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 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 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 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

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

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 study the effect of compound Danshen dripping pills and atorvastatin on restenosis after abdominal aorta angioplasty in rabbits.

METHODSRabbit models of abdominal aorta restenosis after angioplasty were established and treated with saline (group A), compound Danshen dripping pills (group B), atorvastatin (group C), or compound Danshen dripping pills plus atorvastatin (group D). HE staining was used to determine the thickness of arterial intimal hyperplasia and assess the morphological changes of the narrowed artery. Immunohistochemistry was employed to detect the expression of nuclear factor-κB (NF-κB) and monocyte chemoattractant protein-1 (MCP-1).

RESULTSCompared with group A, the 3 treatment groups showed significant increased vascular cavity area and reduced intimal area and percentage of intimal hyperplasia (P<0.05). The vascular cavity area, intimal area and percentage of intimal hyperplasia levels differed significantly between group D and groups B and C (P<0.05). Immunohistochemistry showed a significant reduction of the expression rate of NF-κB and MCP-1 in the 3 treatment groups compared with group A (P<0.05), and the reduction was especially obvious in group D (P<0.05).

CONCLUTIONSCompound danshen dripping pills combined with atorvastatin produces better effects than the drugs used alone in inhibiting vascular smooth muscle cell proliferation in rabbits after abdominal aorta angioplasty possibly due to a decreased expression of MCP-1 as a result of NF-κB inhibition.

Angioplasty ; Animals ; Aorta ; pathology ; Atorvastatin Calcium ; Cell Proliferation ; Chemokine CCL2 ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Heptanoic Acids ; pharmacology ; Hyperplasia ; Myocytes, Smooth Muscle ; drug effects ; NF-kappa B ; metabolism ; Phenanthrolines ; Pyrroles ; pharmacology ; Rabbits ; Salvia miltiorrhiza ; chemistry ; Tunica Intima

OBJECTIVETo investigate the effect of atorvastatin on platelet aggregation and activation in the acute phase following balloon-induced carotid artery injury in rabbits fed cholesterol-enriched diet.

METHODSThirty rabbits were randomly divided into 5 equal groups, namely control group, high-cholesterol group, model group, low-dose (5 mg/kg daily) atorvastatin group, and high-dose (10 mg/kg daily) atorvastatin group. Platelet aggregation rate was measured in the rabbits by turbidimetric platelet aggregometry, and the changes of serum P-selectin and thromboxane B2 (TXB2) levels were detected with enzyme-linked immunosorbent assay (ELISA).

RESULTSCompared with those in the control group, serum P-selectin level increased significantly (P<0.01) but platelet aggregation rate and TXB2 level exhibited no obvious changes in high-cholesterol group. After carotid artery balloon injury, P-selectin and TXB2 levels and platelet aggregation significantly increased in cholesterol-fed rabbits, reaching the peak level at 24 h after the injury (P<0.01). Compared with the model group, low-dose atorvastatin treatment significantly decreased P-selectin and TXB2 levels and inhibited platelet aggregation in cholesterol-fed rabbits following carotid artery balloon injury (P<0.01), and such effects of atorvastatin were more prominent at a higher daily dose of 10 mg/kg (P<0.05).

CONCLUSIONSCarotid artery balloon injury in rabbits fed cholesterol-enriched diet can induce platelet activation and aggregation, which reaches the peak level at 24 h after balloon injury and can be dose-dependently inhibited by atorvastatin in the acute phase following the injury.

Animals ; Atorvastatin Calcium ; Blood Platelets ; Carotid Artery Injuries ; drug therapy ; Cholesterol ; Enzyme-Linked Immunosorbent Assay ; Heptanoic Acids ; pharmacology ; P-Selectin ; metabolism ; Platelet Activation ; Platelet Aggregation ; Pyrroles ; pharmacology ; Rabbits ; Thromboxane B2 ; metabolism

OBJECTIVETo assess the effect of atorvastatin on lipopolysaccharide (LPS)-induced TNF-α production in RAW264.7 macrophages.

METHODSRAW264.7 macrophages were treated in different LPS concentrations or at different time points with or without atorvastatin. TNF-α level in supernatant was measured. Expressions of TNF-α mRNA and protein and heme oxygenase-1 (HO-1) were detected by ELISA, PCR, and Western blot, respectively. HO activity was assayed.

RESULTSLPS significantly increased the TNF-α expression and secretion in a dose- and time-dependent manner. The HO-1 activity and HO-1 expression level were significantly higher after atorvastatin treatment than before atorvastatin treatment and attenuated by SB203580 and PD98059 but not by SP600125, suggesting that the ERK and p38 mitogen-activated protein kinase (MAPK) pathways participate in regulating the above-mentioned effects of atorvastatin. Moreover, the HO-1 activity suppressed by SnPP or the HO-1 expression inhibited by siRNA significantly attenuated the effect of atorvastatin on TNF-α expression and production in LPS-stimulated macrophages.

CONCLUSIONAtorvastatin can attenuate LPS-induced TNF-α expression and production by activating HO-1 via the ERK and p38 MAPK pathways, suggesting that atorvastatin can be used in treatment of inflammatory diseases such as sepsis, especially in those with atherosclerotic diseases.

Adjuvants, Immunologic ; pharmacology ; Animals ; Atorvastatin Calcium ; Enzyme Activation ; drug effects ; Heme Oxygenase-1 ; genetics ; metabolism ; Heptanoic Acids ; pharmacology ; Hydroxymethylglutaryl-CoA Reductase Inhibitors ; pharmacology ; Lipopolysaccharides ; pharmacology ; Macrophages ; drug effects ; Membrane Proteins ; genetics ; metabolism ; Mice ; Pyrroles ; pharmacology ; Tumor Necrosis Factor-alpha ; metabolism