| Background and Objection:In recent years, a growing body of evidence has been reported supporting the hypothesis that tumors are driven and maintained by a minority subpopulation of cells that have the capacity to self-renew (i.e.,give rise to progeny with similar properties as themselves) and to generate the more differentiated progeny which make up the bulk of a tumor.The minority population has been termed cancer stem cells (CSCs), tumorigenic cancer cells, or tumor-initiating cells, by various investigators, to indicate that only they can give rise to new tumors when transplanted into immunodeficient animals. Tumor-initiating cells have been identified in blood, brain, breast cancers,prostate, liver, pancreas, and colon cancers (4-6) through an experimental strategy that combines sorting of tumor cell subpopulations, identified on the basis of the different expression of surface markers, with functional transplantation into appropriate animal models. The subpopulation with the CD24-/low/CD44+ phenotype was shown to identify breast cancer-initiating cells in primary patient specimens which 200 cells were transplanted into NOD/SCID mice and formed tumor by Al Hajj using fluorescence-activated cell sorting (FACS) etc in 2003.Then in 2005,Ponti etc showed that breast tumorigenic cells with stem/progenitor cell properties can be propagated in vitro as nonadherent mammospheres and represent a suitable in vitro model to study breast cancer-initiating cells and to challenge them with molecularly targeted agents specifically interfering with breast cancer-initiating cells self-renewal and survival.According to the hypothesis, CSCs limited number within the bulk of the tumor are not only the source of the tumor but also may be responsible for tumor progression,metastasis,thus leading to subsequent tumor relapse although the primary lesion is eradicated and resistance to therapy; Radiotherapy, as an integral part of the current comprehensive breast cancer treatment regimen, may be used to eradicate remaining cancer cells in the breast, chest wall, or axilla after surgery or to reduce the size of an advanced tumor before surgery.hence, it is imperative to understand the molecular mechanism underlying this radioresistance. A number of candidate genes have been implicated in the response of eukaryotic cells to ionizing radiation, including cell cycle, checkpoint, and DNA repair genes, as well as mediators of apoptosis, such as p53,Bax,Bcl-2, and so forth.PI3K-AKT signaling is an important cascade to promote cell survival in response to exogenous stress factors such as exposure to ionizing radiation. It has been shown that PI-3K pathway plays a critical role in mediating radioresistance.One of the best characterized downstream targets for the PI-3K lipid products is Aktl, which was discovered about a decade ago and which is now known to be a member of a family of closely related, highly conserved cellular homologues that consists of at least two additional members (Akt2 and Akt3). Akt has been shown to be a critical player in oncogenesis. There is increasing evidence indicating that PI-3K/Akt plays an important role in breast cancer tumorigenesis. An elevated level of Akt activity was associated with increased cellular resistance to the treatment with doxorubicin or tamoxifen in breast cancer cell lines.STAT transcription factors were discovered 10 years ago as mediators of interferon-induced gene expression. They now form an important group, comprising seven members, that are activated by virtually every cytokine and growth factor. Cell culture work has further delineated their importance in cellular proliferation, transformation, apoptosis, differentiation and growth control. STAT1,STAT3 and STAT5 may play an important role in breast carcinogenesis. Recently, signal transducer and activator of transcription 1(STAT1)has been implicated in the presence of radioresistance,first demonstrated in a head-and-neck cancer cell line (4).To date, AKT1 and STAT1 involvement in CD24-/low/CD44+ breast cancer-initiating cells in general and their specific relationship with radioresistance of CD24-/low/CD44+ breast cancer-initiating cells have never been explored.This study intend to discover the genes which may play a key role in radioresistence of CD24-/low/CD44+ breast cancer-initiating cells by qRT-PCR and investigate the effects of AKT1 and STAT1 in radiosensitivity by the additional of specific inhibitors.METHODS AND MATERIALS1.Suspension culture combined with radiation to enrich CD24-/low/CD44+breast cancer-initiating cells that have more radioresistance.MCF-7 cultured in SSM and SFM respectively. Then some mammaspheres were cultured with radiation at a dose rate of 300-500 cGy/min by varian2100 C/D. The cells cultured to achieve 80-90% confluence on the day of experiments were stained with CD24-PE and CD44-FITC and analyzed the percentage of CD44+CD24- by flow cytometry.2. The 51 genes chip was designed by Primer 5 software and syntheses by Invitrogen Co.Real-time quantitative reverse transcription polymerase chain reactions performed to detect the mRNA level of 51 genes in MCF-7 and mammaspheres before and after radiation.3.AKT1 and STAT1 mRNA levels were examined d by qRT-PCR repeatedly.4. Evaluated apoptosis by flow cytometrywith Annexin-V100 FITC/PI double staining method and calculated the plating efficiency by the number of colonies/cells seeded and fitted the surviving fraction by GraphPad Prism(4.0) in MCF-7 and mammaspheres before and after AKT1 or STAT1 inhibitor before and after radiation..5.All data are represented as means and differences of the means with 95% confidence intervals (CIs). P values of.05 or less, calculated using a paired two-sided Student's t test or chi-square test, were considered to indicate statistically significant differences.RESULTS1.Flow-cytometric analysis.The percentage of cells from suspension culture combined with radiation higher than those in single suspension culture without radiation and the difference was determined to be statistically significant(P<0.001)2.It was revealed that-mRNA levels of BC1-2, BRCA-1,Statl,Ing1, E2F1, AKT1,HDAC1 and Kit were higher after radiation than those before radiation in mammaspheres.3.The mRNA levels of STAT1 and AKT1 were higher after radiation than those before radiation in mammaspheres and the difference was determined to be statistically significant(P<0.001),whereas,there was no difference between MCF-7 before and after radiation.4. Apoptosis and the surviving fraction had no difference in MCF-7 treated with AKT1 inhibitor or STAT1 inhibitor respectively before and after radiation and mammaspheres with AKT1 inhibitor or STAT1 inhibitor respectively before radiation(P>0.05).However, Apoptosis was enhanced and the surviving fraction decreased in mammaspheres with AKT1 inhibitor or STAT1 inhibitor respectively after radiation.The difference was determined to be statistically significant(P<0.001).Conclusion1.The percentage of CD44+CD24-through suspension culture combined with radiation is up to 98%,stable at about 90%。2.Our data suggest that BC1-2, BRCA-1,Stat1,Ing1,E2F1,AKT1,HDAC1 and Kit may be associated with the radioresistance in breast cancer-initiating cells CD44+/CD24-/low.3.AKT1 and STAT1 may play a key role in radioresistance in breast cancer-initiating cells CD44+/CD24-/low and manipulation of this pathway may enhance the efficacy of radiotherapy.Innovations of our study:1.The enrichment of CD44+CD24-through suspension culture combined with radiation is successful than all the methods previous。2.This is the first study reporting the mRNA level of genes which may be associated with the radioresistance in breast cancer-initiating cells CD44+/CD24-/low by qRT-PCR and suggesting that BC1-2, BRCA-1,Statl,Ing1,E2F1,AKT1,HDAC1 and Kit may be associated with the radioresistance.3.The blockage of PI3K/AKT and JAK/STAT by the addition of a STAT1-specific inhibitor, fludarabine, and AKT1-specific inhibitor, significantly increased the fraction of apoptotic cells.These results confimed that AKT1 and STAT1 may play a key role in radioresistance in breast cancer-initiating cells CD44+/CD24-/low and the targeting of STAT1 and AKT1 could prove to be a promising new therapeutic strategy against breast cancer.4.Genetic studies of radioresistence in breast cancer stem cell has been repoted yet and our study provide a valuable result.
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