Dual targeting of Src and ER prevents acquired antihormone resistance in breast cancer cells

Acquired resistance to endocrine therapies presents a major obstacle to the successful treatment of breast cancer patients. Previously, we have shown that acquisition of resistance to tamoxifen in breast cancer cells is accompanied by an elevation in Src kinase activity which promotes an aggressive, invasive phenotype in vitro. Here, we have explored the potential therapeutic effects of combining Src inhibition with anti-oestrogen treatment on the development of endocrine insensitivity in breast cancer cells. Treatment of MCF7 and T47D cells with tamoxifen alone resulted in an initial growth inhibitory phase followed by the eventual development of tamoxifen resistance together with an elevation of Src kinase activity, which was central to their increased invasive capacity. Chronic exposure of both cell types to the Src inhibitor, AZD0530, as a monotherapy resulted in outgrowth of AZD0530-resistant cells, in which Src kinase activity remained suppressed as did their in vitro invasive nature. Treatment of both MCF7 and T47D cells with AZD0530 in combination with tamoxifen resulted in a reduction of Src activity together with inhibition of focal adhesion kinase phosphorylation and a complete abrogation of their in vitro invasive behaviour. Furthermore, combination therapy significantly suppressed expression of cyclinD1 and c-myc and prevented cell proliferation and the subsequent emergence of a resistant phenotype, with total cell loss occurring by 12 weeks. These data demonstrate that pharmacological targeting of Src kinase, in conjunction with antihormone therapies, effectively prevents antihormone resistance in breast cancer cells in vitro and suggests a potential novel therapeutic benefit of Src kinase inhibitors clinically.     

PH006, a novel and selective Src kinase inhibitor,suppresses human breast cancer growth and metastasis in vitro and in vivo

The central role of Src in tumor progression and metastasis has validated it as an attractive therapeutic target for the treatment of human breast cancer. The aim of this study was to identify potential Src kinase inhibitor, explore its activity, and mechanism of action in human breast cancer. A strategy integrating focused combinatorial library design, virtual screening, chemical synthesis, and high-throughput screening was adopted and a novel 6-hydrazinopurine-based inhibitor of c-Src kinase PH006 was obtained. The kinase enzymatic activities were measured by enzyme-linked immunosorbent assay. The binding mode between PH006 and Src was profiled by surface plasmon resonance approach and molecular simulation. The anti-proliferative activity was evaluated by Sulforhodamin B (SRB) and Colony formation. The anti-invasion and anti-migration activities were assessed by trans-well and wound healing assay. Results indicated that PH006 was an ATP-competitive Src inhibitor, which selectively inhibited c-Src with an IC₅₀ of 0.38 μM among a panel of 14 diverse tyrosine kinases. PH006 potently inhibited c-Src phosphorylation and c-Src-dependent signal transduction, resulting in inhibition of cell proliferation, migration, and invasion in human breast cancer MDA-MB-231 cells. Further study demonstrated that the anti-proliferative activity of PH006 was ascribed to its capability to arrest cells in G1 phase, while its anti-motility activity was related to suppression of MMP2/9 and HGF secretion. Moreover, PH006 exhibited potent activity against tumor growth as well as metastasis of human breast cancer MDA-MB-435 xenograft beard in nude mice, which was accompanied with reduced Src/FAK signaling in tumor tissue. Taken together, PH006 is a novel selective inhibitor of c-Src and possesses potent activity against breast cancer growth and metastasis, which could be potentially developed as a lead candidate against breast cancers with elevated Src tyrosine kinase activity.     

二甲双胍抑制人MDA-MB-231细胞增殖及迁移的体外实验研究

目的： 研究二甲双胍对体外培养的人三阴性乳腺癌MDA-MB-231细胞增殖及迁移的影响。方法： MTT实验分为4个处理组,分别为RPMI 1640培养基对照组,浓度为1、2、4 mmol/L的二甲双胍处理组,在作用48、72 h后测定各组的细胞活力并进行统计学分析;倒置相差显微镜下观察药物处理72 h后人乳腺癌MDA-MB-231细胞与对照组相比的形态变化;Giemsa染色观察4 mmol/L二甲双胍作用48 h后的细胞形态;采用细胞划痕实验和Transwell实验,检测二甲双胍（2、4 mmol/L）作用人乳腺癌MDA-MB-231细胞24 h后,对细胞迁移能力的影响。结果： MTT实验结果显示4 mmol/L二甲双胍作用72 h可显著抑制MDA-MB-231细胞的活力,抑制率为（29.83±2.25）％,与对照组相比,细胞活力降低（P<0.05）。倒置相差显微镜下形态学观察发现,与对照组相比,4 mmol/L二甲双胍处理组细胞密度降低,变圆的细胞数量增加,细胞与细胞之间的联系减少。Giemsa染色结果显示4 mmol/L二甲双胍作用48 h后,部分细胞出现凋亡细胞核碎裂形态。划痕实验结果表明,2、4 mmol/L二甲双胍作用24 h后细胞汇合度明显低于对照组,其中4 mmol/L处理组的划痕愈合率为（52.67±4.48）%,与对照组间的差异显著（P<0.01）;Transwell实验结果表明,2、4 mmol/L二甲双胍作用细胞24 h后穿膜细胞数量减少,穿膜细胞数量分别为（61.6±1.6）、（51.3±2.6）个,均低于对照组的（99.3±18.9）个（P<0.05）。结论： 二甲双胍在体外可抑制人乳腺癌MDA-MB-231细胞的增殖及迁移。     

Green tea polyphenols and its constituent epigallocatechin gallate inhibits proliferation of human breast cancer cells in vitro and in vivo

Tea [Camellia sinensis (Theaceae)] intake is second only to water in terms of worldwide popularity as a beverage. The Green tea polyphenols have been shown to have a protective effect in prostate cancer in various pre-clinical animal models and has been reported to be effective in several other cancer types as well. An inverse association between the risk of breast cancer and the intake of green tea has also been reported in Asian Americans. Several epidemiological studies have shown that breast cancer progression is delayed in the Asian population that consumes green tea on regular basis. In this study, we report the effectiveness of green tea polyphenols (GTP) and its constituent Epigallocatechin Gallate (EGCG) in tumor regression using both in-vitro cell culture models and in vivo athymic nude mice models of breast cancer. The anti-proliferative effect of GTP and EGCG on the growth of human breast cancer MDA-MB-231 cell was studied using a tetrazolium dye-based (MTT) assay. Both GTP and EGCG treatment had the ability to arrest the cell cycle at G1 phase as assessed by flow cytometry. The expression of Cyclin D, Cyclin E, CDK 4, CDK 1 and PCNA were down regulated over the time in GTP and EGCG treated experimental group, compared to the untreated control group as evaluated by western blot analysis for cell cycle proteins, which corroborated the G1 block. Nude mice inoculated with human breast cancer MDA-MB-231 cells and treated with GTP and EGCG were effective in delaying the tumor incidence as well as reducing the tumor burden when compared to the water fed and similarly handled control. GTP and EGCG treatment were also found to induce apoptosis and inhibit the proliferation when the tumor tissue sections were examined by immunohistochemistry. Our results suggest that GTP and EGCG treatment inhibits proliferation and induce apoptosis of MDA-MB-231 cells in-vitro and in-vivo. All together, these data sustain our contention that GTP and EGCG have anti-tumor properties.     

Selective blockade of T-type Ca2+ channels suppresses human breast cancer cell proliferation

We have measured the expression of T-type Ca2+ channel mRNA in breast cancer cell lines (MCF-7 (ERalpha+) using Western blot and quantitative real-time PCR (Q-RT-PCR). These results revealed that the MCF-7 cells express both alpha1G and alpha1H isoforms of T-type Ca2+ channels. In order to further clarify the role of T-type Ca2+ channels in proliferation, we tested the effects of a selective T-type Ca2+ channel inhibitor NNC-55-0396 on cellular proliferation. MCF-7 (ERalpha+) cellular proliferation was inhibited by the compound. In contrast, NNC-55-0396 at same concentration had no effect on the proliferation of MCF-10A cells, a non-cancer breast epithelial cell line. We also found that message expression of the T-type Ca2+ channels were only expressed in rapidly growing non-confluent cells but not in the cytostatic confluent cells. Knocking down the expression of T-type Ca2+ channels with siRNA targeting both alpha1G and alpha1H resulted in growth inhibition as much as 45%+/-5.0 in MCF-7 cells as compared to controls. In conclusion, our results suggest that T-type Ca2+ channel antagonism/silencing may reduce cellular proliferation in mitogenic breast cells.     

Targeting Src in breast cancer

The clinical benefit of blocking oncogenic pathways in breast cancer and other malignancies has validated this approach and ushered in the era of molecularly targeted therapeutics. Src and its family members make up the largest group of nonreceptor tyrosine kinases. In laboratory models, these proteins have been shown to play a critical role in cellular growth and proliferation, angiogenesis, and invasion and metastasis. In addition, Src plays an important role in osteoclast activation and bone resorption, which are often aberrantly activated in the setting of bone metastases. Given its role in these functions, blocking Src kinase would be predicted to have a broad therapeutic benefit in patients with Src-dependent cancers. In this review, we highlight the rationale for targeting Src in breast cancer, including laboratory and clinical data implicating it in these signaling pathways, and review small-molecule tyrosine kinase inhibitors currently in clinical development. Identifying which patients should be selected for Src-directed therapies will be important to the clinical success of these agents. Importantly, recent preclinical data support a role for this class of inhibitors in basal-type/triple-negative breast cancer, which represents a group of patients with limited effective treatment options.     

Interleukin-2 differentially affects the proliferation of a hormone-dependent and a hormone-independent human breast cancer cell line in vitro and in vivo

Direct in vitro and in vivo effects of the lymphokine, interleukin-2 (IL-2), on hormone-dependent (MCF-7) and- independent (MDA-231) human breast cancer cell proliferation were investigated. In vitro, picomolar concentrations of IL-2 directly inhibited MCF-7 cell proliferation after 12 days of culture, while nanomolar doses of IL-2 significantly stimulated MCF-7 cell growth over the same time period. In addition, micromolar concentrations of IL-2 had virtually no effect on the in vitro proliferation of MCF-7 cells. In parallel in vitro growth experiments, the hormone-independent cells, MDA-231, were not affected by IL-2 regardless of concentration. IL-2 treatment of overiectomized, estrogen-treated nude mice, burdened with MCF-7 or MDA-231 tumors, inhibited MCF-7 tumor growth, but had no effect on MDA-231 tumors. Examination of T, B and natural killer (NK) cell function in these animals indicated that the interleukin-2-mediated effect on MCF-7 cell growth in vivo is independent of the proliferative abilities of these lymphoid cells, suggesting that IL-2 may directly affect the growth of these hormone-dependent human breast cancer cells.     

In vivo blockade of the estradiol-binding-protein (EBP) by clomiphene citrate in human breast cancer

The effect of clomiphene citrate given in vivo upon the in vitro uptake of labeled estradiol (tritiated-E2) was investigated in a 60-year-old patient with breast cancer who had had a mastectomy 10 months earlier followed by radiotherapy. Multiple subcutaneous metastatic nodules and enlargement of the liver were present but bone metastases could not be shown. A biopsy from a subcutaneous nodule, taken prior to present treatment, showed 86 fmol estradiol binding sites per mg of cytoplasmic protein with a dissociation constant of the estradiol-estradiol binding protein interaction of 2.8 X 10 -10 M. The patient was treated with 200 mg clomiphene citrate daily. Subjective symptoms improved and a reduction of skin nodule size and of liver enlargement followed. The serum enzymes alkaline phosphatase, nucleotidase, and phosphohexoseisomerase were diminished. A 2nd biopsy taken at Day 26 of treatment with clomiphene citrate showed complete inhibition of labeled estradiol tritiated-E2 uptake by the cytosol protein. This finding is thought to show the absence of free binding sites after clomiphene citrate therapy. Microscopic studies of biopsy material were unchanged. These results are thought to be the first to record human in vivo inhibition of trititated-E2 uptake for EBP by an antiestrogen compound, although similar in vitro observations have been made in human tumor specimens. There is thought to be a potential value of antiestrogenic agents, alone or with inhibitors of prolactin secretion, to replace endocrine ablations and to predict the response to endocrine therapy.     

Inhibition of pentraxin 3 in glioma cells impairs proliferation and invasion in vitro and in vivo

Pentraxin 3 (PTX3) is an inflammatory molecule that is involved in immune responses, inflammation, and cancer. Recent evidence suggests that PTX3 plays a critical role in tumor progression; however, its impact on the biological function of gliomas remains unknown. In the present study, immunohistochemical staining showed that patients with high-grade gliomas exhibited increased expression levels of PTX3 compared to those with low-grade gliomas (P?<?0.001). Furthermore, knockdown of PTX3 in GBM8401 cells inhibits proliferation, increases p21 protein levels, and decreases cyclin D1 protein levels, resulting in cell cycle arrest at the G0/G1 phase. In addition, knockdown of PTX3 significantly decreases GBM8401 cell migration and invasion through the downregulation of matrix metalloproteinase-1 and -2 (MMP-1 and MMP-2) expression. In a GBM8401 xenograft animal model, PTX3 knockdown decreases tumor growth in vivo. In conclusion, PTX3 plays an important role in glioma cell proliferation and invasion, and may thus serve as a novel potential therapeutic target in the treatment of gliomas.     

A rapid in vitro method for measuring cell proliferation in human breast cancer.

A method has been developed for studying in vitro the cell proliferation kinetics of human breast cancer, Surgical specimens from primary tumors were studied in 56 patients. Viable cell suspensions for assay were obtained by the dissociation of tumor tissue with collagenase. Mean Labeling indices of 2.43 +/- S.D 2.05 and 4.48 +/- S.D. 3.73, respectiviely, were found after incubation with 3HTdR for 2 hours and 24 hours. Mean S-times of 21.9 +/- S.D. 4.3 hours were estimated by 3H and 14C-TdR double-labeling. The kinetic data have been validated by parallel labeling studies in vivo and in vitro in four patients. The processing of autoradiographs using gold latensification provided slides for kinetic analysis within 3 days. The assay offers a method that is useful in the planning and monitoring of drug therapy.     

NF-kappaB activity blockade impairs the angiogenic potential of human pancreatic cancer cells

The effect of blockade of NF-kappaB activity on human pancreatic cancer angiogenesis was determined in an orthotopic xenograft model. Highly metastatic L3.3 human pancreatic cancer cells, which expressed an elevated level of constitutive NF-kappaB activity, were transfected with a mutated IkappaBalpha (IkappaBalphaM). After implantation in the pancreas of nude mice, parental (L3.3) and control vector-transfected (L3.3-Neo) cells produced rapidly growing tumors and liver metastases, whereas IkappaBalphaM-transfected (L3.3-IkappaBalphaM) cells had decreased tumorigenicity and metastatic potential. NF-kappaB signaling blockade significantly inhibited the in vitro and in vivo expression of the major proangiogenic molecules vascular endothelial growth factor and interleukin-8 and decreased tumor vascular formation. These events were correlated with retarded tumor growth and suppression of metastasis. Collectively, these data suggest that suppression of tumorigenicity and metastasis by NF-kappaB blockade is due to impaired angiogenic potential of tumor cells.     

Effects of phenylbutyrate on proliferation and apoptosis in human prostate cancer cells in vitro and in vivo

Phenylbutyrate (PB) is a potent differentiating agent and currently under investigation for the treatment of prostate cancer (CaP) and other malignancies. We have studied the impact of PB in vitro and in vivo on differentiation, proliferation and apoptosis in the LNCaP and LuCaP 23.1 prostate cancer xenograft models. In vitro we found that i) PB increased PSA secretion/cell, ii) inhibited cell proliferation in a time- and dose-dependent manner resulting in a cell cycle arrest in G1-phase and iii) induced apoptosis at concentrations of 2.5 mM after 3 days of treatment. In PB treated animals tumor growth stabilized or regressed. Combination of castration and PB treatment had a synergistic antiproliferative effect. The growth-inhibitory and differentiating properties and a low toxicity profile of PB provide rationale for further clinical studies in patients with CaP.     

Enhancement of ionizing radiation response by histamine in vitro and in vivo in human breast cancer

The radioprotective potential of histamine on healthy tissue has been previously demonstrated. The aims of this work were to investigate the combinatorial effect of histamine or its receptor ligands and gamma radiation in vitro on the radiobiological response of 2 breast cancer cell lines (MDA-MB-231 and MCF-7), to explore the potential molecular mechanisms of the radiosensitizing action and to evaluate the histamine-induced radiosensitization in vivo in a triple negative breast cancer model. Results indicate that histamine significantly increased the radiosensitivity of MDA-MB-231 and MCF-7 cells. This effect was mimicked by the H₁R agonist 2-(3-(trifluoromethyl)phenyl)histamine and the H₄R agonists (Clobenpropit and VUF8430) in MDA-MB-231 and MCF-7 cells, respectively. Histamine and its agonists enhanced radiation-induced oxidative DNA damage, DNA double-strand breaks, apoptosis and senescence. These effects were associated with increased production of reactive oxygen species, which correlated with the inhibition of catalase, glutathione peroxidase and superoxide dismutase activities in MDA-MB-231 cells. Histamine was able also to potentiate in vivo the anti-tumoral effect of radiation, increasing the exponential tumor doubling time. We conclude that histamine increased radiation response of breast cancer cells, suggesting that it could be used as a potential adjuvant to enhance the efficacy of radiotherapy.     

VEGF-C短发夹RNA对人乳腺癌细胞的体内外抑制作用的实验研究

目的:探讨靶向血管内皮生长因子-C(VEGF-C)的短发夹RNA(shRNA)质粒载体在体内外对乳腺癌细胞株MCF-7生物学活性的抑制作用。方法:倒置荧光显微镜及流式细胞术检测细胞的转染率,RT-PCR及免疫印迹法检测转染细胞内VEGF-C mRNA及蛋白的表达变化,MTT法检测细胞增殖抑制率,人工基底膜侵袭实验检测细胞侵袭能力。MCF-7裸鼠皮下尾静脉注射VEGF-C shRNA表达载体,观察其对乳腺癌生长的抑制作用。ELISA方法检测VEGF-C在肿瘤组织中的蛋白浓度,免疫组织化学分析肿瘤组织VEGFR-3阳性脉管密度。结果:VEGF-C shRNA能高效转染入MCF-7细胞中,细胞VEGF-C mRNA及蛋白表达水平显著下降,生长增殖受抑(F24 h=515.51,F48 h=859.97,F72 h=992.91,P均=0.000 1)。体外侵袭人工基底膜能力减弱(F=133.36,P=0.000 1)。全身系统性给予VEGF-C shRNA后,VEGF-C shRNA组肿瘤体积缩小(F=498.05,P=0.000 1);瘤组织VEGF-C浓度降低(t=30.75,P=0.001);免疫组化分析VEGF-C shRNA组肿瘤组织中VEGFR-3阳性脉管生成减少(F=265.49,P=0.000 1)。结论:VEGF-C在乳腺癌增殖、侵袭转移和VEGFR-3阳性脉管形成中发挥重要作用,通过RNA干扰技术实现VEGF-C沉默在乳腺癌的基因治疗中具有较好的应用前景。     

Effects of ecto-5'-nucleotidase on human breast cancer cell growth in vitro and in vivo

Ecto-5'-nucleotidase (CD73) is an essential enzyme that generates adenosine, an essential molecule for cell growth. CD73 increases significantly in many breast cancers. In this study, alpha,beta-methylene adenosine-5'-diphosphate (APCP), a specific CD73 inhibitor was used to block the hydrolase's activity. Effects of CD73 were examined on human breast cancer cells MDA-MB-231 in culture for proliferation, cell cycle progression, and apoptosis before and after APCP treatment. The in vivo effect of CD73 was examined on MDA-MB-231 tumor xenograft growth in nude mice. Cell growth curve, cell cycle and apoptosis were observed with MTT assays and flow cytometry, respectively. Microvessel density (MVD) and lymph vessel density (LVD) of implanted tumor tissues was analyzed by immunohistochemistry for CD31 and VEGFR-3 staining respectively. Our results showed that APCP inhibited MDA-MB-231 viability in a dose-dependent manner. APCP (12 microM) increased the percentage of G0/G1 phase cells from 49.75 to 59.16% while it decreased S phase and G2/M cells from 24.85 and 18.65% to 21.65 and 12.55%, respectively. The percentages of early and late apoptotic cells were also decreased after APCP treatment. However, APCP treatment did not affect the percentage of normal cells. Xenograft of MDA-MB-231 cells in the APCP treatment group had lower volume and weight than those of control group (2.70+/-1.14 vs 1.41+/-0.39 cm(3) and 2.7+/-0.5 vs 1.3+/-0.2 g), accompanied with less vessel formation with a MVD of 5+/-1 compared to the control group's 10+/-2 and an LVD of 4+1 vs 7+2. Our results suggest that CD73 may promote tumor growth and serve as a marker of breast cancer progression.     

MicroRNA-301 mediates proliferation and invasion in human breast cancer

Several microRNAs have been implicated in human breast cancer but none to date have been validated or utilized consistently in clinical management. MicroRNA-301 (miR-301) overexpression has been implicated as a negative prognostic indicator in lymph node negative (LNN) invasive ductal breast cancer, but its potential functional impact has not been determined. Here we report that in breast cancer cells, miR-301 attenuation decreased cell proliferation, clonogenicity, migration, invasion, tamoxifen resistance, tumor growth, and microvessel density, establishing an important oncogenic role for this gene. Algorithm-based and experimental strategies identified FOXF2, BBC3, PTEN, and COL2A1 as candidate miR-301 targets, all of which were verified as direct targets through luciferase reporter assays. We noted that miR-301 is located in an intron of the SKA2 gene which is responsible for kinetochore assembly, and both genes were found to be coexpressed in primary breast cancer samples. In summary, our findings define miR-301 as a crucial oncogene in human breast cancer that acts through multiple pathways and mechanisms to promote nodal or distant relapses.