Expression of bone sialoprotein and osteopontin in breast cancer bone metastases

Bone sialoprotein (BSP) and osteopontin (OPN) are prominent, mineral-associated proteins in the extracellular matrix of bone that have been implicated in the metastatic activity of cancer cells. The expression of BSP, which is normally restricted to mineralizing tissues, has been observed in cancers with a high propensity for forming bone metastases. To investigate the relationship between BSP expression and the formation of bone metastases we have conducted an initial study of the expression of BSP in 10 intraductal breast carcinoma bone metastases using immunostaining and in situ hybridization, and compared the expression with OPN. The metastases were characterized by the infiltration of tumour cells into bone with extensive bone resorption evident. Moderate to strong staining for BSP was observed in all (100%) carcinomas, which also expressed BSP mRNA as determined by in situ hybridization. Variable staining for BSP was also observed in the mineralized bone and expression of BSP mRNA could be observed in osteoblastic cells on the bone surface and in some osteocytes at sites of bone remodelling. Contrary to a previous report, BSP expression could be demonstrated by PCR in three breast cancer cell lines, MCF-7, T47-D and MDA-MB-231. Moreover, in sub-cutaneous tumours formed by MDA-MB-231 breast cancer cells injected into athymic mice, higher immunostaining for BSP was seen in large ulcerating tumours in which mineral deposits were formed. In contrast to BSP, staining for OPN in bone metastases was generally restricted to the interface between tumor cells and bone surface of the carcinomas. While OPN staining was also observed in the cytoplasm of osteoclasts, which showed strong hybridization to a digoxygenin-labelled OPN cRNA probe, expression of OPN was not clearly detectable in the tumour cells. These studies provide the first demonstration of BSP expression by tumour cells in bone metastases and support the concept that BSP may have a role in targeting metastatic cells to bone. Expression of OPN in bone metastases appears to be related to increased bone resorptive activity by osteoclasts. This revised version was published online in July 2006 with corrections to the Cover Date.     

Differential expression of osteopontin and bone sialoprotein in bone metastasis of breast and prostate carcinoma

Breast and prostate cancer often metastasise to the skeleton. Interestingly, the histopathological characteristics of the bone lesions that arise from these two cancer types differ. Breast tumours give rise to metastases in the skeleton with a mixed lytic/sclerotic pattern, whereas a predominantly sclerotic pattern is seen in metastases from prostate tumours. Osteopontin (OPN) and bone sialoprotein (BSP) are bone matrix proteins that have been implicated in the selective affinity of cancer cells for bone. In the present study, 21 patient cases with skeletal metastasis and their respective primary tumours (12 with breast cancer, 9 with prostate cancer) were investigated by immunohistochemistry in order to assess the level of OPN and BSP. Moderate to strong OPN expression was found in 42% of all breast tumours and in 56% of all prostate tumours. Significantly more breast cancer bone metastases exhibited high OPN expression, 83%, as compared with prostate tumour bone metastases, 11% (P=0.0019). In contrast, moderate to strong BSP expression was found in 33% of breast tumours and in 89% of prostate tumours. In the bone lesions, only 33% of breast tumour metastases showed moderate/strong BSP expression compared to 100% of prostate tumour metastases (P=0.0046). This divergent pattern of OPN/BSP expression could be an important determinant for the different characteristics of these two types of bone metastasis, i.e., lytic vs. sclerotic, consistent with the proposed role of OPN in differentiation and activation of osteoclasts and of BSP as a stimulator of bone mineralisation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Nanotexturing of titanium-based surfaces upregulates expression of bone sialoprotein and osteopontin by cultured osteogenic cells

Bone formation around implants is influenced by surface geometry. Since cell/matrix/substrate interactions associated with cell signaling occur in the nanoscale dimension, we have evaluated the influence of nanotexturing of titanium-based surfaces on the expression of matrix proteins by cultured osteogenic cells at initial time points. Cells were obtained by enzymatic digestion of newborn rat calvaria and grown on titanium and titanium alloy discs with nanotextured or machined surfaces, and on glass coverslips for periods of 6 h, 1 day, and 3 days, under standard culture conditions. Cultures were processed for single or dual immunolabeling with monoclonal and/or polyclonal antibodies against bone sialoprotein (BSP), fibronectin (FN), osteopontin (OPN), type-I pro-collagen, or tubulin, followed by corresponding fluorophore-conjugated secondary antibodies. Some samples were processed for scanning electron microscope analysis of morphology and immunogold labeling. After 6 h, nanotextured surfaces exhibited up to a nine-fold increase in the proportion of cells with peripheral OPN labeling. At day 3, the proportion of OPN and BSP labeled cells was higher, and the intensity of immunoreactivity dramatically increased. No significant differences were observed in the expression pattern and the proportion of cells immunoreactive for FN or type-I pro-collagen. Our results demonstrate that nanotexturing of titanium-based surfaces upregulates the early expression of BSP and OPN in osteogenic cell cultures.     

Increased expression of osteonectin and osteopontin, two bone matrix proteins, in human breast cancer.

Microcalcifications are a common phenomenon associated with breast cancer and are often the only mammographic sign of a malignant breast disease. Although microcalcifications are not restricted to breast cancer and can be also associated with benign lesions, it is noteworthy that they are composed exclusively of hydroxyapatite in breast carcinoma. Hydroxyapatite is the bone-associated phosphocalcic crystal the deposition of which in bone tissue requires the coordinated expression of several molecules such as osteonectin (OSN) and osteopontin (OPN), synthesized by cells of the osteoblastic lineage. In this study, we evaluated the expression of these two bone matrix proteins, using an immunoperoxidase technique and specific antibodies, in 79 breast lesions including 28 benign and 51 cancerous specimens. We found that normal mammary tissue associated with the lesions examined expressed generally undetectable or lightly detectable (0 or 1+) amounts of OSN and OPN (92 and 81%, respectively). Benign breast lesions, including fibroadenoma and fibrocystic dysplasia, were generally weakly stained (0 or 1+) with both anti-OSN and anti-OPN antibodies (96.4 and 60.7%, respectively). Interestingly, the majority of both in situ and invasive breast carcinoma lesions showed a strong expression (2+ or 3+) for OSN or OPN (74.5 and 84.3%, respectively). High expression of these two bone matrix proteins was associated with frequent microcalcification deposition in the lesion. This study is the first extensive study of OSN and OPN expression in mammary cancers. Our data suggest that OSN and OPN could play a role in the formation of ectopic microcalcifications often associated with breast cancer. It is also tempting to speculate that the expression of these two glycoproteins by breast cancer cells play a role in the preferred bone homing of breast metastases.     

Binding of bone sialoprotein, osteopontin and synthetic polypeptides to hydroxyapatite.

The phosphorylated acidic glycoproteins bone sialoprotein (BSP) and osteopontin (OPN) bind to hydroxyapatite (HA) crystals and may be involved in the regulation of bone mineralization. The HA-binding properties of these proteins have been attributed to glutamic acid-rich sequences in BSP and aspartic acid-rich sequences in OPN. The present study examines the roles of these polycarboxylate sequences in the binding of BSP and OPN to HA. Porcine BSP, OPN and the synthetic polypeptides poly-L-glutamic acid [Poly(Glu)] and poly-L-aspartic acid [Poly(Asp)] were labeled with fluorescein isothiocyanate and their binding to HA determined by fluorimetry. From the binding isotherms, dissociation constants (KDs) for all the reagents tested were determined to be in the micromolar range. The saturation binding capacities of HA for Poly(Glu), Poly(Asp), BSP and OPN were similar (500-600 micrograms/m2). To investigate the role of glutamic acid-rich and aspartic acid-rich sequences in the binding to HA of BSP and OPN, respectively, competitive binding studies with Poly(Glu) and Poly(Asp) were performed. Poly(Glu) was able to displace a maximum of 100% of Poly(Glu), 81% of OPN, 68% of BSP and 65% of Poly(Asp). Poly(Asp) was able to displace a maximum of 100% of Poly(Glu), 99% of Poly(Asp), 95% of OPN and 89% of BSP. These results are consistent with the view that BSP and OPN bind to HA via their polycarboxylate sequences, but suggest a complex mode of interaction between polyelectrolytes and ionic crystals.     

Correlation between calcification and bone sialoprotein and osteopontin in papillary thyroid carcinoma

The correlation between calcification and papillary thyroid carcinoma has received increasing attention. We investigated the ability of bone sialoprotein (BSP) and osteopontin (OPN) protein levels to diagnose papillary thyroid carcinoma (PTC), and explored the correlation between BSP and OPN protein levels and calcification in PTC. Archival PTC specimens from patients with PTC with calcification and lateral cervical lymph node metastasis (LNM) were included in this retrospective immunohistochemical study. The protein levels of BSP and OPN were analysed immunohistochemically using routinely prepared tissue sections. PTC specimens from 66 patients with PTC were reviewed retrospectively (25 patients with histological calcification seen in paraffin sections, 41 patients without calcification; 35 patients with lateral cervical LNM, 31 patients without LNM). The percentage of samples that had cells that demonstrated positive protein staining differed significantly between PTC specimens, benign thyroid nodules, and adjacent normal follicular epithelium (BSP: 87.88%, 55.00%, and 42.50%, respectively; OPN: 83.33%, 70.00% and 50.00%, respectively). There was a significant difference in the immunohistochemical score (IHS) for BSP and OPN protein staining between PTC specimens with and without calcification (P < 0.05). The level of BSP protein staining was found to be significantly correlated with the level of OPN protein staining in PTC specimens. We conclude that the strong correlation between BSP and OPN and PTC suggests a role for BSP and OPN in calcification and tumor progression of PTC. BSP and OPN might be useful tumour markers for the diagnosis of PTC with limited value, because both of them had low specificity.     

IFN-gamma induction of osteopontin expression in human monocytoid cells.

Osteopontin (OPN) is an arginine-glycine-aspartate (RGD)-containing cytokine that increases macrophage expression of the Th1 cytokines interferon-gamma (IFN-gamma) and interleukin-12 (IL-12), and downregulates macrophage expression of IL-10. OPN has been implicated in the clearance of mycobacterial infection and in granuloma formation. We previously showed decreased OPN expression in the granulomas of patients with disseminated mycobacterial infection in the setting of mutation of the IFN-gamma receptor-1 (IFNGR-1). These data suggested that IFN-gamma critically regulates OPN expression during mycobacterial infection. Herein, we characterize the effect of IFN-gamma stimulation on OPN expression by human monocytoid cells. By Western blot, ELISA, and Northern blot analysis, IFN-gamma treatment of THP-1 cells was shown to induce OPN mRNA and protein expression in a time-dependent and dose-dependent manner. The human OPN promoter was amplified by PCR using human genomic DNA as a template and transfected into THP-1 cells. Compared with control, OPN promoter activity increased by 15-fold after treatment with IFN-gamma. Similar induction of OPN was seen in IFN-gamma-stimulated primary human blood monocytes. These data show that IFN-gamma stimulates OPN expression from monocytoid cells and suggest that OPN may function in a positive feedback loop in Th1 inflammation, increasing expression of IFN-gamma, which itself upregulates the OPN gene.     

Adhesion of MC3T3-E1 cells to bone sialoprotein and bone osteopontin specifically bound to collagen I

Bone sialoprotein (BSP) and bone osteopontin (OPN) are members of the SIBLING (small integrin-binding ligand, N-linked glycoproteins) family of proteins commonly found in mineralized tissues. Previously, OPN was shown to exhibit a preferential orientation for MC3T3-E1 cell adhesion when it was specifically bound to collagen. In this work, the orientation of BSP under similar circumstances is examined and compared with OPN. Radiolabeled adsorption isotherms were obtained for BSP bound to both tissue culture polystyrene (TCPS) and collagen-coated TCPS. The results show that collagen has the capacity to bind almost twice as much OPN under identical conditions. An in vitro MC3T3-E1 cell adhesion assay was then performed to compare the cell binding ability of BSP on either TCPS or collagen-coated TCPS with identical amounts of adsorbed protein. It was found that there is no significant difference in the cell binding ability of BSP on either of the substrates. For cell binding studies on collagen-coated TCPS, it was shown that there are a greater number of cells bound to substrates with adsorbed OPN as compared with BSP. The preferable orientation of OPN for cell binding coupled with the higher binding capability of collagen for OPN indicates that OPN is more important than BSP for osteoblast adhesion to the collagen matrix. In addition, a cell inhibition assay was performed to show that all of the cell binding that occurred throughout these studies was dependent upon integrin interactions with the RGD cell binding moiety.     

Expression of osteopontin and osteonectin in breast cancer.

Osteopontin (OP) and osteonectin (ON) are bone matrix proteins produced by mammary and other cancers. These proteins may play a role in tumor invasion and metastasis through integrin-mediated signal transduction. We evaluated expressions of OP and ON in 253 resected infiltrating ductal carcinoma of the breast, using immunohistochemical staining and follow-up data. OP and ON were detected 87.4% and 54.2% of 253 cases, respectively. The OP and ON positive staining were localized in the cytoplasm of carcinoma cells. OP and ON did not correlate with various clinicopathological parameters, such as age, lymph node involvement, tumor size, histologic grade, expression of p53 and estrogen receptor (ER). In the multivariate model, lymph node involvement and histologic grade were statistically significant prognostic factors. Assessed by a log rank test, the 5-year-survival rates of OP and ON positive groups and their negative groups were not statistically different. In conclusion, OP and ON immunopositivity of infiltrating ductal carcinomas of the breast provide no additional prognostic information in this study.     

人乳腺癌细胞化疗敏感性与相关基因表达的关系

目的探讨人乳腺癌细胞化疗敏感性与多药耐药相关基因和凋亡调控基因表达的关系。方法MTT法检测5株人乳腺癌细胞株Bcap37、MCF-7、T47D、MDA-MB-231和MDA-MB-435对阿霉素(ADM)、顺铂(DDP)、丝裂霉素(MMC)、5-氟尿嘧啶(5-Fu)、卡氮芥(BCNU)的敏感性。流式细胞术检测多药耐药基因P-糖蛋白(P-GP)、谷胱甘肽-S-转移酶-π(GST-π)、肺耐药蛋白(LRP)、多药耐药相关蛋白(MRP)和甲基鸟嘌呤甲基转移酶(MGMT)等耐药相关基因和凋亡调控基因FAS、BCL-2、P53和P16的表达。结果不同人乳腺癌细胞株对同一药物敏感性差异较大。相关分析表明,细胞株对5-Fu的敏感性与P16表达呈正相关(P<0.01),对其余药物的敏感性与所检基因的表达水平无关。结论人乳腺癌细胞株对5-Fu的敏感性与P16表达有关。     

Nonhematopoietic cells are the primary source of bone marrow-derived lung epithelial cells

Previous studies have demonstrated that bone marrow (BM)-derived cells differentiate into nonhematopoietic cells of multiple tissues. To date, it remains unknown which population(s) of BM cells are primarily responsible for this engraftment. To test the hypothesis that nonhematopoietic stem cells in the BM are the primary source of marrow-derived lung epithelial cells, either wild-type hematopoietic or nonhematopoietic BM cells were transplanted into irradiated surfactant-protein-C (SPC)-null mice. Donor-derived, SPC-positive type 2 pneumocytes were predominantly detected in the lungs of mice receiving purified nonhematopoietic cells and were absent from mice receiving purified hematopoietic stem and progenitor cells. We conclude that cells contained in the nonhematopoietic fraction of the BM are the primary source of marrow-derived lung epithelial cells. These nonhematopoietic cells may represent a primitive stem cell population residing in adult BM.     

LKB1 protein expression in human breast cancer.

Peutz-Jeghers syndrome is caused by germline mutations in the LKB1/STK11 gene. Peutz-Jeghers syndrome is associated with an increased risk of developing intestinal and extraintestinal cancers, including pancreatic, lung, and breast carcinomas. LKB1 gene inactivation has recently been demonstrated in a subset of sporadic pancreatic and lung carcinomas. The role of the LKB1 gene in sporadic breast carcinomas remains unclear, though recent studies suggest inactivation only within papillary carcinomas. Using a commercially available polyclonal antibody that has been shown to mirror LKB1 genetic status in gastrointestinal and pulmonary carcinomas, the authors performed IHC on a large series of breast cancers using tissue microarrays (TMAs). All abnormal TMA results were confirmed using whole sections; specifically, whole sections from the donor blocks of lesions demonstrating diminished or absent LKB1 protein expression on TMA were evaluated to compare labeling of the lesion with that of the surrounding normal breast. In all cases, normal breast epithelium demonstrated strong cytoplasmic labeling (providing an internal positive control), whereas the stroma was nonreactive. Luminal cells typically labeled more strongly than myoepithelial cells. Among 70 invasive ductal carcinomas, 3 (4.3%) showed complete loss of LKB1 labeling, whereas 6 others (8.6%) showed diminished labeling. Of the eight intraductal carcinoma lesions adjacent to these invasive carcinomas, one (12.5%) showed complete loss of LKB1 labeling and one other (12.5%) showed diminished labeling; these results were identical to those of the adjacent invasive carcinomas. One of 10 (10%) hematogenous metastases of mammary carcinoma showed loss of LKB1 labeling. Nine of the 10 invasive carcinomas and both of the ductal carcinoma in situ (DCIS) cases showing loss of or diminished LKB1 expression were of high grade. In contrast, all 13 pure nonpapillary DCIS lesions, all 5 invasive lobular carcinomas and 3 accompanying lobular carcinoma in situ lesions, all 7 papillary DCIS lesions, and all 3 papillomas evaluated showed intact LKB1 labeling. Therefore, although frequent methylation of the LKB1 gene has been reported in papillary carcinomas of the breast, the authors did not find loss of protein expression in these lesions. Instead, it was found that loss of LKB1 protein expression occurs in a subset of high-grade in situ and invasive mammary carcinomas. The authors found LKB1 gene methylation in several of these invasive carcinomas. Given recent Western blot results indicating that diminished LKB1 expression in breast carcinomas correlates with shorter relapse-free survival, LKB1 IHC merits evaluation as a potential prognostic marker for breast carcinoma.     

骨桥蛋白基因真核表达载体构建及对结肠癌细胞的作用

目的：构建骨桥蛋白（Osteopontin,OPN）基因真核表达载体,转染人结肠癌细胞SW480,分析其对SW480细胞株增殖及生存能力的作用。方法：构建重组表达载体pEGFP-N1/OPN,经测序鉴定无误后,转染人结肠癌SW480细胞。RT-PCR检测SW480细胞转染后OPN mRNA表达;Western blot检测SW480细胞转染后OPN蛋白表达量;Cell Counting Kit-8（CCK-8）测定细胞增殖;软琼脂克隆形成实验观察细胞的锚定非依赖性生长能力。结果：pEGFP-N1/OPN转染SW480后,OPN基因获得很好转录,OPN蛋白表达增高,转染OPN后SW480细胞增殖率（光吸收值）显著高于转染阴性组（P〈0.05）,软琼脂克隆形成速度增快,数量明显多于对照组和空载体组（P〈0.05）。结论：OPN基因真核表达载体pEGFP-N1/OPN构建成功,OPN具有促进SW480细胞增殖、存活的作用,为进一步研究OPN作用机制奠定了重要基础。     

Tumor antigens and antigen-presenting capacity in breast cancer.

AIMS: Cancer cells frequently express antigens capable of being recognized by the host immune system; however, any resultant immune response is often ineffective. This may be related in part to tumor-induced defects in antigen presentation. We screened for dendritic cell infiltration, tumor MHC II expression and associated lymphocytic reaction in the context of three established breast tumor antigens. METHODS: Forty primary breast tumors were evaluated by immunohistochemical techniques for expression of her2/neu, p53, and MUC1 and MHC class II molecules. Twenty-five samples were further analyzed for p53 mutations by PCR-SSCP analysis and DNA sequencing. The phenotype of tumor-infiltrating inflammatory cells was evaluated using the following markers: CD1a, MHC Class II, CD3, CD45, and CD45RO. RESULTS: Tumors with p53 mutations and overexpression, but not her2/neu or MUC1 overexpressing tumors, more frequently harbored marked CD1a+ dendritic cell infiltrates. An overall correlation between CD1a+ cell infiltrates and HLA class II expression on tumor cells (p = 0.0008) was also observed and these tumors had greater CD45RO+ lymphocytic infiltrates. CONCLUSIONS: In breast cancer, p53 mutations may present a more visible signal to the immune system and hence provide a better target for immunotherapy. Infiltrating CD1a positive cells are associated with a more dense tumor lymphocytic infiltrate and tumor cell expression of MHC II molecules.     

Increased levels of alpha6 integrins are associated with the metastatic phenotype of human breast cancer cells.

Integrins play an important role in interactions between cells and the extracellular matrix, and thus have a potential role in metastasis. Expression levels of alpha6, beta1 and beta4 integrin sub-units were measured in a panel of human breast cancer cell lines by RT/PCR, immunoprecipitation and flow cytometry. All the lines expressed alpha6, with the highest levels in the MDA-MB-231 and MDA-MB-435 cells. These grew the most aggressively and were metastastic in nude mice. Low levels of alpha6 protein were measured in breast cancer cells that were poorly tumorigenic and non-metastatic in nude mice, and there was an inverse relationship between ER and alpha6 expression. RT/PCR revealed that all lines expressed the 2 isoforms of alpha6, with the alpha6A isoform generally more abundant than alpha6B isoform. Clones of MDA-MB-435 were isolated by sterile sorting for cells with high or low alpha6 expression, and two variants established from metastases in nude mice were found to differ in alpha6 expression. When injected into nude mice. the alpha6-high variants produced significantly more lung metastases than the alpha6-low variants. beta1was abundant in all lines, while beta4 was not detected in MDA-MB- 134 cells, and in the MDA-MB-435 cells an alternately spliced variant of beta4 was identified. Sequencing of the alternate variant revealed a novel sequence from a splicing event in the cytoplasmic tail of beta4. None of the cells with this variant mRNA expressed detectable levels of beta4 protein. Our results suggest that high alpha6 expression in human breast cancer cells is associated with tumorigenicity and metastatic potential.     

HE4 suppresses the expression of osteopontin in mononuclear cells and compromises their cytotoxicity against ovarian cancer cells

Ovarian cancers are known to evade immunosurveillance and to orchestrate a suppressive immune microenvironment. Here we examine the role of human epididymis protein 4 (HE4), an ovarian cancer biomarker, in immune evasion. Through modified subtractive hybridization analyses we have characterized the gene targets of HE4 in human peripheral blood mononuclear cells (PBMCs), and established a preliminary mechanism for HE4‐mediated immune failure in ovarian tumours. Upon exposure of purified PMBCs to HE4, osteopontin (OPN) and dual‐specificity phosphatase 6 (DUSP6) emerged as the most suppressed and up‐regulated genes, respectively. SKOV3 and OVCAR8, human ovarian carcinoma cell lines, exhibited enhanced proliferation in conditioned media from HE4‐exposed PBMCs, an effect that was attenuated by the addition of recombinant OPN or OPN‐inducible cytokines [interleukin (IL)‐12 and interferon (IFN)‐?]. Additionally, upon co‐culture with PBMCs, HE4‐silenced SKOV3 cells were found to be more susceptible to cytotoxic cell death. The relationship between HE4 and OPN was reinforced further through the analysis of serous ovarian cancer patient samples. In these biopsy specimens, the number of OPN⁺ T cells correlated positively with progression free survival (PFS) and inversely with serum HE4 level. Taken together, these findings show that HE4 enhances ovarian cancer tumorigenesis by compromising OPN‐mediated T cell activation.     

芪三酚与人乳腺癌MDC1基因关系的研究

目的探讨芪三酚（Res）对人乳腺癌MDA—MB-231细胞增殖抑制的相关效应及其与MDCl基因的关系。方法以人乳腺癌MDA—MB-231细胞株为研究对象,采用MTS方法测定细胞增殖,应用吖啶橙荧光染色观察Res对乳腺癌MDA—MB-231细胞的影响,用RT-PCR与免疫印迹方法测定MDCl基因与蛋白表达水平,用小RNA干扰MDCl基因后,用流式细胞仪检测细胞的凋亡并观察其对Res的敏感性影响。结果40μmol／L以上的Res可显著抑制乳腺癌MDA—MB-231细胞的增殖（P〈0．05）,给予0、60、120μmol／LRes能明显降低MDCl基因和蛋白的表达（P〈0．05）。用小RNA干扰MDC1基因后,流式细胞术分析显示,实验组（MDCl．siRNA）的细胞凋亡率[（45．13±6．2）％]较阴性对照组[（24．34±2．6）％]和未处理组[（17．69±4．9）％]明显上升（P〈0．05）,MTS结果显示MDCl基因干扰后细胞对Res的敏感性增加。结论40μmol／L以上的Res可以抑制MDA-MB-231细胞的增殖,Res可以有效降低MDC基因和蛋白的表达并促进细胞的凋亡。用小RNA干扰MDCl基因（MDCl-siRNA）后,MDA-MB-231细胞对Res的敏感性增加。