription-polymerase chain reaction. The protein expressions of MKP-1 and phosphorylated ERK2 (p-ERK2) were examined by Western blotting. The plasma contents of nitric oxide (NO) and cyclic guanosine 3',5'-monophosphate (cGMP) were also determined.

RESULTSCompared with the model group, Rut treatment significantly decreased intimal thickening and ameliorated endothelial injury (P<0.05 or P<0.01). The positive expression rate of PCNA was decreased, while the expression rate of SM α-actin obviously increased in the vascular wall after Rut (50 and 75 mg/kg) administration (P<0.05 or P<0.01). Furthermore, the mRNA expressions of c-myc, ERK2 and PCNA were downregulated while the expressions of eNOS and MKP-1 were upregulated (P<0.05 or P<0.01). The protein expressions of MKP-1 and the phosphorylation of ERK2 were upregulated and downregulated after Rut (50 and 75 mg/kg) administration (P<0.05 or P<0.01), respectively. In addition, Rut dramatically reversed balloon injury-induced decrease of NO and cGMP in the plasma (P<0.05 or P<0.01).

CONCLUSIONRut could inhibit the balloon injury-induced carotid intimal hyperplasia in rats, possibly mediated by promotion of NO production and inhibiting ERK2 signal transduction pathways.

Actins ; metabolism ; Animals ; Carotid Arteries ; drug effects ; metabolism ; pathology ; Carotid Artery Injuries ; drug therapy ; genetics ; pathology ; Cyclic GMP ; blood ; Disease Models, Animal ; Gene Expression Regulation ; drug effects ; Hyperplasia ; Indole Alkaloids ; pharmacology ; therapeutic use ; Male ; Nitric Oxide ; blood ; Phosphorylation ; drug effects ; Proliferating Cell Nuclear Antigen ; metabolism ; Quinazolines ; pharmacology ; therapeutic use ; RNA, Messenger ; genetics ; metabolism ; Rats, Sprague-Dawley ; Tunica Intima ; drug effects ; pathology

PURPOSE: Rearrangement of the proto-oncogene rearranged during transfection (RET) has been newly identified potential driver mutation in lung adenocarcinoma. Clinically available tyrosine kinase inhibitors (TKIs) target RET kinase activity, which suggests that patients with RET fusion genes may be treatable with a kinase inhibitor. Nevertheless, the mechanisms of resistance to these agents remain largely unknown. Thus, the present study aimed to determine whether epidermal growth factor (EGF) and hepatocyte growth factor (HGF) trigger RET inhibitor resistance in LC-2/ad cells with CCDC6-RET fusion genes. MATERIALS AND METHODS: The effects of EGF and HGF on the susceptibility of a CCDC6-RET lung cancer cell line to RET inhibitors (sunitinib, E7080, vandetanib, and sorafenib) were examined. RESULTS: CCDC6-RET lung cancer cells were highly sensitive to RET inhibitors. EGF activated epidermal growth factor receptor (EGFR) and triggered resistance to sunitinib, E7080, vandetanib, and sorafenib by transducing bypass survival signaling through ERK and AKT. Reversible EGFR-TKI (gefitinib) resensitized cancer cells to RET inhibitors, even in the presence of EGF. Endothelial cells, which are known to produce EGF, decreased the sensitivity of CCDC6-RET lung cancer cells to RET inhibitors, an effect that was inhibited by EGFR small interfering RNA (siRNA), anti-EGFR antibody (cetuximab), and EGFR-TKI (Iressa). HGF had relatively little effect on the sensitivity to RET inhibitors. CONCLUSION: EGF could trigger resistance to RET inhibition in CCDC6-RET lung cancer cells, and endothelial cells may confer resistance to RET inhibitors by EGF. E7080 and other RET inhibitors may provide therapeutic benefits in the treatment of RET-positive lung cancer patients.
Adenocarcinoma/drug therapy/*genetics ; Cell Line, Tumor ; Cetuximab/pharmacology ; Drug Resistance, Neoplasm/drug effects/*genetics ; Epidermal Growth Factor/metabolism/*pharmacology ; *Gene Rearrangement ; Hepatocyte Growth Factor/*pharmacology ; Humans ; Indoles/pharmacology ; Lung Neoplasms/drug therapy/*genetics ; MAP Kinase Signaling System ; *Mutation ; Niacinamide/analogs & derivatives/pharmacology ; Phenylurea Compounds/pharmacology ; Piperidines/pharmacology ; Protein Kinase Inhibitors/therapeutic use ; Proto-Oncogene Proteins c-ret/*antagonists & inhibitors/genetics ; Pyrroles/pharmacology ; Quinazolines/pharmacology ; RNA, Small Interfering/pharmacology ; Receptor, Epidermal Growth Factor/genetics/metabolism ; Signal Transduction/drug effects ; fms-Like Tyrosine Kinase 3/metabolism

Specific gene expressions of host cells by spontaneous STAT6 phosphorylation are major strategy for the survival of intracellular Toxoplasma gondii against parasiticidal events through STAT1 phosphorylation by infection provoked IFN-γ. We determined the effects of small molecules of tyrosine kinase inhibitors (TKIs) on the growth of T. gondii and on the relationship with STAT1 and STAT6 phosphorylation in ARPE-19 cells. We counted the number of T. gondii RH tachyzoites per parasitophorous vacuolar membrane (PVM) after treatment with TKIs at 12-hr intervals for 72 hr. The change of STAT6 phosphorylation was assessed via western blot and immunofluorescence assay. Among the tested TKIs, Afatinib (pan ErbB/EGFR inhibitor, 5 µM) inhibited 98.0% of the growth of T. gondii, which was comparable to pyrimethamine (5 µM) at 96.9% and followed by Erlotinib (ErbB1/EGFR inhibitor, 20 µM) at 33.8% and Sunitinib (PDGFR or c-Kit inhibitor, 10 µM) at 21.3%. In the early stage of the infection (2, 4, and 8 hr after T. gondii challenge), Afatinib inhibited the phosphorylation of STAT6 in western blot and immunofluorescence assay. Both JAK1 and JAK3, the upper hierarchical kinases of cytokine signaling, were strongly phosphorylated at 2 hr and then disappeared entirely after 4 hr. Some TKIs, especially the EGFR inhibitors, might play an important role in the inhibition of intracellular replication of T. gondii through the inhibition of the direct phosphorylation of STAT6 by T. gondii.
Antiparasitic Agents/pharmacology ; Blotting, Western ; Cell Line ; Enzyme Activation/drug effects ; Fluorescent Antibody Technique ; Humans ; Janus Kinase 1/metabolism ; Janus Kinase 3/metabolism ; Phosphorylation/drug effects ; Quinazolines/*pharmacology ; STAT6 Transcription Factor/*metabolism ; Signal Transduction/*drug effects ; Toxoplasma/*drug effects/physiology ; Toxoplasmosis/physiopathology

Gefitinib is used as a first-line treatment for advanced non-small cell lung cancer (NSCLC). Unfortunately, most NSCLC patients inevitably develop gefitinib resistance during treatment. In addition to EGFR mutation status, the mechanisms involved are largely unknown. In this study, we showed that miR-124, a tumor suppressor, was significantly down-regulated in gefitinib-resistant NSCLC patients and cell lines compared with gefitinib-sensitive patients and cell lines. In addition, the miR-124 depletion induced gefitinib resistance, and miR-124 overexpression sensitized gefitinib-resistant cells to gefitinib. Mechanistic analysis revealed that miR-124 decreased SNAI2 and STAT3 expression by directly targeting their 3'UTRs and that knocking down SNAI2 or STAT3 partly reversed the gefitinib resistance induced by miR-124 depletion. Our data demonstrate that the miR-124 plays a new critical role in acquired resistance to gefitinib and that the manipulation of miR-124 might provide a therapeutic strategy for reversing acquired gefitinib resistance.
3' Untranslated Regions ; Antineoplastic Agents ; pharmacology ; therapeutic use ; Carcinoma, Non-Small-Cell Lung ; drug therapy ; genetics ; metabolism ; Cell Line, Tumor ; Drug Resistance, Neoplasm ; genetics ; HEK293 Cells ; Humans ; Lung Neoplasms ; drug therapy ; genetics ; metabolism ; MicroRNAs ; genetics ; Quinazolines ; pharmacology ; therapeutic use ; STAT3 Transcription Factor ; genetics ; metabolism ; Snail Family Transcription Factors ; genetics ; metabolism

OBJECTIVETo determine the effect of the combination of lapatinib with chlorogenic acid on metastasis of breast cancer in mouse model.

METHODSThe classical macrophage M2 polarization model induced by interlukin13in vitro was adopted in the study. Flow cytometric analysis was performed to detect the expression of M2 marker CD206. The transcription of M2-associated genes was measured by RT-PCR. HE staining was used to analyze the metastatic nodes of breast cancer in lungs of MMTV-PyVT mice. Immunostaining analysis was used to detect the expression of related proteins in breast cancer.

RESULTSThe combination of lapatinib and chlorogenic acid inhibited the expression of CD206 induced by IL-13[(42.17%±2.59%) vs (61.15%±7.58%), P<0.05]. The combination more markedly suppressed expression of M2-associated gene Ym1 than lapatinib alone[(0.9±0.1) vs (1.8±0.0), P<0.05]. The combination of lapatinib and chlorogenic acid significantly reduced metastatic nodes in lung[P<0.05], and also significantly decreased the percentage of CD206(+) cells in breast cancer compared to controls[(6.08%±2.60%) vs(29.04%±5.86%), P<0.05].

CONCLUSIONThe combination of lapatinib and chlorogenic acid can effectively inhibit macrophage M2 polarization and metastasis of breast cancer.

Animals ; Chlorogenic Acid ; pharmacology ; Female ; Lung Neoplasms ; drug therapy ; secondary ; Macrophages ; drug effects ; Mammary Neoplasms, Experimental ; drug therapy ; Mice ; Neoplasm Metastasis ; drug therapy ; Quinazolines ; pharmacology

OBJECTIVETo observe the inhibitory effect of gefitineb on the proliferation and its inducing effect on the apoptosis of mouse I-10 Leydig testicular cancer cells in vitro.

METHODSWe treated I-10 Leydig testicular cancer cells of mice with gefitineb at 0, 1.25, 2.5, 5, 10, 20, and 40 µmol/L. Then we determined the inhibitory effect of gefitineb on the growth of the cells by MTT, detected their early and late apoptosis by Annexin V-FITC/propidium iodide double staining and Hoechst 33258 nuclear staining, respectively, and observed the expressions of apoptosis-related proteins Bcl-2, Bax and caspase 3/9 by Western blot.

RESULTSCompared with the blank control group, gefitineb significantly inhibited the proliferation of the I-10 cells at 10 and 20 µmol/L (P < 0.05). The survival rate of the cells was (32.4 ± 2.8)% (P < 0.01) and their early and late apoptosis rates were (26.7 ± 4.2)% and (59.33 ± 10.2)% in the 40 µmol/L group, significantly different from those in the control (P < 0.05 and P <0.01). In comparison with the blank control group, gefitineb at 10, 20, and 40 µmol/L increased the expression of pro-apoptotic protein Bax by (41.9 ± 7.1), (60.1 ± 9.8), and (69.0 ± 11.3)% (all P < 0.05), decreased that of apoptosis-inhibitory protein Bcl-2 by (50.3 ± 8.9), (63.9 ± 6.9), and (88.7 ± 13.9)% (all P < 0.05), and elevated that of the cleft proteins caspase-3 by (69.0 ± 6.9)% (P < 0.05), (71.5 ± 8.1)% (P < 0.05), and (110.9 ± 14.2)% (P < 0.01) and caspase-9 by (51.8 ± 4.9), (54.7 ± 6.7), and (43.8 ± 11.8)% (all P < 0.05).

CONCLUSIONGefitineb can increase the cytotoxicity of I-10 Leydig testicular cancer cells of mice and induce their apoptosis via the mitochondria-mediated apoptosis signaling pathway.

Animals ; Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; metabolism ; Caspase 3 ; metabolism ; Caspase 9 ; metabolism ; Cell Proliferation ; drug effects ; Cell Survival ; Leydig Cell Tumor ; drug therapy ; metabolism ; pathology ; Male ; Mice ; Neoplasm Proteins ; metabolism ; Neoplasms, Germ Cell and Embryonal ; drug therapy ; metabolism ; pathology ; Quinazolines ; pharmacology ; Testicular Neoplasms ; drug therapy ; metabolism ; pathology ; bcl-2-Associated X Protein ; metabolism