Antiangiogenic cancer therapy: why do mouse and human patients respond in a different way to the same drug?

The tumor vasculature is an increasingly attractive target for development of anticancer drugs. The fundamental principle for antiangiogenic cancer therapy is based on the inhibitory effect of chemical compounds, proteins or nucleotides on tumor angiogenesis. Indeed, in almost all preclinical tumor models, antiangiogenic monotherapy with different agents shows potent effects on suppression of tumor growth. However, antiangiogenic monotherapy has barely produced any clinical benefits in cancer patients. Although in combination with chemotherapy some antiangiogenic drugs demonstrate survival improvement in patients with certain types of cancers, the overall benefits by addition of antiangiogenic drugs (ADs) to chemotherapy remain modest. The disparity of AD responses between preclinical models and clinical cancer patients has raised important issues, which include: 1) Are current animal tumor models appropriate for assessing the therapeutic efficacy of ADs for clinical development? 2) What are the key differences between mouse tumor models and human cancer patients? 3) Are anti-VEGF drugs off target in cancer patients? 4) What are alternative options for improvement of the clinical benefits of ADs? In this short review, I discuss these critical issues in relation to the clinical practice of ADs.     

Identification of canine circulating miRNAs as tumor biospecific markers using Next-Generation Sequencing and Q-RT-PCR

Delay in cancer diagnosis often results in metastasis and an inability to successfully treat the tumor. The use of broadly cancer-specific biomarkers at an early stage may improve cancer treatment and staging. This study has explored circulatory exosomal miRNAs as potential diagnostic biomarkers to identify cancer patients. Secretory exosomal miRNAs were isolated from 13 canine cancer cell lines (lymphoma, mast cell tumor, histiocytic cell line, osteosarcoma, melanoma, and breast tumor) and were sequenced by Next-Generation sequencing (NGS). We have identified 6 miRNAs (cfa-miR-9, -1841, ?1306, ?345, ?132, and ?26b) by NGS that were elevated in all cancer cell types. The miRNAs identified by NGS were then examined by Q-RT-PCR. The PCR data demonstrated similar expression patterns to those seen with NGS but provided fold differences that were much lower than those seen for NGS. Cfa-miR-9 was found to be the most consistently elevated miRNA in NGS and PCR, making it the most likely miRNA to prove diagnostic. In this study, we have demonstrated that it is possible to identify exosomal miRNAs with elevated secretion across multiple tumor types that could be used as circulatory diagnostic biomarkers for liquid biopsy in the future.     

IL-6 Signaling Promotes Tumor Growth in Colorectal Cancer

Recent investigations support an important role for TGF-? in the development of colorectal cancer. However, the molecular consequences ofTGF-? signaling in the colon remains incompletely understood. In a recent study in Immunity, we analyzed the role of TGF-? in a murine model of colon cancer. Using transgenic mice overexpressing TGF-? or a dominant negative TGF-? receptor II under control of the CD2 minigene, we show that TGF-? signaling in tumor infiltrating T lymphocytes regulates the growth of dysplastic colon epithelial cells, as determined by histology and a novel system for high resolution chromoendoscopy in vivo. At the molecular level, TGF-? signaling in T cells regulated STAT-3 activation in tumor cells via IL-6. IL-6 signaling required tumor cell derived soluble IL-6R rather than membrane bound IL-6R and suppression of such TGF-?-dependent IL-6 trans-signaling prevented tumor progression in vivo. Similar to these observations in mice, here we show that human colon cancer tissue expressed only low amounts of membrane bound IL-6R. In contrast, expression and activity of the matrix metalloproteinase TACE were increased. In summary, our data provide novel insights into the role of TGF-? signaling in colorectal cancer and suggest novel therapeutic approaches for colorectal cancer based on an inhibition of TGF-?-dependent IL-6 trans-signaling.     

CD40 pathway activation reveals dual function for macrophages in human endometrial cancer cell survival and invasion

Reproductive malignancies are a major cause of cancer death in women worldwide. CD40 is a TNF receptor family member, which upon activation may mediate tumor regression. However, despite the great potential of CD40 agonists, their use as a therapeutic option for reproductive cancers has never been investigated. Because CD40 ligation is a potent pathway of macrophage activation, an in vitro model of pro-inflammatory type-1 (M?-1) and anti-inflammatory type-2 (M?-2) macrophages was developed to determine whether and how macrophage CD40 pathway activation might influence endometrial tumor cell behavior. Analysis of tumor growth kinetic in the endometrial cancer xenograft model indicates that, when injected once into the growing tumors, CD40-activated M?-1 greatly reduced, while CD40-activated M?-2 increased tumor size when compared to control isotype-activated M?-1 and M?-2, respectively. In vitro assays indicated that CD40-activated M?-2 increased cell viability but failed to promote cell invasion. CD40-activated M?-1, in contrast, decreased cell survival but greatly increased cell invasion in tumor cells less susceptible to cell death by apoptosis; they also induced the expression of some pro-inflammatory genes, such as IL-6, LIF, and TNF-α, known to be involved in tumor promotion and metastasis. The presence of IFN-γ is minimally required for CD40-activated M?-1 to promote tumor cell invasion, a process that is mediated in part through the activation of the PI3K/Akt2 signaling pathway in tumor cells. From these results, we speculate that some functions of CD40 in tumor-associated M?s might limit the therapeutic development of CD40 agonists in endometrial cancer malignancies.     

Prognostic utility of circulating transforming growth factor beta 1 in breast cancer patients

Transforming growth factor betas (TGF-?s) are multifunctional cytokines with a biphasic role in breast tumorigenesis, acting as tumor suppressors at early stages while stimulating tumor progression at later stages (TGF-? switch). Among the 3 human isoforms, TGF-?1 is known to be overexpressed in several tumor types including breast tumors. TGF-? signaling and "crosstalk" in the tumor microenvironment presents a unique challenge and an opportunity to develop novel therapies. We assessed circulating TGF-?1 levels by ELISA in blood samples from 117 previously untreated breast cancer patients in this prospective study to explore the TGF-? switch at the forefront. The levels were correlated with clinicopathological prognosticators like age, menopausal status, nodal status, histological type, histological grade, necrosis, stromal involvement, and survival. Higher mean preoperative serum TGF-?1 was observed in early-stage patients than controls (p=0.05) as revealed by receiver operating characteristic (ROC) analysis. Elevation of TGF-?1 was evident in patients with advanced-stage breast cancer compared with those having early-stage disease (p=0.0001). Prognosticators of an aggressive phenotype were associated with higher TGF-?1 levels, and higher levels thus announced the likelihood of relapse, marking the role of TGF-?1 as a tumor promoter and evidencing the existence of a TGF-? switch. Moreover, higher levels of TGF-?1 shortened the overall survival in breast cancer patients (p=0.010). The results indicate that circulating TGF-?1 may be used as a predictive and prognostic marker in breast carcinoma.     

Low-dose paclitaxel enhances the anti-tumor efficacy of GM-CSF surface-modified whole-tumor-cell vaccine in mouse model of prostate cancer

Chemotherapy combined with a tumor vaccine is an attractive approach in cancer therapy. This study was designed to investigate the optimal schedule and mechanisms of action of a novel GM-CSF (granulocyte?Cmacrophage colony-stimulating factor) surface-modified tumor-cell vaccine in combination with paclitaxel in the treatment of mouse RM-1 prostate cancer. First, the anti-tumor efficiencies of various dosage of paclitaxel (4, 20, 40?mg/kg) in combination with the vaccine in different administration sequences were examined in the mouse RM-1 prostate cancer model. Then, the in vivo and in vitro effects of various dosage of paclitaxel on RM-1 cells, T cells, and DCs (dendritic cells) were evaluated. The results showed that: (a) the GM-CSF-surface-modified tumor-cell vaccine was more potent at inducing the uptake of tumor antigens by DCs than irradiated tumor cells plus free GM-CSF; (b) 4?mg/kg paclitaxel combined with the GM-CSF-surface-modified tumor-cell vaccine was the most effective at enhancing tumor regression in RM-1 prostate cancer mice when the vaccine was administrated 2?days after paclitaxel; and (c) administration of 4?mg/kg paclitaxel followed by the vaccine induced the highest degree of CD8⁺ T-cell infiltration in tumor tissue, suggesting that the induction of tumor-specific immune response had occurred. These findings suggested that the GM-CSF-surface-modified tumor-cell vaccine may have potential clinical benefit for patients with prostate cancer when it is combined with paclitaxel. Furthermore, the effect of immunochemotherapy depends on careful selection of paclitaxel dosage and the sequence of paclitaxel/vaccine administration.     

Cytokines and proteases in invasive processes: molecular similarities between inflammation and cancer.

Tumor-derived serine proteinases and metalloproteinases have been associated with invasion and metastasis of cancer cells. Leukocytes, particularly monocytes/macrophages and neutrophils, actively synthesize and store these proteolytic enzymes. The production by tumor cells of chemotactic factors that attract white blood cells raises questions that are important for the basic researcher as well as the clinical scientist. Are the proteinases, which have the capacity to dissolve the extracellular matrix and by this solubilization promote cell migration, the same in tumor cells as in normal cells? Is the production of chemotactic factors by tumor cells a coincident epiphenomenon of the malignant state or a selective way to parasitize the host? Does the early attraction of leukocytes to the tumor site contribute to early host defense against cancer? Does our knowledge about mechanisms of action of cytokines have implications for therapy of the cancer patient? Recent experimental data give hints to the answers to these questions and make it possible to deduce a fundamental model of cytokine mediated proteolysis in tissue remodelling.     

Regulation of PD-L1 expression by matrix stiffness in lung cancer cells

Expression of programmed death-ligand 1 (PD-L1) in tumor cells such as lung cancer cells plays an important role in mechanisms underlying evasion of an immune check point system. Lung cancer tissue with increased deposition of extracellular matrix is much stiffer than normal lung tissue. There is emerging evidence that the matrix stiffness of cancer tissue affects the phenotypes and properties of cancer cells. Nevertheless, the effects of substrate rigidity on expression of PD-L1 in lung cancer cells remain elusive. We evaluated the effects of substrate stiffness on PD-L1 expression in HCC827 lung adenocarcinoma cells by using polyacrylamide hydrogels with stiffnesses of 2 and 25?kPa. Expression of PD-L1 protein was higher on the stiffer substrates (25?kPa gel and plastic dish) than on the soft 2?kPa gel. PD-L1 expression was reduced by detachment of cells adhering to the substrate. Interferon-γ enhanced expression of PD-L1 protein cultured on stiff (25?kPa gel and plastic dishes) and soft (2?kPa gel) substrates and in the cell adhesion-free condition. As the stiffness of substrates increased, formation of actin stress fiber and cell growth were enhanced. Transfection of the cells with short interfering RNA for PD-L1 inhibited cell growth without affecting stress fiber formation. Treatment of the cells with cytochalasin D, an inhibitor of actin polymerization, significantly reduced PD-L1 protein levels. Taken together, a stiff substrate enhanced PD-L1 expression via actin-dependent mechanisms in lung cancer cells. It is suggested that stiffness as a tumor environment regulates PD-L1 expression, which leads to evasion of the immune system and tumor growth.     

Hypoxia,angiogenesis and cancer therapy: To breathe or not to breathe?

As an expanding tumor conquers space within the host, it calls out for an increased oxygen supply. This demand is rarely matched by tumor blood vessels because neo-angiogenesis generates a structurally aberrant and functionally impaired vasculature. As a result of this unbalance, tumor progression is invariably associated with cancer cell hypoxia. Insufficient oxygenation appears to have opposing effects on cancer biology: on one hand, it limits tumor cell division; on the other, it selects for more malignant cells and it induces a series of cellular adaptations that sustain and foster tumor invasion. When designing a therapeutic strategy, how should we resolve this dichotomy? Should we cut oxygen supply, thereby halting neoplastic expansion, or should we let the tumor breathe, in order to prevent its malignant conversion? Recent studies using angiogenesis inhibitors or oxygen transporters finally provide some clue.     

Buforin IIb induced cell cycle arrest in liver cancer

The inhibitory effect of buforin IIb on different types of cancer, although not liver cancer, has been demonstrated previously. The aim of the present study was to investigate the effects of buforin IIb on the progression of liver cancer. The human liver cancer cell line HepG2 was treated with purified buforin IIb and the cell activity was determined by MTT, colony formation and transwell assays. The protein expression levels of cyclin-dependent kinases (CDKs) and cyclins were analyzed by western blotting and immunofluorescent cell staining. A tumor growth model was constructed using nude mice, and buforin IIb treatment was administered. The levels of CDK2 and cyclin A in the tumor tissues were detected by western blotting. Buforin IIb treatment depressed cell viability and colony formation and induced apoptosis significantly, and 1.0?µM concentration of buforin IIb was found to be the optimal dosage. The cell cycle was arrested at the G2/M phase following buforin IIb treatment. CDK2 and cyclin A were downregulated by treatment of the cells with 1.0?µM buforin IIb for 24?h. Treatment with buforin IIb also inhibited the migration of liver cancer cells in vitro. Furthermore, 50?nmol buforin IIb injection suppressed HepG2 cell subcutaneous tumor growth in the nude mouse model. Similar to the in vitro results, buforin IIb injection reduced the expression of CDK2 and cyclin A in the tumor tissue. these results demonstrate that buforin IIb inhibited liver cancer cell growth via the regulation of CDK2 and cyclin A expression.     

Survival or death: disequilibrating the oncogenic and tumor suppressive autophagy in cancer

Autophagy (macroautophagy) is an evolutionarily conserved lysosomal degradation process, in which a cell degrades long-lived proteins and damaged organelles. Recently, accumulating evidence has revealed the core molecular machinery of autophagy in carcinogenesis; however, the intricate relationship between autophagy and cancer continue to remain an enigma. Why does autophagy have either pro-survival (oncogenic) or pro-death (tumor suppressive) role at different cancer stages, including cancer stem cell, initiation and progression, invasion and metastasis, as well as dormancy? How does autophagy modulate a series of oncogenic and/or tumor suppressive pathways, implicated in microRNA (miRNA) involvement? Whether would targeting the oncogenic and tumor suppressive autophagic network be a novel strategy for drug discovery? To address these problems, we focus on summarizing the dynamic oncogenic and tumor suppressive roles of autophagy and their relevant small-molecule drugs, which would provide a new clue to elucidate the oncosuppressive (survival or death) autophagic network as a potential therapeutic target.     

Molecular genetics of cancer and tumorigenesis: Drosophila models

Why do some cells not respond to normal control of cell division and become tumorous? Which signals trigger some tumor cells to migrate and colonize other tissues? What genetic factors are responsible for tumorigenesis and cancer development? What environmental factors play a role in cancer formation and progression? In how many ways can our bodies prevent and restrict the growth of cancerous cells?How can we identify and deliver effective drugs to fight cancer? In the fight against cancer,which kills more people than any other disease,these and other questions have long interested researchers from a diverse range of fields.To answer these questions and to fight cancer more effectively,we must increase our understanding of basic cancer biology.Model organisms,including the fruit fly Drosophila melanogaster,have played instrumental roles in our understanding of this devastating disease and the search for effective cures.Drosophila and its highly effective,easy-touse,and ever-expanding genetic tools have contributed toand enriched our knowledge of cancer and tumor formation tremendously.     

Genomic instability en route to and from cancer stem cells

Cancer is caused by successive gene mutations that amount to confer malignant phenotype. Genomic instability (GIN) is considered a key endogenous mechanism for accumulation of mutations, and therefore, has been proposed as an engine of tumorigenesis. Recently, cancer stem cells, or tumor initiating cells, have been identified in a variety of human cancers. These cancer stem cells (CSCs) are believed to be responsible for the initiation of malignant growth and metastasis of some, and perhaps all cancer types. How are these two engines of tumorigenesis related to each other? Is GIN a driving force in the genesis of cancer stem cells? Is the genome in CSCs inherently unstable? Could GIN in CSC be the cause of the observed cancer cell heterogeneity? In this article, we will discuss some early clues indicating that these two driving forces of tumorigenesis appear to be intimately connected.     

Systemic Cancer Progression and Tumor Dormancy: Mathematical Models Meet Single Cell Genomics

Metastatic progression is thought to result from genetically advanced ?fully-malignant“ tumor cells. Within the concept the prevailing view holds that such cells disseminate mostly from large tumors and are capable of growing into metastases once they arrive at a distant site. Support for this scenario comes from numerous mouse models in which transplanted tumor cells grow into metastases within days or weeks. However, the assumption of such fully-malignant disseminating cells in human cancer is misleading and is neither supported by mathematical modeling of survival data from cancer patients nor by ex-vivo genomic data from disseminated cancer cells. For example, in breast cancer the growth of metastases is highly homogeneous and takes on average six years, the number of disseminated tumor cells before diagnosis of metastasis is similar for different tumor stages, and the genomic aberrations of disseminated cancer cells do rarely correspond to those in the primary tumor. Since these facts question conventional concepts of metastatic progression we provide a model of cancer progression in which time considerations and direct ex-vivo data form a starting point. In the proposed model tumor dormancy is a characteristic of almost all migrated tumor cells and metastatic growth is a rare, stochastic, evolutionary process of selection and mutation of cells that often disseminate shortly after transformation at the primary site.     

Linking stemness with colorectal cancer initiation,progression, and therapy

The discovery of cancer stem cells caused a paradigm shift in the concepts of origin and development of colorectal cancer. Several unresolved questions remain in this field though. Are colorectal cancer stem cells the cause or an effect of the disease? How do cancer stem cells assist in colorectal tumor dissemination to distant organs? What are the molecular or environmental factors affecting the roles of these cells in colorectal cancer? Through this review, we investigate the key findings until now and attempt to elucidate the origins, physical properties, microenvironmental niches, as well as the molecular signaling network that support the existence, self-renewal, plasticity, quiescence, and the overall maintenance of cancer stem cells in colorectal cancer. Increasing data show that the cancer stem cells play a crucial role not only in the establishment of the primary colorectal tumor but also in the distant spread of the disease. Hence, we will also look at the mechanisms adopted by cancer stem cells to influence the development of metastasis and evade therapeutic targeting and its role in the overall disease prognosis. Finally, we will illustrate the importance of understanding the biology of these cells to develop improved clinical strategies to tackle colorectal cancer.     

Hypertonic stress induces VEGF production in human colon cancer cell line Caco-2: inhibitory role of autocrine PGE₂

Vascular Endothelial Growth Factor (VEGF) is a major regulator of angiogenesis. VEGF expression is up regulated in response to micro-environmental cues related to poor blood supply such as hypoxia. However, regulation of VEGF expression in cancer cells is not limited to the stress response due to increased volume of the tumor mass. Lipid mediators in particular arachidonic acid-derived prostaglandin (PG)E? are regulators of VEGF expression and angiogenesis in colon cancer. In addition, increased osmolarity that is generated during colonic water absorption and feces consolidation seems to activate colon cancer cells and promote PGE? generation. Such physiological stimulation may provide signaling for cancer promotion. Here we investigated the effect of exposure to a hypertonic medium, to emulate colonic environment, on VEGF production by colon cancer cells. The role of concomitant PGE? generation and MAPK activation was addressed by specific pharmacological inhibition. Human colon cancer cell line Caco-2 exposed to a hypertonic environment responded with marked VEGF and PGE? production. VEGF production was inhibited by selective inhibitors of ERK 1/2 and p38 MAPK pathways. To address the regulatory role of PGE? on VEGF production, Caco-2 cells were treated with cPLA? (ATK) and COX-2 (NS-398) inhibitors, that completely block PGE? generation. The Caco-2 cells were also treated with a non selective PGE? receptor antagonist. Each treatment significantly increased the hypertonic stress-induced VEGF production. Moreover, addition of PGE? or selective EP? receptor agonist to activated Caco-2 cells inhibited VEGF production. The autocrine inhibitory role for PGE? appears to be selective to hypertonic environment since VEGF production induced by exposure to CoCl? was decreased by inhibition of concomitant PGE? generation. Our results indicated that hypertonicity stimulates VEGF production in colon cancer cell lines. Also PGE? plays an inhibitory role on VEGF production by Caco-2 cells exposed to hyperosmotic stress through EP? activation.     

DNA and adenovirus tumor vaccine expressing truncated survivin generates specific immune responses and anti-tumor effects in a murine melanoma model

Survivin is overexpressed in major types of cancer and is considered an ideal "universal" tumor-associated antigen that can be targeted by immunotherapeutic vaccines. However, its anti-apoptosis function raises certain safety concerns. Here, a new truncated human survivin, devoid of the anti-apoptosis function, was generated as a candidate tumor vaccine. Interleukin 2 (IL-2) has been widely used as an adjuvant for vaccination against various diseases. Meanwhile, the DNA prime and recombinant adenovirus (rAd) boost heterologous immunization strategy has been proven to be highly effective in enhancing immune responses. Therefore, the efficacy of a new cancer vaccine based on a truncated form of survivin, combined with IL-2, DNA prime, and rAd boost, was tested. As prophylaxis, immunization with the DNA vaccine alone resulted in a weak immune response and modest anti-tumor effect, whereas the tumor inhibition ratio with the DNA vaccine administered with IL-2 increased to 89?% and was further increased to nearly 100?% by rAd boosting. Moreover, complete tumor rejection was observed in 5 of 15 mice. Efficacy of the vaccine administered therapeutically was enhanced by nearly 300?% when combined with carboplatin. These results indicated that vaccination with a truncated survivin vaccine using DNA prime-rAd boost combined with IL-2 adjuvant and carboplatin represents an attractive strategy to overcoming immune tolerance to tumors and has potential therapeutic benefits in melanoma cancer.     

盐酸洛拉曲克在体内、外对S-180细胞株的抗增殖作用

研究合合成新型胸苷合成酶抑制剂盐酸洛拉曲克在体内、外对S－180细胞株及正常人胚肾HEK293细胞的抗增殖作用;使用MTT法测定抑制率,以提高荷腹水瘤小鼠存活时间及荷实体瘤瘤重减轻情况为指标考察盐酸洛拉曲克对S－180所致肿瘤的治疗作用。结果表明：在体外盐酸洛拉曲克对S－180肿瘤细胞株有较强的细胞毒作用,对正常细胞HEK293抑制作用较弱（P＜0.05）;体内实验显示盐酸洛拉曲克可明显提高荷腹水瘤小鼠的存活时间,减轻荷实体瘤小鼠的瘤重,疗效与氟尿嘧啶（10mg/kg）相当（P＞0.05）或更好（P＜0.05）。可见,盐酸洛拉曲克在体内、外对S－180肿瘤细胞有显著的抗增殖作用,在体外对肿瘤细胞 具有选择性的抗增殖作用。     

Anticancer Effects of Chemokine Receptor 4(CXCR4) Gene Silenced by CXCR4-siRNA in Nude Mice Model of Ovarian Cancer

The aim is to study the anticancer effect of CXCR4 gene knockdown by CXCR4-siRNA in nude mice model of ovarian cancer. Injecti the SW626 tumor cells which had been transfected by vectors to make nude mouse model of ovarian cancer. The model mice were divided into interference group, negative control group, and blank control group. When the level of target genes were knocked down, the tumor volume was monitored and the tumor quality was measured; the expression of CXCR4 gene in the xenograft tumor was detected by RT-PCR, Western blot, and immunohistochemical staining. Nude mice model with implanted tumor were built successfully, after observing for 20 days. While the CXCR4 was knocked down, the abilities of invasion were weakened; the tumor volume and the tumor quality were also decreased. The CXCR4 mRNA and protein of the interference group decreased significantly (P < 0.05). The animal experiment was confirmed that silencing of CXCR4 gene by siRNA can obviously inhibit the tumorigenesis of ovarian cancer. Our work will provide the theoretical basis for genes interference therapy of ovarian cancer in future.     

Formononetin induces cell cycle arrest of human breast cancer cells via IGF1/PI3K/Akt pathways in vitro and in vivo

Formononetin is one of the main components of red clover plants, and is considered as a typical phytoestrogen. This study further investigated that formononetin inactivated IGF1/IGF1R-PI3K/Akt pathways and decreased cyclin D1 mRNA and protein expression in human breast cancer cells in vitro and in vivo. MCF-7 cells were treated with different concentrations of formononetin. The proliferation of the cells treated with formononetin was tested by MTT assay. The cell cycle in the treated cells was examined by flow cytometry. The levels of p-IGF-1?R, p-Akt, and cyclin D1 protein expression and cyclin D1?mRNA expression in the treated cells were determined by Western blot and RT-PCR, respectively. In addition, the antitumor activity of formononetin was evaluated in nude mice bearing orthotopic tumor implants. Compared with the control, formononetin inhibited the proliferation of MCF-7 cells and effectively induced cell cycle arrest. The levels of p-IGF-1?R, p-Akt, cyclin D1 protein expression, and cyclin D1?mRNA expression were also downregulated. On the other hand, formononetin also prevented the tumor growth of human breast cancer cells in nude mouse xenografts. These results show that formononetin causes cell cycle arrest at the G0/G1 phase by inactivating IGF1/IGF1R-PI3K/Akt pathways and decreasing cyclin D1?mRNA and protein expression, indicating the use of formononetin in the prevention of breast cancer carcinogenesis.