Insulin,Insulin-like Growth Factor-I and Breast Cancer Risk in Japanese Women

To evaluate the effects of glucose metabolism related factors, such as insulin and insulin-like growth-factors ‍(IGFs), on breast cancer development among Japanese women, we conducted a case-referent study comparing 187 ‍women presenting with operable breast cancer and 190 women of the same age having no breast cancer. Odds ratios ‍(OR) and 95% confidence intervals (95%CI) were determined by multiple logistic regression analysis. ‍ In the present study, no association in risk was observed with increasing levels of IGF-I or IGF binding protein- ‍3 (IGFBP-3), before or after adjustment these factors. However, a suggestion of a positive association of an increased ‍breast cancer risk was evident in postmenopausal women with elevated plasma insulin levels, particularly those with ‍BMI>23.07. The OR for plasma insulin in the top tertile was 4.48 (95%CI:1.07-18.7) compared to the bottom tertile. ‍For C-peptide, there was a similar positive association, with a corresponding OR of 2.28. In addition, we observed ‍strong links between plasma insulin, C-peptide levels and estrogen receptor (ER) negative breast cancer, with ORs ‍of 2.79(95%CI:1.09-7.16), and 2.52 (95%CI:0.91-6.97) respectively, for the top versus bottom tertiles. In conclusion, ‍the present study suggested that plasma insulin level is a predictor of postmenopausal breast cancer in obese women ‍and ER negative breast cancer. Additional studies are needed to clarify the role of glucose metabolism pathways in ‍breast cancer development and interaction of IGF systems.     

MicroRNA-664 Targets Insulin Receptor Substrate 1 to Suppress Cell Proliferation and Invasion in Breast Cancer

A large number of microRNAs (miRNAs) have been previously demonstrated to be dysregulated in breast cancer (BC), and alterations in miRNA expression may affect the initiation and progression of BC. This study showed that miR-664 expression was obviously reduced in BC tissues and cell lines. Resumption of the expression of miR-664 attenuated the proliferation and invasion of BC cells. The molecular mechanisms underlying the inhibitory effects of BC cell proliferation and invasion by miR-664 were also studied. Insulin receptor substrate 1 (IRS1) was identified as a novel and direct target of miR-664. In addition, siRNAmediated silencing of IRS1 expression mimicked the suppressive effects of miR-664 overexpression in BC cells. Rescue experiments demonstrated that recovered IRS1 expression partially antagonized the inhibition of proliferation and invasion of BC cells caused by miR-664 overexpression. Thus, miR-664 may serve as a tumor suppressor in BC by directly targeting IRS1. Moreover, miR-664 downregulation in BC may contribute to the occurrence and development of BC, suggesting that miR-664 may be a novel therapeutic target for patients with BC.     

RNA干扰MDM2下调人乳腺癌细胞中VEGF的合成并抑制肿瘤血管的生成

目的 探讨小干扰RNA（small interfering RNA, siRNA）抑制人乳腺癌细胞中鼠双微粒体2 （murine double minute 2, MDM2）的表达对癌细胞中血管内皮生长因子（vascular endothelial growth factor, VEGF）合成以及裸鼠移植瘤组织内血管生成的影响。方法 根据MDM2已知的cDNA序列设计并转录合成特异性siRNA,转染高表达MDM2的人乳腺癌MDA-MB-468细胞,低氧培养后应用蛋白质印迹法和ELISA法检测肿瘤细胞及其上清液中VEGF的水平。构建裸鼠乳腺癌移植瘤模型,观察MDM2沉默后肿瘤生长情况,蛋白质印迹法、免疫组织化学及ELISA方法检测荷瘤组织和裸鼠血清标本中的VEGF含量,并以CD34标记血管内皮细胞计数微血管密度（microvessel density, MVD）。结果 siRNA抑制MDM2表达后,MDA-MB-468细胞分泌的VEGF蛋白显著减少（P=0.006）。裸鼠移植瘤模型显示,封闭MDM2表达使荷瘤组织生长变慢（P=0.008）,且荷瘤小鼠血清VEGF水平明显减低（P=0.008）,荷瘤组织内MVD也明显降低（P=0.003）。结论 MDM2 siRNA能有效减低乳腺癌细胞中VEGF的合成,并抑制裸鼠移植瘤组织内新生血管的生成,为MDM2/VEGF途径抗肿瘤血管生成作用的研究及靶向药物开发提供了新的思路。     

Targeting Epidermal Growth Factor Receptor by MiRNA-145 Inhibits Cell Growth and Sensitizes NSCLC Cells to Erlotinib

Background: Despite effective activity of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), such as erlotinib, all non-small cell lung cancer (NSCLC) patients eventually acquire resistance to these agents. Studies have demonstrated that down-regulation of miRNA-145 leads to enhancement of EGFR expression, cell proliferation and metastasis. The aim of this study was to investigate the effect of miRNA-145 on sensitivity of the A549 NSCLC cells to erlotinib. Methods: Quantitative real-time PCR was used to examine the effect of miRNA-145 on EGFR expression. The effect of miRNA-145 on cell growth and sensitivity the lung cancer cells to erlotinib was examined by trypan blue and MTT assays, respectively. The combination index was calculated using the non-constant method of Chou-Talalay. Apoptosis was determined by ELISA cell death assay. Results: We found that miRNA-145 was markedly suppressed the expression of EGFR and inhibited the cancer cell growth, relative to blank control and negative control miRNA (p<0.05). Pretreatment with miRNA-145 synergistically enhanced the sensitivity of the lung cancer cells to erlotinib. Results of apoptosis assay revealed that miRNA-145 can induce apoptosis and increase the erlotinib-mediated apoptosis. Conclusions: Our data demonstrate that miRNA-145 play a critical role in the lung cancer cell growth, survival and EGFR-TKIs resistance possibly by regulation of EGFR. Therefore, miRNA-145 replacement therapy can become a new therapeutic strategy in lung cancer.     

Positive Expression of Insulin-Like Growth Factor-1 Receptor Is Associated with a Positive Hormone Receptor Status and a Favorable Prognosis in Breast Cancer

Purpose

Insulin-like growth factor 1 receptor (IGF-1R) is commonly expressed in primary breast cancers. Understanding the role of IGF-1R signaling in the different subtypes of breast cancer is important because each subtype has a different outcome and requires different treatment modalities. However, the precise biological significance of IGF-1R expression in cancer cells is still unclear. In this study, we examined the expression of IGF-1R in the different molecular subtypes of breast cancer. The effects of IGF-1R expression on the survival rates and outcomes of breast cancer were also examined.

Methods

IGF-1R expression was evaluated immunohistochemically in tissue microarray blocks constructed from 1,198 invasive breast cancer samples collected from six medical institutions. IGF-1R expression was interpreted according to the human epidermal growth factor receptor 2 (HER2)/neu immunohistochemistry scoring system. Scores of 2+ and 3+ were considered positive.

Results

Positive IGF-1R expression was observed in 65.4% of invasive breast cancer samples. IGF-1R expression was detected in all cancer subtypes (luminal A, 84.4%; luminal B, 75.9%; HER2, 21.2%; triple-negative, 46.6%) and was found to be associated with a positive hormone receptor status and the absence of HER2 amplification (p<0.001). Positive IGF-1R expression was significantly associated with high survival rates (p=0.014). However, a multivariate analysis revealed that the expression levels of IGF-1R did not achieve statistical significance. In the triple-negative cancer subtype, IGF-1R expression was found to be associated with a lower disease-free survival rate (p=0.031).

Conclusion

Positive IGF-1R expression is associated with a favorable prognosis in breast cancer. IGF-1R is frequently expressed in the luminal A/B subtypes of breast cancer, and its expression is related to the hormone receptor status.     

Sulforaphane Inhibits Growth of Human Breast Cancer Cells and Augments the Therapeutic Index of the Chemotherapeutic Drug,Gemcitabine

Phytochemicals are among the natural chemopreventive agents with most potential for delaying, blocking orreversing the initiation and promotional events of carcinogenesis. They therefore offer cancer treatment strategiesto reduce cancer related death. One such promising chemopreventive agent which has attracted considerableattention is sulforaphane (SFN), which exhibits anti-cancer, anti-diabetic, and anti-microbial properties. Thepresent study was undertaken to assess effect of SFN alone and in combination with a chemotherapeutic agent,gemcitabine, on the proliferative potential of MCF-7 cells by cell viability assay and authenticated the resultsby nuclear morphological examination. Further we analyzed the modulation of expression of Bcl-2 and COX-2on treatment of these cells with SFN by RT-PCR. SFN showed cytotoxic effects on MCF-7 cells in a dose- andtime-dependent manner via an apoptotic mode of cell death. In addition, a combinational treatment of SFNand gemcitabine on MCF-7 cells resulted in growth inhibition in a synergistic manner with a combination index(CI)<1. Notably, SFN was found to significantly downregulate the expression of Bcl-2, an anti-apoptotic gene, andCOX-2, a gene involved in inflammation, in a time-dependent manner. These results indicate that SFN inducesapoptosis and anti-inflammatory effects on MCF-7 cells via downregulation of Bcl-2 and COX-2 respectively.The combination of SFN and gemcitabine may potentiate the efficacy of gemcitabine and minimize the toxicityto normal cells. Taken together, SFN may be a potent anti-cancer agent for breast cancer treatment.     

Perillyl Alcohol Inhibits Human Breast Cancer Cell Growth in vitro and in vivo

The effect of monoterpene perillyl alcohol (POH) on cell growth, cell cycle progression, and expression of cell cycle-regulatory proteins in estrogen receptor (ER)-positive (KPL-1 and MCF-7) and ER-negative (MKL-F and MDA-MB-231) human breast cancer cell lines was examined. POH inhibited cell proliferation in a dose-dependent manner in all cell lines tested. POH at a dose of 500 micro M had a cytostatic effect, in which growth inhibition was due to accumulation of cells in G1-phase. Cell cycle progression was preceded by a decrease in G1 cyclins (cyclin D1 and E), followed by an increase in p21(Cip1/Waf1) and a decrease in proliferating cell nuclear antigen level. Levels of p53 and cyclin A were unchanged. POH at a dose of 75 mg/kg administered intraperitoneally three times a week throughout the entire 6-week experimental period suppressed orthotopically transplanted KPL-1 tumor cell growth and regional lymph node metastasis in a nude mouse system. POH inhibited both ER-positive and -negative human breast cancer cell growth in vitro, and suppressed growth and metastasis in vivo.     

Expression and Clinical Significance of Sushi Domain- Containing Protein 3 (SUSD3) and Insulin-like Growth Factor-I Receptor (IGF-IR) in Breast Cancer

Background: To investigate the expression of insulin-like growth factor-I receptor (IGF-IR) and sushi domaincontaining protein 3 (SUSD3) in breast cancer tissue, and analyze their relationship with clinical parameters and the correlation betweenthe two proteins. Materials and Methods: The expression of IGF-IR and SUSD3 in 100 cases of breast cancer tissues and adjacent normal breast tissues after surgery was detected by immunohistochemical technique MaxVisionTM, and the relationship with clinical pathological features was further analyzed. Results: The positive rate of IGF-IR protein was 86.0% in breast cancer, higher than 3.0% in adjacent normalbreast tissue (P<0.05) .The positive expression rate of SUSD3 protein was 78.0% in breast cancer, higher than 2.0% in adjacent normal breast tissue (P<0.05). The expression of IGF-IR and SUSD3 was related to estrogen receptor and pathological types (P<0.05),but not with age, stage, the expression of HER-2 and Ki-67 (P>0. 05). The expression of IGF-IR and SUSD3 in breast cancer tissue was positively related (r= 0.553, P<0.01). Conclusions: The expression of IGF-IR and SUSD3 may be correlated to the occurrence and development of breast cancer. The combined detection of IGF-IR, SUSD3 and ER may play an important role in judging prognosis and guiding adjuvant therapy after surgery of breast cancer.     

MiR-124靶向调控PKM2基因表达抑制乳腺癌细胞的增殖和转移

摘 要：［目的］ 探讨miR-124对乳腺癌MDA-MB231细胞增殖和转移的影响。［方法］ 运用实时荧光定量PCR法、免疫印迹实验检测25例乳腺癌乳腺癌组织及对应癌旁组织中的miR-124、丙酮酸激酶同工酶（PKM2）相对表达量。将pre-miR-124、抗miR-124转染至乳腺癌MDA-MB231细胞中,同时设置阴性对照组,检测三组细胞中miR-124、PKM2基因及蛋白表达量。运用CCK-8法及克隆形成实验检测转染后的MDA-MB231细胞增殖和转移。［结果］ 乳腺癌组织中miR-124（0.92±0.24）的相对表达量显著性少于癌旁组织（1.55±0.28）,PKM2的相对表达量更高（0.69±0.18）。pre-miR-124转染MDA-MB231细胞呈现miR-124过表达（0.37±0.08）,且其可以反向调控PKM2基因（0.52±0.11）及蛋白表达量（0.19±0.04）,抑制细胞的增殖与转移能力。抗miR-124转染MDA-MB231细胞呈现miR-124抑制表达（0.06±0.03）,其PKM2基因（2.09±0.08）及蛋白表达量（0.47±0.10）显著性上升,促进细胞增殖与转移（P＜0.05）. ［结论］ miR-124可以通过下调PKM2基因的表达,从而抑制乳腺癌MDA-MB231细胞的增殖和转移。     

Synuclein Gamma Inhibits the Mitotic Checkpoint Function and Promotes Chromosomal Instability of Breast Cancer Cells

Summary Aberrant expressions of the neuronal protein synuclein gamma (SNCG) in malignant mammary epithelial cells are strongly associated with the progression of breast cancer. SNCG is not expressed in normal breast tissues but abundantly expressed in a high percentage of invasive and metastatic breast carcinomas. Several studies have demonstrated that SNCG expression significantly stimulates proliferation, invasion, and metastasis of breast cancer cells. To elucidate the molecular and cellular mechanisms underlying the tumorigenic functions of SNCG, we investigated the effects of SNCG expression on the mitotic checkpoint function of breast cancer cells. By conducting several different lines of investigations, we now demonstrate that SNCG expression in breast cancer cells overrides the mitotic checkpoint control and confers the cellular resistance to anti-microtubule drug-caused apoptosis. We further show that the inhibitory effects of SNCG on mitotic checkpoint can be overthrown by enforced overexpression of the mitotic checkpoint protein BubR1 in SNCG-expressing cells. These new findings combined with our previous observation that SNCG intracellularly associates with BubR1 together suggest that SNCG expression compromises the mitotic checkpoint control by inhibition of the normal function of BubR1, thereby promoting genetic instability. Genetic instability is recognized as an important contributing factor in tumorigenesis. Hence, our studies gain insight into the mechanisms whereby SNCG expression advances breast cancer disease progression and fasters tumor metastasis.     

Fenugreek Induced Apoptosis in Breast Cancer MCF-7 Cells Mediated Independently by Fas Receptor Change

Trigonella foenum in graecum (Fenugreek) is a traditional herbal plant used to treat disorders like diabetes,high cholesterol, wounds, inflammation, gastrointestinal ailments, and it is believed to have anti-tumor properties,although the mechanisms for the activity remain to be elucidated. In this study, we prepared a methanol extractfrom Fenugreek whole plants and investigated the mechanism involved in its growth-inhibitory effect on MCF-7 human breast cancer cells. Apoptosis of MCF-7 cells was evidenced by investigating trypan blue exclusion,TUNEL and Caspase 3, 8, 9, p53, FADD, Bax and Bak by real-time PCR assays inducing activities, in thepresence of FME at 65 μg/mL for 24 and 48 hours. FME induced apoptosis was mediated by the death receptorpathway as demonstrated by the increased level of Fas receptor expression after FME treatment. However,such change was found to be absent in Caspase 3, 8, 9, p53, FADD, Bax and Bak, which was confirmed by atime-dependent and dose-dependent manner. In summary, these data demonstrate that at least 90% of FMEinduced apoptosis in breast cell is mediated by Fas receptor-independently of either FADD, Caspase 8 or 3, aswell as p53 interdependently.     

FRZB is Regulated by the Transcription Factor EGR1 and Inhibits the Growth and Invasion of Triple-Negative Breast Cancer Cells by Regulating the JAK/STAT3 Pathway

BackgroundTo explore the expression of frizzled related protein (FRZB) in triple-negative breast cancer (TNBC) and role of FRZB in TNBC cell growth and invasion.MethodsBreast cancer clinical data were downloaded from the Cancer Genome Atlas. FRZB and early growth response 1 (EGR1) mRNA levels in TNBC were measured by quantitative real-time polymerase chain reaction. FRZB protein level was measured by immunohistochemistry and western blot. Proliferation, migration, and invasion of TNBC cells were detected by colony formation, wound healing, and transwell assay, respectively. The protein levels of EGR1, E-cadherin, N-cadherin, Snail, p-JAK1/JAK1, p-JAK2/JAK2, and p-STAT3/STAT3 were measured by western blot. JASPAR was used to predict the binding site of FRZB and EGR1. The binding ability of FRZB and EGR1 was verified by dual-luciferase reporter gene assay and chromatin immunoprecipitation assay.ResultsFRZB was low expressed in TNBC tissues and cells. Silencing FRZB promoted cell proliferation, migration, invasion, and EMT and activated JAK/STAT pathway in MDA-MB-468 and MDA-MB-231 cells, but overexpression of FRZB acted opposite effects in MDA-MB-468 and MDA-MB-231 cells. EGR1 was low expressed in TNBC samples and positively correlated with FRZB. Moreover, EGR1 could recover the promotion of silencing FRZB on cell proliferation, migration, invasion, and JAK/STAT pathway in MDA-MB-468 cells, but silencing EGR1 led to the opposite results in MDA-MB-231 cells.ConclusionFRZB was low expressed in TNBC and was regulated by EGR1, and FRZB inhibited TNBC cell growth and invasion by regulating the JAK/STAT3 pathway.     

Luteolin inhibits proliferation induced by IGF-1 pathway dependent ERα in human breast cancer MCF-7 cells

The growth of many breast tumors is stimulated by IGF-1, which activates signal transduction pathways inducing cell proliferation. ERα is important in this process. The aim of the study was to investigate relationships in vitro among inhibitory effects of luteolin on the growth of MCF-7 cells, IGF-1 pathway and ERα. Our results showed that luteolin could effectively block IGF-1-stimulated MCF-7 cell proliferation in a dose- and time- dependent manner and block cell cycle progression and induce apoptosis evidenced by the flow cytometric detection of sub-G1DNA content. Luteolin markedly decreased IGF-1-dependent IGF-1R and Akt phosphorylation without affecting Erk1/2 phosphorylation. Further experiments pointed out that ERα was directly involved in IGF-1 induced cell growth inhibitory effects of luteolin, which significantly decreased ERα expression. Knockdown of ERα in MCF-7 cells by an ERα-specific siRNA decreased the IGF-1 induced cell growth inhibitory effects of luteolin. ERα is thus a possible target of luteolin. These findings indicate that the inhibitory effect of luteolin on the growth of MCF-7 cells is via inhibiting IGF-1 mediated PI3K-Akt pathway dependent on ERα expression.     

Synergistic Growth Inhibitory Effects of Chrysin and Metformin Combination on Breast Cancer Cells through hTERT and Cyclin D1 Suppression

Objective: To explore the possibility of a novel chemopreventive strategy for improving breast cancer treatment,the anticancer effects of a combination two natural compounds, Chrysin and Metformin, against T47D breast cancercells were investigated. Materials and Methods: After treatment of T47D cells with Metformin, Chrysin and the twodrugs in combination, toxicity to cancer cells was evaluated by MTT assay. Real time PCR was then used to determinethe expression levels of hTERT and cyclin D1 genes. Results: The MTT test findings showed that the combination ofmetformin and chrysin had high synergistic effects in killing cancer cells. In addition PCR demonstrated a significantdecrease in cyclin D1 and hTERT gene expression in the T47D breast cancer cell line. Conclusion: The conmbinationof metformin and chrysin suppressing hTERT and cyclin D1 gene expression might offer an appropriate approach forbreast cancer therapy.