Folic acid conjugated palygorskite/Au hybrid microgels: Temperature, pH and light triple-responsive and its application in drug delivery.

Herein, folic acid conjugated poly (NIPAM-co-functional palygorskite-Au-co-acrylic acid) (FA-PNFA) hybrid microgels were fabricated by emulsion polymerization. The introduction of acrylic acid can increase the low critical solution temperature (LCST) of FA-PNFA from 36 °C at pH 5.5-42 °C at pH 7.4. Doxorubicin hydrochloride (DOX) was chosen as the load drug, the results show that the DOX release behavior is driven by temperature, pH and light. Cumulative drug release rate can reach 74 % at 37 °C and pH 5.5 while only 20 % at 37 °C and pH 7.4, which effectively avoided the early leakage of the drug. In addition, by exposing FA-PNFA hybrid microgels to laser irradiation, the cumulative release rate was increased by 5 % compared to the release rate under dark conditions. Functional palygorskite-Au as physical crosslinkers not only improves the drug loading content of microgels but also promotes the release of DOX through light drive. Methyl thiazolyl tetrazolium bromide (MTT) assay demonstrated that the FA-PNFA are nontoxic up to 200 µg mL-1 towards 4T1 breast cancer cell. Meanwhile, DOX-loaded FA-PNFA show more significant cytotoxicity than the free DOX. Confocal laser scanning microscope (CLSM) revealed that the DOX-loaded FA-PNFA could be efficiently taken by 4T1 breast cancer cells. FA-PNFA hybrid microgels not only improve the LCST of PNIPAM, but also endow the microgels with photostimulation responsiveness, which can release drugs in response to the triple stimulation response of temperature, pH and light, thus effectively reducing the activity of cancer cells, making them more promising for wider medical applications.

Fms related tyrosine kinase 1 (Flt1) functions as an oncogene and regulates glioblastoma cell metastasis by regulating sonic hedgehog signaling.

Studies have shown that the abnormal expression of Fms related tyrosine kinase 1 (Flt1) is associated with multiple malignancies, yet its role in glioblastoma pathology remains to be elucidated. In this study, we investigated the role of Flt1 in regulating proliferation, migration and invasion of glioblastoma cells by establishing glioblastoma cell strains with constitutively silenced or elevated Flt1 expression. We demonstrate that ectopic expression of Flt1 promotes glioblastoma cells migration, invasion through cell scratching and Transwell assays. Further study has indicated that Flt1 knockdown prevents the spread of glioblastoma cells in vivo. Conversely, we also show that suppression of Flt1 expression inhibits migration and invasion of glioblastoma cells. Finally, our findings demonstrate that Flt1 promotes invasion and migration of glioblastoma cells through sonic hedgehog (SHH) signaling pathway. Our study suggests that galectin-1 represents a crucial regulator of glioblastoma cells metastasis. Thus, the detection and targeted treatment of Flt1-expressing cancer serves as a new therapeutic target for glioblastoma.

Overexpression of Protease Serine 8 Inhibits Glioma Cell Proliferation, Migration, and Invasion via Suppressing the Akt/mTOR Signaling Pathway.

Protease serine 8 (PRSS8), a serine peptidase, has a widespread expression in normal epidermal cells. Recently, many researchers demonstrated downregulation of PRSS8 in cancer tissues as well as its tumor suppressor role in cancer development. However, the biological functions of PRSS8 in glioma remain unclear. In the current study, we demonstrated a decreased expression of PRSS8 in glioma tissues and cell lines. PRSS8 upregulation inhibited glioma cell proliferation, migration, and invasion. In addition, xenograft experiments showed that PRSS8 overexpression suppressed glioma cell growth in vivo. We also found that upregulated PRSS8 reduced the protein expression levels of p-Akt and p-mTOR in glioma cells. Taken together, our study demonstrated that overexpression of PRSS8 inhibited glioma cell proliferation, migration, and invasion via suppressing the Akt/mTOR signaling pathway. Therefore, PRSS8 may act as a novel therapeutic target for glioma.

[Effect of 5-aminolevulinic acid-mediated photodynamic therapy on human gastric cancer xenografts in nude mice in vivo].

OBJECTIVE: To investigate the effect of 5-aminolevulinic acid (ALA)-mediated photodynamic therapy (PDT) on human gastric cancer xenografts in vivo and to explore its potential tumoricidal mechanism. METHODS: Cultured MGC-803 human gastric cancer cells were injected below the skins of the nude mice to develop the tumor model. The tumor-bearing nude mice were examined under the Leica LT-9 MACIMSYSPULS to detect the fluorescence. The tumor volume of day 1, 3, 7, 14, 21 after treatment were measured, and its histological changes were also studied. The tissues of the tumors in nude mice of the control group, light group, 5-ALA group and PDT group were examined with the electron microscope and apoptosis was detected by TUNEL assay. RESULTS: The tumor model was successfully developed. The tumor in the nude mice emitted the red fluorescence under the Leica LT-9 MACIMSYSPULS. The tumor volumes were (0.189+/-0.010) cm(3), (0.183+/-0.011) cm(3), (0.185+/-0.019)cm(3), (0.182+/-0.015)cm(3) for the control group, light group, 5-ALA group, PDT group, respectively at day 1 after treatment, while at day 3, (0.294+/-0.010) cm(3), (0.280+/-0.013) cm(3), (0.278+/-0.016) cm(3), (0.183+/-0.014) cm(3); at day 7, (0.409+/-0.016) cm(3), (0.411+/-0.009) cm(3), (0.407+/-0.015) cm(3), (0.221+/-0.008) cm(3); at day 14, (0.970+/-0.055) cm(3), (0.976+/-0.054) cm(3), (0.981+/-0.032)cm(3), (0.318+/-0.005) cm(3); at day 21, (1.495+/-0.059) cm(3), (1.513+/-0.057) cm(3), (1.524+/-0.063) cm(3), (0.446+/-0.042) cm(3) (F=1003.086, P=0.000). The histology demonstrated that most tumor blood vessels were congested and necrosis developed after PDT while not in the control group, light group and 5-ALA group. Necrosis and apoptosis were observed in the cells of the tumors of the PDT group examined by TUNEL and electron microscope while not in the cells of the tumors of the other groups. CONCLUSIONS: 5-aminolevulinic acid-mediated photodynamic therapy (PDT) can induce injury to human gastric cancer xenografts and inhibit the tumor growth while light only and 5-ALA only can not. 5-aminolevulinic acid-mediated photodynamic therapy (ALA- PDT) appears to be a promising therapy for human gastric cancer, whose mechanism involves in the destruction of the tumors partly by apoptosis other than necrosis.

Effect of photodynamic therapy with 5-aminolevulinic acid on human gastric cancer cells in vitro.

OBJECTIVE: To investigate the effect of 5-aminolevulinic acid (5-ALA)-mediated photodynamic therapy (PDT) on MGC-803 human gastric cancer cells in vitro. METHODS: MGC-803 human gastric cancer cells were treated with 5-ALA at various concentrations followed by laser irradiation. The cells were also treated with 5-ALA at the same concentration before laser exposure at various doses. PDT-induced phototoxicity of the cells was determined by MTT assay. RESULTS: After laser exposure of the cells at the same dose (25.0 J/cm(2)), the cell survival rates decreased significantly with incubation of the cells with 5-ALA at 0.25, 0.5, 1.0, 2.0 and 4.0 mmol/L, respectively (F=266.39, P<0.001), but 2.0 and 4.0 mmol/L ALA showed no significant difference in lowering the cell survival rates (P>0.05). Following treatment with the same 5-ALA concentration (1 mmol/L), the cell survival rates decreased in response to increased laser doses (at 6.25, 12.5, 25.0, 50.0, and 100 J/cm(2), respectively, F=226.31, P<0.0001). Without laser exposure, the survival rate of the cells did not significantly change for different 5-ALA concentrations (F=0.79, P=0.5383), nor did it undergo obvious variation in response to different laser doses without 5-ALA incubation (F=0.61, P=0.6551). CONCLUSIONS: The damage of MGC-803 cells by PDT increases with 5-ALA concentration within a relative lower range and is proportional to the laser doses delivered. Without 5-ALA treatment, the laser at the chosen dose cannot produce photodynamic effect and ALA itself is nontoxic. ALA-mediated PDT appears to be a promising therapy for gastric cancer.

Construction of eukaryotic expression vector with brain-derived neurotrophic factor receptor trkB gene.

OBJECTIVE: To construct an eukaryotic expression vector carrying rat brain-derived neurotrophic factor receptor trkB gene. METHODS: Using the total RNA isolated from rat brain as template, the trkB gene was amplified by reverse-transcription-polymerase chain reaction (RT-PCR) with a pair of specific primers which contained the restrictive sites of EcoR I and BamH I. The amplified fragment of trkB gene was digested with EcoR I and BamH I, and then subcloned into cloning vector pMD18-T and expression vector pEGFP-C2 respectively. The recombinant plasmids were identified by restriction endonuclease enzyme analysis and PCR. RESULTS: The amplified DNA fragment was about 1461 bp in length. Enzyme digestion and PCR analysis showed that the gene of trkB had been successfully cloned into vector pMD18-T and pEGFP-C2. CONCLUSIONS: The trkB gene of rat has been amplified and cloned into the eukaryotic expression vector pEGFP-C2.

Time window characteristics of cultured rat hippocampal neurons subjected to ischemia and reperfusion.

OBJECTIVE: To explore cell death and apoptosis in rat hippocampal neurons at different time points after ischemia, hypoxia and reperfusion injury and to elucidate time window characteristics in ischemia neuronal injury. METHODS: Hippocampal neurons were obtained from rat embryo and were cultured in vitro. The ischemia and reperfusion of cultured rat hippocampal neurons were simulated by oxygen-glucose deprivation (OGD) and recovery. OGD at different time points (0.25 h to 3.0 h) and then the same recovery (24 h) were prepared. Annexin V-PI staining and flow cytometry examined neuron death and apoptosis at different time after injury. RESULTS: After OGD and recovery, both necrosis and apoptosis were observed. At different times after OGD, there were statistically significant differences in neuron necrosis rate (P < 0.05), but not in apoptosis rate (P > 0.05). At recovery, survival rate of hippocampal neurons further decreased while apoptosis rate increased. Furthermore, apoptosis rates of different time differed greatly (P < 0.05). Apoptosis rate gradually increased with significant difference among those of different time points (P < 0.05). However, 2 h after ischemia, apoptosis rate decreased markedly. CONCLUSIONS: Apoptosis is an important pathway of delayed neuron death. The therapeutic time window should be within 2 h after cerebral ischemia and hypoxia.

[Induced differentiation of rabbit bone marrow stromal cells into neuron-like cells].

OBJECTIVE: To study whether the neuron-like cells derived from bone marrow stromal cells (BMSCs) may excrete amino acids with neurobiological activities and possess the biochemical characteristics of neurons. METHOD: Under sterile condition, BMSCs from New Zealand rabbits were purified by gradient density centrifugation, and were induced to differentiate into neural stem cells and neuronal-like cells in the culture medium for neural stem cells containing retinoic acid (RA, 0.5 microg/ml) and glial-derived neurotrophic factor (GDNF, 20 ng/ml). The differentiated cells were then examined with immunocytochemical method and high-performance liquid chromatograpy (HPLC). RESULTS: The round and enlarged BMSCs on day 10 of cell culture were positive for nestin, and on day 20, the cells with RA+GDNF stimulation differentiated into neuron-like cells with long protrusions and presented neuron-specific enolase (NSE) antigen. HPLC identified high levels of amino acids like Asp, Glu, Gly and Ala in the differentiated cells (P<0.01). CONCLUSIONS: Rabbit BMSCs may proliferate in vitro as from nestin-positive cells and differentiate into NSE-positive cells containing high levels of excitatory and inhibitory amino acid neurotransmitters. RA and GDNF are important promoters for in vitro differentiation of the BMSCs toward neural stem cells.