Lapatinib耐药性HER2阳性乳腺癌细胞株的建立及鉴定

目的:为研究HER2阳性乳腺癌细胞获得拉帕替尼耐药性的机制,建立稳定具有拉帕替尼耐药性的细胞株。方法:采用2μmol/L的拉帕替尼处理HER2阳性乳腺癌细胞BT-474,维持12个月,使细胞获得稳定耐药性。然后分别利用MTT、平板克隆和软琼脂克隆形成能力分析等方法,评价所建立耐药细胞的耐药能力。结果:所建立的耐药细胞在细胞增殖能力、平板克隆形成能力和软琼脂克隆形成能力等方面均具有耐药性。结论:建立的拉帕替尼耐药性细胞BT-474具有稳定的耐药性,为后续机制研究奠定基础。     

拉帕替尼和紫杉醇在食管癌细胞中的抗肿瘤活性及其作用机制

目的 探讨拉帕替尼、紫杉醇和拉帕替尼联合紫杉醇对食管癌EC109细胞的抗肿瘤活性及作用机制。方法 MTT法检测拉帕替尼（1、2、4、8 μmol/L）、紫杉醇（5、10、20、40 μg/L）及联合使用对食管癌EC109细胞增殖的影响;分别检测拉帕替尼 2 μmol/L、紫杉醇10 μg/L及联合使用对EC109细胞的侵袭、周期、凋亡和EGFR、HER2及其下游信号通路的影响。结果 MTT结果显示：拉帕替尼联合紫杉醇可协同抑制EC109细胞增殖,联合作用指数均大于1.15;拉帕替尼联合紫杉醇组发生侵袭的细胞数62.0±9.5个,明显少于拉帕替尼152.4±16.1个和紫杉醇组103.6±12.7个（P<0.05）;拉帕替尼联合紫杉醇组G2/M期细胞（（43.4±3.1）%）,高于拉帕替尼（（20.3±2.5）%）和紫杉醇组（（26.6±2.8）%）（P<0.05）;拉帕替尼联合紫杉醇组的细胞凋亡率为（47.3±8.4）%,高于拉帕替尼（（12.7±2.3）%）和紫杉醇组（（21.4±5.2）%）（P<0.05）;Western blot结果显示：拉帕替尼联合紫杉醇可协同抑制磷酸化EGFR、HER2及AKT蛋白的表达。结论 拉帕替尼联合紫杉醇通过抑制食管癌细胞EC109的增殖和侵袭、阻滞细胞周期、诱导细胞凋亡、抑制EGFR、HER2下游信号通路的转导发挥协同抗肿瘤活性。     

拉帕替尼联合顺铂抗食管鳞癌的作用及机制

目的 探讨拉帕替尼联合顺铂抗食管鳞癌的体外活性及其作用机制。方法 MTT法检测拉帕替尼、顺铂单独及二者联合对食管鳞癌细胞的增殖抑制作用，并计算两药联合作用指数。碘化丙啶（PI）染色或Annexin V-FITC/PI双染结合流式细胞术检测细胞周期进程及凋亡，Western blot法检测两药单独及联合处理后食管鳞癌细胞EGFR和HER2的磷酸化及下游信号分子的表达变化。结果 拉帕替尼联合顺铂可协同抑制食管鳞癌细胞的增殖，二者之间的联合作用指数小于1。拉帕替尼联合顺铂可使食管鳞癌细胞阻滞于G2/M期，并可显著诱导细胞凋亡。联合用药可显著抑制EGFR和HER2的磷酸化，并进而抑制两个主要的下游信号分子ERK和AKT的活化。结论 拉帕替尼联合顺铂在体外具有协同抗食管鳞癌活性，对EGFR和HER2过表达食管鳞癌是一种有前景的治疗策略。     

circ-ERBB2通过抑制miR-136-5p维持乳腺癌中的HER2下游信号促进曲妥珠单抗耐药

目的:探讨circ-ERBB2/miR-136-5p轴是否参与曲妥珠单抗耐药乳腺癌的耐药性分子机制。方法:qPCR法检测曲妥珠单抗敏感和耐药乳腺癌组织,以及HER2-和HER2+乳腺癌组织中circ-ERBB2和miR-136-5p的表达水平。建立小鼠异种移植瘤模型,评估敲低/过表达circ-ERBB2联合过表达/敲低miR-136-5p对乳腺癌细胞成瘤性及对曲妥珠单抗耐药性的影响。生物信息学和双荧光素酶报告基因分析circ-ERBB2与miR-136-5p的序列互作位点。CCK-8细胞增殖能力测定敲低/过表达circ-ERBB2联合过表达/敲低miR-136-5p以及给予曲妥珠单抗治疗对于乳腺癌常规或曲妥珠单抗耐药BT-474（Trast-resist）细胞增殖能力的影响。Western blot法检测敲低/过表达circ-ERBB2联合过表达/敲低miR-136-5p后,对常规或BT-474（Trast-resist）细胞内HER2、p-Akt、p-ERK1/2表达水平的影响。结果:与曲妥珠单抗敏感乳腺癌组织相比,曲妥珠单抗耐药乳腺癌组织中circ-ERBB2的表达水平显著升高,miR-136-5p表达水平显著降低（P<0.01）。无论激素受体表达为阳性或是阴性,与HER2-乳腺癌组织相比,在HER2+乳腺癌组织中circ-ERBB2表达显著上调,miR-136-5p表达水平被显著抑制（P<0.01）。敲低circ-ERBB2的表达或过表达miR-136-5p均能够抑制BT-474（Trast-resist）细胞增殖及其胞内HER2、p-Akt和p-ERK1/2的表达水平（P<0.01）。结论:circ-ERBB2能够通过抑制miR-136-5p参与维持乳腺癌中的HER2下游信号并促进曲妥珠单抗耐药。     

淋巴瘤细胞膜P糖蛋白、信号转导与转录激活因子3及bcl-2表达水平与化疗耐药的相关研究

 目的　探讨淋巴瘤细胞膜P糖蛋白（P-gp）、细胞胞内信号转导与转录激活因子3（STAT3）、bcl-2、白细胞介素6（IL-6）及白细胞介素10（IL-10）表达水平与淋巴瘤患者化疗耐药的相关性。方法　对疑诊淋巴瘤的18例患者,手术活检淋巴结,应用流式细胞术（FCM）测定细胞膜P-gp 、胞内STAT3、抗凋亡蛋白bcl-2、细胞因子IL-6及 IL-10表达水平,前瞻性研究与化疗疗效的关系。其中,化疗耐药组10例,化疗敏感组8例,以10例炎性淋巴结为正常对照。结果　化疗耐药淋巴瘤患者P-gp和 bcl-2表达水平均高于化疗敏感患者（P＝0.01和P＝0.039）,而STAT3、IL-6及IL-10表达水平差异无统计学意义（P＞0.05）。淋巴瘤细胞膜P-gp表达水平高于炎性淋巴结增生（P＝0.01）,STAT3表达水平明显低于炎性淋巴结增生（P＝0.04）,淋巴瘤与炎性淋巴结增生bcl-2、IL-6及IL-10表达水平差异无统计学意义（P＞0.05）。结论　bcl-2、P-gp 表达水平的高低与恶性淋巴瘤化疗疗效密切相关,STAT3参与了淋巴瘤细胞的信号转导,是否参与了多药耐药信号转导尚不能肯定,而胞内IL-6、IL-10的表达与化疗疗效未见相关。     

安罗替尼在宫颈癌HeLa/DDP细胞中的作用及逆转顺铂耐药性机制研究

目的:探究安罗替尼对HeLa/DDP(宫颈癌顺铂耐药细胞)细胞增殖和凋亡的影响及其逆转顺铂耐药的机制。方法:不同浓度顺铂及安罗替尼处理HeLa/DDP细胞,CCK-8法检测细胞增殖能力;流式细胞术检测细胞凋亡;Western Blot检测P-gp的表达水平差异。结果:顺铂对HeLa/DDP细胞的增殖具有抑制作用,并呈剂量及时间依赖性。安罗替尼对HeLa/DDP细胞的增殖抑制作用呈浓度依赖性。安罗替尼、安罗替尼联合顺铂可以抑制HeLa/DDP细胞增殖(P<0.05)。流式细胞术结果发现安罗替尼组及安罗替尼联合顺铂组细胞凋亡率均升高(P<0.05)。安罗替尼联合顺铂显著抑制细胞增殖,诱导凋亡。Western Blot提示安罗替尼联合顺铂组与对照组和顺铂组及安罗替尼单药组相比,P-gp蛋白表达上调(P>0.05)。结论:安罗替尼可抑制宫颈癌HeLa/DDP细胞增殖,诱导凋亡;安罗替尼联合顺铂抑制细胞增殖、诱导凋亡作用更显著;安罗替尼可增加宫颈癌HeLa/DDP顺铂耐药株对顺铂的敏感性,逆转耐药性,但不能介导耐药相关蛋白P-gp表达下调。     

阿帕替尼逆转P-gp转运体介导的乳腺癌化疗多药耐药性的作用及其机制

目的 探讨阿帕替尼对人乳腺癌化疗多药耐药性的逆转作用及其机制。方法 不同浓度的阿帕替尼作用于体外培养的人乳腺癌MCF-7及MCF-7/ADR细胞48 h,MTT法检测阿帕替尼对两种细胞的细胞毒性;低毒浓度的阿帕替尼与化疗药物紫杉醇及阿霉素联用,探讨阿帕替尼对两种细胞化疗耐药性的影响;采用流式细胞术检测阿帕替尼对罗丹明123在MCF-7及MCF-7/ADR细胞内蓄积的影响;采用Pgp-Glo? Assay Systems试剂盒观察阿帕替尼对多药耐药相关蛋白P-gp的ATPase活性的影响;采用Western blot法检测阿帕替尼对P-gp表达及AKT磷酸化的影响。结果 阿帕替尼可浓度依赖性地逆转乳腺癌MCF-7/ADR细胞对紫杉醇及阿霉素的多药耐药性（P<0.05）、增加罗丹明123在MCF-7/ADR细胞内的蓄积（P<0.05）及激活P-gp转运体的ATPase活性（P<0.05）,而对P-gp的表达没有影响;此外,阿帕替尼不改变AKT的磷酸化水平。结论 阿帕替尼可能通过抑制P-gp转运体的外排功能逆转P-gp转运体介导的乳腺癌化疗多药耐药性。     

EML4-ALK融合基因阳性表达的人肺腺癌H2228细胞对克唑替尼耐药后BIM信号通路的影响

目的 建立人肺腺癌H2228克唑替尼耐药细胞株,探讨克唑替尼（crizotinib）诱导EML4-ALK融合基因阳性表达的人肺腺癌细胞H2228在BIM信号通路中的作用。 方法 克唑替尼按50 nmol/L、100 nmol/L、200 nmol/L、500 nmol/L、1 000 nmol/L的浓度逐步递增法诱导人肺腺癌H2228细胞株获得性耐药;分别采用MTT法和流式细胞术检测H2228和H2228/CR细胞在不同浓度克唑替尼作用后的IC50和细胞凋亡率;采用Western blot法检测H2228和H2228/CR细胞在BIM信号通路关键分子ALK、p-ALK、ERK、p-ERK及BIM蛋白表达的变化。 结果 成功诱导克唑替尼耐药细胞株H2228/CR;MTT法检测H2228/CR细胞和H2228细胞的IC50分别为3 418 nmol/L、335 nmol/L,H2228/CR细胞的耐药指数为10.20,H2228/CR细胞在不同浓度克唑替尼作用下的增殖抑制率低于H2228细胞（P＜0.05）;流式细胞术检测显示克唑替尼对H2228细胞有促凋亡作用,且呈时间依赖性（P＜0.05）,H2228/CR细胞的凋亡率明显低于H2228细胞的凋亡率（P＜0.05）;Western blot法检测显示H2228/CR细胞的p-ALK、p-ERK的表达不受抑制,BIM蛋白表达无上调趋势。 结论 实验表明H2228/CR耐药细胞中的BIM蛋白的表达与其耐药有关。初步阐明EML4-ALK阳性表达的非小细胞肺癌克唑替尼耐药产生的可能机制,BIM在诱导EML4-ALK阳性表达的人肺腺癌细胞株H2228对克唑替尼产生耐药发挥重要作用,提示BIM可作为克服克唑替尼耐药的一个靶点。     

丙泊酚抑制IL-6诱导的A549肺癌细胞上皮间质转化及其机制探讨

目的:探究丙泊酚对白细胞介素-6（IL-6）诱导的A549肺癌细胞上皮间质转化（EMT）的作用及机制。方法:将A549细胞随机分成四组:对照组、IL-6组（50 ng/ml重组IL-6蛋白）、IL-6+丙泊酚低剂量组（50 ng/ml重组IL-6蛋白和5 μmol/L丙泊酚）、IL-6+丙泊酚高剂量组（50 ng/ml重组IL-6蛋白和10 μmol/L丙泊酚）。MTT法检测细胞活力，Transwell检测细胞迁移情况，Real-time PCR方法检测EMT相关基因（E-cadherin、Vimentin和Snail1）mRNA的表达水平，Western blot检测EMT相关蛋白及JAK2和STAT3的磷酸化表达水平。使用0.5 μmol/L STAT3激活剂colivelin处理细胞，检测其对丙泊酚调节的IL-6诱导的A549细胞活力、迁移和EMT的影响。结果:与对照组相比，IL-6组中细胞的活力、迁移、EMT和JAK2/STAT3的活化均增加（P均<0.05）；与IL-6组相比，IL-6+丙泊酚组中细胞活力、迁移、EMT和JAK2/STAT3的活化均降低（P均<0.05），这些变化均具有剂量依赖性。STAT3激活剂能够减弱丙泊酚对IL-6诱导的A549细胞活力、迁移和EMT的影响（P均<0.05）。结论:丙泊酚能够抑制IL-6诱导的A549肺癌细胞EMT进程，这种作用是通过抑制JAK2/STAT3的活化发挥作用的。     

c-Src参与RANKL诱导的乳腺癌BT-474细胞迁移的机制研究

目的：核因子-κB受体活化因子配体（RANKL）/核因子κB受体活化因子（RANK）通路在肿瘤骨定向性迁移中发挥重要的作用,但具体信号传导机制尚不清楚。本文探讨非受体酪氨酸激酶c-Src在RANKL诱导的乳腺癌BT474细胞迁移中的作用。方法：Western blot检测BT-474细胞表面受体RANK蛋白的表达及RANKL刺激后细胞p-Src及c-Src的表达;Transwell法测定细胞迁移能力。采用SPSS 16.0统计学软件分析实验数据。结果：BT-474细胞表达RANK蛋白,RANKL诱导BT-474细胞迁移能力增强。应用RANKL的圈套受体OPG可阻断RANKL诱导的细胞迁移。RANKL刺激后BT-474细胞p-Src表达升高,应用c-Src激酶抑制剂PP2可显著抑制RANKL诱导的细胞迁移。结论：c-Src信号通路参与RANKL诱导的乳腺癌BT-474细胞迁移。     

Downregulation of Plk1 Expression By Receptor-Mediated Uptake of Antisense Oligonucleotide-Loaded Nanoparticles

Human serum albumin (HSA) nanoparticles represent a promising tool for targeted drug delivery to tumor cells. The coupling of the antibody trastuzumab to nanoparticles uses the capability of human epidermal growth factor receptor 2 (HER2)-positive cells to incorporate agents linked to HER2. In our present study, we developed targeted nanoparticles loaded with antisense oligonucleotides (ASOs) against polo-like kinase 1 (Plk1). We evaluated the receptor-mediated uptake into HER2-positive and -negative breast cancer and murine cell lines. We performed quantitative real-time PCR and Western blot analyses to monitor the impact on Plk1 expression in HER2-positive breast cancer cells. Antibody-conjugated nanoparticles showed a specific targeting to HER2-overexpressing cells with cellular uptake by receptor-mediated endocytosis and a release into HER2-positive BT-474 cells. We observed a significant reduction of Plk1 mRNA and protein expression and increased activation of Caspase 3/7. Thus, this is the first report about ASO-loaded HSA nanoparticles, where an impact on gene expression could be observed. The data provide the basis for the further development of carrier systems for Plk1-specific ASOs to reduce off-target effects evoked by systemically administered ASOs and to achieve a better penetration into primary and metastatic target cells. Treatment of tumors using trastuzumab-conjugated ASO-loaded HSA nanoparticles could be a promising approach to reach this goal.     

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.     

HER2/neu increases the expression of Wilms' Tumor 1 (WT1) protein to stimulate S-phase proliferation and inhibit apoptosis in breast cancer cells

High levels of the Wilms' Tumor 1 (WT1) protein and mRNA had been associated with aggressive phenotypes of breast tumors. Here we report that the HER2/neu oncogene increases WT1 expression. Approximately threefold higher levels of WT1 protein were observed in MCF-7 breast cancer cells transfected with the HER2/neu oncogene than in parental MCF-7 cells. Conversely, inhibition of HER2/neu with the anti-HER2/neu trastuzumab (Herceptintrade mark) antibody decreased WT1 protein levels in HER2/neu-overexpressing BT-474 and SKBr3 cells. We also found that HER2/neu engages Akt to regulate WT1 levels since inhibition of Akt reduced WT1 levels. Decreased expression of WT1 protein led to cell cycle arrest at the G1 phase and increased apoptosis in HER2/neu-overexpressing cells, which is correlated with decreased cyclin D1 and Bcl-2 levels. Our data indicate that HER2/neu engages Akt to increase WT1 expression, and that WT1 protein plays a vital role in regulating cell cycle progression and apoptosis in HER2/neu-overexpressing breast cancer cells.     

Fatty acid synthase phosphorylation: a novel therapeutic target in HER2-overexpressing breast cancer cells

Introduction

The human epidermal growth factor receptor 2 (HER2) is a validated therapeutic target in breast cancer. Heterodimerization of HER2 with other HER family members results in enhanced tyrosine phosphorylation and activation of signal transduction pathways. HER2 overexpression increases the translation of fatty acid synthase (FASN), and FASN overexpression markedly increases HER2 signaling, which results in enhanced cell growth. However, the molecular mechanism and regulation of HER2 and FASN interaction are not well defined. Lapatinib is a small-molecule tyrosine kinase inhibitor that blocks phosphorylation of the epidermal growth factor receptor and HER2 in breast cancer cells, resulting in apoptosis. We hypothesized that FASN is directly phosphorylated by HER2, resulting in enhanced signaling and tumor progression in breast cancer cells.

Methods

Using mass spectrometry, we identified FASN as one of the proteins that is dephosphorylated by lapatinib in SKBR3 breast cancer cells. Immunofluorescence, immunoprecipitation, Western blotting, a kinase assay, a FASN enzymatic activity assay, an invasion assay, a cell viability assay and zymography were used to determine the role of FASN phosphorylation in invasion of SKBR3 and BT474 cells. The FASN inhibitor C75 and small interfering RNA were used to downregulate FASN expression and/or activity.

Results

Our data demonstrated that FASN is phosphorylated when it is in complex with HER2. FASN phosphorylation was induced by heregulin in HER2-overexpressing SKBR3 and BT474 breast cancer cells. Heregulin-induced FASN phosphorylation resulted in increased FASN enzymatic activity, which was inhibited by lapatinib. The FASN inhibitor C75 suppressed FASN activity by directly inhibiting HER2 and FASN phosphorylation. Blocking FASN phosphorylation and activity by lapatinib or C75 suppressed the activity of matrix metallopeptidase 9 and inhibited invasion of SKBR3 and BT474 cells.

Conclusions

FASN phosphorylation by HER2 plays an important role in breast cancer progression and may be a novel therapeutic target in HER2-overexpressing breast cancer cells.     

Feedback activation of STAT3 mediates trastuzumab resistance via upregulation of MUC1 and MUC4 expression

Although HER2-targeting antibody trastuzumab confers a substantial benefit for patients with HER2-overexpressing breast and gastric cancer, overcoming trastuzumab resistance remains a large unmet need. In this study, we revealed a STAT3-centered positive feedback loop that mediates the resistance of trastuzumab. Mechanistically, chronic exposure of trastuzumab causes the upregulation of fibronection (FN), EGF and IL-6 in parental trastuzumab-sensitive breast and gastric cells and convergently leads to STAT3 hyperactivation. Activated STAT3 enhances the expression of FN, EGF and IL-6, thus constituting a positive feedback loop which amplifies and maintains the STAT3 signal; furthermore, hyperactivated STAT3 signal promotes the expression of MUC1 and MUC4, consequently mediating trastuzumab resistance via maintenance of persistent HER2 activation and masking of trastuzumab binding to HER2 respectively. Genetic or pharmacological inhibition of STAT3 disrupted STAT3-dependent positive feedback loop and recovered the trastuzumab sensitivity partially due to increased apoptosis induction. Combined trastuzumab with STAT3 inhibition synergistically suppressed the growth of the trastuzumab-resistant tumor xenografts in vivo. Taken together, our results suggest that feedback activation of STAT3 constitutes a key node mediating trastuzumab resistance. Combinatorial targeting on both HER2 and STAT3 may enhance the efficacy of trastuzumab or other HER2-targeting agents in HER2-positive breast and gastric cancer.     

二甲基塞来昔布通过活性氧诱导乳腺癌细胞凋亡

目的:探讨二甲基塞来昔布（2,5-dimethyl celecoxib,DMC）对乳腺癌细胞凋亡的作用。方法:用DMC处理人乳腺癌BT-20和MCF-7细胞,检测细胞生存率、细胞凋亡情况、细胞内ROS的产生;Caspase凋亡通路相关蛋白表达水平;预处理ROS抑制剂NAC、Caspase通路抑制剂Z-VAD-Fmk,检测细胞内Caspase凋亡通路蛋白表达水平。结果:DMC对BT-20和MCF-7细胞呈现浓度及时间依赖性杀伤作用,并可以明显增加细胞的凋亡,且呈现浓度依赖性趋势;DMC可以显著增加BT-20和MCF-7细胞内ROS的产生和凋亡相关蛋白cleaved caspase-7、9的表达水平;另一组实验证明,预处理NAC和Z-VAD-Fmk,可以明显降低cleaved caspase-7、9的表达水平,且抑制剂NAC组的效果大于Z-VAD-Fmk组。结论:DMC通过ROS途径诱导乳腺癌细胞发生凋亡。     

Genistein sensitizes inhibitory effect of tamoxifen on the growth of estrogen receptor-positive and HER2-overexpressing human breast cancer cells

Although tamoxifen (TAM) is used for the front-line treatment and prevention of estrogen receptor-positive (ER+) breast tumors, nearly 40% of estrogen-dependent breast tumors do not respond to TAM treatment. Moreover, the positive response is usually of short duration, and most tumors eventually develop TAM-resistance. Overexpression of HER2 gene is associated with TAM-resistance of breast tumor, and suppression of HER2 expression enhances the TAM activity. Soy isoflavone genistein has been shown to have anti-cancer activities and suppress expression of HER2 and ERalpha. The objective of this study was to test the hypothesis that genistein may sensitize the response of ER+ and HER2-overexpressing breast cancer cells to TAM treatment. The combination treatment of TAM and genistein inhibited the growth of ER+/HER2-overexpressing BT-474 human breast cancer cells in a synergistic manner in vitro. Determination of cellular markers indicated that this synergistic inhibitory effect might be contributed in part from combined effects on cell-cycle arrest at G(1) phase and on induction of apoptosis. Further determination of the molecular markers showed that TAM and genistein combination synergistically induced BT-474 cell apoptosis in part by synergistic downregulation of the expression of survivin, one of the apoptotic effectors, and downregulation of EGFR, HER2, and ERalpha expression. Our research may provide a novel approach for the prevention and/or treatment of TAM insensitive/resistant human breast cancer, and warrants further in vivo studies to verify the efficacy of genistein and TAM combination on the growth of ER+/HER2-overexpressing breast tumors and to elucidate the in vivo mechanisms of synergistic actions.