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

OBJECTIVETo explore whether MG-132 could enhance the anti-tumor activity of obatoclax against esophageal cancer cell line CaES-17.

METHODSMTT assay was used to determine the cytotoxicity of obatoclax and MG-132 in CaES-17 cells. The IC(50) of obatoclax and MG-132 were used to determine the molar ratio (1:2.4) of the two drugs for combined treatment of the cells. The concentrations of obatoclax and MG-132 ranged from 1/8 IC(50) to 4 IC(50) after serial dilution, and their combination index (CI) was calculated using CompuSyn software. The expression of ubiquitin and the cleavage of PARP, caspase-9, phospho-histone H3 and phospho-aurora A/B/C in the exposed cells were examined with Western blotting; the cell apoptosis was measured by flow cytometry with Annexin V staining, and the percentage of cells in each cell cycle phase was also determined by flow cytometry.

RESULTSThe CI of obatoclax and MG-132 was 0.296 for a 50% inhibition of Caes-17 cells and was 0.104 for a 95% inhibition. The cells treated with obatoclax or MG-132 alone showed increased expression of ubiquitin and cleavage of PARP and caspase-9. Compared with the cells treated with obatoclax or MG-132 alone, the cells with a combined treatment exhibited significantly increased expression of ubiquitin, cleavage of PARP and caspase-9, and expression of phospho-Histone H3 (P<0.05). The combined treatment of the cells also resulted in significantly increased expression of phospho-Aurora A/B/C compared with obatoclax treatment alone. The cells with the combined treatment showed significantly higher percentages of apoptotic cells and cells in sub-G(1) and G(2)/M phases compared with the cells treated with either of the drugs (P<0.05).

CONCLUSIONObatoclax combined with MG-132 shows a significant synergistic anti-tumor effect against esophageal cancer CaES-17 cells by inducing apoptosis and cell cycle arrest.

Apoptosis ; Caspase 9 ; metabolism ; Cell Cycle Checkpoints ; Cell Line, Tumor ; drug effects ; Esophageal Neoplasms ; pathology ; Histones ; metabolism ; Humans ; Leupeptins ; pharmacology ; Poly (ADP-Ribose) Polymerase-1 ; Poly(ADP-ribose) Polymerases ; metabolism ; Pyrroles ; pharmacology

OBJECTIVETo investigate the efficacy and safety of sunitinib as first-line therapy for metastatic renal cell carcinoma (mRCC) on a 2 weeks on/1 week off intermittent dosing schedule.

METHODSA total of 11 mRCC patients were enrolled to receive sunitinib 50 mg/day in 2 weeks on/1 week off schedule per 6 weeks till disease progression or intolerable toxicity occurred. The primary end point was progression free survival (PFS), the secondary end points were overall survival (OS), incidence of adverse effects and objective response.

RESULTSThe objective response rate in the 11 cases was 45.5% and disease control rate 72.7% (partial response n = 5, stable disease n = 3). Till the last follow up on Dec 2013, the median PFS was 17.0 months (95% CI 7.3 to 26.7 months), and median OS 26.0 months (95% CI 2.2 to 49.8 months). The common adverse events included leucopenia, thrombocytopenia, diarrhea, mucositis and hand-foot skin reaction. Dose reduction to 37.5 mg was seen only in 2 patients without discontinuation.

CONCLUSIONSSunitinib on an intermittent dosing schedule 2 weeks on /1 week off as first-line therapy for mRCC patients shows a good efficacy and tolerance, with less grade 3-4 drug-related toxicities and a tendency of prolonged PFS in mRCC patients.

Antineoplastic Agents ; administration & dosage ; pharmacology ; therapeutic use ; Carcinoma, Renal Cell ; drug therapy ; Disease-Free Survival ; Drug Administration Schedule ; Humans ; Indoles ; administration & dosage ; pharmacology ; therapeutic use ; Kidney Neoplasms ; drug therapy ; Pilot Projects ; Pyrroles ; administration & dosage ; pharmacology ; therapeutic use ; Treatment Outcome

OBJECTIVETo investigate whether SIS3, a specific inhibitor of Smad3 phosphorylation, can reverse the stemness of multidrug-resistant(MDR) hepatocellular carcinoma cells.

METHODSMDR HCC Huh7.5.1/ADM cell lines were developed by exposing parental cells to stepwise increasing concentrations of ADM. CCK-8 assay was used to determine the cellular sensitivity of various anticancer drugs. Flow cytometry (FCM) was used to analyze the expression level of cancer stem cell marker CD133. Clone formation assay and mouse subcutaneous xenograft tumors were used to investigate the tumorigenicity in vitro and in vivo. Western blotting (WB) was used to analyze the changes of expressions of CD133, Smad3, Bcl-2, Bax and p-Smad3 in different conditions.

RESULTSADM treatment of HCC cells in vitro resulted in a development of subline, Huh7.5.1/ADM cells, with CSC phenotypes: stable MDR phenotype (besides ADMc Huh7.5.1/ADM cells were also more resistant to some other anticancer drugs including VCR, MMC and CTX ) (IC50: 0.215 ± 0.018 vs. 0.123 ± 0.004, 0.145 ± 0.009 vs. 0.014 ± 0.002, 1.021 ± 0.119 vs. 0.071 ± 0.006, 27.007 ± 1.606 vs. 1.919 ± 0.032) (unit: µg/ml) (P<0.05). Huh7.5.1/ADM cells enriched the cancer stem-like cell fraction (CD133-positive subpopulation) (76.06 ± 2.948% vs. 25.38 ± 4.349%) (P<0.05), had stronger tumorigenicity in vivo and colony formation ability, and activated the Smad3 activity. Inhibition of Smad3 activity by SIS3 decreased stemness of the Huh7.5.1/ADM cells: CD133-positive subpopulation (48.49 ± 2.304% vs. 76.06 ± 2.948%) (P<0.05); ADM IC50: (0.112 ± 0.019 vs. 0.215 ± 0.018), VCR IC50 (0.065 ± 0.013 vs. 0.145±0.009), MMC IC₅₀ (0.749 ± 0.121 vs. 1.021 ± 0.119), CTX IC50 (10.576 ± 1.248 vs. 27.007 ± 1.606) (unit: µg/ml) (P<0.05), and decreased tumorigenicity and colony formation ability.

CONCLUSIONSIS3 as a specific inhibitor of Smad3 signal is involved in the stemness of multidrug resistant hepatocellular carcinoma cells.

AC133 Antigen ; Animals ; Antibiotics, Antineoplastic ; pharmacology ; Antigens, CD ; metabolism ; Carcinoma, Hepatocellular ; drug therapy ; metabolism ; pathology ; Doxorubicin ; pharmacology ; Drug Resistance, Neoplasm ; Glycoproteins ; metabolism ; Heterografts ; Humans ; Isoquinolines ; pharmacology ; Liver Neoplasms ; drug therapy ; metabolism ; pathology ; Mice ; Neoplasm Proteins ; metabolism ; Neoplastic Stem Cells ; drug effects ; Peptides ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Pyridines ; pharmacology ; Pyrroles ; pharmacology ; Smad3 Protein ; antagonists & inhibitors ; metabolism ; Tumor Stem Cell Assay ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo investigate the effect of sunitinib on the migration of ovarian cells and its mechanism of the negative regulation TGF-β mediated of epithelial-mesenchymal transition(EMT) by sunitinib to inhibit ovarian cancer metastasis.

METHODSThe migration of human ovarian cancer cells SKOV3 was evaluated by wound-healing and transwell assays. The effects of sunitinib on TGF-β-induced E-cadherin expression was assessed by Western-blotting, real time RT-PCR and immunofluorescence assay. The protein levels of Snail and the transcriptional activity of Smad in sunitinib-treated cells were examined by Western-blotting and SBE-luciferase assay.

RESULTSSunitinib suppressed the migration of SKOV3 cells in a concentration-dependent manner. TGF-β stimulation reduced E-cadherin protein level, which was attenuated by sunitinib. Sunitinib inhibited the up-regulation of Snail protein level induced by TGF-β treatment. The SBE reporter was constructed by linking the Smad-binding elements promoter upstream of luciferase reporter gene. A remarkable increment of transcriptional activity of Smads complexes was observed in SKOV3 cells exposed to TGF-β, which was significantly prohibited by sunitinib.

CONCLUSIONSunitinib can inhibit the migration of SKOV3 cells and attenuate the down-regulation of E-cadherin protein level induced by TGF-β. Sunitinib can abolish TGF-β-induced up-regulation of Snail protein and decrease the transcriptional activity of Smad complexes. The results indicate that sunitinib suppresses migration of ovarian cancer cells through negative modulation of TGF-β-mediated epithelial-mesenchymal transition.

Cadherins ; metabolism ; Cell Line, Tumor ; drug effects ; Cell Movement ; drug effects ; Down-Regulation ; Epithelial-Mesenchymal Transition ; Female ; Humans ; Indoles ; pharmacology ; Ovarian Neoplasms ; pathology ; Pyrroles ; pharmacology ; Smad Proteins ; metabolism ; Snail Family Transcription Factors ; Transcription Factors ; metabolism ; Transforming Growth Factor beta ; pharmacology ; Up-Regulation

OBJECTIVETo investigate the growth-inhibitory effect of sunitinib malate on human bladder transitional cell carcinoma (TCC) in vitro.

METHODSHuman bladder TCC cell line T24 was cultured and exposed to graded concentrations of sunitinib malate for 72 hours in vitro to determine the sensitivities to drug. Cell viability was measured by MTT assay. Cell apoptotic morphology was observed by fluorescence microscope following DAPI staining. Band expressions of Fas, Fas ligand, poly (ADP-ribose) polymerase (PARP) and β-actin were analyzed by Western blot. Wound healing process of T24 cells exposed to sunitinib malate was assayed.

RESULTSSunitinib malate exerted a concentration-dependent and time-dependent inhibitory effect on the T24 cell lines. Fluorescence microscopy showed that small vacuoles appeared in the nuclei of T24 cells and the vacuoles were bigger with higher drug concentrations. The expressions of Fas ligand and PARP in T24 cells treated with sunitinib malate exhibited a concentration-dependent increase. Moreover sunitinib malate suppressed the wound healing process in a concentration-dependent manner.

CONCLUSIONSunitinib malate exerted marked inhibitory activity against bladder cancer cell line T24.

Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Carcinoma, Transitional Cell ; metabolism ; pathology ; Cell Line, Tumor ; Fas Ligand Protein ; metabolism ; Humans ; In Vitro Techniques ; Indoles ; pharmacology ; Poly(ADP-ribose) Polymerases ; metabolism ; Pyrroles ; pharmacology ; Urinary Bladder Neoplasms ; metabolism ; pathology ; Wound Healing ; drug effects ; fas Receptor ; metabolism

The ocean continues to provide a plethora of unique scaffolds capable of remarkable biological applications. A large number of pyrroloiminoquinone alkaloids, including discorhabdins, epinardins, batzellines, makaluvamines, and veiutamine, have been isolated from various marine organisms. A class of pyrroloiminoquinone-related alkaloids, known as bispyrroloquinones, is the focus of this review article. This family of marine alkaloids, which contain an aryl substituted bispyrroloquinone ring system, includes three subclasses of alkaloids namely, wakayin, tsitsikammamines A-B, and zyzzyanones A-D. Both wakayin and the tsitsikammamines contain a tetracyclic fused bispyrroloiminoquinone ring system, while zyzzyanones contain a fused tricyclic bispyrroloquinone ring system. The unique chemical structures of these marine natural products and their diverse biological properties, including antifungal and antimicrobial activity, as well as the potent, albeit generally nonspecific and universal cytotoxicities, have attracted great interest of synthetic chemists over the past three decades. Tsitsikammamines, wakayin, and several of their analogs show inhibition of topoisomerases. One additional possible mechanism of anticancer activity of tsitsikammamines analogs that has been discovered recently is through the inhibition of indoleamine 2, 3-dioxygenase, an enzyme involved in tumoral immune resistance. This review discusses the isolation, synthesis, and evaluation of bioactivities of bispyrroloquinone alkaloids and their analogs.
Alkaloids ; chemistry ; pharmacology ; Animals ; Anti-Infective Agents ; chemistry ; pharmacology ; Antineoplastic Agents ; chemistry ; pharmacology ; Biological Products ; chemistry ; pharmacology ; Humans ; Indole Alkaloids ; chemistry ; pharmacology ; Indoles ; chemistry ; pharmacology ; Pyrroles ; chemistry ; pharmacology ; Quinolines ; chemistry ; pharmacology ; Quinones ; chemistry ; pharmacology

OBJECTIVETo investigate the effects of Pyrroloquinoline quinine (PQQ) on hydrogen peroxide-induced apoptosis of Schwann cells (SCs) and its mechanism.

METHODSSCs were isolated and cultured in vitro, and identified by S-100 immunofluorescence staining. The cultured SCs were divided into control group, hydrogen peroxide-treated group, hydrogen peroxide and PQQ treated groups. The intracellular superoxide dismutase (SOD) and malondialdehyde (MDA) content was detected; the apoptotic rate of SCs induced by hydrogen peroxide was determined by flow cytometry assay. The Hoechst33342 staining was used to detect the nuclear fragmentation and apoptotic nuclear condensation of SCs; the Rhodamine123 staining was used to detect the changes of mitochondrial membrane potential in SCs, the Western blot analysis was used to detect the expression of Bcl-2 in hydrogen peroxide induced SCs.

RESULTSThe SOD activity was significantly decreased and MDA level was increased in H2O2 induced SCs (P < 0.05), after addition of PQQ, the SOD content increased and MDA content decreased (P < 0.05). Flow cytometry results showed that the early apoptotic rate was 58.8% in H2O2 induced SCs, which has significant difference compared with the control group (P < 0.05), after addition of 10, 50, 100 nmol/L PQQ, the apoptotic rates were reduced to 33.7%, 18.7%, 3.9% respectively, showing significantly different with injured group (P < 0.05). Hoechst 33342 staining showed that H2O2 induced SCs had typical morphological characteristics, such as uptake of nuclear chromatin, nuclear shrinkage, nuclear fragmentation phenomenon. The proportion of apoptotic cells after PQQ treatment reduced. Rhodamine staining results showed that the H2O2 induced mitochondrial membrane potential reduction in SCs, which was reversed by addition of PQQ. Western blot analysis showed that the expression of Bcl-2 was decreased in H2O2 induced SCs, while it increased significantly after addition of PQQ (P < 0.05).

CONCLUSIONPQQ has a protective effect on oxidative stress-induced apoptosis of SCs.

Apoptosis ; drug effects ; Benzimidazoles ; Cell Nucleus ; drug effects ; DNA Fragmentation ; Fluorescent Dyes ; Humans ; Hydrogen Peroxide ; pharmacology ; Malondialdehyde ; metabolism ; Oxidants ; pharmacology ; Oxidative Stress ; Pyrroles ; pharmacology ; Quinine ; pharmacology ; Quinolines ; pharmacology ; Schwann Cells ; cytology ; drug effects ; Superoxide Dismutase ; metabolism

OBJECTIVETo evaluate whether atorvastatin inhibits oxidized low-density lipoproteins (Ox-LDL)-stimulated foam cell formation from THP-1 macrophages by regulating the activation of peroxisome proliferator-activated receptor γ (PPARγ) and nuclear factor-κB (NF-κB). Methods THP-1 macrophages were pretreated with 10, 20, or 40 µmol/L atorvastatin for 2 h, and after washing with PBS twice, the cells were incubated with 60 µg/ml of Ox-LDL for 48 h. The quantity of intracellular lipid of the cells was detected with Oil red O staining and enzymatic fluorometric method. The expression of the scavenger receptors of CD36 and SRA were analyzed with Western blotting. We also examined the effect of atorvastatin on adenosine triphosphate (ATP)-binding cassette transporter A1 (ABCA1) expression and the activation of PPARγ and p-iκB, and further assessed the capacity of the macrophages to bind to Dil-oxLDL.

RESULTSAtorvastatin potently inhibited ox-LDL-induced macrophage-derived foam cell formation, down-regulated the expression of CD36 and SRA, and up-regulated the expression of ABCA1. Atorvastatin markedly suppressed the activation of PPARγ and p-iκB in ox-LDL-stimulated THP-1 macrophages (P<0.05) and significantly decreased the Dil-oxLDL-binding capacity of the macrophages (P<0.05).

CONCLUSIONAtorvastatin as an effective anti-atherosclerosis agent can suppress the activation of PPARγ and p-iκB to reduce lipid accumulation in macrophages.

ATP Binding Cassette Transporter 1 ; metabolism ; Atorvastatin Calcium ; Cell Line ; Foam Cells ; cytology ; drug effects ; Heptanoic Acids ; pharmacology ; Humans ; I-kappa B Proteins ; metabolism ; Lipoproteins, LDL ; metabolism ; Macrophages ; cytology ; drug effects ; NF-kappa B ; metabolism ; PPAR gamma ; metabolism ; Pyrroles ; pharmacology ; Signal Transduction ; drug effects ; Transcriptional Activation ; Up-Regulation

OBJECTIVETo investigate the effect of atorvastatin on exercise tolerance in patients with diastolic dysfunction and exercise-induced hypertension.

METHODSA randomized, double-blind, placebo-controlled prospective study was performed. Sixty patients with diastolic dysfunction (mitral flow velocity E/A <1) and exercise-induced hypertension (SBP>200 mm Hg) treated with atorvastatin (20 mg q.d) or placebo for 1 year. Cardiopulmonary exercise test and exercise blood pressure measurement were performed. Plasma B-natriuretic peptide (BNP) concentration at rest and at peak exercise, plasma high sensitive-C reaction protein (hs-CRP) and endothelin (ET) concentration were determined at baseline and after treatment.

RESULTSAfter treatment by atorvastatin, the resting SBP, pulse pressure, the peak exercise SBP and BNP were significantly decreased; and the exercise time, metabolic equivalent, maximal oxygen uptake and anaerobic threshold were increased. All of these parameters had significant differences with baseline levels (P<0.05) and the rest pulse pressure, the peak exercise SBP and BNP, and the exercise time had significant differences compared with placebo treatment (P<0.05). Plasma concentrations of hs-CRP and ET were markedly reduced by atorvastatin treatment compared with baseline and placebo (P<0.05). No difference in above parameters was found before and after placebo treatment (P>0.05).

CONCLUSIONIn patients with diastolic dysfunction at rest and exercise-induced hypertension, atorvastatin can effectively reduce plasma hs-CRP and ET level, lower blood pressure and peak exercise SBP, decrease peak exercise plasma BNP concentration, and ultimately improve exercise tolerance.

Aged ; Atorvastatin Calcium ; C-Reactive Protein ; metabolism ; Double-Blind Method ; Endothelins ; blood ; Exercise Tolerance ; drug effects ; Female ; Heart Failure ; complications ; drug therapy ; physiopathology ; Heptanoic Acids ; pharmacology ; Humans ; Hypertension ; complications ; drug therapy ; physiopathology ; Male ; Middle Aged ; Natriuretic Peptide, Brain ; blood ; Prospective Studies ; Pyrroles ; pharmacology