Study Of The Relationship And Machenism Between Pten-PI3K/Akt Cell Signaling Pathwany And Multidrug Resistance In Breast Cancer

Background: The multidrug resistance (MDR) of the tumors is the most important obstacle in clinical therapy. And it is also the main reason that leads to the clinical chemotherapy failure and results in ralapse and/or distant metastasis of breast cancer. Primary breast cancer is much sensitive to chemotherapy and most of the patients can alleviate by using different structural cytoxious drugs, but most of them come into being multidrug resistants soon which lead to high relapse rate and poor prognosis of breast cancer. So the scientist put more attention to the mechanism of drug resistance and reversal strategy. The mechanism of MDR is much complex. Beside the mdr1 gene and its protein P-gp, the relationship between the cell signaling pathway and MDR becomes a new light. The two most important cell signaling pathways are MAPK pathway and PI3K/Akt pathway which involved in cell survival, growth and proliferation of the cells. Since PI3K has the call in signaling, it is very interesting to investigate the relationship between MDR and PI3K/Akt signaling pathway.PTEN is a new tumor suppressor gene confirmed in recent years and has dual specific phosphotase. And it is the most important tumor suppressor gene after P53. PTEN can exerts its tumor suppression effect through suppress PI3K/Akt pathway. PI3K/Akt cell signaling pathway is the mainly pathway to lead cell survival and Akt is the key factor in this pathway which induce cell survival through regulating the phoshorylation status of multiple downstream proteins. It has been confirmed that lossand mutation of PTEN gene occurs in many human malignant tumors. The LOH rate of PTEN gene in human breast cancer is about 30%-50% which suggests loss of PTEN protein expression is a common event in breast cancer.lt suggests that PTEN may play a very important role in development and progression of human breast cancer. However, the expression and the clinical significance of PTEN in breast cancer tissues are not very clear yet and many results are inconsistent or conflict. Loss of PTEN expression leads to activition of PI3K/Akt cell signaling pathway. Especially the expression and function of phospho-Akt are usually increased greatly. Although many studies focus on the function of Akt in cells in vivo, the expression of p-Akt in breast cancer detected by immunohistochemistry are not discussed fully.Several studies has suggested that the tumor suppressor gene PTEN and Akt are related to chemosensitivity of tumor cells. For example, it has been found that cells expressing PTEN are more sensitive to adriamycin than cells expressiong mutated/no PTEN. Overexpression of PTEN by transfection in vivo increases the chemosensitivity to cytoxious drugs in bladder cancer cells and prostate cancer cells. These results suggest that PTEN may sensitize tumor cells to drugs and it can become a new target gene to reverse multidrug resistance. Activation of Akt lead to cell survival, cell growth, cell proliferation and reduce the sensitivity to drugs. And many chemotherapy drugs can active PI3K/Akt cell signaling transduction pathway temporarily or persistently. Many studies showed that the specific inhibitor of phosphoralytion of Akt can preferably induce apoptosis in MDR cells but has no significantly effect in sensitive cells.Besides that, the phosphorylation of p-Akt in MDR cells is higher than that of drug sensitive cells which also suggests that the appearance of MDR is tightly related to the activation of PI3K/Akt cell signaling pathway. Although it has been studied that PTEN and PI3K/Akt can change chemosensitivity in cultured cells in vivo, it has not be noticed that whether they have some relationship with typical multidrug resistance and what is the main regulation mechanism and pathway of it. It is very valuble to investigate in this field. This study on the relationship between cell signaling pathway and multidrug resistance will find new mechanism of MDR and will provide newtargets of reversion of MDR and new method way to therapy in breast cancer.Traditional reversion drugs have great side-effects to patients at its reversal dose and are limited in clinical therapy. So it is urgent and pivotal to find new means and drugs to reverse MDR. In recent years, gene transduct and gene therapy have become very valuable and important method to conquer tumor or MDR. The breakthrough in gene therapy relys on finding more efficient new targets. It crys for new pertinence target genes to treat human breast cancer. Cell signal pathway is a new important target to gene therapy. And PI3K plays a central role in multiple cell signaling and in tumor genesis. A good many studies have found that PTEN is often loss and Akt is always activated in breast cancer. So we can consider the PTEN-PI3K/Akt pathway is a pertinent target for breast cancer gene therapy.Objective: To study the expression and significance of PTEN, p-Akt and P-gp in human node negative breast cancer and investigate the relation between PTEN-POK/Akt signaling pathway and MDR. Investigate the inhibitory effects, apoptosis induce effects, cell cycle arrest effects and MDR reverse effects of the tumor suppressor gene wide-type PTEN by transient transduction in drug sensitive MCF-7 cells and MDR MCF-7/ADR cells through Lipofectamine2000. Analyze the relationship and regulation mechanism between PDK/Akt signaling pathway and drug resistance and study the effect on the expression of mdrl and P-glycoprotein. And at the same time, we design to show the effect on cell transcriptional fators NF- k B by transfection of expressing vectors which encode active or dominant negative Akt. This study aim to provides a basic theory and evidence to gene therapy and multidrug resistance reversion of breast cancer.Methods:1. 81 cases of LNN breast cancers and corresponding adjacent tissues were detected the expression of active p-Akt, PTEN and P-gp by immunohistochernistry staining. Results were analyzed with the clinical pathologic parameters and survival.2. The eukaryotic expression plasmid pEGFP-Ci-PTEN containing wild-type PTEN and two mutated type PEGFP-C1-C124S and PEGFP-Cl-G129E(inactive in lipid phophotase) was transfected into MCF-7 and MCF-7/ADR by Lipofectamine2000. The effects of growth suppression in cell apoptosis and cell cycle were observed through flow cytometry, growth curve, clone formation assay, phase-microscope, MTT, and DNA agarose gel electrophoresis. We also evaluated the expression of anti-apoptosis protein Bcl-2 by flow cytometry, pro-apoptosis protein Caspase3 and Caspase9 by immunocytochemistry and western blot to detect the detailed apoptosis pathway.3. The reversal effects of wild type PTEN of MDR in MCF-7 and MCF-7/ADR were evaluated by MTT, intracellular adriamycin accumulation assay and Rhodanmin 123 exclusive assay. The expression changes of mdrl gene in m RNA level and protein level were evaluated by RT PCR, Western blot and immunocytochemistry respectively. Through the experiment mentioned above we can analyze the reversal effect of multidrug resistance in breast cancer cells.4. Gag-Akt (expressiong constitutive active Akt) and AAA-Akt( expressing dominant negative Akt ) were transfected into MCF-7 and MCF-7/ADR by Lipofectamine 2000. We detected Akt phosphoralytional level by western blot. And we detected the intracellular adriamycin accumulation by cytometry and the chemosensitivity of the cells to adriamycin by MTT. The expression of mdrl and P-glycoprotein in mRNA and protein level by RT-PCR, western blot and immunocytochemistry respectively. Analyzed the relationship and the mechanism between the PDK/Akt cell signaling pathway and multidrug resistance. Meanwhile, we studied the reversal effect of PI3K inhibitor LY294002 and Akt phosphorylation inhibitor UCN-01 on multidrug resistance in breast cancer MDR cells.5. We detected the change of expression level of the transcriptional factors NF-k B by western blot in order to confirm that PTEN-PI3K/Akt cell signaling pathwayregulate MDR through transcriptional factor NF- k B.Results:1. A marked positive activity (27.2%) of p-Akt was observed in tumors which always were low differation and ER negative (P<0.05). However, no obvious immunoactivities were detected in adjacent breast tissues. 37.0% tumors were PTEN absent/reduced and 39.5% were positive expression of P-gp. In p-Akt positivesubgroup, 81.8% tumors were loss/reduced expression of PTEN (/>< 0.05) . The positive rate of P-gp in p-Akt positive subgroup (68.2%)was higher than that of negative group (23.6%). Overexpression of p-Akt, reduced/loss of PTEN and overexpression of P-gp had a significant correlation with decreased OS in the multivariate Cox regression analysis (P<0.05) .2. The present data showed that the overexpression of PTEN could lead to mophalogical changes of apoptosis and Gl arrest in MCF-7 and MCF-7/ADR.The differences were significant. The cells showed typical apoptosis morphological changes microscope: smaller, rounder and appear some buds. It was observed that apoptosis rate and Go/Gi cell population of wild type PTEN overexpression cells was much higher than those of mutation type and vetor control. The clonogenic survival rate of cells transfected with PTEN was significantly decreased after doxorubicin treatment compared with control. Meanwhile, G129E also had the growth suppression in the two cell lines. We also detected down regulation of Bcl-2 and caspase3 active fragment.3.Wild type PTEN could partly reverse MDR in MCF-7/ADR cell and increase sensitive to doxorubicin compared with the control cells. And the IC50 decreased to 1/4 of that of the vector control(p<0.01). The adriamycin accumulation and Rodamine 123 exclusive effect were significantly decreased. The protein P-gp expression was also decreased in not only transcriptional level but also in translation level.4..Constitutive active Akt could lead to drug resistance to adriamycin in drug sensitive cell MCF-7 and IC50 to adriamycin increased to 4 fold of that of the control cells. Akt could induced the expression of the gene mdrl, but this effect in MDR cells were not so distinct than in sensitive cells. On the contrary, the effect of the dominant negative Akt was inverse compared with active Akt. Inhibitor of PI3K, LY294002 and inhibitor of Akt phosphorylation UCN-01 could specially inhibited this pathway and preferably induced MDR cells to apoptosis. And both of the two drugs could increase the sensitivity to adriamycin and downregulate P-gp expression.5.The expression level of the cell transcriptional factors NF-k B was involved in regulation course of PI3K/Akt pathway and mdrl.Conclusions:l.The PTEN-PI3K/Akt signaling pathway is aberrant in approximately 1/3 of LNN breast tumors and determination of its status which may be of value in identifying high-risk patients. This pathway may be a very important machenism inducing multidrug resistance of breast cancer cells. It gives a rationale to investigate new therapy strategies by specific inhibiting the signaling pathway in breast cancer.2. PTEN exerts its tumor-suppressive effects in MCF-7 cells through blocking cell cycle progression and inducing cell apoptosis. The induction of apoptosis is through activating caspase3 and down regulating Bcl-2. PTEN has dominant anticancer effect in breast cancer cells through inhibiting PI3K/AKT pathway. Its lipid phosphotase is the main mechanism of its growth suppression in breast cancer cells.3.Wild-type PTEN could reverse multidrug resistance in breast cancer cells partly. It could increase the chemosensitivity to adriamycin and also decreased the expression of mdrl and P-gp in MCF-7/ADR cells. PTEN reverse MDR in breast cancer cells through suppression of PI3K/Akt pathway.4.The constitutive active Akt could significantly increase the level of phosphorylated Akt and induce the resistance to adriamycin. Gag-Akt can decrease the adriamycin accumulation in cells and up-regulate the expression of mdrl and P-gp which suggests that activation of PI3K/Akt is a new mechanism of multidrug resistance phenotype. The small molecular inhibitor of PDK/Akt pathway LY294002 and UCN-01 can reverse MDR partly as well. It suggests that the inhibition of PI3K/Akt is a new kind of MDR reversion strategy.5.PTEN-PI3K/Akt cell signaling pathway regulates the growth, survival and drug resistance in breast tumor cells through changing the expression of the transcriptional factors -NF- k B.