Proliferation Inhibited And Apoptosis Induced Of Transfected Noxa Gene On Human Breast Cancer Cell Line MCF-7

BackgroundThe development of breast cancer is a multi—gene, multi—step process of evolution. According to statistics, the breast cancer accounts for 7—10% in a variety of malignant tumors. With the acceleration of urbanization of China's population, breast cancer incidence and mortality have increased year by year, particularly in China's coastal cities. The incidence of breast cancer is often associated with the incidence of inherited. Women have a higher prevalence from 40 to 60 years old than other ages. The breast cancer serious impact on women's physical and mental health and threaten patients life. At present, the threpy of breast cancer is surgery, induced tumor cell death and apoptosis(including chemotherapy, radiotherapy, and gene therapy) and endocrine therapy. These methods have been difficult to achieve greater progress in recent years, Therefore, people try to seek new means of breast cancer treatment from the pathogenesis. Recently years, with the development of relation between tumor and cell apoptosis, more and more new anticancer methods have been reported from the mechanism of induced apoptosis. From the result of research about cell apoptosis, we should seek new means of anti—cancer from induced cell apoptosis. Effective anti—cancer therapy is induce tumor cells apoptosis more than inhibition of tumor cell proliferation or use toxic drugs to kill tumor cells. Apoptosis, the process of programmed cell death, is generally characterized by distinct morphological characteristics that is pyknosis, karyorrhexis, nuclear chromatin fragments, the formation of apoptotic bodies and intact plasma membrane. On histologic examination with hematoxylin and eosin stain, apoptosis involves that the cells are smaller in size, the cytoplasm is dense, eosinophilic staining is strengthened and formed apoptotic bodies. Apoptosis involves single cells or small clusters of cells that separat from surrounding cell, there is essentially no inflammatory reaction associated with the process of apoptosis. Apoptosis is a fine—tuned mechanism to eliminate the harmful, seriously damaged, unnecessary, or virus—or bacteria—infected cells in multi—cellular organisms. Huge lines of evidence indicate that the mitochondria act as critical regulators of cell death. Mitochondrial function supports cell survival by generating ATP and sequestering pro—apoptotic proteins such as cytochrome c, AIF which are released into cytosol during cell death. Also mitochondria are dynamic organelles and continuously change their morphology through fusion and fission in response to environmental status of cells. During cell death, the interconnected mitochondrial tubular networks in healthy cells are disintegrated into small fragmented (or condensed) mitochondria. Mitochondrial integrity is controlled by the Bcl—2 family proteins. A recent report demonstrated that mitochondrial dynamics is regulated by Bax and Bak in healthy cells, suggesting that Bcl—2 family proteins may contribute mitochondrial morphogenesis.Currently, there are two means of induced apoptosis in mammalian cell, the extrinsic or death receptor pathway and the intrinsic or mitochondrial pathway through Bcl—2 protein family. Bcl—2 members can be divided in three different classes of proteins based on their Bcl—2 homology(BH) domains. These include four BH domains antiapoptotic proteins (Bcl—2, Bcl—xL, Mcl—1, Bcl—w and Al), multidomain proapoptotic proteins (Bak and Bax) and BH3—only proapoptotic proteins which include Bid, Bad, Bim, Puma, Noxa and others, the proapoptotic BH3—only proteins are the sponsor of the mitochondrial pathway. Noxa, or APR(ATL—2 derived PMA—2 responsive gene), or PMAIP1(phorbol—12 myristate—13—acetate—induced protein 1), was first found in 2000, is BH3—only subgroup of proapoptotic Bcl—2 family members. It is one target gene of p53. The human gene is located to chromosome 18q21 and encodes a 54—amino—acid protein containing one BH3 motif. Noxa belongs to the pro—apoptotic proteins "BH3—only" branch of the Bcl-2 protein family, lower express in the hunman body. Noxa inhibits Mcl—1 and Al which is an anti—apoptotic member of Bcl—2 family by specific interaction with Mcl—land Al through BH3 domain. The mitochondrial targeting domain (MTD) of Noxa is the region responsible for targeting to mitochondria, Noxa are the most apical regulators of apoptosis, and once activated, it regulate the ability of the multi—BH domain subfamily members Bax and Bak to undergo a conformational switch and to oligomerize in the outer mitochondrial membrane. Activated Bax and Bak then induce mitochondrial membrane permeabilization and subsequent release of proapoptotic factors, such as Cytochrome c, from the intermembrane space into the cytosol. Cytosolic Cytochrome c band with apoptotic protease—activating factor(Apaf21) and caspase29 lead to formation of the apoptosome and ultimately activate caspase23 to triggers execution of the intrinsic caspase signaling cascade, induce apoptosis.As far as we know, in the context of gene therapy, the p53 tumor—suppressor gene has been extensively studied. This gene is functionally inactivated in approximately 50% ofhuman tumors and its function in the induction of apoptosis in cancerous cells is considered as a major mechanism in tumor suppression. When p53 is activated by noxious stimuli such as DNA damage, hypoxia, and oncogenes, it mediates two types of cellular response, induction of cell cycle arrest followed by DNA repair and induction of apoptosis. It is thought that the fine balance of these two types of cellular response is a key event in the. determination of cell fate in tumor suppression. How p53 mediates these cellular responses has been extensively studied; activation of its target genes is considered an essential mechanism. The induction of cell—cycle arrest requires induction of the cyclin dependent kinase (CDK) inhibitor p21WAF1/Cip 1, whereas the induction of apoptosis requires the induction of at least two genes for BH3—only proteins of the Bcl—2 family, namely, Noxa. Thus, reinstatement of p53 function is an attractive tumor specific therapeutic strategy, and p53 gene therapies for malignant tumors such as lung, breast, and head and neck cancer have been widely performed in animal experiments and clinical trials. In previous clinical, trials, such tumors ceased their progression. after intratumoral injection of an adenoviral vector, termed Ad—p53, bearing the p53 gene, and they seemingly became more susceptible to other therapies such as radiation or chemotherapy. In many studies, however, tumors shrank only in the case of combining p53 expression with other therapies.Noxa, a p53-target gene, is a key point mediate gene. Upstream promoter sequence have target site of p53. When receive stimulus of apoptosis single, p53 can combine with target site to activate Noxa transcription and expresstion protein. When DNA damage, p53 can combine with recognition site of Noxa promoter as a transcription factor to induce pro—apoptosis through activated Noxa gene to transcript. Noxa, a downstream p53—target gene, not only mediate expression through p53 response element of Noxa promoter, but also induce apoptosis though p53—independment. So, when suffer noxious stimuli, such as DNA damage and hypoxia, Noxa can prominently induce tumour cell apoptosis in a p53—dependent manner or in a p53—independent manner. It is important role in the tumor development and treatment. Ectopic expression of Noxa in HeLa cells with an adenovirus—mediated gene expression system caused apoptosis in.90% of the cells after virus infection. Drugs that imitation BH domain of Noxa also can induce tumor cell apoptosis.Based on the low—level in human body and current research, the recombinant expression plasmid with Noxa gene was transiently transfected into human breast cancer cell line MCF—7 with lipofectamine. Then, it can obtain higher expression. The expression of Noxa mRNA and protein were detected by RT—PCR and Western blot respectively. The inhibition of cell proliferation was evaluated by MTT assay. Changes in cell cycle and cell apoptosis were detected by flow cytometry(FCM). The cell apoptosis was studied by Hoechst 33342 staining.ObjectivesThe recombinant eukaryotic expression plasmid pIRES2—EGFP—Noxa was transiently transfected into human breast cancer cell line MCF—7 with lipofectamine2000. The expressions of Noxa mRNA and protein were detected respectively. To explore the effect of transfected Noxa gene on proliferation and apoptosis of human breast cancer cell line MCF—7. These research provide a theoretical and experimental basis for Gene therapy of breast cancer.Methods1. The recombinant eukaryotic expression plasmid pIRES2—EGFP—Noxa was transiently transfected into human breast cancer cell line MCF—7 with lipofectamine; After transfected 6h,12h and 48h, Cell morphology were observed after transfection by fluorescent microscopy.2. The experiment was divided into control group, negative control group and positive control group. The expressions of Noxa mRNA were detected by RT—PCR after transfected 48h. The results were compared among different groups by one—way ANOVA.3. The experiment was divided into control group, negative control group and positive control group. The expressions of Noxa protein were detected by Western bolt after transfected 48h. The results were compared among different groups by one—way ANOVA.4. The experiment was divided into control group and positive control group. The inhibition of cell proliferation was evaluated by MTT assay after transfected 24h, 48h and 72h. The results were compared among different groups by Univariate.5. The experiment was divided into control group and positive control group. Changes in cell apoptosis were detected by flow cytometry(FCM) after transfected 48h. The results were compared among different groups by Independent—Sample T Test.6. The experiment was divided into control group and positive control group. The cell apoptosis was studied by Hoechst 33342 staining after transfected 48h. The results were compared among different groups by Independent—Sample T Test.Results1. According to the characteristic of pIRES2—EGFP, MCF—7 cell can express EGFP after transfected pIRES2—EGFP. Also, MCF—7 cell can express EGFP-Noxa after transfected pIRES2—EGFP—Noxa. We can observe green fluorescence in everu phase. Green fluorescence of pIRES2—EGFP lighter than pIRES2—EGFP—Noxa.2. Noxa mRNA expression upgrade gradually after transfected 48h. The expression of Noxa in mRNA of MCF—7 cells was significantly different after transfected 48h between three groups. The control group(0.298±0.028) and negative control group (0.295±0.006) have lower expression of Noxa mRNA than positive control group(0.605±0.034).3. Noxa protein expression upgrade gradually after transfected 48h. Noxa in protein of MCF—7 cells was significantly different after transfected 48h than other groups. The control group(0.192±0.088) and negative control group (0.200±0.097)have lower expression of Noxa protein than positive control group(0.493±0.026).4. MTT revealed that there are statistical differences of the cell proliferation among different time points(Ftime=8.842, P<0.05) and different groups(Fgroup=58.574, P<0.05), and there is interaction effect between time and group(FIE=6.669, P<0.05). There is statistical difference between 48h and 72h in three groups(P<0.05), and it is not happen in 24h of three groups (P> 0.05). There are significant differences between different time points in positive control group (P<0.05), and it is not happen in negative control group and control group (P>0.05).5. FCM indicate that Noxa can induce apoptosis. Apoptosis rate of positive control group(21.02±2.34)% significantly different compared with the control group(3.58±0.76)%(t=-15.843, P<0.05).6. Apoptosis detected by hoechst 33342 staining after transfected 48h into control group and positive group was (4.00±2.54)% and (21.00±3.80)%, respectively. There were significant difference (t=—8.295, P<0.05).Conclusions1. The Noxa mRNA and protein express successfully after transfected into human breast cancer cell line MCF—7.2. Transfection of Noxa gene can effectively inhibit the proliferation and promote the apoptosis of breast cancer cells.3. Noxa gene can effectively inhibit the proliferation and promote the apoptosis of breast cancer cells. It will become a new research direction in gene therapy of breast cancer.