| Every year,1.3million women world wide were diagnosed and40thousand died from breast cancer making this the most common cancer in women and the leading cause of death. With the change of living environment, lifestyle and dietary patterns, the incidence of breast cancer is increasing notably around the world. The tendency of breast cancer patient is getting younger. In the past ten years, the incidence of breast cancer has increased27%in our country. In the great cities, just like Beijing, Shanghai, Tianjin, the most common malignancy in female is breast cancer. Breast cancer is becoming the worst threaten to women’s lives and health. With the development of modern molecular biology techniques, it gets insight into understanding of breast cancer. Breast cancer is a highly heterogeneous biological characteristic of malignant tumors. According to the histological, immunohistochemical, and molecular biology techniques, it can be divided into different subtypes.Triple-negative breast carcinomas (TNBC) are defined as tumors lacking the expression of oestrogen receptor, progesterone receptor and HER2.The evaluation of genetic knowledge shows that the triple-negative subgroup of breast cancers is very heterogeneous. TNBC are associated with aggressive clinical behaviors. They account for10-17%of all breast carcinomas. Several studies suggested that the prognoses for patients with TNBC are worse than for other types of breast cancers taken together. Most TNBC show a basal like phenotype. Triple-negative breast cancers and basal-like breast cancers share the same morphological features:pushing border of invasion, lymphocytic response, high nuclear grade, high mitotic count, metaplastic features, geographical tumour necrosis, central scar or sclerosis. Both triple-negative and basal-like cancers have a tendency to affect younger patients (<50years).The immunohistochemical panel proposed by Nielsen seems to be the most appropriate, in which basal-like breast cancers are defined as tumours lacking oestrogen receptor and HER2expression and expressing cytokeratin, CK5/6, and epidermal growth factor receptor, EGFR.CK5/6is a high molecular weight cytokeratin that is expressed in normal basal/myoepithelial cells of the breast and is currently regarded by many as the most useful marker to identify basal-like breast cancers. Other markers that have been associated with a basal-like phenotype are CK14, CK17, p53, smooth muscle actin, c-kit and P-cadherin. In addition, some studies suggest that deleterious BRCA1mutations may be present in at least25%of triple-negative tumors, compares with an incidence of approximately2%in other types of breast carcinomas. This evidence points out that the relationship between BRCA1and triple-negative breast cancer. Although most basal-like breast cancers are triple negative, they cannot be considered as one and the same entity. Previous studies have shown that basal-like breast cancers sometimes express oestrogen receptor, progesterone receptor or HER2. Furthermore, triple negative tumours do not always express basal markers and nontriple-negative tumours sometimes do.There are therapy strategies for triple-negative breast cancer:chemotherapy, gen therapy, radiation therapy, targeted therapies and so on. Keeping on exploring the effective therapy strategy for triple-negative breast cancers is still in urgent.The Beclinl gene encodes a60-kDa coiled-coil protein that interacts with the prototypic apoptosis inhibitor Bcl-2. The complete cDNA sequence of beclin1encodes a2098-bp transcript, with a120-bp5’UTR,1353-bp coding region, and625-bp3’UTR. The Beclin1gene comprises12exons ranging from61(exon9) to794bp (exon12) extending over a12kb region of the genome, with introns ranging from106bp to-2kb in length.Beclin1was rediscovered independently in a yeast two-hybrid screen of an adult mouse brain library performed to identify proteins that interact with the apoptosis inhibitor and on coprotein Bcl-2. On the basis of genomic localization to the minimal deletion unit on chromosome17q21, Beclin1is a candidate tumor suppressor for sporadic breast and ovarian cancer.Beclin1is a60-kDa protein containing a Bcl-2homology domain (BH3), a coiled-coil domain (CCD) and an evolutionary conserved domain (ECD); these domains enable multiple protein interactions. Through its ECD, Beclin1interacts with the class III phosphatidylinositol3-kinase (PI3K), Vps34, which is required for PI3KC3-dependent generation of PI3K and the subsequent recruitment of additional Atg proteins that orchestrate autophagosome formation. Through its BH3, Beclinl binds Bcl-2/Bcl-XL to form Bcl-2/Bcl-XL-Beclin1interactions. Bcl-2promotes cell survival by inhibiting induction of apoptosis. Bcl-2overexpression is a common aberration in many types of human cancer.Beclinl dually regulates both apoptosis and autophagy molecules.When Beclinl is overexpressed in mammalian cells, it can stimulate autophagy. Autophagy is a catabolic process involved in recycling of amino acids, nucleotides and lipids:superfluous proteins and organelles are sequestered in double-membrane vesicles (autophagosomes) and degraded in lysosomes. In this way, autophagy also leads to the removal of misfolded proteins, protein aggregates and damaged organelles, such as mitochondria and endoplasmic reticulum, which could harm the cell. It is often activated in response to nutrient deprivation, when it leads to recycling of organelles and other cytoplasmic substances to provide metabolic precursors. Failure to activate autophagy in response to nutrient deprivation, or its constitutive activation in response to stress, can lead to cell death. For this reason, autophagy is sometimes referred to as a second form of programmed cell death.The level of Beclin1gene has down-regulated in many carcinomas, including50%of breast cancers,75%of ovarian cancers,40%of prostate cancers, gastric cancer, colorectal cancer and so on.To confirm that Beclinl is frequently deleted in human breast cancer, FISH analysis was performed by Aita on chromosomes from22breast carcinoma-derived cell lines and one nontumorigenic breast epithelial cell line using p452O8and chromosome17centromeric probes. The absence of p452O8probe signal on any chromosome with17centromeric probe signals was used as criterion to identify deletions of the genomic region containing beclinl. Using this criterion,9of22(41%) of the breast cancer cell lines displayed deletions of beclin1. Their FISH analyses confirm that beclin1is one of the genes within this region that are commonly deleted in human breast carcinoma cell lines.Beclinl is a candidate tumor suppressor gene. To evaluate whether levels of Beclin1protein expression are decreased in human breast carcinoma. Liang analyzed by western blot human breast carcinoma-derived cell lines and matched normal breast and breast carcinoma tissue from patients with invasive sporadic breast carcinoma. Out of11human breast carcinoma cell lines examined, only3had detectable Beclin1protein expression. Out of17pairs of equivalent volumes of matched normal breast and breast carcinoma tissue,15samples had higher levels of Beclin1in normal than in tumour breast tissue. In contrast,16tumour samples had higher expression of control proteins than in the matched normal tissue samples. Therefore, specific expression of Beclin1protein is usually decreased in breast carcinomas.Furuya showed that overexpression of Beclin1in MKN28human gastric cancer cells augmented cisdiamminedichloroplatinum (CDDP)-induced apoptosis. Conversely,"knockdown" of Beclin1by a small inhibitory RNA in MKN1cells attenuated this cytotoxicity. Furthermore, not only caspase-3/7activities, but also caspase-9activity was increased in Beclin1gene transfectants treated with CDDP, Thus, Beclin1augments CDDP-induced apoptosis through enhancing caspase-9activity and functions as a pro-apoptotic molecule. They also examined the sensitivity to doxorubicin-induced apoptosis in these transfectants.Overexpression of Beclin1in MKN28cells augmented doxorubicin-induced apoptosis and down-regulation of Beclin1in MKN1cells attenuated this cytotoxicity. These evidences can characterize Beclin1as a pro-apoptotic molecule. Their findings indicated that Beclin1enhances not only autophagy but also apoptosis.Beclin1promote autophagy in breast cancer cells, however, the effect of Beclin1on the apoptotic signaling remained unclear, especially in triple-negative breast cancer cell. In view of above mentioned, exploring the relationship between Beclin1and triple-negative breast cancer will bring the therapy strategy for breast cancers.Objective:The deletion of autophagy gene Beclin1results in several malignant transformation, including breast cancers, ovarian cancers, prostate cancers. Currently, a lot of researches have showed that Beclinl gene regulate autophagy.By restricting instability of chromosome,preventing accumulation of oncogenic mutations, mitigating the responsibility to hypoxia, autophagy can induce programmed cell death of cancer cell (different from apoptosis). Some studies have showed that Beclinl not only regulate autophagy, but also increase chemotherapy drug doxorubicin induced apoptosis in gastric cancer cell. It has not yet reported that Beclinl gene whether can promote the apoptosis of triple negative breast cancer cell. Our research analyze the effect of overexpression Beclinl on the growth of triple-negative breast cancer cell line BT-549,and explore the role of Beclin1gene on BT-549cell when it’s treated with doxorubicin.Methods:(1) Plasmid pDsRed-Cl-Beclinl was transected via lipofectamine into triple-negative breast cancer BT-549cells; and plasmid pDsRed-C1was used as control. The experimental cells were classified into3groups:pDsRed-C1-Beclin1group, pDsRed-C1group and blank group. Three groups were incubated in normal environment or treated with doxorubicin after transfection.(2)Detection half inhibitory concentration of doxorubicin to BT-549cell:Five different concentration of doxorubicin was setted;5μl of doxorubicin with every concentration was added to BT-549cells under normal environment; the method of MTT was used to detect optical density in each concentration and then calculate half inhibitory concentration.(3)Acridine orange staining was used to determined the autophagy of BT-549cell under normal environment:The cells were seeded in6well plates. The method of liposome was used to transfect the cells when they were growing the proportion of60-80%.After tranfection, the cells were still cultured in1640medium containing10%FBS.The cells were washed twice with PBS.And then, two drops of acridine orange solution were added into the cells in dark environment. The stuation of staining was observed after the cells incubated at37℃for15minutes.(4)The proliferation of the cells under two environments were analyzed by MTT assay:The cells were seeded in96well plates. The method of liposome was used to transfect the cells when they were growing the proportion of60-80%. After tranfection, the cells were still cultured in1640medium containing10%FBS.MTT solution was added into each sample at different time. After the supernatant removed, 150μl DMSO was added. The optical density at490nm was detected on Multifunctional microplate reader.(5)Real time-polymerase chain reaction was used for detecting expression of Beclinl mRNA:The cells were seeded in6well plates. The method of liposome was used to transfect the cells when they were growing the proportion of60-80%.After tranfection,the cells were still cultured in1640medium containing10%FBS for72h.And then collect the mRNA of each group. Reverse transcription and amplification were done after determination of the concentration. An amplification product was used to electrophoresis by2%agarose gel.(6)Western blot was used for detecting expression of Beclinl protein in the transected cells. The protein of every group was collected after72h.The results were observed in visualizer after every step prepared.(7)Flow cytometry (FCM) was employed to observe the effect of transfection on the apoptosis and cycle of BT-549cells. The cells were seeded in6well plates. After tranfection. the cells were still cultured in1640medium containing10%FBS for72h.Cells were collected and added Binding Buffer, AnnexinV-FITC and PI.The samples was placed on ice. Apoptosis was detected by FCM in one hour. Cycle was detected by the similar method.(8)Statistical analysis:Data are presented as means±standard deviation. The SPSS13.0system software was used to test the data for normality. Analysis of variance(ANOVA)was used to analyze data that were normally distributed. Homogeneity of variance was tested by Levene test. In accordance with the homogeneity of variance can be used by LSD test, while not it can be used by Dunnett’T3test.Repeated measures analysis of variance was used to determine the statistical differences between cell proliferation.P value of<0.05Was considered significant. Results:(1) Eukaryon expression of pDsRed-Cl-Beclinl significantly improved the expression of Beclinl mRNA and protein in BT-549cells whatever treated with doxorubicin or not.[The Relative gray value of normal environment: pDsRed-C1-Beclin1group (0.70±0.03), pDsRed-C1group (0.34±0.04) and blank group (0.32±0.07); The Relative gray value of treated with doxorubicin: pDsRed-C1-Beclin1group (0.76±0.07), pDsRed-C1group (0.33±0.05) and blank group (0.29±0.05).every P<0.05].(2) The half inhibitory concentration of doxorubicin to BT-549cell measured by MTTis1.4μg/ml.(3) The curve of cell proliferation is upward before treated with doxorubicin. However, the curve of Beclinl group is more gentle than other two groups.[The OD value of normal environment at96h:pDsRed-Cl-Beclinl group(1.73±0.27), pDsRed-C1group(2.65±0.32),blank group(2.71±0.22).every P<0.05] The Beclinl gene inhibit the proliferation of BT-549cell. The curve of every group show a trend toward declining after treated with doxorubicin.[The OD value after treated with doxorubicin at96h:pDsRed-Cl-Beclinl group(0.08±.05),pDsRed-C1group (0.35±0.14), blank group(0.41±0.08).every P<0.05](4)FCM investigation showed that the apoptotic rate in the normal environment was (8.81±1.45)%of pDsRed-Cl-Beclinl group, significantly higher than the (3.06±0.31)%in the pDsRed-C1group and (2.87±0.18)%in the blank group (P <0.05).Beclinl gene can promote apoptosis of BT-549cell. The apoptotic rate under treated with doxorubicin was (14.3±1.09)%of pDsRed-Cl-Beclinl group,(4.81±0.7)%in the pDsRed-C1group and (4.58±0.7)%in the blank group (P<0.05).Beclinl gene can enhance sensitivity of BT-549cells to doxorubicin.(5)The cell cycle analysis showed that the percentage of G1-phase cells of pDsRed-C1-Beclin1group was significantly higher than other groups whatever treat with doxorubicin or not.Beclinl gene can inhibit mitosis of BT-549cells.(6)Red spot was observed in the cell transected with pDsRed-C1-Beclin1in the normal environment in acridine orange staining.Conclusions:Autophagy gene Beclinl overexpression can inhibit the proliferation and growth of BT-549cells in vitro.The proliferation curve declined faster in the cell transected with pDsRed-C1-Beclin1than other groups. The apoptotic rate is also higher in the cell transected with pDsRed-Cl-Beclinl than other groups. So, Beclinl gene can raise sensitivity of BT-549cell to doxorubicin. So it might be one of new gene therapy strategies for triple-negative breast carcinoma. |
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