乳腺癌细胞AP-2α表达上调机制

目的探讨乳腺癌中转录因子2α（AP-2α）蛋白高表达的机制。方法采用Western blot、Southern blot、liT—PCR方法,从蛋白水平、基因水平、mRNA水平等方面系统研究乳腺癌细胞中AP-2α的高表达。结果HER-2癌基因高表达的转移性乳腺癌细胞系MDA—MB-453和SK—BR-3中AP-2α蛋白水平高,但未检测到AP-2α基因扩增,AP-2αmR—NA水平也没有显著上调。结论翻译后修饰水平的异常可能导致在转移性乳腺癌细胞系MDA-MB-453和SK-BR-3中AP-2α蛋白水平高,AP-2α可能作为转录因子促进HER-2癌基因在这些细胞中的高表达而致癌。     

Anti-Her-2/neu antibody induces apoptosis in Her-2/neu overexpressing breast cancer cells independently from p53 status.

Anti-Her-2/neu antibody is known to induce apoptosis in HER-2/neu overexpressing breast cancer cells. However, exact regulatory mechanisms mediating and controlling this phenomenon are still unknown. In the present study, we have investigated the effect of anti-Her-2/neu antibody on apoptosis of HER-2/neu overexpressing human breast cancer cell lines SK-BR-3, HTB-24, HTB-25, HTB-27, HTB-128, HTB-130 and HTB-131 in relation to p53 genotype and bcl-2 status. SK-BR-3, HTB-24, HTB-128 and HTB-130 cells exhibited mutant p53, whereas wild type p53 was found in HTB-25, HTB-27 and HTB-131 cells. All seven cell lines weakly expressed bcl-2 protein (10-20%). Anti-Her-2/neu antibody, irrespective of p53 and bcl-2 status, induced apoptosis in all 7 cell lines dose- and time-dependently and correlated with Her-2/neu overexpression. In addition, incubation of cell lines with anti-Her-2/neu antibody did not alter p53 or bcl-2 expression. Anti-HER-2/neu antibody did not induce apoptosis in HER-2/neu negative HBL-100 and HTB-132 cell lines. Our results indicate that within the panel of tested breast cancer cell lines, anti-Her-2/neu antibody-induced apoptosis was independent from the presence of intact p53.     

HER2-specific T lymphocytes kill both trastuzumab-resistant and trastuzumab-sensitive breast cell lines in vitro

Objective: Although the development of trastuzumab has improved the outlook for women with human epidermal growth factor receptor 2 (HER2)-positive breast cancer, the resistance to anti-HER2 therapy is a growing clinical dilemma. We aim to determine whether HER2-specific T cells generated from dendritic cells (DCs) modified with HER2 gene could effectively kill the HER2-positive breast cancer cells, especially the trastuzumab-resistant cells. Methods: The peripheral blood mononuclear cells (PBMCs) from healthy donors, whose HLA haplotypes were compatible with the tumor cell lines, were transfected with reconstructive human adeno-association virus (rhAAV/HER2) to obtain the specific killing activities of T cells, and were evaluated by lactate dehydrogenase (LDH) releasing assay. Results: Trastuzumab produced a significant inhibiting effect on SK-BR-3, the IC50 was 100ng /ml. MDA-MB-453 was resistant to trastuzumab even at a concentration of 10,000 ng/ml in vitro. HER2-specific T lymphocytes killed effectively SK-BR-3 [(69.86±13.41)%] and MDA-MB-453 [(78.36±10.68)%] at 40:1 (effector:target ratio, E:T), but had no significant cytotoxicity against HER2-negative breast cancer cell lines MDA-MB-231 or MCF-7 (less than 10%). Conclusion: The study showed that HER2-specific T lymphocytes generated from DCs modified by rhAAV/HER2 could kill HER2-positive breast cancer cell lines in a HER2-dependent manner, and result in significantly high inhibition rates on the intrinsic trastuzumab-resistant cell line MDA-MB-453 and the tastuzumab-sensitive cell line SK-BR-3. These results imply that this immunotherapy might be a potential treatment to HER2-positive breast cancer.     

Silencing of the HER2/neu gene by siRNA inhibits proliferation and induces apoptosis in HER2/neu-overexpressing breast cancer cells

In eukaryotes, double-stranded (ds) RNA induces sequence-specific inhibition of gene expression referred to as RNA interference (RNAi). We exploited RNAi to define the role of HER2/neu in the neoplastic proliferation of human breast cancer cells. We transfected SK-BR-3, BT-474, MCF-7, and MDA-MB-468 breast cancer cells with short interfering RNA (siRNA) targeted against human HER2/neu and analyzed the specific inhibition of HER2/neu expression by Northern and Western blots. Transfection with HER2/neu-specific siRNA resulted in a sequence-specific decrease in HER2/neu mRNA and protein levels. Moreover, transfection with HER2/neu siRNA caused cell cycle arrest at G0/G1 in the breast cancer cell lines SK-BR-3 and BT-474, consistent with a powerful RNA silencing effect. siRNA treatment resulted in an antiproliferative and apoptotic response in cells overexpressing HER2/neu, but had no influence in cells with almost no expression of HER2/neu proteins like MDA-MB-468 cells. These data indicate that HER2/neu function is essential for the proliferation of HER2/neu-overexpressing breast cancer cells. Our observations suggest that siRNA targeted against human HER2/neu may be valuable tools as antiproliferative agents that display activity against neoplastic cells at very low doses.     

Inhibitory effects of the combination of HER-2 antisense oligonucleotide and chemotherapeutic agents used for the treatment of human breast cancer.

Poor response to chemotherapy in patients with breast cancer is often associated with overexpression of HER-2/neu. Interference with HER-2 mRNA translation by means of antisense oligonucleotides might improve the efficacy of chemotherapy. To test this hypothesis, eight breast cancer cell lines and a normal human fibroblast cell line were examined for their level of HER-2 expression, their sensitivity to phosphorothioate antisense oligonucleotides (AS HER-2 ODN), and to various chemotherapeutic agents, and the combination of the two. No correlation was found between the intrinsic HER-2 level and either the sensitivity to a particular chemotherapeutic agent alone, or the amount of growth inhibition observed with a specific AS HER-2 ODN concentration. Although sequence specificity and extent of AS HER-2 ODN inhibition of HER-2 synthesis were somewhat higher in the HER-2 overexpressing MDA-MB-453 and SK-BR-3 cells, we found that antisense treatment significantly sensitized all of the breast cancer cells, even MDA-MB-231 and MDA-MB-435 cells, with approximately basal levels of HER-2, to various chemotherapeutic agents. In addition, the combination of AS HER-2 ODN and taxol was shown to synergistically induce apoptosis in MDA-MB-435. These results demonstrate that overexpression of HER-2 would not be a prerequisite for the effective use of AS HER-2 ODN as a combination treatment modality for breast cancer and suggest that the use of AS HER-2 ODN, as part of a combination treatment modality, need not be limited to breast tumors that display elevated levels of HER-2.     

维生素E琥珀酸酯诱导HER-2过表达乳腺癌细胞凋亡机制的研究

目的:探讨维生素E琥珀酸酯(vitamin E succinate,VES)对HER-2过表达乳腺癌细胞的生长抑制和诱导凋亡作用。方法:MTT法测定VES对乳腺癌MDA-MB-231和MDA-MB-453细胞增殖的抑制作用，应用Western blot法筛选HER-2过表达乳腺癌细胞系，同时检测VES处理后GSK-3、NF-κBp65、caspase 9蛋白在乳腺癌MDA-MB-231和MDA-MB-453细胞中的表达水平，划痕实验与侵袭实验观察VES对乳腺癌细胞体外迁移能力的影响，流式细胞术检测VES对乳腺癌细胞凋亡的作用。结果:VES处理后GSK-3、NF-κBp65、caspase 9蛋白在乳腺癌MDA-MB-453细胞中的表达明显高于在乳腺癌MDA-MB-231细胞中的表达，VES作用于HER-2低表达乳腺癌细胞使其迁移及侵袭能力明显增强，却能够抑制HER-2高表达乳腺癌细胞的迁移及侵袭。VES以直接杀伤方式作用MDA-MB-231细胞，而对MDA-MB-453则以诱导细胞凋亡方式杀伤，VES使HER-2高表达乳腺癌MDA-MB-453细胞发生G1/G0期阻滞。结论:VES促进乳腺癌细胞凋亡是通过影响多条信号传导途径完成的，VES作用于不同HER-2表达乳腺癌细胞其迁移及侵袭能力明显不同，其机制与细胞表面蛋白表达有关。     

SUCI02选择性抑制HER-2/neu受体酪氨酸激酶磷酸化及其对HER-2/neu过表达乳腺癌细胞生长的影响

背景与目的：HER-2／neu受体过度表达与肿瘤的发生发展、对化疗的敏感性以及患者预后有关;我们通过大量筛选,发现SUCI02[N-(4-乙氧苯基)-2-羟基酸胺]能抑制HER2／neu受体酪氨酸激酶磷酸化。本研究拟探讨SUCI02对HER-2／nell过度表达的肿瘤细胞生长的影响。方法：用免疫沉淀、免疫印迹法检测：HER-2／neu受体酪氨酸激酶磷酸化、酪氨酸激酶蛋白水平的变化;MTT法检测SUCI02对乳腺癌细胞的抑制作用。结果：SUCI02能抑制HER-2／neu受体的自身磷酸化,在乳腺癌MDA—MB．453m1细胞中,SUCI02对HER-2／neu受体自身磷酸化的IC50为4．34μg／ml,对HER-2／neu的表达没有任何影响。在用SUCI02处理MDA-MB-453ml细胞30min后,用培养液洗掉药物,继续培养不同时间,结果可见洗掉药物后30min,SUCI02对HER-2／neu受体磷酸化的抑制就开始恢复。SUCI02处理MDA-MB-453ml细胞后其下游靶分子MAPK和AKT激活明显受抑制,呈现剂量依赖性。SUCI02对EGFR受体酪氨酸磷酸化在最高剂量用到40μg／ml下没有明显的抑制作用。应用MTT法检测了SUCI02对过度表达HER-2／neu的MDA-MB-453ml细胞的生长抑制作用,同时应用过表达EGFR的乳腺癌MDA—MB-468细胞作为对照,结果可见SUCI02对HER-2／neu过表达的肿瘤细胞相对于过表达EGFR的肿瘤细胞有更明显的抑制作用。结论：SUCI02选择性抑制HER-2／neu受体酪氨酸激酶磷酸化,阻断其下游信号途径,对过度表达HER-2／neu的肿瘤细胞具有更强的生长抑制作用。     

Soluble HER-2/neu neutralizes biologic effects of anti-HER-2/neu antibody on breast cancer cells in vitro

Amplification and over-expression of the HER-2/neu proto-oncogene are associated with poor prognosis in women with both node-positive and node-negative breast cancer. Therefore, the encoded surface glycoprotein represents an attractive target for cancer immunotherapies. Furthermore, the extracellular domain of HER-2/neu is released from the cell surface by proteolytic cleavage. In the present experiments, we investigated the potential biologic effects of soluble HER-2/neu with particular emphasis on its interaction with anti-HER-2/neu antibodies. A monoclonal antibody specific for the extracellular domain of HER-2/neu dose dependently inhibited the proliferation of highly HER-2/neu-expressing SK-BR-3 and BT-474 breast cancer cells but had no effect on the proliferation of weakly to moderately HER-2/neu-expressing MCF-7, HBL-100 and ZR-75-1 breast cells. Addition of SK-BR-3 or BT-474 cell supernatants with high concentrations of soluble HER-2/neu led to a neutralization of anti-HER-2/neu antibody–mediated inhibition of proliferation due to a binding of soluble HER-2/neu by the antibody, which could be demonstrated by immunoprecipitation. Furthermore, the ability of anti-HER-2/neu antibodies to mediate antibody-dependent cellular cytotoxicity (ADCC) by lymphokine-activated killer cells was assessed. Cytolysis of SK-BR-3 tumor cells was increased significantly in the presence of anti-HER-2/neu antibodies. Similar to the proliferation inhibition, ADCC was neutralized by addition of soluble HER-2/neu-containing supernatants. Our data suggest that tumors rich in HER-2/neu might thus escape certain steps of immunologic control by neutralizing biologic activities of anti HER-2/neu antibodies due to the presence of soluble HER-2/neu. Int. J. Cancer 73:875–879, 1997. © 1997 Wiley-Liss, Inc.     

Detection of genetic instability at HER-2/neu and p53 loci in breast cancer cells sing Comet-FISH

A proportion of breast cancers acquire genetic alterations at 17q11.2-q12 (HER-2/neu), 20q13.2 (ZNF217 gene) and 17p13.1 (p53). We describe a unique technique (Comet-FISH) in which we documented relative genetic instability at p53 and HER-2/neu gene loci within a panel of malignant breast cancer cell lines (MCF-7; MDA-MB-468 and CRL-2336). Furthermore, Comet-FISH data were consistent with preferential repair of the p53 locus following gentoxic insult and suggest that this assay may be quite useful for the study of genetic instability.     

Determination of HER-2/neu amplification and expression in tumor tissue and cultured cells using a simple, phenol free method for nucleic acid isolation.

A rapid, simple and non-toxic procedure for the simultaneous isolation of DNA and RNA from tumor tissue and cells grown in vitro is described. Guanidinium isothiocyanate was used for homogenization of tumor tissue and for cell lysis. Separation of proteins, DNA and RNA was carried out by isopycnic centrifugation in cesium trifluoroacetate. DNA was further purified by salting out residual protein. Nucleic acids prepared by this method from 47 primary human carcinomas and 17 human cell lines were analysed for amplification and expression of the HER-2/neu proto-oncogene. 2- to 10-fold amplification of HER-2/neu was noted in 7/22 mammary carcinomas (32%) and in 4/14 ovarian carcinomas (28%). No amplification of the proto-oncogene was found in 4 laryngeal carcinomas, 1 pharyngeal carcinoma, 2 retrolingual carcinomas, 3 gastric carcinomas and 1 kidney carcinoma. HER-2/neu overexpression was observed in 6/22 of mammary carcinomas (27%) and 7/14 of ovarian carcinomas (50%). No overexpression was found in all other carcinomas studied. Concordance between amplification and overexpression was noted in 3 mammary and 4 ovarian carcinomas, respectively. 3 mammary and 3 ovarian carcinomas showed overexpression without amplification. 5 human mammary carcinoma cell lines showed both amplification and overexpression of HER-2/neu. In two mammary carcinoma cell lines (MDA MB-453 and ZR 75-1) overexpression was noted without amplification of the proto-oncogene. These data combine to suggest that mechanisms other than gene amplification may also lead to overexpression of the HER-2/neu protooncogene in cancer cells.     

Effect of resveratrol on the development of spontaneous mammary tumors in HER-2/neu transgenic mice

Resveratrol (Res) has been reported to possess cancer chemopreventive activity on the basis of its in vitro effects on tumor cells and in vivo experimental models of rodents transplanted with parental tumors or treated with carcinogens. We investigated the effects of Res on the development of mammary tumors appearing spontaneously in HER-2/neu transgenic mice at an early age. The mechanisms involved in the Res antitumor effect were evaluated by studying the immune effectiveness, tumor apoptosis and expression of mRNA and protein for HER-2/neu in tumoral mammary glands from Res-treated mice and in tumor cell lines. Res supplementation delayed the development of spontaneous mammary tumors (p < 0.001), reduced the mean number and size of mammary tumors (p < 0.0001) and diminished the number of lung metastases in HER-2/neu transgenic mice. The effects of Res were associated with downregulation of HER-2/neu gene expression and increased apoptosis both in tumoral mammary glands and in murine (N202) and human (SKBr3) tumor cell lines. Neither the basal, the IL-2-induced NK activity nor the lymphocyte number and proliferation was modified in Res-supplemented compared to control mice. Our results demonstrate that Res supplementation delays the development and reduces the metastasizing capacity of spontaneous mammary tumors in HER-2/neu transgenic mice. The antitumor effect of Res might be related to the downregulation of HER-2/neu expression and the induction of apoptosis in tumor cells.     

Synergistic inhibition of breast cancer cell lines with a dual inhibitor of EGFR-HER-2/neu and a Bcl-2 inhibitor

The epidermal growth factor receptor (EGFR) (ErbB1) and HER-2/neu (ErbB2) are members of the ErbB family of receptor tyrosine kinases. These receptors are overexpressed in a variety of human tumors and overexpression generally correlates with poor prognosis and decreased survival. Lapatinib, a reversible inhibitor of both EGFR and HER-2/neu, has shown some success in achieving clinical responses in heavily pretreated advanced cancer patients. GW2974 is a reversible dual inhibitor similar to lapatinib, but GW2974 was not progressed to clinical trials due to pharmacokinetic issues. Bcl-2, an anti-apoptotic protein, is also overexpressed in a number of human tumors. Bcl-2 inhibitors induce apoptosis and sensitize cancer cells to other therapies. The purpose of this study was to assess the effects of combining ErbB and Bcl-2 inhibitors on the growth of human breast cancer cell lines. EGFR/HER-2/neu tyrosine kinase inhibitors (lapatinib and GW2974) were combined with Bcl-2 inhibitors (HA14-1 or GX15-070) and the anti-proliferative effects were determined by the MTT tetrazolium dye assay. Combinations were tested in MCF-7 human breast cancer cells, a HER-2/neu transfected MCF-7 cell line (MCF/18), and a tamoxifen-resistant MCF-7 cell line (MTR-3). A synergistic inhibitory effect was observed with the combination of inhibitors of EGFR-HER-2/neu (lapatinib or GW2974) and Bcl-2 (GX15-070 or HA14-1) on the growth of the MCF-7, MCF/18, and MTR-3 human breast cancer cell lines. This study suggests that simultaneously blocking the ErbB family of receptor tyrosine kinases and Bcl-2 family of proteins may be a benefit to breast cancer patients.     

GRB-7 facilitates HER-2/Neu-mediated signal transduction and tumor formation

Growth factor receptor-bound protein-7 (GRB-7), an adaptor molecule, can interact with multiple signal transduction molecules. GRB-7 is amplified concurrently with HER-2/Neu in most, if not all, of breast cancer with chromosome 17q11-21 amplification. GRB-7 gene amplification is associated with RNA over-expression. We show GRB-7 protein is over-expressed by immunoblotting in breast cancer cell lines and primary breast tumors with HER-2/Neu protein over-expression. Over-expression of GRB-7 in MCF-7 breast cancer cells that over-express HER-2/Neu leads to activation of tyrosine phosphorylation of HER-2/Neu. Knockdown of GRB-7 expression in SKBR-3 breast cancer cells with naturally occurring HER-2/Neu gene amplification decreases tyrosine phosphorylation of HER-2/Neu. Activation of HER-2/Neu phosphorylation is associated with increase in tyrosine phosphorylation of phosphoinositide-specific lipase C-gamma-1 (PLC-gamma-1) and recruitment of PLC-gamma-1 to HER-2/Neu protein molecule. Activation of downstream protein kinase C (PKC) pathway is evidenced by increase in the phosphorylation of a common PKC substrate-myristoylated alanine-rich protein kinase C substrate (MARCKS). In addition, over-expression of GRB-7 in MCF-7 breast cancer cells that over-express HER-2/Neu leads to activation of AKT phosphorylation. Knockdown of GRB-7 expression in MB-453 and SKBR-3 breast cancer cells results in decrease in AKT phosphorylation. GRB-7 over-expression therefore facilitates activation of phosphorylation of HER-2/Neu and AKT in breast cancer cells with HER-2/Neu over-expression. GRB-7 over-expression in MCF-7 cells over-expressing HER-2/Neu leads to morphologic change of cells and promotes tumor xenograft growth in nude mice. GRB-7 over-expression therefore plays pivotal roles in activating signal transduction and promoting tumor growth in breast cancer cells with chromosome 17q11-21 amplification.     

腺病毒E1A下调HER2/neu原癌基因表达及其抗瘤效应研究

目的　研究腺病毒E1A基因对HER2 /neu高表达肿瘤细胞生长的抑制作用及增强其化疗敏感性的作用。方法　以腺病毒Ⅴ型为载体 ,经体、内外对HER2 /neu高表达及低表达的肿瘤细胞株转染腺病毒E1A基因后 ,观察E1A基因对肿瘤细胞生长抑制作用 ;用MTT法检测E1A增强肿瘤细胞化疗敏感性作用。结果　E1A能显著抑制HER2 /neu高表达的肿瘤细胞在体内外的生长 ,延长荷瘤裸鼠的生存期。免疫印迹及免疫组织化学分析显示 ,转染E1A的HER2 /neu高表达肿瘤细胞 ,其HER2 /neu基因产物p185蛋白表达明显降低 ;经AdE1A 处理的HER2 /neu高表达的乳腺癌细胞株能明显增强对TaxotereTM的化疗敏感性。结论　E1A通过下调HER2 /neu原癌基因的表达 ,抑制HER2 /neu高表达的肿瘤细胞生长 ,增强肿瘤细胞对化疗药的敏感性。     

Hormonal modulation of HER-2/neu protooncogene messenger ribonucleic acid and p185 protein expression in human breast cancer cell lines

Recent work has suggested that overexpression of the HER-2/neu protooncogene may play a role in the aggressive clinical behavior of some breast tumors. Since hormones are also known to change the proliferation rate and invasiveness of these cells, we have studied the effect of sex steroid hormones and antihormones on levels of the HER-2/neu mRNA and protein in human breast cancer cell lines using complementary DNA and antibody probes. In MCF-7 cells, which contain high levels of estrogen receptor and an estradiol (E2)-inducible progesterone receptor (PR), 1 nM E2 caused a rapid drop in HER-2/neu mRNA (4.8 kilobases), to 40% of control values by 6 h, and a more gradual decrease in HER-2/neu protein, to 50% by 24 h. HER-2/neu protein and mRNA levels remained reduced throughout 1 week of E2 treatment. The effect of E2 was dose dependent, with the maximal effect seen with concentrations of 10(-10) M E2 and above, and antiestrogen partly reversed the E2-induced decrease in HER-2/neu expression. These characteristics suggest that the observed modulation of HER-2/neu is an estrogen receptor-mediated process. In contrast, progestins did not change HER-2/neu mRNA or protein levels in E2-primed MCF-7 cells that contain high levels of PR; in T47D cells, which contain low levels of ER and high levels of PR, addition of E2 or the progestin R5020 or the antiprogestin RU38,486 had no significant effect on HER-2/neu mRNA or protein levels over 6 days of treatment. These results indicate that estrogen but not progestin modulates HER-2/neu protooncogene expression in these breast cancer cell lines and suggest that aggressiveness associated with high levels of HER-2/neu mRNA and protein may be uncoupled from estrogen-stimulated proliferation in these cells.     

Restoration of estrogen responsiveness by blocking the HER-2/neu pathway

HER-2/neu gene amplification or protein overexpression is evident in 20-30% of primary breast cancers. Its amplification correlates with poor prognosis. There appears to be an association between HER-2/neu overexpression and estrogen independence. The MCF-7 human breast carcinoma cell line is estrogen-dependent and sensitive to the anti-estrogen, tamoxifen (TAM). This line, when transfected with the HER-2/neu gene, becomes estrogen-independent and resistant to TAM. Blockade of the HER-2/neu receptor with 1-5 nM of the humanized HER-2/neu antibody, Herceptin, restored estrogen, as well as TAM, sensitivity. These results suggest that Herceptin or similar drugs may restore estrogen sensitivity and the administration of a HER-2/neu inhibitor with an anti-estrogen to premenopausal patients should be considered.     

FSIP1促进乳腺癌细胞迁移和侵袭并导致患者不良预后

目的 探讨乳腺癌组织中纤维鞘相互作用蛋白1(FSIP1)表达对乳腺癌细胞侵袭和迁移能力的影响及其与乳腺癌患者预后的关系,从而为乳腺癌的诊断和治疗提供一定的理论参考。方法 收集2004年1月—2018年12月于哈尔滨医科大学附属肿瘤医院确诊的404例乳腺癌患者的乳腺组织样本和病例资料,对收集的乳腺癌患者资料进行回顾性分析并采用Kaplan-Meier方法绘制生存曲线,采用免疫组织化学方法分析FSIP1在乳腺癌和癌旁组织中的表达情况,取乳腺癌细胞系MCF-7、MDA-MB-231、MDA-MB-435、SK-BR-3、T-47D及正常乳腺上皮细胞(HMECs)MCF-10A进行细胞培养,采用CRISPR/CAS9技术敲除乳腺癌细胞系MDA-MB-231和SK-BR-3中的FSIP1基因,通过Western blot实验检测各乳腺癌细胞系中FSIP1蛋白的表达情况并对FSIP1基因敲除结果进行检测,通过细胞迁移和侵袭实验评估FSIP1蛋白敲除对乳腺癌细胞迁移和侵袭能力的影响。结果 与正常乳腺上皮细胞(MCF-10A)相比,乳腺癌细胞系MCF-7、MDA-MB-231、MDA-MB-435、S...     

Growth inhibitory effects of trastuzumab and chemotherapeutic drugs in gastric cancer cell lines

The various treatments for advanced gastric cancer have limitations and induce only marginal survival benefit. HER-2/neu protein is overexpressed in several types of human cancers and its amplification is associated with poor prognosis. Recombinant humanized anti-HER-2/neu antibody (trastuzumab) not only inhibits the proliferation of HER-2/neu overexpressing tumor cells but also augments the cytotoxicity of concomitant chemotherapeutic agents in metastatic breast cancer. In this study, we evaluated the growth inhibitory effects of trastuzumab in gastric cancer cells. HER-2/neu protein was evaluated by immunohistochemical analysis in seven gastric cancer cell lines. MTT assay was performed to evaluate the growth inhibitory effects of trastuzumab and three chemotherapeutic agents, doxorubicin, cisplatin and paclitaxel, both alone and in combinations. The changes of cell cycle after trastuzumab treatment were analyzed by flow cytometry. Four of the cell lines, YCC-2 with strong positivity of HER-2/neu expression, NCI-N87 with moderate positivity, YCC-3 with weak positivity, and SK-BR-3 as a positive control, were selected. After in vitro MTT assay for 1-day and 5 consecutive days' treatment of trastuzumab at various concentrations, growth inhibition was not observed in any cancer cell lines. However, there was variable dose-dependent sensitivity to doxorubicin, cisplatin and paclitaxel. YCC-2 and SK-BR-3 cancer cells were more sensitive to three chemotherapeutic drugs, constantly (P<0.05). The combination of 5 consecutive days' treatment of trastuzumab with 1-day doxorubicin treatment showed significant growth inhibition only in YCC-2 and NCI-N87 gastric cancer cells. After 1-day trastuzumab treatment, the S-phase fraction was decreased by 52 and 70% in YCC-2 and SK-BR-3, respectively. In conclusion, the expressions of HER-2/neu protein in gastric cancer cells are variable, and concomitant treatments of trastuzumab with doxorubicin increase cytotoxicity. This suggests that trastuzumab-based biologic therapy with chemotherapeutic agents can be applied in gastric cancer treatment.