| Background:Breast cancer is a most common malignant tumor that serious damage to the women's health. Over the past decades years, its incidence increased year by year. Around the world about 1.15 million people are diagnosed with breast cancer in every year, and about 410000 people die of this disease [1]. In Europe and the United States national breast cancer accounts for 25%- 25% of female malignant tumor, according to the American cancer society estimates in the United States each year, 170000 new cases of breast cancer, the number of persons death from breast cancer are about 40000 [2]. Our country is not in high incidence of breast cancer, but average annual growth rate is1 ~ 2% higher than other country, which is still increasing at an annual rate of 3%. In many big cities, breast cancer incidence has raised for the first in the female malignant tumor, becoming the greatest threat to the health of women. In the Beijing, Tianjin, Shanghai and some coastal cities the incidence of this disease is higher, such as the incidence in Shanghai is worst and Tianjin breast cancer incidence in women malignant tumor raised to second, behind in lung cancer. Even worse the incidence of breast cancer and age of patients don shows trend into more young people.Surgery, radiotherapy and chemotherapy are still the main way for treatment of breast cancer. By using of auxiliary chemical treatment in early breast cancer survival rate was increased by 30%, but breast cancer drug resistance is a major cause of treatment failure and death in patients. According to the drug-resistant characteristics of tumor cells, drug resistance can be divided into Primary drug resistance(PDR) and multi-resistant Multidrug resistance(MDR). Multi drug resistance and metastasis are the main causes of failure and death in clinical cancer patients. In the past few decades, although many research have been widely take on these two characteristics of malignant tumors, most of them are only focus on the biological behavior. Recently more and more research shows that there indeed build a functional relationship between the two phenotypes: First, screened out from the parental cells,the resistant cell line has the stronger ability of invasion and metastasis. In addition, compared to the primary tumor, resistance drug was found enhanced significantly in the metastasis tumor cell foci, which show the result observed the occurrence of metastasis of tumor with poor effect of chemotherapy and chemotherapy in the treatment of tumor patients are prone to metastasis 1. Therefore, tumor cells of multidrug resistance have ability to metastasis, in the process of occurrence and development of tumor, which show the progress of drug resistance and metastasis correlating with each other. There have more direct contact between the two processes. Therefore, the correct understanding of the relationship between drug resistance and metastasis of tumor cells can lead to more effective treatment process.Breast cancer is the most common malignant tumors which serious harm to women's health and one of the main cause of poor prognosis because of the recurrence and metastasis. 1/3 of patients with breast cancer will recur and metastases or death within five years, unless being taken timely and effective treatment in early stage by detection of recurrence and metastasis, then part of patients can acquire better curative effect and prolong survival time. Mechanism of recurrence in metastasis of breast cancer is a very complex.There are a lot of factors.In the first part of the research, the association between recurrence of breast cancer and the the multi-resistant metastasis is discussed according to literatures in breast cancer recurrence.Human epididymis protein 4(HE4) is a tumor marker for the early detection of ovarian cancer in recent years, which have advantage in the diagnosis of the pelvic tumor disease of department of gynaecology. the U.S. food and drug administration(FDA) has approved HE4 for clinical monitoring of ovarian cancer recurrence and progress. The role of the HE4 in breast cancer is rarely reported. Although It has been proved that HE4 is also expressed in breast cancer, the specific role and mechanism are still unclear. So this study is to explore human epididymis protein 4(HE4) in its mechanism in the development of breast cancer research. Objective:Part I:The development of multidrug resistance greatly impedes effective cancer therapy. Recent advances in cancer research have demonstrated that acquisition of multidrug resistance by cancer cells is usually accompanied by enhanced cell invasiveness. Several lines of evidence indicated that cross activation of other signaling pathways during development of drug resistance may increase invasive potential of multidrug-resistant(MDR) cancer cells. However, the accurate mechanism of this process is largely undefined. In this study, to better understand the associated molecular pathways responsible for cancer progression induced by drug resistance, a MDR human breast cancer cell line SK-BR-3/EPR with P-glycoprotein over expression was established using stepwise long-term exposure to increasing concentration of epirubicin. The SK-BR-3/EPR cell line exhibited decreased cell proliferative activity, but enhanced cell invasive capacity. We showed that the expression of metastasis-related matrix metalloproteinase(MMP)-2/9 was elevated in SK-BR-3/EPR cells. Moreover, SK-BR-3/EPR cells showed elevated activation of STAT3. Activation of STAT3 signaling is responsible for enhanced invasiveness of SK-BR-3/EPR cells through upregulation of MMP-2/9. STAT3 is a well-known oncogene and is frequently implicatedin tumorigenesis and chemotherapeutic resistance. Our findings augment insight into themechanism underlying the functional association between MDR and cancer invasiveness.Part II:Reverse transcription quantitative PCR(RT-qPCR) was used to detect the HE4 mRNA expression levels in primary breast cancer tissues and the paired adjacent normal tissues. The HE4 mRNA and protein expression was also detected in breast cancer or breast epithelial cell lines by RT-qPCR and western blot, respectively. The RNAi was used to knock down the HE4 expression in HE4 high expression level cell line SK-BR-3, CCK-8 and colony formation assays were performed to analyze the proliferation ability. Flow cytometry was used to detect the apoptotic cells in HE4-depleted cells. Method:Part I:Introduction of drug resistant by using cell culture, which half inhibitory concentration is used to determine resistance index; Using reverse transcription and real-time quantitative PCR detection mRNA expression level; Western Blotting(protein imprinting method) detection of MMP- 2/9, STAT3, P- STAT3, ERK1/2, P-ERK1/2 protein expression levels, immunofluorescence assay for detection of SK-BR- 3 cells and expression of P glycoprotein of SK- BR- 3 / EPR in the nepirubicin free culture medium; Cell proliferation and clone formation experiment were taken in SK- BR- 3 / EPR cell; Cell invasion experiment verify the change of ability of cell invasion.Part II:Reverse transcription quantitative PCR(RT-qPCR) was used to detect the HE4 mRNA expression levels in primary breast cancer tissues and the paired adjacent normal tissues. The HE4 mRNA and protein expression was also detected in breast cancer or breast epithelial cell lines by RT-qPCR and western blot, respectively. The RNAi was used to knock down the HE4 expression in HE4 high expression level cell line SK-BR-3, CCK-8 and colony formation assays were performed to analyze the proliferation ability. Flow cytometry was used to detect the apoptotic cells in HE4-depleted cells. The phosphorylation expression levels of Erk and Akt was evaluated by western blot. Result:Part I:Evaluating the expression of P-glycoprotein, SK-BR-3/EPR cancer cell will get multi-resistant phenotype; SK-BR-3/EPR were continuous cultured in epirubicin free medium six weeks which also has the multi-resistant phenotype; SK-BR-3/EPR cells in cell proliferation rate is significantly reduced, and show the enhancement in invasive ability; Expression of MMP2/9 in SK-BR-3/EPR raised; SK-BR-3/EPR cells phosphorus acylation and the activation of STAT3 increased; the expression of MMP2/9 was downgraded and the invasion ability of the cells were also inhibited when STAT3 of the SK-BR-3/EPR cells was knocked down.Part II:The HE4 mRNA was up-regulated in primary breast cancer tissues compared to the adjacent normal breast tissues. Depletion of HE4 expression suppresses the proliferation and promotes the apoptosis in SK-BR-3 breast cancer cell line. Furthermore, the phosphorylation expression levels of Erk and Akt were reduced in HE4-depleted SK-BR-3 cells. Conclusion:1. An MDR breast cancer cell line SK-BR-3/EPR with P-glycoprotein overexpression was established.2. In this study, SK-BR-3/EPR cells exhibited enhanced cell invasiveness and upregulation of MMP-2/9.3. we showed that elevated STAT3 signaling-mediated upregulation of MMP-2/9 is responsible for the enhanced invasive properties in SK-BR-3/EPR breast cancer cells.4. The expression of HE4 has been relationed with proliferation and apoptosis of SK-BR-3 cell.5. The expression of HE4 influenced the Erk and Akt phosphorylation level. |
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