Niclosamide enhances the antitumor effects of radiation by inhibiting the hypoxia-inducible factor-1α/vascular endothelial growth factor signaling pathway in human lung cancer cells.

Lung cancer is one of the leading causes of cancer-associated mortality, worldwide. The overall survival rate remains low, but progress has been made in improving the diagnosis and treatment of lung cancer over the past decades. Niclosamide, a salicylanilide derivative used for the treatment of tapeworm infections, is safe, well tolerated, inexpensive and readily available. Previous studies have identified niclosamide as a potential anticancer agent. The present study demonstrated that niclosamide enhanced the effect of irradiation by inhibiting the hypoxia-inducible factor-1α/vascular endothelial growth factor signaling pathway. These findings suggest that niclosamide may be a promising candidate for clinical evaluation as part of a combined regimen for the treatment of non-small cell lung cancer.

Combination of gambogic acid with cisplatin enhances the antitumor effects on cisplatin-resistant lung cancer cells by downregulating MRP2 and LRP expression.

Cisplatin resistance is a main clinical problem of lung cancer therapy. Gambogic acid (GA) could prohibit the proliferation of a variety of human cancer cells. However, the effects of GA on cisplatin-resistant lung cancer are still unclear. The objective of the present study was to find out the antitumor effects of GA on cisplatin-resistant human lung cancer A549/DDP cells and further explore its underlying mechanisms. Cell Counting Kit-8 assay was used to observe the impacts of GA and/or cisplatin on the proliferation of lung cancer cells; flow cytometry was used to detect the effects of GA on cell cycle and apoptosis; Western blot was used to examine the effects of GA on the expression of lung resistance protein (LRP) and multidrug resistance-associated protein 2 (MRP2) protein in A549/DDP cells. Our results showed that GA dose- and time-dependently prohibited the proliferation and induced significant cell apoptosis in A549 and A549/DDP cells. GA also induced G0/G1 arrest in both A549/DDP and A549 cells. Moreover, GA upregulated protein expression level of cleaved caspase-3 and Bax and downregulated protein expression level of pro-caspase-9 and Bcl-2 in time- and dose-dependent way in A549/DDP cells. GA combined with cisplatin enhanced the cells apoptotic rate and reduced the cisplatin resistance index in A549/DDP cells. In addition, GA reduced the MRP2 and LRP protein expression level in A549/DDP cells. GA inhibits the proliferation, induces cell cycle arrest and apoptosis in A549/DDP cells. Combination of GA with cisplatin enhances the antitumor effects on cisplatin-resistant lung cancer cells by downregulating MRP2 and LRP expression.

Targeted delivery of chemically modified anti-miR-221 to hepatocellular carcinoma with negatively charged liposomes.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death. Gene therapy was established as a new strategy for treating HCC. To explore the potential delivery system to support the gene therapy of HCC, negatively charged liposomal delivery system was used to deliver miR-221 antisense oligonucleotide (anti-miR-221) to the transferrin (Tf) receptor over expressed HepG2 cells. The liposome exhibited a mean particle size of 122.5 nm, zeta potential of -15.74 mV, anti-miR-221 encapsulation efficiency of 70%, and excellent colloidal stability at 4°C. Anti-miR-221-encapsulated Tf-targeted liposome demonstrated a 15-fold higher delivery efficiency compared to nontargeted liposome in HepG2 cells in vitro. Anti-miR-221 Tf-targeted liposome effectively delivered anti-miR-221 to HepG2 cells, upregulated miR-221 target genes PTEN, P27(kip1), and TIMP3, and exhibited greater silencing efficiency over nontargeted anti-miR-221 liposome. After intravenous injection into HepG2 tumor-bearing xenografted mice with Cy3-labeled anti-miR-221 Tf-targeted liposome, Cy3-anti-miR-221 was successfully delivered to the tumor site and increased the expressions of PTEN, P27(kip1), and TIMP3. Our results demonstrate that the Tf-targeted negatively charged liposome could be a potential therapeutic modality in the gene therapy of human HCC.

G-patch domain containing 2, a gene highly expressed in testes, inhibits nuclear factor-κB and cell proliferation.

G-patch domain containing 2 (GPATC2), a human gene that is highly expressed in the testes, was implicated as a novel cancer/testis antigen. The present study investigated GPATC2 expression in a number of human cell lines and rat tissues, and its potential biological function in 293T cells. Semi­quantitative reverse transcription-polymerase chain reaction analysis showed that GPATC2 was widely expressed in 15 human cell lines (representing different lineages) and in 11 different rat tissues, and that the GPATC2 mRNA relative expression level was significantly higher in the testis than it was in other tissues. 293T cells were transiently transfected with GPATC2-p enhanced green fluorescent protein (EGFP)­N1 or GPATC2-pEGFP-C3 and the nuclei were stained with 4',6'­diamidino­2­phenylindole. The results showed that GPATC2 is predominantly expressed in the nucleus of 293T cells. Overexpression of GPATC2 may inhibit transcription of the NF-κB reporter gene. The role of GPATC2 in proliferation was analyzed with cell counting kit-8, colony-forming efficiency and flow cytometry assays. The results indicated that over­expression of GPATC2 in 293T cells significantly inhibited cell proliferation by decreasing the number of cells in S phase. By contrast, GPATC2 knockdown by RNA interference exhibited the opposite effect, suggesting that GPATC2 may be involved in inhibiting G1-S phase transition in 293T cells. In conclusion, these results provide novel insight into the breadth of expression of GPATC2 and its role in cell proliferation.

MicroRNA profiles and potential regulatory pattern during the early stage of spermatogenesis in mice.

Spermatogenesis is a complicated and poorly understood process that relies on the precise regulation of the self-renewal and differentiation of spermatogonia. In many organisms, microRNAs (miRNAs) are involved in multiple developmental processes as critical regulators of transcriptional and post-transcriptional gene silencing. This study investigated the expression pattern of miRNAs in type B spermatogonia cells (BSc) and primary spermatocytes (PSc) of mice, using a high-throughput small RNA sequencing system. The results revealed that the expression levels of Let-7 family miRNAs were remarkably high in both cell types. Furthermore, the expression levels of miR-21, miR-140-3p, miR-103, miR-30a, miR-101b and miR-99b were decreased during the transformation from BSc to PSc. These miRNAs target vital genes that participate in apoptosis, cell proliferation and differentiation, junction assembly and cell cycle regulation. These results highlight the indispensable role of miRNAs in spermatogenesis.

A novel hydrolysis-resistant lipophilic folate derivative enables stable delivery of targeted liposomes in vivo.

Instability of targeting ligand is a roadblock towards successful development of folate targeted liposomes. Folate ligands have been linked to polyethylene glycol (PEG) and cholesterol by an amide bond to form folate-CONH-PEG-CONH-Cholesterol (F-CONH-PEG-CONH-Chol), which is subject to hydrolysis. To increase the stability of folate ligands and promote the long circulation and targeting effects, we synthesized a chemically stable lipophilic folate derivative, folate-CONH-PEG-NH-Cholesterol (F-CONH-PEG-NH-Chol), where the amide bond was replaced by a C-N bond, to deliver liposomal doxorubicin (Dox). Its physical stability, cellular uptake, cellular toxicity, pharmacokinetics, distribution, anti-tumor efficacy, and cardiac toxicity were investigated. Our results indicate that F-CONH-PEG-NH-Chol conjugated liposomes are taken up selectively by folate receptor-positive HeLa and KB cells. Compared with F-CONH-PEG-CONH-Chol with two carbonate linkages, F-CONH-PEG-NH-Chol better retained its drug entrapment efficiency and folate receptor-targeting activity during prolonged circulation. F-CONH-PEG-NH-Chol thus represents a physically stable and effective ligand for delivering folate receptor-targeted liposomes, with prolonged circulation time and efficient tissue distribution, as well as higher efficacy and less cardiac toxicity. Collectively, these results suggest that this novel conjugate can serve as a promising derivative for the delivery of anti-tumor therapeutic agents.

Co-delivery of doxorubicin and Bmi1 siRNA by folate receptor targeted liposomes exhibits enhanced anti-tumor effects in vitro and in vivo.

Bmi1 gene overexpression is found in various human tumors and has been shown as a potential target for gene treatment. However, siRNA-based treatments targeting Bmi1 gene have been restricted to limited delivery, low bioavailability and hence relatively reduced efficacy. To overcome these barriers, we developed a folate receptor targeted co-delivery system folate-doxorubicin/Bmi1 siRNA liposome (FA-DOX/siRNA-L). The FA-DOX/siRNA-L was prepared through electrostatic interaction between folate doxorubicin liposome (FA-DOX-L) and Bmi1 siRNA. In vitro and in vivo studies showed that FA-DOX/siRNA-L inhibited tumor growth by combinatory role of Bmi1 siRNA and doxorubicin (DOX). Co-delivery of Bmi1 siRNA and DOX by FA-DOX/siRNA-L showed significantly higher efficacy than sole delivery of either DOX or Bmi1 siRNA. Real-time PCR and western blot analysis showed that FA-DOX/siRNA-L silenced the expression of Bmi1 gene. In addition, higher accumulation of the siRNA and DOX in tumor cells indicated that folate ligand displayed tumor targeting effect. These results suggest that Bmi1 is an effective therapeutic target for siRNA based cancer treatment that can be further improved by co-delivery of DOX through targeted liposomes.

Folate receptor-targeted liposomes enhanced the antitumor potency of imatinib through the combination of active targeting and molecular targeting.

PURPOSE: Imatinib inhibits platelet-derived growth factor receptor (PDGFR), and evidence shows that PDGFR participates in the development and progression of cervical cancer. Although imatinib has exhibited preclinical activity against cervical cancer, only minimal clinical therapeutic efficacy was observed. This poor therapeutic efficacy may be due to insufficient drug delivery to the tumor cells and plasma protein binding. Therefore, the purpose of this study was to explore a novel folate receptor (FR)-targeted delivery system via imatinib-loaded liposomes to enhance drug delivery to tumor cells and to reduce plasma protein binding. METHODS: Imatinib was remote-loaded into FR-targeted liposomes which were prepared by thin film hydration followed by polycarbonate membrane extrusion. Encapsulation efficiency, mean size diameter, and drug retention were characterized and cellular uptake, cell cytotoxicity, and cell apoptosis on cervical cancer HeLa cells were evaluated. Comparative pharmacokinetic studies were also carried out with FR-targeted imatinib liposomes, simple imatinib liposomes, and free imatinib. RESULTS: High encapsulation efficiency (>90%), appropriate mean particle size (143.5 nm), and zeta potential (-15.97 mV) were obtained for FR-targeted imatinib liposomes. The drug release profile showed minimal imatinib leakage (<5%) in phosphate-buffered saline (PBS) at pH =7.4 within 72 hours of incubation, while more leakage (>25%) was observed in PBS at pH =5.5. This indicates that these liposomes possess a certain degree of pH sensitivity. Cytotoxicity assays demonstrated that the FR-targeted imatinib liposomes promoted a six-fold IC50 reduction on the non-targeted imatinib liposomes from 910 to 150 µM. In addition, FR-targeted imatinib liposomes enhanced HeLa cell apoptosis in vitro compared to the non-targeted imatinib liposomes. Pharmacokinetic parameters indicated that both targeted and non-targeted liposomes exhibited long circulation properties in Kunming mice. CONCLUSION: These findings indicate that the nano-sized FR-targeted PDGFR antagonist imatinib liposomes may constitute a promising strategy in cervical cancer therapy through the combination of active targeting and molecular targeting.

Novel mixed ligand di-n-butyltin(IV) complexes derived from acylpyrazolones and fluorinated benzoic acids: synthesis, characterization, cytotoxicity and the induction of apoptosis in Hela cancer cells.

Twenty one novel mixed ligand di-n-butyltin(IV) complexes [(n)Bu2SnAL] (A = substituted 4-acyl-5-pyrazolone, and L = fluorinated benzoic acid) were prepared by condensation of di-n-butyltin(IV) oxide with HL and HA in 1:1:1 molar ratio in refluxing methanol. All of the complexes were characterized by elemental analyses, IR, NMR ((1)H, (13)C, (119)Sn) and in four cases by X-ray diffraction. Cytotoxicity of the compounds was studied against two human cancer cell lines (KB and Hela) by means of the MTT assay compared to cisplatin, featuring IC50 values in the low micromolar range. Hela cancer cell apoptosis-induced by 2 was examined by flow cytometry analysis, and preliminary results showed that 2 at concentrations of more than 1.0 µM can induce apoptosis.

In vitro and in vivo antitumor effects of folate-targeted ursolic acid stealth liposome.

The antitumor efficacy of ursolic acid (UA) was limited by poor hydrophilicity and low bioavailability. To overcome this issue, UA was encapsulated in liposomes modified with folate conjugates for better solubility and bioavailability. This novel agent was prepared by a thin-film dispersion method and characterized by mean diameter, zeta potential, and entrapment efficiency (160.1 nm, -21.2 mV, and 88.9%, respectively). In vitro, cellular uptake efficiency, cytotoxicity, apoptosis, and cell cycle analyses were performed to show that folate-receptor (FR) positive cells endocytose more FR-targeted liposome (FTL-UA) than nontargeted PEGylated liposome (PL-UA) and that FTL-UA induced more cytotoxicity and higher apoptosis than PL-UA. Pharmacokinetic assessments showed advantages of systemic bioavailability of FTL-UA (AUC = 218.32 mg/L·h, t1/2 = 7.61 h) over free UA (AUC = 36.88 mg/L·h, t1/2 = 0.78 h). In vivo, FTL-UA showed significantly higher human epidermoid carcinoma (KB) inhibition in Balb/c nu/nu mice compared to PL-UA or free UA. The results indicate the great potential of FTL-UA against KB tumor.