Anti-GPC3 Antibody-Conjugated BEZ235 Loaded Polymeric Nanoparticles (Ab-BEZ235-NP) Enhances Radiosensitivity in Hepatocellular Carcinoma Cells by Inhibition of DNA Double-Strand Break Repair.

AIM: To assess AB-BEZ235-NP potential as a radio-sensitizer in hepatocellular carcinoma models. METHOD: By comparing hepatocellular carcinoma cell with simple radiation or combined AB-BEZ235-NP therapy, the HCC apoptosis and self-repair level have significant differences in mortality rates and cell migration abilities. RESULTS: Cell proliferation and DNA damage increased by pretreatment with AB-BEZ235-NP after irradiation; further studies on the repair pathway indicated that AB-BEZ235-NP inhibited the important pathway of DSB repair. Our results further show that AB-BEZ235-NP significantly inhibits the phosphorylation of the canonical protein, γ-H2AX, in the NHEJ DSB repair pathway and Serine Protein Kinase (SPK) ATM, and TP53-Binding Protein one. More importantly, AB-BEZ235-NP increased the mount of mean γ-H2AX Foci in irradiated cells, indicating that AB-BEZ235-NP can selectively inhibit DSB repair in HCC cells. Therefore, these results clearly eludicate that treatment with AB-BEZ235-NP is a potential promising therapy which can increase the radiosensitivity to HCC.

The PI3K/mTOR dual inhibitor BEZ235 nanoparticles improve radiosensitization of hepatoma cells through apoptosis and regulation DNA repair pathway.

Polymer materials encapsulating drugs have broad prospects for drug delivery. We evaluated the effectiveness of polyethylene glycol-poly (lactic-co-glycolic acid) (PLGA-PEG) encapsulation and release characteristics of PI3K/mTOR inhibitor NVP-BEZ235 (BEZ235). We proposed a strategy for targeting radiosensitization of liver cancer cells. The biocompatibility, cell interaction, and internalization of Glypican-3 (GPC3) antibody-modified, BEZ235-loaded PLGA-PEG nanoparticles (NP-BEZ235-Ab) in hepatoma cells in vitro were studied. Also, the cell killing effect of NP-BEZ235-Ab combined with γ-ray cell was evaluated. We used confocal microscopy to monitor nanoparticle-cell interactions and cellular uptake, conducted focus-formation experiments to analyze the synergistic biological effects of NP-BEZ235-Ab and priming, and studied synergy in liver cancer cells using molecular biological methods such as western blotting. We found that PLGA-PEG has good loading efficiency for BEZ235 and high selectivity to GPC3-positive HepG2 liver cancer cells, thus documenting that NP-BEZ235-Ab acts as a small-molecule drug delivery nanocarrier. At the nominal concentration, the NP-BEZ235-Ab nanoformulation synergistically kills liver cancer cells with significantly higher efficiency than does the free drug. Thus, NP-BEZ235-Ab is a potential radiosensitizer.

Resistance of hepatocellular carcinoma to sorafenib can be overcome with co-delivery of PI3K/mTOR inhibitor BEZ235 and sorafenib in nanoparticles.

Background: The combination of BEZ235 with sorafenib (SFB) enhances anti-hepatocellular carcinoma (HCC) efficacy of the two agents. However, pharmacokinetic profiles in vivo and different endocytosis abilities of these two drugs hinder their therapeutic application.Research design and methods: In this work, we developed d-α-tocopheryl polyethylene glycol 1000 succinate - polycaprolactone polymer nanoparticles (NPs) for co-delivery of SFB and BEZ235 (SFB/BEZ235-NPs). Explored the anti-proliferative and pro-apoptotic effects of SFB/BEZ235-NPs through in vitro and in vivo experiments.Results: Stabilized SFB/BEZ235-NPs were prepared with optimized drug ratio, yielding high encapsulation efficiency, low polydispersity, and enhanced cellular internalization in HepG2 cells. Synergistic cytotoxicity and pro-apoptotic ability were documented. In vivo pharmacokinetic results revealed extended circulation and bioavailability of SFB/BEZ235-NPs compared with those of free drugs. SFB/BEZ235-NPs enhanced antitumor effectiveness in SFB-resistant HCC xenograft mouse models.Conclusion: Taken together, the results of this study describe a promising strategy using SFB and BEZ235 in a nanoparticle formulation for treatment of SFB-resistant HCC.

Albumin-bilirubin and platelet-albumin-bilirubin grades for hepatitis B-associated hepatocellular carcinoma in Child-Pugh A patients treated with radical surgery: A retrospective observational study.

Child-Pugh (CP) grade A patients with early stage hepatocellular carcinoma (HCC) are candidates for curative surgery, while some patients still have a poor outcome. The aim of this study was to investigate the prognostic values of 2 new evaluation models for liver function, named albumin-bilirubin (ALBI) and platelet-albumin-bilirubin (PALBI) grades, in CP grade A patients with HCC.In this retrospective cohort study, we reviewed 134 cases of CP grade A patients with hepatitis B-associated HCC who underwent radical surgery. ALBI and PALBI grades were calculated based on preoperative serologic examinations. Overall survival (OS) and recurrence-free survival (RFS) were estimated by Kaplan-Meier curve and Cox regression. The prognostic performances of the models were estimated by using the concordance index (C-index).During a median follow-up time of 27 months, 27.6% (37/134) of patients died and 26.1% (35/134) experienced recurrence. Kaplan-Meier analyses showed that ALBI and PALBI grades were significantly associated with OS and RFS. Multivariate analyses further revealed that both ALBI and PALBI grades were independent predictors for survival. Furthermore, the prognostic values of the combination of tumor size with ALBI (C-index = 0.754, 95% confidence interval [CI]: 0.675-0.849) or with PALBI (C-index = 0.762, 95% CI: 0.664-0.844) may be comparable with both Barcelona Clinic Liver Cancer and Cancer of Liver Italian Program staging systems.The ALBI and PALBI grades, in particular the combination with tumor size, are effective models for discriminating survival in CP grade A patients with HCC.

BEZ235 increases sorafenib inhibition of hepatocellular carcinoma cells by suppressing the PI3K/AKT/mTOR pathway.

BACKGROUND: Sorafenib is an oral multi-kinase inhibitor that inhibits hepatocellular carcinoma (HCC) via the Ras/Raf/MAPK pathway. However, sorafenib loses effectiveness because most tumors acquire drug resistance over time. As the PI3K/AKT/mTOR signaling pathway is also activated abnormally in HCC, we evaluated the effect of sorafenib, in combination with a dual PI3K/mTOR inhibitor, BEZ235, on HCC cell proliferation and survival in vitro. MATERIALS AND METHODS: Biological phenotypes were analysed in HCC cell lines, parental and sorafenib-resistant HepG2 cells (HepG2 and HepG2R), treated with sorafenib or BEZ235, alone or in combination. HCC cellular proliferation and apoptosis were investigated, and perturbations of the Ras/Raf/MAPK and PI3K/AKT/mTOR signaling/survival pathways were evaluated by western blot analysis. RESULTS: BEZ235 enhanced sorafenib inhibition of cellular proliferation, migration, and promotion of apoptosis in HepG2 and HepG2R cells. The combined effects were associated with inhibition of phosphorylation of AKT, mTOR and S6K in the PI3K/AKT/mTOR pathway, whereas the combination of sorafenib and BEZ235 did not significantly alter the Ras/Raf/MAPK pathway compared with the effect of sorafenib alone. CONCLUSION: Sorafenib/BEZ235 combination has potent anti-HCC cell activity. This anti-tumor activity is most likely multi-factorial, mainly involving PI3K down-regulation and AKT, mTOR and S6K dephosphorylation. Combined inhibition of PI3K/AKT/mTOR and Ras/Raf/MAPK pathways enhances sorafenib inhibition of HCC. The results of these in vitro studies suggest that trials of combined sorafenib and BEZ235 in the treatment of HCC should be considered.

Surgical removal of adult recurrent neuroblastoma located in the posterior mediastinum and retroperitoneum: A case report.

RATIONALE: Adult recurrent neuroblastoma is extremely rare, especially in the posterior mediastinum and retroperitoneal cavity. The surgical treatment of this special part of the tumor is also a clinical difficulty. PATIENT CONCERNS: This study reports a case of a 24-year-old man with a history of treated posterior mediastinal neuroblastoma. Enhanced computed tomography found a heterogeneously enhancing mass occupying the retroperitoneal and posterior mediastinum, and the initial impression was recurrent neuroblastoma. DIAGNOSES: The patient was diagnosed with recurrent neuroblastoma based on his medical history and histopathological results. INTERVENTIONS: The young adult underwent radical resection of recurrent neuroblastoma in posterior mediastinum and retroperitoneum through thoracoabdominal incision. OUTCOMES: The young patient recovered to normal within 10 days after surgery and had no relapse for following-up 12 months. LESSONS: Despite the difficulty of surgery, it is feasible to remove the tumor in the posterior mediastinum and retroperitoneal cavity safely.

Lentivirus-mediated overexpression of HSDL2 suppresses cell proliferation and induces apoptosis in cholangiocarcinoma.

BACKGROUND: Cholangiocarcinoma (CCA) is a malignant tumor of the bile duct epithelium, including intrahepatic, perihilar, and distal CCA based on anatomical location. Hydroxysteroid dehydrogenase-like 2 (HSDL2) belongs to the SDR subfamily of oxidoreductases, and it is involved in glioma oncogenesis, as it can promote cell proliferation and inhibit cell apoptosis. The purpose of this study was to explore the underlying molecular mechanisms of HSDL2 in the process of CCA. METHODS: HSDL2 expression levels were observed in CCA and adjacent (normal control) tissues by analyzing data from The Cancer Genome Atlas and Gene Expression Omnibus databases. A receiver operating characteristic curve analysis was carried out. In vitro, we overexpressed HSDL2 in RBE cells (a human CCA cell line) using a stable lentivirus-mediated transduction strategy. We then used quantitative real-time-PCR and Western blotting methods to detect the efficiency of HSDL2 overexpression. Cell proliferation was assessed using a Celigo Image Cytometer, MTT assays, and the expression of PCNA. Cell apoptosis was assessed by flow cytometry analysis, caspase3/7 activity, and the expression of the apoptotic markers BCL-2 and BAX. RESULTS: We observed a downregulation of HSDL2 in CCA tissues based on The Cancer Genome Atlas and Gene Expression Omnibus data analysis. The receiver operating characteristic curve analysis showed that HSDL2 could be an excellent efficacy biomarker for CCA. In vitro, HSDL2 overexpression largely suppressed the proliferation of RBE cells. In addition, apoptosis was induced by HSDL2 overexpression. CONCLUSION: The results of the data analysis indicated that, compared with adjacent tissues, HSDL2 was downregulated in CCA tissues, and overexpressing HSDL2 in CCA cells suppressed growth and proliferation, which involved activating apoptosis. This helps to understand the underlying HSDL2-related molecular mechanisms in the process of CCA.

Enhanced delivery of sorafenib with anti-GPC3 antibody-conjugated TPGS-b-PCL/Pluronic P123 polymeric nanoparticles for targeted therapy of hepatocellular carcinoma.

Although sorafenib (SFB) showed improved efficacy and much reduced the side effects in clinical liver cancer therapy, its therapeutic efficacy was still greatly limited due to short half-life in vivo as well as drug resistance. To solve these problems, we developed a novel SFB-loaded polymeric nanoparticle for targeted therapy of liver cancer. This polymeric nanoparticle, referred to NP-SFB-Ab, was fabricated from self-assembly of biodegradable block copolymers TPGS-b-poly(caprolactone) (TPGS-b-PCL) and Pluronic P123 and drug SFB, followed by conjugating the anti-GPC3 antibody. NP-SFB-Ab showed robust stability and achieve excellent SFB release in cell medium. The CLSM demonstrated that the Ab-conjugated NP exhibited much higher cellular uptake in HepG2 human liver cells than non-targeted NP. The MTT assay also confirmed that NP-SFB-Ab caused much greater cytotoxicity than non-targeted NP-SFB and free SFB. Finally, NP-SFB-Ab was proved to greatly inhibit the tumor growth of HepG2 xenograft-bearing nude mice without obvious side effects. Therefore, this NP-SFB-Ab provides a promising new approach for targeted therapy of hepatocellular carcinoma.

Neuroendocrine tumor of the common bile duct: a case report and review of the literature.

We report a rare case of neuroendocrine tumor (NET) in the common bile duct (CBD). The patient is a 56-year-old female who presented to our department with symptoms of fever but without jaundice. A preoperative examination showed a tumor in the CBD. The tumor volume was almost 5.5 × 4.5 × 4 cm3, which is the biggest NET in the CBD reported on PubMed. The imaging results (computed tomography [CT] and magnetic resonance imaging [MRI]) were not consistent with CBD adenocarcinoma. The tumor appeared to oppress the growth of the CBD rather than originate in the bile duct wall; combined with the low blood bilirubin index and lack of jaundice symptoms, the preoperative diagnosis was not clear. We performed a radical resection of the cholangiocarcinoma. The patient recovered well before going home. The pathology was NET (Grade 2). The patient showed no recurrence to date, without intravenous chemotherapy (8 months).

Genistein-loaded nanoparticles of star-shaped diblock copolymer mannitol-core PLGA-TPGS for the treatment of liver cancer.

The purpose of this research is to develop nanoparticles (NPs) of star-shaped copolymer mannitol-functionalized PLGA-TPGS for Genistein delivery for liver cancer treatment, and evaluate their therapeutic effects in liver cancer cell line and hepatoma-tumor-bearing nude mice in comparison with the linear PLGA nanoparticles and PLGA-TPGS nanoparticles. The Genistein-loaded M-PLGA-TPGS nanoparticles (MPTN), prepared by a modified nanoprecipitation method, were observed by FESEM and TEM to be near-spherical shape with narrow size distribution. The nanoparticles were further characterized in terms of their size, size distribution, surface charge, drug-loading content, encapsulation efficiency and in vitro drug release profiles. The data showed that the M-PLGA-TPGS nanoparticles were found to be stable, showing almost no change in particle size and surface charge during 3-month storage of their aqueous solution. In vitro Genistein release from the nanoparticles exhibited biphasic pattern with burst release at the initial 4days and sustained release afterwards. The cellular uptake efficiency of fluorescent M-PLGA-TPGS nanoparticles was 1.25-, 1.22-, and 1.29-fold higher than that of the PLGA-TPGS nanoparticles at the nanoparticle concentrations of 100, 250, and 500µg/mL, respectively. In the MPTN group, the ratio of apoptotic cells increased with the drug dose increased, which exhibited dose-dependent effect and a significant difference compared with Genistein solution group (p<0.05). The data also showed that the Genistein-loaded M-PLGA-TPGS nanoparticles have higher antitumor efficacy than that of linear PLGA-TPGS nanoparticles and PLGA nanoparticles in vitro and in vivo. In conclusion, the star-shaped copolymer M-PLGA-TPGS could be used as a potential and promising bioactive material for nanomedicine development for liver cancer treatment.

[Effect of MSX2 interference on epithelial-mesenchymal transitions of pancreatic cancer cell line PANC-1].

OBJECTIVE: To investigate the effect of MSX2 interference on epithelial-mesenchymal transitions (EMT) of pancreatic cancer cell line PANC-1. METHODS: Three vectors containing short hairpin RNAs (shRNAs) of MSX2 (shMSX2-1, shMSX2-2, and shMSX2-3) and the empty vector (negative control) were transfected separately into PANC-1 cell line with Lipofectamine2000. Real-time RT-PCR and Western blotting were used to observe changes in the expressions of MSX2, E-cadherin, and vimentin in the cells. CCK-8 assay was used to assess the changes in the cell growth, and wound scratch assay and Transwell assay were employed to evaluate the cell invasion and metastasis after the transfection. RESULTS: Among the 3 shRNA, shMSX2-1 showed the highest interference efficiency. MSX2 knockdown by the specific shRNA of MSX2 significantly increased E-cadherin expressions, lowered vimentin expressions, and suppressed the invasion, metastasis and proliferation of the cells (P<0.05). MSX2 knockdown also resulted in morphological changes of the cells into cobblestone-like cells in close contact. RT-PCR results revealed significantly reduced mRNA expressions of the transcription factors snail and twist (P<0.05) without affecting slug and zeb1 expressions in the cells with MSX2 knockdown. Conclusion MSX2 knockdown can reverse EMT and induce MET in PANC1 cells, in which process the transcription factors snail and twist may play a role.