Downregulated AKT Expression Research Articles

Uncovering the Key Targets and Therapeutic Mechanisms of Qizhen Capsule in Gastric Cancer through Network Pharmacology and Bioinformatic Analyses.

Objective. This study is aimed at screening out effective active compounds of Qizhen capsule (QZC) and exploring the underlying mechanisms against gastric cancer (GACA) by combining both bioinformatic analysis and experimental approaches. Weighted gene coexpression network analysis (WGCNA), network pharmacology, molecular docking simulation, survival analysis, and data-based differential gene and protein expression analysis were employed to predict QZC's potential targets and explore the underlying mechanisms. Subsequently, multiple experiments, including cell viability, apoptosis, and protein expression analyses, were conducted to validate the bioinformatics-predicted therapeutic targets. The results indicated that luteolin, rutin, quercetin, and kaempferol were vital active compounds, and TP53, MAPK1, and AKT1 were key targets. Molecular docking simulation showed that the four abovementioned active compounds had high binding affinities to the three main targets. Enrichment analysis showed that vital active compounds exerted therapeutic effects on GACA through regulating the TP53 pathway, MAPK pathway, and PI3K/AKT pathway. Furthermore, data-based gene expression analysis revealed that TP53 and JUN genes were not only differentially expressed between normal and GACA tissues but also correlated with clinical stages. In parallel, in vitro experimental results suggested that QZC exerted therapeutic effects on GACA by decreasing IC50 values, downregulating AKT expression, upregulating TP53 and MAPK expression, and increasing apoptosis of SGC-7901 cells. This study highlights the potential candidate biomarkers, therapeutic targets, and basic mechanisms of QZC in treating GACA, providing a foundation for new drug development, target mining, and related animal studies in GACA.

Splicing factor SRSF3 promotes the progression of cervical cancer through regulating DDX5.

Aberrant alternative splicing (AS) profoundly affects tumorigenesis and cancer progression. Serine/arginine-rich splicing factor 3 (SRSF3) regulates the AS of precursor mRNAs and acts as a proto-oncogene in many tumors, but its function and potential mechanisms in cervical cancer remain unclear. Here, we found that SRSF3 was highly expressed in cervical cancer tissues and that SRSF3 expression was correlated with prognosis after analyses of the The Cancer Genome Atlasand GEO databases. Furthermore, knockdown of SRSF3 reduced the proliferation, migration, and invasion abilities of HeLa cells, while overexpression of SRSF3 promoted proliferation, migration, and invasion of CaSki cells. Further studies showed that SRSF3 mediated the variable splicing of exon 12 of the transcriptional cofactor DEAD-box helicase 5(DDX5). Specifically, overexpression of SRSF3 promoted the production of the pro-oncogenic spliceosome DDX5-L and repressed the production of the repressive spliceosome DDX5-S. Ultimately, both SRSF3 and DDX5-L were able to upregulate oncogenic AKT expression, while DDX5-S downregulated AKT expression. In conclusion, we found that SRSF3 increased the production of DDX5-L and decreased the production of DDX5-S by regulating the variable splicing of DDX5. This, in turn promoted the proliferation, migration, and invasion of cervical cancer by upregulating the expression level of AKT. These results reveal the oncogenic role of SRSF3 in cervical cancer and emphasize the importance of the SRSF3-DDX5-AKT axis in tumorigenesis. SRSF3 and DDX5 are new potential biomarkers and therapeutic targets for cervical cancer.

Akt Inhibition Enhanced the Growth Inhibition Effects of Low-Dose Heavy-Ion Radiation via the PI3K/Akt/p53 Signaling Pathway in C6 Glioblastoma Cells.

BackgroundGlioma has one of the highest mortality rates of all tumors of the nervous system and commonly used treatments almost always fail to achieve tumor control. Low-dose carbon-ion radiation can effectively target cancer and tumor cells, but the mechanisms of growth inhibition induced by heavy-ion radiation via the PI3K/Akt signaling pathway are unknown, and inhibition by heavy-ion radiation is minor in C6 cells.MethodsCarbon-ion radiation was used to investigate the effects of heavy-ion radiation on C6 cells, and suppression of Akt was performed using perifosine. MTT assays were used to investigate optimal perifosine treatment concentrations. Clone formation assays were used to investigate the growth inhibition effects of carbon-ion radiation and the effects of radiation with Akt inhibition. Lactate dehydrogenase release, superoxide dismutase activity, and malondialdehyde content were assessed to investigate oxidative stress levels. Expression levels of proteins in the PI3K/Akt/p53 signaling pathway were assessed via western blotting.ResultsThe 10% maximum inhibitory concentration of perifosine was 19.95 μM. In clone formation assays there was no significant inhibition of cell growth after treatment with heavy-ion irradiation, whereas perifosine enhanced inhibition. Heavy-ion radiation induced lactate dehydrogenase release, increased the level of malondialdehyde, and reduced superoxide dismutase activity. Akt inhibition promoted these processes. Heavy-ion radiation treatment downregulated Akt expression, and upregulated B-cell lymphoma-2 (Bcl-2) expression. p53 and Bcl-2 expression were significantly upregulated, and Bcl-2-associated X protein (Bax) expression was downregulated. The expression profiles of pAkt, Bcl-2, and Bax were reversed by perifosine treatment. Caspase 3 expression was upregulated in all radiation groups.ConclusionsThe growth inhibition effects of low-dose heavy-ion irradiation were not substantial in C6 cells, and Akt inhibition induced by perifosine enhanced the growth inhibition effects via proliferation inhibition, apoptosis, and oxidative stress. Akt inhibition enhanced the effects of heavy-ion radiation, and the PI3K/Akt/p53 signaling pathway may be a critical component involved in the process.

Alliin inhibits adipocyte differentiation by downregulating Akt expression: Implications for metabolic disease.

Obesity is currently an important health problem and is associated with an increased likelihood of various diseases. The efficacies of various natural treatments have been assessed for their utility in treating obesity. Alliin (S-allyl-L-cysteine sulfoxides) is considered the major component of garlic and has a wide range of natural antioxidant properties. However, the direct effects of alliin on obesity have not been well clarified. The present study investigated the effects and possible mechanisms of alliin on adipocyte differentiation. The 3T3-L1 cells were treated with alliin (0-40 µg/ml) during adipogenic differentiation. The effect of alliin on lipid accumulation was evaluated by Oil red O staining. Reverse transcription-quantitative PCR was performed to investigate the expression levels of adipogenic differentiation-related genes. The accumulation of lipid droplets was markedly inhibited following alliin treatment. The expression levels of multiple adipogenic transcription markers, such as CCAAT/enhancer-binding protein (C/EBP) β, C/EBP α and peroxisome proliferation-activity receptor γ, were markedly decreased following treatment with alliin during adipogenic differentiation. Expression levels of several adipocyte-related genes were subsequently suppressed. Additionally, alliin suppressed PKB/Akt and PI3K expression. These results suggested that alliin exhibits anti-adipogenic activity by downregulating major adipogenic differentiation-related genes and Akt/PI3K expression. Alliin may have a potential therapeutic effect on metabolic disease.

Metformin and asarone inhibit HepG2 cell proliferation in a high glucose environment by regulating AMPK and Akt signaling pathway

BackgroundMetabolic dysregulation is one of the hallmarks of tumor cell proliferation. Evidence indicates the potential role of the 5′adenosine monophosphate-activated protein kinase (AMPK) and protein kinase B/Akt signaling pathway in regulating cell proliferation, survival, and apoptosis. The present study explores the effect of metformin HCl and the combination of α- and β-asarone on the proliferation of HepG2 cells in the presence of high glucose levels simulating the diabetic-hepatocellular carcinoma (HCC) condition.ResultsThe metformin and asarone reduced HepG2 cell viability in a dose-dependent manner and induced morphological changes as indicated by methyl thiazolyl tetrazolium (MTT) assay. The metformin and asarone arrested the cells at the G0/G1 phase, upregulated the expression of AMPK, and downregulated Akt expression in high glucose conditions as identified by the flow cytometry technique. Further, the upregulated AMPK led to a decrease in the expression of phosphoenolpyruvate carboxykinase-2 (PCK-2) and sterol regulatory element-binding protein-1 (SREBP-1).ConclusionThe anti-proliferative effect of metformin and asarone in the diabetic-HCC condition is mediated via AMPK and Akt pathway.

Four and a Half LIM Domains 2 (FHL2) Contribute to the Epithelial Ovarian Cancer Carcinogenesis.

Epithelial ovarian cancer (EOC) is one of the most lethal gynecologic malignancies. To date, the etiology of this deadly disease remains elusive. FHL2, a member of the four and a half LIM domain family, has been shown to serve either as an oncoprotein or as a tumor suppressor in various cancers. Our previous study showed that FHL2 plays a critical role in the initiation and progression of ovarian granulosa cell tumor via regulating AKT1 transcription. However, direct and systematic evidence of FHL2 in the initiation and progression of EOC remains unclear. In the present study, immunohistochemical analysis from EOC patient tissues showed that positivity and intensity of FHL2 immunosignal were up-regulated in the EOC tissues compared with normal ovary tissues. Knockdown of FHL2 in SKOV-3 cell line reduced cell growth and cell viability, blocked cell cycle progression, and inhibited cell migration. Ectopic expression of FHL2 in IGROV-1 cells which have low endogenous FHL2, promoted cell growth, improved cell viability and enhanced cell migration. Additionally, knock down of FHL2 in the SKOV-3 cell line significantly inhibited anchorage-independent growth indicated by the soft agar assay. In comparison, overexpression of FHL2 in IGROV-1 cell improved the colonies growth in soft agar. Western blot data showed that knockdown of FHL2 downregulated AKT expression level, and upregulated apoptosis related proteins such as cleaved PARP, and cleaved-lamin A. Finally, by employing stable SKOV-3/FHL2 stable knock down cell line, our data clearly showed that knockdown of FHL2 inhibited EOC xenograft initiation in vivo. Taken together, our results showed that FHL2, via regulating cell proliferation, cell cycle, and adhesion, has a critical role in regulating EOC initiation and progression. These results indicate that FHL2 could be a potential target for the therapeutic drugs against EOC.

Secoisolariciresinol Diglucoside Exerts Anti-Inflammatory and Antiapoptotic Effects through Inhibiting the Akt/IκB/NF-κB Pathway on Human Umbilical Vein Endothelial Cells.

Inflammation is a key regulator in the progression of atherosclerosis (AS) which extremely affects people's health. Secoisolariciresinol diglucoside (SDG), a plant lignan, is relevant to angiogenesis and cardioprotection against ischemia-reperfusion injury and improves vascular disorders. However, the effect of SDG on cardiovascular disorder is not clear. In the present study, we aimed to investigate the effects of SDG on lipopolysaccharide- (LPS-) stimulated Human Umbilical Vein Endothelial Cells (HUVECs) and elucidate the underlying mechanism. The LPS-stimulated HUVEC cellular model was established. The cell viability, the cell tube formation activity, the nitric oxide (NO) release, the levels of inflammatory cytokine interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), the activation of nuclear factor kappa-B (NF-κB) pathway, and the expression of protein kinase B (Akt) were determined using Cell Counting Kit-8, cell tube-formation assay, western blotting, and enzyme-linked immunosorbent assay. Our results revealed that SDG reduces the angiogenic capacity of HUVECs and inhibited LPS-mediated HUVEC injury and apoptosis. In addition, SDG increased NO release and decreased the levels of IL-1β, IL-6, and TNF-α in LPS-treated HUVECs. Meanwhile, SDG inhibited the NF-κB pathway and downregulated Akt expression in LPS-induced HUVECs. Our results indicated that SDG relieves LPS-mediated HUVEC injury by inhibiting the NF-κB pathway which is partly dependent on the disruption of Akt activation. Therefore, SDG exerts its cytoprotective effects in the context of LPS-treated HUVECs via regulation of the Akt/IκB/NF-κB pathway and may be a potential treatment drug for cardiovascular disease.

GLI3 repressor determines Hedgehog pathway activation and is required for response to SMO antagonist glasdegib in AML

AbstractThe Hedgehog (Hh) signaling pathway is activated in many cancers and is a promising target for therapeutic development. Deletions in the receptor Patched (PTCH) or activating mutations in Smoothened (SMO) have been reported in basal cell carcinoma and medulloblastoma, but are largely absent in most tumor types. Therefore, the mechanism of pathway activation in most cancers, including hematological malignancies, remains unknown. In normal tissues, Hh pathway activation via PTCH/SMO causes an increase in the downstream transcriptional activator GLI1 and a decrease in the GLI3 transcriptional repressor (GLI3R). In this article, we confirm that the Hh pathway is active in acute myeloid leukemia (AML), however, this activity is largely independent of SMO. Epigenetic and gene expression analysis of The Cancer Genome Atlas AML data set reveals that GLI3 expression is silenced in most AML patient samples, and the GLI3 locus is abnormally methylated. We show that GLI3R is required for the therapeutic effect of SMO antagonists in AML samples and restoration of GLI3R suppresses the growth of AML. We additionally demonstrate that GLI3R represses AML growth by downregulating AKT expression. In summary, this study provides the first evidence that GLI3R plays an essential role in SMO-independent Hh signaling in AML, and suggests that GLI3R could serve as a potential biomarker for patient selection in SMO antagonist clinical trials. Furthermore, these data support rational combinations of hypomethylating agents with SMO antagonists in clinical trials.

Dihydromyricetin induces mitochondria-mediated apoptosis in HepG2 cells through down-regulation of the Akt/Bad pathway

The plant flavonol dihydromyricetin (DHM) was reported to induce apoptosis in human hepatocarcinoma HepG2 cells. This study was undertaken to elucidate the underlying molecular mechanism of action of DHM. In the study, DHM down-regulated Akt expression and its phosphorylation at Ser473, up-regulated the levels of mitochondrial proapoptotic proteins Bax and Bad, and inhibited the phosphorylation of Bad at Ser136 and Ser112. It also inhibited the expression of the antiapoptotic protein Bcl-2 and enhanced the cleavage and activation of caspase-3 as well as the degradation of its downstream target poly(ADP-ribose) polymerase. Our results for the first time suggest that DHM-induced apoptosis in HepG2 cells may come about by the inhibition of the Akt/Bad signaling pathway and stimulation of the mitochondrial apoptotic pathway. Dihydromyricetin may be a promising therapeutic medication for hepatocellular carcinoma.

Flaxseed and its components differentially affect estrogen targets in pre-neoplastic hen ovaries

Flaxseed has been studied for decades for its health benefits that include anti-cancer, cardio-protective, anti-diabetic, anti-inflammatory properties. The biologically active components that mediate these effects are the omega-3 fatty acids and the lignan, secoisolariciresinol diglucoside. We have previously shown that whole flaxseed supplemented diet decreases the severity and incidence of ovarian cancer while a 15% dose of flaxseed is most protective against inflammation and estrogen-induced chemical and genotoxicity. The objective of this study was to dissect the independent effects of the two flaxseed components on estrogen signaling and metabolism. Two and half year old hens were fed either a control diet, 15% whole flaxseed diet, defatted flax meal diet or 5% flax oil diet for 3 months after which the animals were sacrificed and blood and tissues were harvested. Whole flaxseed diet caused a decrease in expression of ERα. ERα target gene expression was assessed using RT2 profiler PCR array. Some targets involved in the IGF/insulin signaling pathway (IRS1, IGFBP4, IGFBP5) were downregulated by flaxseed and its components. Flaxseed diet also downregulated AKT expression. A number of targets related to NF-kB signaling were altered by flaxseed diet including a series of targets implicated in cancer. Whole flaxseed diet also affected E2 metabolism by increasing CYP1A1 expression with a corresponding increase in the onco-protective E2 metabolite, 2-methoxyestradiol.The weak anti-estrogens, enterolactone, enterodiol and 2-methoxyestradiol, might be working synergistically to generate a protective effect on the ovaries from hens on whole flaxseed diet by altering the estrogen signaling and metabolism.

Schistosoma japonicum soluble egg antigens facilitate hepatic stellate cell apoptosis by downregulating Akt expression and upregulating p53 and DR5 expression.

BackgroundThe induction of hepatic stellate cell (HSC) apoptosis has potential as a potent strategy to diminish the progression of liver fibrosis. Previous studies have demonstrated the ability of soluble egg antigens (SEA) from schistosomes to inhibit HSC activation and to induce apoptosis in vitro. In this study, we aimed to explore the mechanism of SEA-induced apoptosis in HSCs.Methodology/Principal FindingsIn this study, we found that SEA could upregulate p53 and DR5 and downregulate the p-Akt. The apoptosis of HSCs induced by SEA could be reduced in HSCs that were treated with p53-specific siRNA and in HSCs that were treated with DR5-specific shRNA. In addition, GW501516, which enhances the expression of Akt, could also decrease the SEA-induced HSC apoptosis. We also found that the increased expression of p53 and DR5 induced by SEA through Mdm2 were reduced by GW501516.Conclusions/SignificanceOur data suggest that SEA can induce HSC apoptosis by downregulating Akt expression and upregulating p53-dependent DR5 expression.

Salinomycin inhibits Akt/NF-κB and induces apoptosis in cisplatin resistant ovarian cancer cells

BackgroundDespite advances in treatment, ovarian cancer is the most lethal gynecologic malignancy. Therefore significant efforts are being made to develop novel strategies for the treatment of ovarian cancer. Salinomycin has been shown to be highly effective in the elimination of cancer stem cells both in vitro and in vivo. The present study focused on investigating important cell signaling molecules such as Akt and NF-κB during salinomycin-induced apoptosis in cisplatin resistant ovarian cancer cells (A2780cis). MethodsMTT assay was performed to determine cell viability. Flow cytometry and DNA fragmentation assay were performed to analyze the effect on cell cycle and apoptosis. The expression of apoptosis related proteins was evaluated by Western blot analysis. ResultsThe cell viability was significantly reduced by salinomycin treatment in a dose dependent manner. The flow cytometry result showed an increase in sub-G1 phase. Salinomycin inhibited the nuclear transportation of NF-κB, and downregulated Akt expression. Declined Bcl-2, activation of caspase-3 and increased PARP cleavage triggered apoptosis. Moreover, DNA fragmentation assay also revealed apoptotic induction. ConclusionThe result suggested that salinomycin-induced apoptosis in A2780cis was associated with inhibition of Akt/NF-κB. It may become a potential chemotherapeutic agent for the cisplatin resistant ovarian cancer therapy.

Abstract 1423: Effects of DFMO-based combination therapy in advanced stage neuroblastoma

Abstract Neuroblastoma (NB) is the most common extra-cranial pediatric tumor. The survival probability for NB patients with disseminated disease and chemo-resistant tumors are despairingly low. Therefore, understanding the mechanisms underlying NB metastasis and chemo-resistance will be critical for developing effective chemotherapeutic strategies for NB in the future. The goal of the present study was to determine the efficacy of DFMO-based combination therapy in NB with MYCN over-expression. MYCN is a transcription factor that directly regulates the expression of ornithine decarboxylase (ODC), a polyamine biosynthesis enzyme that has been shown to be critical for the development of MYCN-amplified NB. Our previous studies have shown that the ODC inhibitor, alpha-difluoromethylornithine (DFMO), activates two opposing pathways in NB cells, one that induced G1 cell cycle arrest by regulating p27kip1 expression and phosphorylation, and a second pathway that activated Akt/PKB signaling to promote cell survival (Cancer Res, 2008, 68:9825-31). Therefore, in the present study, we investigated the efficacy of DFMO-based combinations with PI3K/Akt inhibitors for the treatment of NB. Intracellular polyamines were depleted with the ODC inhibitor α-difluoromethylornithine (DFMO), and the effects of the PI3K and Akt inhibitors on NB cell proliferation, migration and invasion were examined in NB cells. Our results show that DFMO and PI3K/Akt inhibitors were able to significantly inhibit NB proliferation, migration and invasion. However, the inhibitory effects were significantly augmented when DFMO was combined with either the PI3K or Akt inhibitor. In addition, these inhibitors, when administered alone, were able to induce p27kip1 protein accumulation, an effect that was enhanced when DFMO was combined with either the PI3K or Akt inhibitor. These combination treatments appeared to be more effective in NB cells over-expressing MYCN. Furthermore, down-regulating Akt expression also attenuated the anti-apoptotic effect induced by DFMO. The results suggest that the combination of DFMO with PI3K/Akt inhibitors is particularly effective at inhibiting cell proliferation, migration and invasion of NB cells over-expressiong MYCN, and the mechanism by which these processes are regulated involve p27kip1. Overall, this study provides further evidence that targeting both the polyamine synthesis pathway and the PI3K/Akt signaling pathway simultaneously may be an effective treatment strategy for NB, particularly because this combination inhibits multiple steps in the malignant progression of NB. In addition, this drug combination appears to be more effective in cells over-expressing MYCN, suggesting that the efficacy of this combination treatment may be greater in advanced stage NB with MYCN gene amplification. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1423. doi:10.1158/1538-7445.AM2011-1423

Synergistic induction of apoptosis in HeLa cells by the proteasome inhibitor bortezomib and histone deacetylase inhibitor SAHA

Proteasome inhibitors and histone deacetylase (HDAC) inhibitors are two promising groups of anti-cancer agents. In this study, we examined the apoptotic effects of the proteasome inhibitor bortezomib and HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) in human cervical carcinoma HeLa cells. Compared to treatment with bortezomib or SAHA alone, co-exposure with these two agents synergistically resulted in the massive apoptosis of HeLa cells, consistent with a significant increase in caspase-3 activation. We then investigated the mechanisms underlying this effect. The combination of bortezomib and SAHA caused an increase in the ratio of bax/bcl-2 expression, inhibited the nuclear transportation of NF-κB, and down-regulated Akt expression and phosphorylation in HeLa cells. In conclusion, bortezomib and SAHA cooperatively stimulate apoptosis in HeLa cells through the inhibition of several cyto-protective signalling pathways. This is the first report of synergistic apoptotic effect achieved with a proteasome inhibitor and HDAC inhibitor in HeLa cells. Thus, the results build the framework for clinical trials using combined proteasome and HDAC inhibition in the treatment of human cervical carcinoma.

Two specific inhibitors of the phosphatidylinositol 3-kinase LY294002 and wortmannin up-regulate β1,4-galactosyltransferase I and thus sensitize SMMC-7721 human hepatocarcinoma cells to cycloheximide-induced apoptosis

Previous study indicated that beta1,4-galactosyltransferase I (beta1,4GT1) was up-regulated by cycloheximide (CHX) and thus enhanced apoptosis induced by CHX in SMMC-7721 cells. In this study, we reported that constitutively active Akt protein (myr-Akt) inhibited CHX-induced apoptosis in SMMC-7721 cells through down-regulating beta1,4GT1. However, the two PI3K inhibitors LY294002 and wortmannin treatment up-regulated beta1,4GT1 through enhancing Sp1 protein expression and consequently increased CHX-induced SMMC-7721 cells apoptosis. Besides, our results suggested that beta1,4GT1 and cell surface galactose residues synthesized by elevated beta1,4GT1 played an important role in SMMC-7721 cells apoptosis treated with CHX and PI3K inhibitor together. Moreover, we found that CHX accentuated beta1,4GT1 through down-regulating Akt expression to mediate SMMC-7721 cells apoptosis. Taken together, PI3K inhibitors LY294002 and wortmannin up-regulated beta1,4GT1 and enhanced CHX-induced apoptosis in SMMC-7721 cells, which suggested that PI3K inhibitors might have therapeutic potential when combined with CHX in the treatment of hepatoma.

22. The competitive action of 16β-ethyl estradiol on the binding of estrogen receptor in human breast cancer

Flaxseed has been studied for decades for its health benefits that include anti-cancer, cardio-protective, anti-diabetic, anti-inflammatory properties. The biologically active components that mediate these effects are the omega-3 fatty acids and the lignan, secoisolariciresinol diglucoside. We have previously shown that whole flaxseed supplemented diet decreases the severity and incidence of ovarian cancer while a 15% dose of flaxseed is most protective against inflammation and estrogen-induced chemical and genotoxicity. The objective of this study was to dissect the independent effects of the two flaxseed components on estrogen signaling and metabolism. Two and half year old hens were fed either a control diet, 15% whole flaxseed diet, defatted flax meal diet or 5% flax oil diet for 3 months after which the animals were sacrificed and blood and tissues were harvested. Whole flaxseed diet caused a decrease in expression of ERα. ERα target gene expression was assessed using RT2 profiler PCR array. Some targets involved in the IGF/insulin signaling pathway (IRS1, IGFBP4, IGFBP5) were downregulated by flaxseed and its components. Flaxseed diet also downregulated AKT expression. A number of targets related to NF-kB signaling were altered by flaxseed diet including a series of targets implicated in cancer. Whole flaxseed diet also affected E2 metabolism by increasing CYP1A1 expression with a corresponding increase in the onco-protective E2 metabolite, 2-methoxyestradiol.The weak anti-estrogens, enterolactone, enterodiol and 2-methoxyestradiol, might be working synergistically to generate a protective effect on the ovaries from hens on whole flaxseed diet by altering the estrogen signaling and metabolism.