| Cancer has been one of the biggest killers of human health. Nearly half of the new cases of cancer and deaths appear in China around the world. Traditional cancer treatments are surgery, chemotherapy and radiotherapy. However the chemotherapeutics drugs are lack of tissue specificity, and they will cause serious side effects during long-term usage. The curative effect may reduce when the same drug is used for a long time as the development of drug resistance. In recent years, the targeted therapy drugs are born, which can target and inhibit the cancer cells specifically.Immunotoxin is a kind of typical tumor targeted therapeutic drug, which is mainly composed of three domains: the toxin domain, the linkage domain and the targeting domain. Most of the targeting domain of immunotoxins is cancer-specific monoclonal antibody or ligands(e.g., cytokines, growth factors) on the surface of cancer cells. Monoclonal antibodies can bind to cancer cells specifically, but they have some disadvantages in application. Such as they usually have the large molecular weights, their ability of penetration to cancer is weak and they may cause the immunogenicity easily. Ligand molecule is capable of target the receptors specifically, so it can be used as the target domain to deliver the toxin domain to the cancer cells. Those ligands, which can be used as carrier, are interleukin series, tumor necrosis factor and epidermal growth factor. Ligand molecules have many advantages as a carrier, such as a lower molecular weight, weak immunogenicity, strong ability of penetrating to the tumor tissue, short circulating half-life and the convenient gene operation.At present, some immunotoxins show good effects in the clinical trials and application. They have the weaker non-specific toxicity than traditional drugs. Due to some of the toxin domains come from the bacterial toxins, they will cause high immunogenicity, which is still the bottleneck in the clinical applications. Thus it will weaken the anti-tumor efficacy of immunotoxins, or even interrupt the clinical usage.Onconase is separated and purified from the oocyte and early fertilized eggs of the Rana Pipiens with a molecular weight of 12 k Da. It has got the approval documents of clinical treatment for mesothelioma in the United States and parts of the Europe. During the process of long-term clinical application, it presents the following advantages: less drug resistance, lower allergenic, lower immunogenicity, few side effects and low excitant. These characteristics determine that the Onconase has well medicinal properties and clinical application prospects. In order to obtain a more efficient drug than the original Onconase, this study choose the Onconase as the toxin domain and the V3 sequence as the target domain to constructuring an immunotoxin Onc- V3. In this experiment, we constructed the recombinant expression plasmid p GAPZα AOnc- V3 and expressed the immunotoxin Onc- V3. Then we studied the anti-cancer effect and the mechanism of the Onc- V3 through a series of experiments in vitro.The first part: the construction of the expression plasmid p GAPZα A- Onc-V3.According to preference codon of Pichia pastoris, the recombinant expression plasmid p GAPZα A- Onc- V3 was constructed and transformed in Pichia pastoris X-33 by electroporation. A stably inherited multi-copy Pichia pastoris X-33 strain containing the recombinant plasmid p GAPZα A-Onc-V3 was got by antibiotic selection and identified. The optimal electroporation conditions of the Pichia pastoris X-33 are using the 0.2 cm electroporation cup, at 1.1 k V, 200 Ω and 25μF. The Pichia pastoris X-33 strain Onc4 with the highest expression amount was got and identified by the Western Blot. It proved that the immunotoxin Onc-V3 could be expressed in the Pichia pastoris X – 33 strain correctly. The medium containing Onc-V3 was purified and identified by Tricine electrophoresis. The purification strategy of the recombinant immunotoxin Onc-V3 was established.The second part: the optimization of the expression of Onc- V3.The effects of carbon, nitrogen, p H and temperature of the culture medium for the expression of recombinant proteins Onc-V3 was examined, and the optimal expression conditions in flask was selected. The carbon source was glucose, the nitrogen source was peptone, the initial p H was 6.5-7.0 and temperature was 27-30℃. In the response surface analysis, it could be observed that the content of the glucose and peptone in the culture liquid have a significant effect on the production of Onc-V3. The optimized results were as follows: the carbon source(glucose) was 21.6g / L, the nitrogen(peptone) was 22.25 g / L, the temperature was at 27℃ and the p H was 6.5. The production increased 24% compared with the original conditions. According to this, a 50 liters fermentor was used for the culture of Onc-V3. The Onc-V3 protein solution with an enzyme activity of 4161 U / L was got after the process of centrifugation, ammonium sulfate precipitation, dialysis desalination, affinity chromatography and gel filtration chromatography.The third part: the anti-cancer effect and mechanism of the immunotoxin Onc- V3 to cancer cells.Onc-V3 hemolysis experiment results showed that the Onc-V3 had no hemolysis at a concentration of 100μM, which was more than 200 times compared to the IC50 concentration(0.19 ~ 0.38μM). It revealed that the Onc-V3 was stable in the circulation system. It could be observed that the Onc-V3 had no cytotoxic effect on human normal HEK293 cells. MTT results showed that Onc-V3 had cytotoxic effect to A549 cells, Hep G2 cells, MCF-7 cells, Hela cells and HO-8910 PM cells, and the IC50 varied from 0.19μM to 0.38μM, while the original Onconase was about 6μM. In order to observe the distribution of the FITC-Onc-V3 with the laser confocal microscope, the Mito Tracker® Red CMXRos was used to label the mitochondria and the Hochest 33342 to label the nucleus. Results showed that the nucleus was dyed blue with Hochest 33342 and surrounded by red dyed mitochondria in the HO-8910 PM cells. The green FITC-Onc-V3 was located in the cytoplasm along with the red mitochondrial molecules, indicating that Onc- V3 molecules could target cancer cells and played its role into the cytoplasm. The HO-8910 PM cells slice was cultured with 0.4μM Onc-V3 medium for 48 h and stained with DAPI. Under the fluorescence microscopy, it showed shrinkage, grainy, condensed blue fluorescence nucleus compared with the control group. Results of flow cytometry showed that apoptosis occurred in the HO-8910 PM cells when treated with Onc-V3. This apoptotic effect was significantly dose-dependent, and at the dose of 1.60μM the apoptosis rate was up to more than 90%. The HO-8910 PM cell line was a kind of highly metastatic human ovarian carcinoma cells, thus this cell line was used in the wound healing and Transwell invasion assay. Wound healing assay showed that the high dose of Onc-V3 inhibited the cancer cell migration. The Transwell invasion assay showed that Onc-V3 could inhibit the invasion of the cancer cell to the basement membrane. Western Blot tests found that the contents of PARP, procaspase-9 and procaspase-3 significantly decreased in the apoptotic cancer cell induced by the Onc- V3, while the caspase-9 and caspase-3 increased. The Onc- V3 could induce the cancer cell apoptosis by the caspase-9 and caspase-3 signal pathway, which was consistent with the literature reports.In summary, this study constructed the recombinant expression plasmid p GAPZαA-Onc-V3 and expressed immunotoxin Onc-V3 in Pichia pastoris X-33 successfully, then observed the Onc- V3 inhibitory effect to cancer cells and the mechanism systematically by a series of experiments in vitro. This study demonstrated that Onc-V3 had the better cytotoxic effect to many kinds of cancer cells compared with the original Onconase. It laid the foundation for further research and application of Onc-V3. |
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