| BackgroundThe morbidity of breast cancer is increasing in the past hundred years. Although our country is belong to the low morbidity country, the increase rate of the breast cancer has exceeded the West Country, especially in big city, and breast cancer has become the number 1,2 within the female malignant tumor. Moreover, metastasis is the main death reason and the essential biological characteristic of malignant tumor. Degradation of extracellular matrix is a crucial mechanism for tumor growth, parenchymal invasion, metastasis, and angiogene-sis. Matrix metalloproteinases (MMPs) are a family of more than 20 zinc - dependent neutral endopeptidases. They are crucial effectors in the steps leading to tumor development because they are capable of degrading essentially all ECM components. Increased expression of MMPs is related to progression of breast carcinoma and may be a promising target for therapy.Recently, a novel MMP inhibitor, reversion - inducing cysteine - rich protein with Kazal motifs ( RECK) , was identified by screening a fibroblast expression library for cDNAs that changed the round morphology in v - Ki - ras transformed NIH 3T3 cells into the nontransformed flat morphology. RECK is a membrane anchored Glycoprotein of approximately 110 kilodaltons that contains multiple epidermal growth factor like repeats and serine protease inhibitor - like domains , but has no structural homology with TIMPs. RECK is expressed in various normal human tissues but was described as undetectable in oncogenically transformed cells. It inhibits MMP - 9, MMP - 2, and membrane Type 1 ( MT1) - MMP ( MMP -14 ) secretion and activity by an as yet unknown mech-anism. Probably via this function, RECK is vital to developmental vasculogene-sis. Down -regulation of RECK has been implicated in tumor angiogenesis and, hence, tumor progression. In support of this hypothesis, increased transcriptional expression of RECK in tumor cells compared with concomitant normal tissue cells was correlated with prolonged survival in patients with hepatocellular carcinoma (HCC).The control of cell proliferation is believed to depend mainly on extracellular signals such as growth factors and intracellular signals that modify the activity of proteins controlling cell cycle progression. In the past decade, a variety of studies have suggested that K channel activity is also a key determinant for cell progression through the G1 phase of mitosis. This conclusion is based on three complementary observations, namely: (1) K channel blockers inhibit mitogene-sis; (2) mitogens increase K channel expression or activity; and (3) K channel openers stimulate cell proliferation. There is substantial evidence that K+ currents through K channels in the plasma membrane are required for proliferating cells to pass through the G1 phase of the cell cycle. This requirement might, in fact, represent a checkpoint in G, phase at which communication must occur between K channels in the plasma membrane and the cytosolic signaling pathways that regulate the cell cycle. Changes in membrane potential or cell volume produced by K+ currents might stimulate or suppress specific cell cycle regulatory signals, and in a reciprocal manner, the activation of K channels might be controlled by signaling pathways that coordinate activation of the channels with other cell cycle regulatory signals. It appears that no single type of K channel u-biquitously regulates the cell cycle, and putative regulatory roles have been proposed for diverse types of K channels, including voltage - gated, Ca2+ - activated and ATP - sensitive K channels.Docetaxel is a second - generation taxane derived from the needles of the European yew tree. Early in vitro studies revealed that docetaxel has a wide spectrum of antitumor activity. The primary mechanism of action for the docetaxel is to promote microtubulin assembly and stabilize the polymers against depoly-merization, thereby inhibiting microtubule dynamics. Impairment of mitotic progression leading to cell cycle arrest is considered to be a principal component ofdocetaxel's mechanism of action. This blocks progression of a cell through its natural division cycle and, consequently, inhibits cell proliferation.Objective1. To determine the invasive capacity of human breast non - tumor cell line, HBL - 100, breast adenocarcinoma cell line, MCF - 7 and breast ductal cancer cell line, MDA - MB -435S.2. To test the mRNA and protein expression level of RECK gene and the protein expression level of MMP - 2 and MMP - 9 gene in those three cell lines, in order to determine the relations among the invasive capacity, RECK gene and MMP-2, MMP-9 gene.3. To determine the effect of Docetaxel on the proliferation and invasive capacity of the HBL -100, MCF - 7 and MDA - MB -435S ceU lines.4. To determine whether there is any difference between MCF -7 and MDA - MB - 435S breast cancer cell lines with different invasive capacity in Ik andIkATP current density.5. To determine the effect of Docetaxel on the Ik and IkATP current density of MCF -7 and MDA - MB -435S breast cancer cell lines.Methods1. Transwell method was used to determine the invasive capacity of human HBL -100, MCF - 7 and MDA - MB - 435S ceU lines.2. RT - PCR, western blotting (WB) and immunocytochemistry (IC) were used to determine the mRNA and protein expression level of RECK gene and the protein expression level of MMP - 2 and MMP - 9 gene.3. MTT assay and Transwell method were used to determine the effect of Docetaxel on the proliferation and invasive capacity of the HBL - 100, MCF -7 and MDA - MB -435S ceU lines.4. Whole cell patch clamp technique was used to determine whether there is any difference between MCF - 7 and MDA - MB - 435 S breast cancer celllines in Ik and IkATP current, and was used to determine the effect of Docetaxel on the Ik and IkATP current of those two cell lines.Results1. The invasive capacity of MDA - MB -435S is the strongest (425. 20 ± 54.09) , that of the MCF -7 is the second(239. 00 ±91. 39) , and that of the HBL - 100 is the weakest( 101.00 ±63. 88). Been treated by 1.0PPC Docetaxel for 24h, the invasive capacity of MDA - MB -435S and MCF -7 are18. 2 ± 4. 32 and 58.4 ±50.53 respectively, which are lower than that of untreated control group significantly( P <0.01) 。 The inhibiting eflFects of Docetaxel on proliferation of those three cell lines are concentration and time dependent.2. The protein expression level of RECK gene in HBL - 100 cell line is the highest( IC:3188. 24 ± 894. 86; WB:3. 32 ±0. 25) , no expression in MDA -MB-435S cell line, and that in MCF - 7 cell line is (IC :1058.92 ±336. 53, p< 0.01; WB: 2.23 ±0. 59 ,p <0.05 compared to MDA - MB -435S cell line) 。 Moreover, in IC, the protein expression level of RECK gene is negative correlation to that of MMP -2 and MMP -9 gene. The mRNA level of RECK gene in HBL - 100 cell line is highest(2. 32 ±0. 02) , and no expression in MDA - MB-435S cell line.3. The Ik current density is gradually increased following the increase of the test potential. The Ik current density of the MCF -7 cell line is (2.60 ±0.375) pA/pF (141. 359 ± 7. 2258 ) pA/pF, whereas, that of the MDA - MB - 435S ceU line is(4.202 ±0. 8082)pA/pF (102. 600 ±5. 8584) pA/pF. Above -30mV, under the same test potential, the Ik current of the MCF -7 cell line is higher than that of the MDA - MB -435S cell lines( P <0.01).4. Following the increase of the test potential, the IkATP current density of the MCF -7 and MDA - MB -435S cell lines are( -45.273 ±1.2346) pA/pF- (79.427 ±5.7441 )pA/pF and ( -58. 523 ± 1.7346) pA/pF (80. 491 ± 7. 3531 )pA/pF, respectively. Within -30mV - 10 mV, under the same test potential , the IkATP current of the MCF - 7 cell line is higher than that of the MDA- MB -435S cell lines(P <0. 05) , and under other test potential, there is no-MB -435S cell lines(P <0. 05) , and under other test potential, there is no significant difference between them( P > 0.05 ).5. The effects of Docetaxel on the Ik and IkATP current of MCF -7 cell line: The slopes of current - voltage curve of Ik and IkATP are decreased following the increase of the concentration of Docetaxel, which means Docetaxel has the inhibition effect on Ik and IkATP. Under the same command potential, the Ik and IkATP current density decrease following the increase of the Docetaxel concentration, which means the influences of Docetaxel on Ik and IkATP are concentration dependent. Under +60mV, before treated, the current density of Ik is 141.36 ± 7. 23( pA/pF) , but it decrease from 124. 03 ± 8.43 (pA/pF) to 75. 16 ± 9.10 (pA/pF) , when the concentration of the docetaxel is increased from 1 x 10" mol · L-1 to 1 × 10-5 mol · L-1 . The differences of current density between the untreated cells to all the treated cells with different Docetaxel concentration are significant P<0. 01). For IKATP, before treated, under +90mV, the current density is 79.42 ±5.74(pA/pF). It decrease from 63.16 ± 10. 56(pA/pF) to 50.56 ±6. 77(pA/pF) , when the concentration of the docetaxel is increased from 1 ×10-7mol · L-1 to 1 ×10-5mol · L-1. However, only at the concentration of 1 × 10-5 mol · L-1 , the difference between the treated cells to untreated cells is significant ( P < 0.05 ).6. The effects of Docetaxel on the Ik and IkATP current of MDA - MB -435S cell line: The slopes of current - voltage curve of Ik and IkATP are decreased following the increase of the concentration of Docetaxel, which means Docetaxel has the close effect on Ik and IkATP. Under the same command potential, the Ik and IkATP current density decrease following the increase of the Docetaxel concentration , which means the influences of Docetaxel on Ik and IkATP are concentration dependent. Under +60mV, before treated, the current density of Ik is 102. 60±5. 86(pA/pF) , but it decrease from 81.66 ±9. 84(pA/pF)to 31. 89 ±7. 72 (pA/pF) , when the concentration of the docetaxel is increased from 1 × 10-7 mol · L-1 to 1 ×l0-5mol · L-1. For IKATP, before treated, under +90mV, the current density is 80.49 ±7.35(pA/pF). It decrease from 70.13 ±5. 69( pA/ pF)to 44. 90 ±5. 91 (pA/pF) , when the concentration of the docetaxel is increased from 1 × 10-7mol · L-1 to 1 × 10-5mol· L-1. However, only at the 1 x...
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