The Effect Of Notch Signaling On The Proliferation Of Breast Cancer Cells And Involved Mechanism

Sectionâ… : The expression and activation of Notch signaling in human breast cancer and the effect ofγ-secretase inhibitor on the proliferation of breast cancerBackground:Notch signaling is a pathway highly conserved through evolution which regulates various physiological processes,including stem cell maintenance, differentiation,proliferation and apoptosis.In mammals,key components of the Notch pathway include four transmembrane receptors(Notch 1-4)and five ligands(Delta-like 1,Delta-like 3,Delta-like 4,Jagged 1,Jagged 2).Direct binding of a ligand from a signaling cell to a Notch receptor on the membrane of the receiving cell initiates two successive proteolytic cleavages by TACE(TNF-α-converting enzyme)and theγ-secretase/presenilin complex,which ultimately results in the release of the intracellular domain(N-IC).N-IC then translocates into the nucleus and directly interacts with the DNA binding protein CBF 1/Su(H)/Lag 1(CSF)that activates the transcription of target genes including the Hes(hairy/enhancer-of-split)and hey.It was found recently that Notch signaling is related with the etiology of breast cancer. The first indication that Notch signaling might play a role in neoplastic development of the mammary gland came from the characterization of a common insertion site for the mouse mammary tumor virus in Czechâ…¡mice.In 20%of these tumors,the mouse mammary tumor virus was inserted within the Notch4/Notch1 locus.At both loci,insertion of the provirus leads to expression of Notch protein that consists of the transmembrane and intracellular domains only,suggesting that deregulated Notch signaling related to tumor.Several studies show that over expression of consititutively activated notch1 or notch4 in normal human breast epithelial cells can induce transformation in vitro.Notch1 protein was highly expressed in four breast tumors that over expressed H-ras,and the expression was associated with adverse prognosis.Aim:To detect systematically the expression of Notch signaling moleculars and the aberrant activation of Notch signaling in human breast cancer.Explore the role ofγ -secretase inhibitor on the proliferation of breast cancer cells.Meterial and Methods:1.Collect breast cancer specimens and normal breast tissues from the margin of tumor sections as control.Total RNA was extracted by TRIZOL according to the manufacturers instructions.Synthesis of first-strand cDNA was carried out with Revert Aid^TMFirst Strand cDNA Synthesis Kit.Notch1,Notch3,Notch4 and Jagged1,Delta-like4 were amplified.Immunostaining was used to detect the activated form of Notch1(N-IC)in formalin-fixed,paraffin-embedded tissue sections2.Cell culture.Human breast cancer cell lines MDA-MB-231,MDA-MB-435 were cultured in RPMI medium 1640.RT-PCR was used to detect the expression of. Notch 1,Notch3,Notch4 and Jagged 1,Delta-like4.Immunostaining was used to detect the activated form of Notch1(N-IC)in these cell lines.3.Immunostaining.10 of breast cancer tissues and 4 of normal breast tissues at the margin of breast cancer were collected.Formalin-fixed,paraffin-embedded tissue sections(5μm thick)were dewaxed,rehydrated and optimized antigen retrieval.Alternatively,cultured cells grown on cover slides were fixed in 95%ethanol. Sections were then used to detect Notch1-IC using Histostain-plus kit according to the manufacturer's instructions.4.Inhibition assay byγ-secretase inhibitor.4×10⁴ of MDA-MB-231 cells and MDA-MB-435 cells were plated in a 96-well plate and allowed to proliferate overnight.The cells were then treated with increasing concentration ofγ-secretase inhibitor.24 hours after treatment,the proliferation of the cells were measured by MTT method.5.Analysis of Cell cycle.Briefly,1×10⁶ cells were plated in 100 ml culture flasks and allowed to proliferate till 70%-80%confluent.Then,the cells were treated withγ-secretase inhibitor(3μmol/L)or DMSO(15μl)as a control,and after 24h, cells were harvested and stained with PI.The DNA content was analyzed by FACS Calibur6.Detection of apoptosis.Cells were treated withγ-secretase inhibitor(3μmol/L)or DMSO(15μl)as described above.24 hours after treatment,cells were collected and labeled with 1:500 Annexin V-biotin conjugated with fluorescent isothiocyanate(FITC)followed by 1:1000 PI.Annexin V-PI were measured by FACS Calibur Results:1.Notch receptors and ligands are over expressed in human breast cancer.The expression rates of Notch1,3,4,Jagged1 and Delta-like4 gene mRNA in cancer specimen and normal breast tissue at the margin of tumor sections are 98%,35%, 8%,15%,81%and 73%,0%,0%,0%,0%,respectively.The expression rate of Notch signaling moleculars mRNA in the cancer specimens is significantly higher than that in the specimens at the margin of tumor sections.Notch1,3 and Jagged1 were all expressed in human breast cancer cell lines MDA-MB-231 and MDA-MB-435.2.Notch1 is activated in human breast cancer.We used the antibody which recognizes only the activated form of Notch1(N1-ICD,the cleaved intracellular form)to detect its activation,we saw accumulation of N1-ICD in most(9 in 10) tumor samples analyzed,only 1 sample showed negative staining.Whereas,no N1-ICD staining was detected in all the normal breast tissues.In all the cell lines, we observed a clear accumulation of N1-ICD in both the cytoplasm and nuclear, with the later finding being indicative of Notch activation.3.γ-secretase inhibitor can inhibit the proliferation of breast cancer cells.γ-secretase inhibitor,a pharmacologic agent known to block effectively Notch activation,was used to evaluate the effect of Notch inhibition on breast cancer cells. We observed the proliferation of both cell lines decreased with the increasing dosage ofγ-secretase inhibitor I(1-5μmol/L).4.γ-secretase inhibitor can cause cell cycle arrest in breast cancer cell lines.γ-secretase inhibitor I-treated cells of MDA-MB-231 and MDA-MB-435 showed a higher proportion of cells in G2/M phase compared with control(29.9%vs 15.99%, and 45.5%vs 16.63%in MDA-MB-231 and MDA-MB-435 respectively).5.γ-secretase inhibitor can cause apoptosis in breast cancer cell lines.The fraction of apoptotic cells inγ-secretase inhibitor -treated populations was significantly higher than that observed in controls(0.94%vs 3.03%,0.23%vs 4.33%in MDA-MB-231 and MDA-MB-435 respectively,p＜0.05,data are mean percentage of apoptotic cells from three independent experiments,demonstrating that inhibition of Notch signaling could also induce apoptosis in breast cancer cells.Conclusion:Notch signaling is over expressed and highly activated in human breast cancer. Blockage of Notch signaling byγ-secretaseinhibitor can inhibit the growth of breast cancer cells,causing cell cycle arrest and apoptosis.Thus,Notch signaling can be a therapeutic target for human breast cancer.Sectionâ…¡The effect of down-regualtion of Notch1 mediated by siRNA on the proliferation of breast cancer cellsBackground:RNA interference(RNAi)is a process of gene silencing induced by double strand RNA(dsRNA).Basically,the process of RNAi is triggered by dsRNA precursors. These dsRNA precursors are processed into small interfering RNAs(siRNAs)that vary in length from 21-23 nucleotides(nt).The siRNAs are subsequently incorporated into a multiprotein complex.This complex is known as the RNA-induced silencing complex(RISC).siRNAs in the RISC complex guide degradation that is highly sequence-specific,of the complementary mRNAs.The gene silencing effect is highly efficient,as reported as high as 90%expression can be blocked.The high specificity and efficiency are very useful for human disease treatment as it can specifically knock down its target gene without affecting other gene's expression.Previously we have found that Notch signaling is over expressed and highly activated in human breast cancer,andγ-secretase inhibitor can inhibit the proliferation by causing cell cycle arrest and apoptosis.Butγ-secretase inhibitor is a compound that has many activities including block the activation of Notch signaling.Phase I clinical study showed that it also has severe gastrointestinal side effect.So we further explored more specific method to knock down the expression of Notch1.We use siRNA that can specific binds to Notch1 to down regulate its expression and examine the effect on proliferation of breast cancer cells.Also we detected whether Notch signaling is related to chemosensitivity and the relation between Notch and NK-kappaB.Meterial and Methods:1.SiRNA Transfection.1.2×10⁵ of MDA-MB-231 and MCF-7 Cells were seeded in 6-well plates(or 4000 cells/well in 96-well plates).After overnight incubation,cells (30-50%confluence)were treated with 40nM siRNA/control siRNA.Seventy-two hours after siRNA transfection,cells were used for MTT,real-time PCR,western blot,and other experiments.2.Cell growth inhibition by MTT assay.MDA-MB-231 and MCF-7 Cells were incubated overnight at a density of 4,000 cells/well in 96-well plates,and subsequently transfected with Notch- 1 siRNA or control siRNA.72 hours after tansfection,20μl of MTT was added each well.48 additional hours later,color development was measured on a microplate reader at 570 nm.3.Flow Cytometry and Cell Cycle Analysis.Notchl siRNA,or control siRNA transfected MDA-MB-231 and MCF7 cells were collected and stained with PI. DNA content was detected on a FACS Calibur.4.Apoptosis Assay.72 hours after transfection,Notch1 siRNA/Control siRNA transfected MDA-MB-231 and MCF-7 cells were collected and were labeled with annexin V-biotin followed by PI.Annexin V-PI were measured by FACS Calibur and analyzed with the Modfit Software.For the chemotherapeutic drug assays,24 hours after transfection,cells were exposed to 1.5 nM docetaxel or 75ug/ml doxorubicin respectively for 48 hours, then cells were collected and analyzed as above.5.Western Blot Analysis.Total protein was extrated from transfected/control cells. Total proteins were fractionated using SDS-PAGE and transferred onto nitrocellulose membrane.After incubation with blocking buffer,the membrane was incubated with primary and secondary antibodies,the protein bands were detected using the enhanced chemiluminesence detection system.6.Electrophoresis Mobility Shift Assay for Measuring NF-KappaB Activity.Nuclear extracts from MDA-MB-231 and MCF-7 cells were prepared.For EMSA,32 fmol of labeled probes were incubated for 20 min at 25℃with 2μg of nuclear extracts. Competition experiments were performed using EMSA conditions similar to those described above,except that the protein extracts were incubated with the probe in the presence of 250-fold molar excess of unlabeled double-stranded oligonucleotides as competitors.Subsequently,the DNA-protein complexes were separated from the free probes by electrophoresis through a 5%non-denaturing polyacrylamide gel,the gel was transferred to a PVDF membrane and chemiluminescent detection was performed.Results:1.Notch 1 siRNA effectively down-regulated the expression level of Notch 1 in human breast cancer cells.We observed that Notch1 and Hes1 mRNA decreased by about 90%in both MDA-MB-231 and MCF7 cells.Protein levels were also greatly reduced in Notch1 siRNA transfected cells compared with control siRNA transfected cells2.Down-regulation of Notch1 expression by siRNA inhibited the growth of human breast cancer cells.72 hours after transfection with Notch1 siRNA or control siRNA,cell viability was determined by the MTT assay.We found that the down-regulation of Notch1 expression caused approximately a 40%reduction of cell growth in both breast cancer cell lines.3.Down-regulation of Notch1 expression by siRNA induced S phase cell cycle arrest in MCF7 Cells.The Notch1 siRNA transfected MCF7 cells demonstrated an S phase arrest pattern(38.7%vs 19.54%)at 72 hours after transfection as compared with control cells.No alteration in cell cycle distribution was observed in MDA-MB-231 cells.4.Down-regulation of Notch-1 expression by siRNA led to apoptosis in human breast cancer.72 hours after transfection,cells were stained with Annexin V/PI,and analyzed by flow cytometry.The apoptosis rates were 5.64%and 2.88%in Notch1 siRNA transfected MDA-MB-231 and MCF7 respectively,compared with 1.25% and 0.94%in control siRNA transfected cells.These data suggested that the growth inhibition induced by Notch1 siRNA was partially due to an increase in cell apoptosis.5.Down-regulation of Notch-1 expression by siRNA increased chemosensitivity.We found that down-regulation of Notch-1 expression in combination with docetaxel or doxorubicin treatment led to a 50%or 70%enhancement in growth inhibition respectively as compared to single chemotherapeutic treatment in MDA-MB-231 cells.Similar results were found in MCF7 cells.In addition,the Notch-1 transfected MDA-MB-231 and MCF-7 cells were significantly more sensitive to docetaxel and doxorubicin-induced apoptosis,this may explain the enhanced growth inhibition.6.Down-regulation of Notch-1 expression by siRNA reduces NF-κB DNA-binding activity.Down-regulation of Notch-1 significantly inhibited NF-κB DNA-binding activity compared with control cells in both MDA-MB-231 and MCF7 cells as detected by EMSA.These results provide evidence that cross-talk exists between Notch1 and NF-κB in breast cancer and that Notch signaling is the upstream regulator.Conclusion: Down-regualtion of Notch-1 mediated by siRNA can inhibit the growth of breast cancer cells and increased chemosensitivity to doxorubicin and docetaxel.This effect may through the inactivation of NF-kappaB.Our results suggested that Notch signaling may be a promising target for breast cancer treatment.