Experimental Study On Effect Of Chemotherapy Treatment For Breast Cancer With Gene Targeting

Background:Breast cancer has become the leading cause of cancer-related morbidity in wo men and has the second mortality in the world. According to statistics in 2007,there were 79 thousands new cases of breast cancer each year in the world, which occupy 21% of all women malignant tumors. The world annual morbidity of breast cancer is 30.30 per hundrend thousand, and the world population adjust rate is 32.97 per hundrend thousand. About 60% new cases of breast cancer occur in developed countries.Although morbi dity of breast cancer is different worldwide, but it shows ascendant and younger trend. In some cities in China, breast cancer has become the first most common cancer in women cancers, which is a leading killer of women malignant tumors. So improvement of breast cancer efficacy and cure rate has become urgent problem.As a systemic disease, Comprehensive therapy which combined with surgery, chemotherapy, radiotherapy and endocrine therapy has become the standard treatment of breast cancer. With the establishment of the concept of systemic disease, the role of systemic chemotherapy treatment in comprehensive treatment is taking more and more attention..Xihuan drugs with doxorubicine represented and paclitaxel class chemotherapy drugs with docetaxel represented still play indispensable roles, which can improve prognosis and reduce the recurrence of patients. BCIRG 001 and NSABPB - 28 clinical experiments show that anthracycline-based combinations and taxane combinations (TAC) scheme curative effect is distinct, and they are recommended as standard solutions of adjuvant chemotherapy of breast cancer. However, clinical problems have to face are:Heart toxicity of doxorubicin seriously affected its clinical widespread use; Marrow inhibition of doxorubicin and docetaxel, especially combination chemotherapy often causes IV-degree marrow inhibition which can be deadly when serious. With intensive study of tumor molecular biology in recent years, people understand happening mechanism, development mechanism and signal transmission mechanism of breast cancer, find many genes and proteins closely related with breast cancer development. Tumor targeted therapy with these genetic and molecular as target also has become a hotspot of adjuvant therapy of breast cancer, and some have achieved good results, such as hormone therapy to ER,PR receptors, whose efficiency can be up to 50%-60%. These treatments cannot achieve the effect of chemotherapy and have some limits. For example, the efficiency of ER,PR breast cancer patients treated by hormone therapy is no more than 10%. Molecular target therapy of breast cancer is treatment aimed at breast cancer occurrence, development related cancer gene and its related express product. Molecular targeted drugs block signal transduction of tumor cells or related cells to control gene expression, then to inhibit or kill tumor cells. In numerous genes, against human epidermal growth factor receptor-2 (HER-2) gene is one successful in example the same genes. HER-2 gene is located in 17q21 and has the transmembrane chromosome tyrosine kinase activity growth factor receptor. Clinical studies showed that HER-2 distinct genes amplification and overexpression exist in 20%-30% breast cancers. Overexpression has high grade histological and late stage, which solid visceral transfer is more likely to occur, can rarely gain complete response after chemotherapy. Herceptin is the first targeted molecular drug used breast cancer HER-2 genes as therapeutic targets, which is widely used in clinical. Herceptin with its clear target and no obvious side effects to normal body, has gradually become a indivisible part of the clinical standard treatment for breast cancer, which has incomparable advantage compared to traditional chemotherapy. In recent years, treatment combined the most efficent TAC scheme with Herception to HER-2 positive breast cancer patients has achieved gratifying achievements. N9831 and BCIRG006 experimental research shows that disease-free surial and oerall surial rates of cyclophosphamide and adriamycin sequential Docetaxel then sequential Herceptin improve obviously compared to pure adjuvant chemotherapy. Gene targeting improved the sensitivity of chemotherapy treatment or they are synergistic? There were few experiments to research and explore. If synergistic effect exists, can dose be reduced as chemotherapy effect not decrease to reduce the side effects of the effective chemotherapy regiment Docetaxel+THP(DA) These problems are taken close attention in clinic. Objective:To explore damage function and possible mechanism of breast cancer cells treated by DA combined Herception and compare the differences of damage function with different dosing sequence by MTT and Apoptosis way, then to provide preliminary experiments basis and develop new ideas of current breast cancer treatments DA+Herception.Method:1. Detect the HER-2 expression of the two cell lines M453 and M231 by immunohistochemical method , choose the higher expression of HER-2 cell lines for the study.2. The inhibited rates of human breast cancer cells (M453) treated by Docetaxel and THP for 24h, 48h and 72h were determined with MTT colorimetric assay. Concentration - cell inhibited rate curve was drowed by Compusyn 1.0 software to calculate IC50.3. The inhibited rates of human breast cancer cells (M453) treated by single Herception, combined Docetaxel and THP, sequential Docetaxel and THP for 48h were determined with MTT colorimetric assay. Use Guinness formula to analyse drug merger effect.4. The changes in cell cycles of M453 by flow cytometry treated by single Herception, combined treatment of Herciptin and chemotherapy in different sequences for 48h were determined by PI.5. Apoptosis of M453 by flow cytometry treated by single Herception, combined treatment of Herciptin and chemotherapy in different sequences for 48h were determined by Annexin-V/FITC double marking method.6. Observe the apoptotic cells form through optical microscope after Gimesa dyeing processing.7. Analyse all experimental data by SPSS17.0 software. Use Compusyn 1.0 software to analyse single-agent role and draw concentration-cell inhibiting rate curve. Use Logitic curve regression method and Microsoft Excel to draw inspection concentration–inhibiting rate curve.Results:1. M453 cell overexpresses HER-2 (3+) and M231 cell doesn't express by IHC .2. IC50 of M453 treated by Docetaxel for 24h, 48h and 72h was respectively 80.4372 ug/ml, 53.9763 ug/ml,26.2771 ug/ml, IC₅₀ of M453 treated by THP for 24h, 48h and 72h was respectively 13.9195 ug/ml, 5.8422 ug/ml,1.4973 ug/ml, which shows time concentration dependence effect. 3. IC₅₀ of M453 treated by Docetaxel combined with THP at the concentration of IC₅₀ was 0.95114ug/ml, while Q was 0.87(+). Q was 1.19(++)and 0.83(-)when combined at the concentration of 1/4 IC₅₀ and 2 IC₅₀ .4. The inhibition was 53.35% and Q was 1.08(+) when treated by 1/2 IC₅₀ concentration of Herception combined with IC₅₀ concentration of Docetaxel. The inhibition was 51.01%, 61.95% and Q was 1.21(++), 1.17(++) respectively when treated by 1/4 IC₅₀ concentration of Herception combined with IC₅₀ concentration of Docetaxel, Docetaxel and THP.5. The inhibition was 62.66%, 85.47%,73.61% and Q was 0.94(+), 1.11(+), 0.84(-) respectively when treated by Docetaxel, THP, Docetaxel and THP sequential Herception. The inhibition was 80.23%, 66.85%, 88.11% and Q was 1.08(+), 0.86(+), 0.97(+) respectively when treated by Herception sequential Docetaxel, THP, Docetaxel and THP.6. Single THP or Docetaxel and THP combined with Herception all resulted in the decrease of proportions of G₀/G₁ , S phase and the increase of G₂/M phase. Docetaxel combined with Herception resulted in increase of proportions of G₀/G₁ phase and decrease of S, G₂/M phase. Docetaxel combined with THP and Herception resulted in decrease of proportions of G₀/G₁ and the increase of S, G₂/M phase.7. Herceptin sequential Docetaxel resulted in the increase of proportions of G₀/G₁ phase and the decrease of S, G₂/M phase while Docetaxel sequential Herceptin resulted in the increase of proportions of S phase and decrease of G₂/M phase. THP sequential Herception resulted in further decrease of proportions of G₀/G₁ , S phase and further increase of G₂/M phase while Herception sequential THP resulted in the increase of proportions of G₀/G₁ phase and decrease of S, G₂/M phase. Docetaxel combined with THP sequential Herception resulted in the decrease of proporations of G₀/G₁ phase and increase of G₂/M phase while Herception sequential Docetaxel combined with THP resulted in further decrease of proporations of G₀/G₁ , S phase and further increase of G₂/M phase. Its mechanism may be related to the cell cycle changes.8. The apoptotic cells were different when treated in different ways from Gimesa dyeing and optical microscope observation.Conclusions:1. Different degrees of proliferation inhibition of M453 treated by single Docetaxel, THP or Herceptin appeared, with HER-2 positive cells stronger than HER-2 negative cells treated by Docetaxel. The inhibition of M453 treated by DA combined Herception is stronger than single DA. The inhibiting effect treated by 0.31, 0.75, 1.6 IC₅₀ DA combined Herception is appropriate to 1/2, 1, 2 IC₅₀ DA.2. Herception mainly arrested cell cycle progression of G₂ phase, while Docetaxel G₂/M phase and THP S phase. Docetaxel+THP mainly arrested cell cycle progression of G₂/M phase, Docetaxel+THP conbined Herception arrested cell cycle progression of G₂/M phase furthe more. Apoptosis rate is higher when treated by Docetaxel conbined THP than single Docetaxel, and higher when treated by Docetaxel+THP combined Herception than Docetaxel+THP.3. Inhibiting effects in different sequences are different. Inhibiting effects treated by THP sequential Herception and Herception Docetaxel are stronger than the other two sequential group. Inhibiting effect of Herceptin sequential Docetaxel+THP is stronger than Docetaxel+THP sequential Herceptin. Key words: Breast Cancer;Herceptin;Docetaxel;THP;Chemosensitization;...