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Expression of Class Ⅲβ-tubulin, MAPT, Survivin and Exvivo Chemosensitivity Testing on Predicting Response Towards Paclitaxel Treatment in Gastric Cancer

Author: HeZuoZuo
Tutor: LiuPing
School: Nanjing Medical University
Course: Oncology
Keywords: Stomach tumor Taxol The type Ⅲ β- tubulin Microtubule-associated protein tau Inhibitor of apoptosis protein survivin ATP bioluminescence method
CLC: R735.2
Type: Master's thesis
Year: 2011
Downloads: 36
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Abstract

Background and Objective: Gastric cancer is the most common malignancy in the world, one of the highest morbidity and mortality in the forefront of various malignancies, serious threat to human health. Chemotherapy in the treatment of advanced and metastatic gastric cancer in an important position. New anti-cancer drugs in recent years continues to appear, the majority of patients still can not get effective control, which one of the important reasons is the gastric cancer cell resistance to chemotherapeutic drugs, leading to the failure of chemotherapy. Taxanes as the third generation of anti-cancer drugs, widely used in the treatment of gastric cancer and other tumors, and achieved a certain effect. However, the clinically observed therapeutic effects and toxicity of the drug manifested in different individuals. Therefore, to explore the the biological predictors associated with paclitaxel chemotherapy sensitivity, and is conducive to the formulation of the chemotherapy of gastric cancer, but also provide the basis for individualized treatment of gastric cancer. In vitro susceptibility testing technology is a simple and convenient means of the sensitivity of tumor cells to chemotherapeutic agents, can reflect Although there are some differences, but the mode of action of drugs in vivo and in vitro reaction of tumor cells to drugs even to some extent effect. In recent years, a number of studies found that the tumor cells become resistant to paclitaxel may tubulin isomers abnormal expression of the microtubule-associated protein expression and apoptosis inhibitory protein family of high expression caused apoptosis inhibition. Different individuals on the the paclitaxel treatment effect differences may be related to the different expression levels of these indicators. In this experiment, we detected gastric cancer cell III β-tubulin, microtubule-associated protein tau and the inhibitor of apoptosis protein survivin gene and protein expression of the three, analyze its relationship with taxane sensitivity in order to explore be able to predict a molecular marker of gastric cancer chemosensitivity. Method: 1. Collected 54 cases of fresh gastric tissue will be organized into two parts: part loaded into a sterile vial filled with PBS, then soaked in antibiotic-containing RPMI 1640 culture medium 15 for 25 min and 12 h ATP -TCA detection; another part was cut into small pieces, put into liquid nitrogen at -196 ℃ wrapped with aluminum foil the instantaneous freezing completely and stored at -80 ℃ ultra-low temperature refrigerator later by RT-PCR and Western blot detection. Connective tissue, fiber and fat in the surface of the tumor tissue was removed, the specimens were minced into small pieces, and transferred to a centrifuge tube containing the tissue of digestive juices, and at 37 ° C incubation for 1.5 for 3 h. The digested mixture was filtered, and discarded to the undigested tissue debris. The filtrate was added RPMI 1640 medium, mixing, centrifugation supernatant was to wash the cells. Ficoll Hypaque 1.077 separation liquid gradient centrifugation, purified collection of tumor cells. Obtain single cells were counted, the suspension concentration is adjusted to 2 to 4 × 105 viable cells / ml. The cell suspension was spread into a 96-well plate, by adding a certain concentration paclitaxel dilution, and incubated for 5 to 7 days. By ATP bioluminescence assay the cultured primary gastric cancer cells to paclitaxel in vitro drug sensitivity. Trizol extracted total RNA samples, reverse transcription polymerase chain reaction (RT-PCR) detection of gastric cancer cells within the Class IIIβ-tubulin, MAPT and survivin mRNA expression. Total protein extract gastric cancer specimens, the Western blot technique (Western Blot) detection of gastric cancer cells Class IIIβ-tubulin, MAPT and survivin protein expression levels. Expression analysis of gastric cancer cells in vitro sensitivity to paclitaxel and three biological indicators. Results: The completion of the 54 specimens in vitro susceptibility test results: highly sensitive six cases, some sensitive 13 cases, weak sensitive 10 cases against 25 cases, in general outside the effective rate of 53.70% (29/54). RT-PCR test results: Class IIIβ-tubulin mRNA expression levels of mRNA expression levels of 1.04 ± 0.22, and the degree of differentiation (P = 0.029); MAPT 0.80 ± 0.17; expression levels of survivin mRNA of 1.00 ± 0.19, and the degree of differentiation (P = 0.009). The susceptibility index Class IIIβ-tubulin mRNA (r = -0.372, P = 0.006) and protein level (r = -0.350, P = 0.009) were negatively correlated with MAPT mRNA (r = -0.292, P = 0.032) and protein expression levels (r = -0.289, P = 0.034) was a negative correlation (r = -0.340, P = 0.012) and protein level (r = -0.296, P = 0.030) were negatively correlated; Class of survivin mRNA IIIβ-tubulin and of MAPT the mRNA (r = 0.284, P = 0.037) and protein levels (r = 0.440, P = 0.001) were positively correlated. Conclusion: 1. Gastric cancer cell Class IIIβ-tubulin the MAPT and survivin expression levels of paclitaxel treatment sensitivity may have better predictive value. 2. Class IIIβ-tubulin and survivin mRNA expression with histological grade significant correlation found no Class IIIβ-tubulin, MAPT and survivin mRNA expression levels and patient gender, age, depth of invasion, lymph node metastasis significant correlation. The Class IIIβ-tubulin of MAPT the mRNA and protein expression levels were positively correlated, there may be some level of co-regulation mechanism both in the tumor cells become resistant to the process. ATP bioluminescence method is a feasible better objectivity in vitro susceptibility testing technology.

Expression of Class Ⅲβ-tubulin, MAPT, Survivin and Exvivo Chemosensitivity Testing on Predicting Response Towards Paclitaxel Treatment in Gastric Cancer

Abstract

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