BRCA1 is a multifunctional tumor-suppressive protein. Many functional aspects of BRCA1 are not fully understood. We used a shRNA approach to probe the function of BRCA1 in human breast cancer cells. Knocking down BRCA1 expression by shRNA in the wild-type BRCA1 human breast cancer MCF-7 and MDA-MB-231 cells resulted in an increase in cell proliferation, anchorage-independent growth, cell migration, invasion and a loss of p21/Waf1 and p27 Kip1 expression. In BRCA1 knocked-down cells, the expression of survivin was significantly up regulated with a concurrent decrease in cellular sensitivity to paclitaxel. We also found that cells harboring endogenous mutant or defective BRCA1 (MDA-MB-436 and HCC1937) were highly proliferative and expressed a relatively low level of p21/Waf1 and p27 Kip1 by comparison to wild-type BRCA1 cells. Cells harboring mutated BRCA1 also expressed a high level of survivin and were relatively resistant to paclitaxel by comparison to wild-type cells. Increase resistance to paclitaxel was due to an increase in the expression of survivin in both the BRCA1 knocked-down and mutant BRCA1 cells because knocking down survivin expression by siRNA restored sensitivity to paclitaxel. We conclude that BRCA1 down-modulates the malignant behavior of breast cancer cells, promotes the expression of p21/Waf1, p27 Kip1 and inhibits the expression of survivin. Moreover, loss of BRCA1 expression or function leads to an increase in survivin expression and a reduction in chemosensitivity to paclitaxel. ' UICCKey words: BRCA1; p21/Waf1; p27Kip1; survivin; chemosensitivity Hereditary breast cancer can be caused by germline mutations in 1 of the 2 tumor suppressor genes, BRCA1 and BRCA2 resulting in a loss of gene function. 1,2 Women carrying 1 mutated BRCA1 allele are at increased risk of developing breast cancer but tumor initiation requires a loss of the wild-type allele indicating that BRCA1 is a tumor suppressor gene. 3,4 Hereditary breast cancers account for 5-10% of all breast cancers. 5-8 Breast cancer cumulative risk to age 50 years and 70 years in mutation carriers are 49% and 71%, respectively, for BRCA1. 9 BRCA1 expression is also frequently reduced in sporadic cancer. 10-13 BRCA1 encodes an 1863 amino acid (220 kDa) protein that is targeted to the nucleus by 2 nuclear localization sequences, which interact with the nuclear transport signal receptor. 14-17 BRCA1 protein is multifunctional and interacts with other proteins in the nucleus in transcriptional regulation of gene expression, DNA repair and in the maintenance of genomic stability. 1,18 Thus, loss of BRCA1 function may lead to accumulation of chromosomal damage, abnormalities in growth control and tumorigenesis. 19 BRCA1 is regarded as an important central component in regulating multiple biological pathways. 18,20 The function of BRCA1 intersects with the cyclin-dependent kinase inhibitor p27 Kip1 , cyclin D1, the extracellular matrix [21][22][23] and with the estrogen and progesterone pathways in hormonal driven tumor development and progres...
Cucurbitacin B (CuB) is one of the potential agents for long term anticancer chemoprevention. Cumulative evidences has shown that cucurbitacin B provides potent cellular biological activities such as hepatoprotective, anti-inflammatory and antimicrobial effects, but the precise mechanism of this agent is not clearly understood. We examine the biological effects on cancer cells of cucurbitacin B extracted from a Thai herb, Trichosanthes cucumerina L. The wild type (wt) BRCA1, mutant BRCA1, BRCA1 knocked-down and BRCA1 overexpressed breast cancer cells were treated with the cucurbitacin B and determined for the inhibitory effects on the cell proliferation, migration, invasion, anchorage-independent growth. The gene expressions in the treated cells were analyzed for p21/Waf1, p27Kip1 and survivin. Our previous study revealed that loss of BRCA1 expression leads to an increase in survivin expression, which is responsible for a reduction in sensitivity to paclitaxel. In this work, we showed that cucurbitacin B obviously inhibited knocked-down and mutant BRCA1 breast cancer cells rather than the wild type BRCA1 breast cancer cells in regards to the cellular proliferation, migration, invasion and anchorage-independent growth. Furthermore, forcing the cells to overexpress wild type BRCA1 significantly reduced effectiveness of cucurbitacin B on growth inhibition of the endogenous mutant BRCA1 cells. Interestingly, cucurbitacin B promotes the expression of p21/Waf1 and p27Kip1 but inhibit the expression of survivin. We suggest that survivin could be an important target of cucurbitacin B in BRCA1 defective breast cancer cells.
Breast cancer is a complex disease driven by multiple factors including both genetic and epigenetic alterations. Recent studies revealed that abnormal gene expression induced by epigenetic changes including aberrant promoter methylation plays a critical role in human breast carcinogenesis. Cucurbitacin B has antiproliferative activity against various human breast cancer cells, but the molecular mechanism is not completely understood. In this study, we explore the influence of cucurbitacin B from Trichosanthes cucumerina on the methylation status at the promoter of oncogenes c-Myc, cyclin D1, and survivin in breast cancer cell lines. Growth inhibitory effect of cucurbitacin B on breast cancer cells was assessed by MTT assay and colony formation assay. Methylation status of genomic DNA was determined by methylation-specific PCR. Gene and protein expression levels of all genes studied were analyzed by real-time RT-PCR and western blot. The results indicated that cucurbitacin B could inhibit cell growth in breast cancer cells. The oncogene promoters are usually hypomethylated in cancer cells. Upon cucurbitacin B treatment, upregulation of DNMT1 and obvious heavy methylation in the promoters of c-Myc, cyclin D1, and survivin, which consequently downregulated the expression of all these oncogenes, were observed. Hence, cucurbitacin B proved to be a potential cancer therapeutic agent, in part by inducing hypermethylation and silences the oncogenic activation.
TGFβ exerts a potent tumor-suppressive effect in the human colon carcinoma CBS and Moser cells. However, TGFβ can also function as a tumor promoter. The mechanisms underlying the tumor promoting effect of TGFβ is not understood. Both the CBS and Moser cells were found to express mutant (truncated) APC. Expression of this form of APC did not interfere with the tumor-suppressive function of TGFβ. However, when APC expression was knocked down in these cells, TGFβ function switched from that of tumor suppression to that of tumor promotion. TGFβ stimulated cellular invasion and anchorage-independent growth in APC knocked-down cells. Knocking down APC expression abrogated the ability of TGFβ to induce the expression of the tumor suppressor E-cadherin and the cyclin dependent kinase inhibitor p21/Waf1. TGFβ now stimulated the constitutive TCF transcriptional activation activity associated with the β-catenin/Wnt pathway in the APC knockeddown cells. Thus, the level of APC expression determined the type of TGFβ function in these human colon carcinoma cells.
Anemia is a major complication in over 50% of chronic kidney disease (CKD) patients. One of the main causes of anemia in CKD is the reduction of erythropoietin (EPO) synthesis from renal tubular cells. Therefore, first-line treatment of CKD is EPO administration; however, EPO unresponsiveness in several patients is frequently found. More undefined causes of anemia in CKD are under interest, especially uremic toxins, which are a group of solutes accumulated in CKD patients. The highly detectable protein-bound uremic toxin, indoxyl sulfate (IS) was investigated for its effects on in vitro erythropoiesis in this study. CD34+ hematopoietic stem cells were isolated from human umbilical cord blood and differentiated toward erythrocyte lineage for 14 days in various concentrations of IS (12.5, 25, 50, and 100 µg/mL). The effects of IS on cell proliferation, differentiation, apoptosis, and senescence were determined. Cell proliferation was investigated by manual cell counting. Cell surface marker expression was analyzed by flow cytometry. Wright’s staining was performed to evaluate cell differentiation capacity. Apoptosis and senescence marker expression was measured using reverse transcription polymerase chain reaction (RT-PCR). TUNEL assay was performed to detect apoptotic DNA fragmentation. Our results demonstrated that IS reduced cell proliferation and impaired erythrocyte differentiation capacity. In addition, this study confirmed the effects of IS on cell apoptosis and senescence during erythropoietic differentiation. Therefore, the promotion of apoptosis and senescence might be one of the possible mechanisms caused by uremic toxin accumulation leading to anemia in CKD patients.
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