Bu-Yin-Qian-Zheng Formula Ameliorates MPP+-Induced Mitochondrial Dysfunction in Parkinson's Disease via Parkin.

As a typical traditional Chinese medicine, Bu-Yin-Qian-Zheng Formula (BYQZF) has been shown to have neuroprotective effects in patients with Parkinson's disease (PD), particularly by ameliorating mitochondrial dysfunction and regulating expression of the parkin protein. However, the underlying mechanisms by which BYQZF affects mitochondrial function through parkin are unclear. Accordingly, in this study, we evaluated the mechanisms by which BYQZF ameliorates mitochondrial dysfunction through parkin in PD. We constructed a parkin-knockdown cell model and performed fluorescence microscopy to observe transfected SH-SY5Y cells. Quantitative real-time reverse transcription polymerase chain reaction and western blotting were conducted to detect the mRNA and protein expression levels of parkin. Additionally, we evaluated the cell survival rates, ATP levels, mitochondrial membrane potential (ΔΨm), mitochondrial morphology, parkin protein expression, PINK1 protein expression, and mitochondrial fusion and fission protein expression after treatment with MPP+ and BYQZF. Our results showed that cell survival rates, ATP levels, ΔΨm, mitochondrial morphology, parkin protein levels, PINK1 protein levels, and mitochondrial fusion protein levels were reduced after MPP+ treatment. In contrast, mitochondrial fission protein levels were increased after MPP+ treatment. Moreover, after transient transfection with a negative control plasmid, the above indices were significantly increased by BYQZF. However, there were no obvious differences in these indices after transient transfection with a parkin-knockdown plasmid. Our findings suggest that BYQZF has protective effects on mitochondrial function in MPP+-induced SH-SY5Y cells via parkin-dependent regulation of mitochondrial dynamics.

Contribution to the knowledge of the genus Cucujus Fabricius (Coleoptera, Cucujidae) from China.

Cucujus costatus Zhao Zhang, new species is described from Guangdong, China. This new species can be easily recognized by the longitudinal elevated carinae on elytra and its strongly convex eyes. Additional records for C. kempi Grouvelle, 1913 and C. elongatus Lee Pütz, 2008 are added. A key to the known Chinese species of Cucujus Fabricius is given.

Correction: Yan Chen, et al. Dual Agent Loaded PLGA Nanoparticles Enhanced Antitumor Activity in a Multidrug-Resistant Breast Tumor Eenograft Model. Int. J. Mol. Sci. 2014, 15, 2761-2772.

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Study on biopharmaceutics classification and oral bioavailability of a novel multikinase inhibitor NCE for cancer therapy.

Specific biopharmaceutics classification investigation and study on phamacokinetic profile of a novel drug candidate (2-methylcarbamoyl-4-{4-[3- (trifluoromethyl) benzamido] phenoxy} pyridinium 4-methylbenzenesulfonate monohydrate, NCE) were carried out. Equilibrium solubility and intrinsic dissolution rate (IDR) of NCE were estimated in different phosphate buffers. Effective intestinal permeability (P(eff)) of NCE was determined using single-pass intestinal perfusion technique in rat duodenum, jejunum and ileum at three concentrations. Theophylline (high permeability) and ranitidine (low permeability) were also applied to access the permeability of NCE as reference compounds. The bioavailability after intragastrical and intravenous administration was measured in beagle dogs. The solubility of NCE in tested phosphate buffers was quite low with the maximum solubility of 81.73 µg/mL at pH 1.0. The intrinsic dissolution ratio of NCE was 1 × 10⁻4 mg·min⁻¹·cm⁻². The P(eff) value of NCE in all intestinal segments was more proximate to the high-permeability reference theophylline. Therefore, NCE was classified as class II drug according to Biopharmaceutics Classification System due to its low solubility and high intestinal permeability. In addition, concentration-dependent permeability was not observed in all the segments, indicating that there might be passive transportation for NCE. The absolute oral bioavailability of NCE in beagle dogs was 26.75%. Therefore, dissolution promotion will be crucial for oral formulation development and intravenous administration route will also be suggested for further NCE formulation development. All the data would provide a reference for biopharmaceutics classification research of other novel drug candidates.

Dual agent loaded PLGA nanoparticles enhanced antitumor activity in a multidrug-resistant breast tumor eenograft model.

Multidrug-resistant breast cancers have limited and ineffective clinical treatment options. This study aimed to develop PLGA nanoparticles containing a synergistic combination of vincristine and verapamil to achieve less toxicity and enhanced efficacy on multidrug-resistant breast cancers. The 1:250 molar ratio of VCR/VRP showed strong synergism with the reversal index of approximately 130 in the multidrug-resistant MCF-7/ADR cells compared to drug-sensitive MCF-7 cells. The lyophilized nanoparticles could get dispersed quickly with the similar size distribution, zeta potential and encapsulation efficiency to the pre-lyophilized nanoparticles suspension, and maintain the synergistic in vitro release ratio of drugs. The co-encapsulated nanoparticle formulation had lower toxicity than free vincristine/verapamil combinations according to the acute-toxicity test. Furthermore, the most effective tumor growth inhibition in the MCF-7/ADR human breast tumor xenograft was observed in the co-delivery nanoparticle formulation group in comparison with saline control, free vincristine, free vincristine/verapamil combinations and single-drug nanoparticle combinations. All the data demonstrated that PLGANPs simultaneously loaded with chemotherapeutic drug and chemosensitizer might be one of the most potential formulations in the treatment of multidrug-resistant breast cancer in clinic.

A polymorphism within ErbB4 is associated with risk for hepatocellular carcinoma in Chinese population.

AIM: To investigate the association between hepatocellular carcinoma (HCC) susceptibility and a 12-bp insertion/deletion polymorphism (rs6147150) in the 3'UTR of ErbB4. METHODS: Using a case-control design, the rs6147150 genotypes in 270 patients with HCC and 270 healthy controls were determined by direct polymerase chain reaction and polyacrylamide gel electrophoresis. Logistic regression was used to analyze the association between the polymorphism and cancer risk. RESULTS: Computational modeling suggested that rs6147150 was located in the seed region of hsa-let-7c, a potential target sequence in ErbB4 3'UTR. Logistic regression analysis showed that, compared with individuals homozygous for wild-type, heterozygotes [adjusted odds ratio (OR) = 1.48, 95% confidence interval (CI) = 1.03-2.17, P = 0.034] and individuals homozygous for 12-bp del/del (OR = 2.50, 95% CI = 1.37-4.56, P = 0.001) were at significantly higher risk of HCC. Carriers of the "del" allele of rs6147150 had a 1.59-fold increased risk for HCC (95% CI = 1.22-2.07, P = 0.003). CONCLUSION: rs6147150 may be associated with HCC risk, in part through let-7c-mediated regulation, and may be involved in the pathogenesis of HCC in Chinese populations.

In vitro antigen-specific cytotoxic T cell response against esophageal carcinoma cells induced by HPV18E7-transfected dendritic cells.

Human Papillomavirus (HPV)-associated esophageal carcinoma (EC) is a high incidence tumor worldwide. Dendritic cell (DC)-based tumor vaccine is considered an alternative therapy to treat EC. Here we developed a DC-based vaccine by transfecting cord blood CD34+ stem cell-derived DC with HPV18E7 gene, observed its biological characteristics and the antigen-specific T-cell cytotoxicity on EC cells induced by HPV18E7-DC in vitro. Our results showed that 1) HPV18E7 gene transfer did not change the typical morphology of mature DC, 2) the representative phenotypes of mature DC (CD80, CD86, and CD83) were highly expressed in HPV18E7- DC (81.6%, 80.5%, and 86.6%, respectively), 3) the expression level of 18E7 protein in HPV18E7-DC was 47.5%, and 4) the specific cytotoxicity against EC cells was significantly higher than that in controls (p<0.01). This study indicates the possibility of a DC-based immunotherapy in HPV-associated EC.

Microencapsulated human hair dermal papilla cells: a substitute for dermal papilla?

Dermal papillae (DP) play a pivotal role in hair formation, growth and cycling. However, the number of DP is limited. In this study, we report the production of "reconstructed DP" by enclosing DP cells within an alginate-polylysine-alginate (APA) semipermeable membrane. MTT assay and electron microscopy showed that the microencapsulated dermal papilla cells retained normal activity. The microcapsules were implanted into rat footpads, which lack follicles and sebaceous glands, to assess their inductive properties. Histologic examination showed that numbers of follicle and sebaceous gland structures formed in the footpads within 6-10-week period. At the 10 weeks following transplantation, hair fibers were visible in the footpad. These findings indicate that the DP cell microcapsules retain the capacity to initiate follicle regeneration and could be considered a substitute for fresh isolated DPs.

LHRH-PE40-Induced Vascular Leak Syndrome.

AIM: This study was designed to study the toxicity of LHRH-PE40, a recombinant DNA-derived protein composed of the decapeptide known as luteinizing hormone-releasing hormone and the translocation and catalytic domains of pseudomonas exotoxin A. METHOD: Single-dose and repeat-dose toxicity of intravenous injection of LHRH-PE40 was studied by clinical signs, hematology, blood chemistry and histopathology, and lung permeability to Evans blue dye. Additional study was performed to find the relationship between dexamethasone pretreatment and vascular leak syndromes. RESULTS: Dyspnea, increased hemocrit, low serum total protein, lung edema, and high lung permeability were found on rats treated with single or repeated doses of LHRH-PE40. Dexamethasone pretreatment before LHRH-PE40 administration partly lowered morbidity of rats. CONCLUSION: LHRH-PE40-induced vascular leak syndrome was the chief cause of rats' death. Dexamethasone pretreatment partly reduced the frequency of vascular leak syndrome. Hypotheses about vascular leak syndromes were also formed by reviewing recent literature.

Effects of dendritic cells from cord blood CD34+ cells on human hepatocarcinoma cell line BEL-7402 in vitro and in SCID mice.

AIM: To develop a cancer vaccine of dendritic cells derived from human cord blood CD34+ cells and to investigate its cytotoxicity on human hepatocarcinoma cells in vitro and in severe combined immunodeficiency (SCID) mice. METHODS: Lymphocytes from cord blood or peripheral blood were primed by DCs, which were derived from cord blood and pulsed with whole tumor cell lysates. Nonradiative neutral red uptake assay was adopted to detect the cytotoxicity of primed lymphocytes on human hepatocarcinoma cell line BEL-7402 in vitro. The anti-tumor effect of primed lymphocytes in vivo was detected in SCID mice, including therapeutic effect and vaccination effect. RESULTS: The cytotoxicity of DC vaccine primed lymphocytes from cord blood or peripheral blood on human hepatocarcinoma cell line BEL-7402 was significantly higher than that of unprimed lymphocytes in vitro (44.09% vs 14.69%, 47.92% vs 19.44%, P<0.01). There was no significant difference between the cytotoxicity of primed lymphocytes from cord blood and peripheral blood (P>0.05). The tumor growth rate and tumor size were smaller in SCID mice treated or vaccinated with primed lymphocytes than those with unprimed lymphocytes. SCID mice vaccinated with primed lymphocytes had a lower tumor incidence (80% vs 100%, P<0.05) and delayed tumor latent period compared with mice vaccinated with unprimed lymphocytes (11 d vs 7 d, P<0.01). CONCLUSION: Vaccine of cord blood derived-DCs has an inhibitory activity on growth of human hepatocarcinoma cells in vitro and in SCID mice. The results also implicate the potential role of cord blood derived-DC vaccine in clinical tumor immunotherapy.

Study on immune function of dendritic cells in patients with esophageal carcinoma.

AIM: To investigate the immune function of dendritic cells from both peripheral blood and operated tissues of esophageal carcinoma patients in order to find the relationship between the immune function of dendritic cells and the pathogenesis of esophageal carcinoma. METHODS: The expression of CD83, CD80, and CD86 on the surface of dendritic cells cultured from the peripheral blood of patients was detected compared with that from health donors using flow cytometry. The ability of dendritic cells to induce T lymphocyte proliferation was evaluated by a liquid scintillation counter. The expression of CD80, CD86, CD83, and S-100 proteins was assessed in esophageal carcinoma tissues using immunohistochemical method. RESULTS: Compared with those from healthy donors, dendritic cells cultured from the peripheral blood of patients expressed lower CD80 and CD86. Furthermore, the ability of dendritic cells in patients to induce T lymphocyte proliferation was significantly lower than that of the control group. Compared with the control group, the positive expression ratio and frequencies of CD80, CD86, and S-100 in esophageal carcinoma tissues were significantly down regulated. The expression of CD83 was up-regulated in the pericancerous tissues, but no expression was found in the cancerous nodules. CONCLUSION: The impaired immune function and the decreased number of dendritic cells cause pathogenesis and progression of esophageal carcinoma.

Antitumor immunopreventive and immunotherapeutic effect in mice induced by hybrid vaccine of dendritic cells and hepatocarcinoma in vivo.

AIM: To develop atumor vaccine by fusion of H22 hepatocarcinoma cells and DC, and to study its protective and therapeutical effect against H22 cell. METHODS: H22-DC vaccine was produced by PEG fusion of H22 and DC induced by cytokine released from splenic mononuclear cells, sorted by CD11c magnetic microbead marker. It was injected through the tail vein of the mice and the H(22)-DC oncogenesis was detected in the liver, spleen and lung. In order to study the therapeutical and protective effect of H(22)-DC against tumor H(22), two groups were divided: immune group and therapeutic group. Immune group was further divided into P, D, HD and H subgroups, immunized by PBS, DC, H(22)-DC and inactivated H(22), respectively, and attacked by H(22) cell. The tumor size, tumor weight, mice survival time and tumor latent period were recorded and statistically analyzed; Therapeutical group was divided into three subgroups of P, D and HD, and attacked by H(22), then treated with PBS, DC, and H(22)-DC, respectively. Pathology and flow cytometry were also applied to study the mechanism how the H(22)-DC vaccine attacked on the H(22) cell. RESULTS: 1. No oncogenesis was found in spleen, lung and liver after H22-DC injection. 2. Hybrid vaccine immunized mice had strongest CTL activity. 3. In the immune group, latent period was longer in HD subgroup than that in P, H and D subgroup; and tumor size and weight were smaller in HD subgroup than that in P, H and D subgroup. 4. In therapeutic group, tumor size was smaller in HD subgroup than that in P, D subgroup. CONCLUSION: 1. H22-DC tumor vaccine is safe without oncogenesis in vivo. 2. Hybrid vaccine can stimulate potent specific CTL activity against H22. 3. H22-DC vaccine has distinctive prophylatic effect on tumor H22 and can inhibit the tumor growth.

Antitumor activities of human dendritic cells derived from peripheral and cord blood.

AIM: To observe the biological specialization of human peripheral blood dendritic cells (DC) and cord blood derived DC and its effects on effector cells killing human hepatocarcinoma cell line BEL-7402. in vitro. METHODS: The DC biological characteristics were detected with immunohistochemical and MTT assay. Two antitumor experiment groups are divided: peripheral blood DC and cord blood DC groups. Peripheral blood DC groups used LAK cells as the effector cells and BEL-7402 as target cells, while cord blood DC groups used CTL induced by tumor antigen twice pulsed DC as effector cells and BEL-7402 as target cells, additional peripheral blood DC and cord blood DC are added to observe its stimulating activities to effector cells. The effector's cytotoxicity to tumor cells were detected with neutral red colorimetric assay at two effector/target ratios of 5:1 and 10:1. RESULTS: Peripheral blood DC and cord blood DC highly expressed HLA-ABC, HLA-DR, HLA-DQ, CD54 and S-100 protein. The stimulating activities to lymphocyte proliferation were compared between experimental groups (DC added) and control group (no DC added), in six experiment subgroups,the DC/lymphocyte ratio was sequentially 0.25:100, 0.5:100, 1:100, 2:100, 4:100 and 8:100.A values were sequentially 0.75396+/-0.009, 0.84916+/-0.010, 0.90894+/-0.012, 0.98371+/-0.007, 1.01299+/-0.006 and 1.20384+/-0.006 in peripheral blood DC groups and 0.77650+/-0.005, 0.83008+/-0.007, 0.92725+/-0.007, 1.05990+/-0.010, 1.15583+/-0.011, 1.22983+/-0.011 in cord blood DC groups. A value was 0.59517+/-0.005 in control group. The stimulating activities were higher in experimental groups than in control group (P<0.01), which were increased when the DC concentration was enlarged (P<0.01). Two differently derived DCs had the same phenotypes and similar stimulating activities (P<0.05). In peripheral blood DC groups, the cytotoxicity of the LD groups (experimental groups) and L groups (control group) was 58.16%+/-2.03% (5:1), 46.18%+/-2.25% (10:1) and 38.13%+/-1.29% (5:1) and 65.40%+/-1.56% (10:1) respectively; in cord blood DC groups, TD groups (experimental groups) and T groups (control groups) were 69.71%+/-2.33 % (5:1), 77.64%+/-1.94% (10:1) and 56.89%+/-1.82% (5:1) and 60.99%+/-1.42% (10:1) respectively.The cytotoxicity activities were enhanced with increased effector/target ratio (P<0.01). At the same effector/target ratio, the cytotoxicity of experimental groups were bigger than that of control groups (P<0.01). The cytotoxicity activities of cord blood DC groups were higher than that of peripheral blood DC groups (P<0.01). CONCLUSION: Peripheral blood DC and cord blood DC are mature DC in morphology and function, both can enhance the effector cell killing activities to hepatocarcinoma cells. DC pulsed with tumor antigen can induce higher specific CTL activity than unpulsed DC.