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author:("Jiang, weilin")
1.  Luteolin exerts a marked antitumor effect in cMet-overexpressing patient-derived tumor xenograft models of gastric cancer 
Aberrated activation of cMet in gastric cancer contributes to tumor growth, angiogenesis and metastasis. cMet-overexpressing gastric cancer has a poor prognosis because of high tumor metastasis and limited therapeutic options. Luteolin is a common dietary flavonoid with antitumor properties. However, the antitumor effect of luteolin on cMet-overexpressing gastric cancer remain unclear.
Two cMet-overexpressing patient-derived human tumor xenograft (PDTX) models of gastric cancer were established, and treated with luteolin or vehicle to evaluate the antitumor effects of luteolin. Tumor specimens were subjected to H&E staining and immunohistochemistry. MKN45 and SGC7901 cells that show high cMet expression were treated with varying concentrations of luteolin and evaluated by western blot, cell viability, apoptosis, migration, and invasion assays.
Luteolin inhibited the tumor growth in cMet-overexpressing PDTX models. Immunohistochemistry demonstrated that expression of cMet, MMP9 and Ki-67 were significantly down-regulated. Luteolin inhibited proliferation, promoted apoptosis and reduced the invasiveness of MKN45 and SGC7901 cells. Western blot revealed that luteolin promoted the activation of apoptosis-related proteins, caspase-3 and PARP-1, and down-regulated the invasion-associated protein, MMP9. Further studies demonstrated that luteolin decreased the expression and phosphorylation of cMet, and downstream phosphorylation of Akt and ERK. In addition, luteolin down-regulated phosphorylated Akt independently of cMet. Blocking Akt and/or ERK with the PI3K inhibitor, LY294002, or the ERK inhibitor, PD98059, induced down-regulation of MMP9 and up-regulation of cleaved caspase-3 and PARP-1, resembling the effects of luteolin.
Our findings ,for the first time, demonstrate that luteolin exerts marked antitumor effects in cMet-overexpressing PDTX models of gastric cancer, through a mechanism likely involving cMet/Akt/ERK signaling. These findings indicate that luteolin may act as a potential therapeutic option for cMet-overexpressing gastric cancer.
Electronic supplementary material
The online version of this article (doi:10.1186/s12967-015-0398-z) contains supplementary material, which is available to authorized users.
PMCID: PMC4320638  PMID: 25638174
Luteolin; cMet-overexpressing; Gastric cancer; Patient-derived tumor xenografts
2.  Effects of limiting energy availability via diet and physical activity on mammalian target of rapamycin-related signaling in rat mammary carcinomas 
Carcinogenesis  2012;34(2):378-387.
This study evaluated how different approaches to limiting energy availability (LEA) by 15% affected mammalian target of rapamycin (mTOR)-related signaling in mammary carcinomas. Female Sprague Dawley rats, injected with 50mg 1-methyl-1-nitrosourea per kilogram body weight, were randomized to a control or three LEA interventions: (i) sedentary and restricted rats fed to 85% of energy available to the control or motorized wheel running (37 m/min) for an average of (ii) 1621±55 (WRL) or (iii) 3094±126 (WRH) meters/day with food intake adjusted to provide the same net amount of available energy across LEA interventions. Under these conditions, LEA reduced overall cancer burden by 28% (P = 0.04) and down-regulated mTOR-related signaling (Hotelling multivariate, P = 0.002). Among the regulatory nodes assessed, reduced levels of activated protein kinase B (pAkt) and induction of sirtuin 1 (SIRT1) were the most influential factors in distinguishing between sham control and LEA carcinomas. P-Akt was predictive of observed changes in levels of proteins involved in cell cycle control (r = 0.698, P < 0.0001) and induction of apoptosis (r = –0.429, P = 0.014). Plasma insulin and leptin were strongly associated with carcinoma pAkt levels. Consistent with downregulation of mTOR-related signaling by LEA, evidence of decreased lipid synthesis in carcinomas was observed (Hotelling multivariate, P < 0.001) and was negatively correlated with SIRT1 induction. Despite large differences between control and LEA, effects on mTOR regulation were insufficient to distinguish among LEA intervention groups. Given the modest effects observed on the LKB1/AMP-activated protein kinase regulatory node, NADH and NADPH rather than ATP may be more limiting for tumor growth when LEA is 15%.
PMCID: PMC3564443  PMID: 23125225
3.  Effects of Energy Restriction and Wheel Running on Mammary Carcinogenesis and Host Systemic Factors in a Rat Model 
Limiting energy availability via diet or physical activity has health benefits; however, it is not known if these interventions have similar effects on the development of cancer. Two questions were addressed: 1) does limiting energy availability by increasing physical activity have the same effect on mammary carcinogenesis as limiting caloric intake, and 2) are host systemic factors, implicated as risk biomarkers for breast cancer, similarly affected by these interventions? Female Sprague Dawley rats were injected with 50 mg 1-methyl-1-nitrosourea/kg body weight at 21 days of age and randomized to one of five groups (30 rats/gp): 1) sham running wheel control; 2) restricted fed to 85% of the sham-control, 3 and 4) voluntary running in a motorized activity wheel (37 m/min) to a maximum of 3500 m/day or 1750 m/day, and 5) sedentary ad libitum fed control with no access to a running wheel. The three energetics interventions inhibited the carcinogenic response, reducing cancer incidence (P=0.01), cancer multiplicity (P<0.001), and cancer burden (P<0.001), while prolonging cancer latency (P=0.004) although differences among energetics interventions were not significant. Of the plasma biomarkers associated with the development of cancer, the energetics interventions reduced bioavailable insulin-like growth factor-1(IGF-1), insulin, interleukin-6, serum amyloid protein, tumor necrosis factor-α, and leptin and increased IGF binding protein 3 (IGFBP3) and adiponection. Plasma fasting glucose, C-reactive protein, estradiol, and progesterone were unaffected. The plasma biomarkers of greatest value in predicting the carcinogenic response were: adiponectin > IGF-1/IGFBP-3 > IGFBP-3 > leptin > IGF-1.
PMCID: PMC3294132  PMID: 22246620
physical activity; dietary energy restriction; mammary carcinogenesis; adipokines; insulin like growth factors; chronic inflammation
4.  Wheel Running Induced Changes in Plasma Biomarkers and the Carcinogenic Response in the 1-Methyl-1-Nitrosourea Induced Rat Model for Breast Cancer 
The purpose of this investigation was to identify pathways by which physical activity, implemented as running in an activity wheel, inhibits carcinogenesis. The focus of this analysis was on 20 plasma biomarkers for glucose homeostasis, inflammation, cytokine function and endocrine activity, known to be affected by a physically active lifestyle. Plasma for analysis was obtained from previously reported carcinogenesis experiments in which the effects on mammary carcinogenesis, induced by i.p. injection of 1-methyl-1-nitrosurea, of running on a motorized activity wheel or a non-motorized free wheel were compared to sedentary controls. Wheel running reduced cancer incidence (P=0.0004) and the number of cancers per animal (P=0.005). Principal components analysis (PCA) was used to reduce the 20 plasma biomarkers to a concise index that was significantly different by treatment group assignment (P<0.0001). Statistical analyses provided evidence that supported the hypothesis of a mediational role of these molecules in accounting for the protective effect of wheel running on the carcinogenic process. In addition, the plasma biomarker index derived from PCA was a good discriminator of treatment group assignment (only 4.5 % of animals were misclassified). These findings suggest that the plasma biomarkers evaluated have utility in assessing the breast cancer response to a physical activity intervention. Identification of such biomarkers is critical for clinical studies where evaluating the effects of physical activity on cancer outcomes (diagnosis, recurrence or mortality) is not possible.
PMCID: PMC2988103  PMID: 20876731
Physical activity; plasma biomarkers; mammary carcinogenesis; insulin like growth factor-1; wheel running
5.  Metformin as an energy restriction mimetic agent for breast cancer prevention 
This study examined whether metformin administration inhibited chemically induced mammary carcinogenesis in rats. In cancer prevention, metformin may act (1) indirectly through reducing systemic risk factors; or (2) directly through AMPK-mediated signaling. To begin to delineate clinically relevant mechanisms for breast cancer prevention, metformin was also studied along with dietary energy restriction.
Materials and Methods:
Mammary cancer was induced in female Sprague--Dawley rats (50 mg/kg MNU, i.p.). Metformin was fed alone (AIN93G + 0.05 to 1.0% w/w metformin) or combined with 40% dietary energy restriction. Plasma analytes (e.g., insulin, glucose, IGF-1) and protein expression (e.g., AMPK, mTOR, Akt) in mammary carcinomas and liver were evaluated. Additional studies included (1) aldehyde dehydrogenase flow cytometry, to gauge potential for cancer-initiated cells in mammary carcinomas to respond to metformin; (2) cell culture, to understand dose response (0.02--20 mM) of different cancer cell line molecular subtypes to metformin; and (3) analysis of a rat mammary epithelial cell microarray database, to examine expression of genes related to metformin pharmacokinetics (e.g., organic cation transporters) and pharmacodynamics (e.g., complex I of electron transport).
While a dosing regimen of 1.0%/0.25% metformin-reduced palpable mammary carcinoma incidence, multiplicity, and tumor burden and prolonged latency, lower doses of metformin failed to inhibit carcinogenesis despite effects on plasma insulin. Human breast cancer cell growth inhibition in response to metformin was only observed at high concentrations. Poor in vivo and in vitro response to metformin may be the result of pharmacokinetic (OCT-1 expression was low in rat mammary cells; OCT-3 was downregulated in mammary carcinoma) and pharmacodynamic (complex I transcripts were higher in mammary epithelial cells from carcinomas versus uninvolved gland) effects. In combination with dietary energy restriction, metformin offered protection against new tumor occurrence following release from combined treatment. Flow cytometry indicated the presence of cancer-initiated cells in mammary carcinomas.
As a single agent, metformin possessed limited cancer inhibitory activity. However, metformin may be an effective component of multiagent interventions that target cancer-initiated cells. There is a clear need to identify the conditions under which metformin is likely to benefit prevention and control of breast cancer.
PMCID: PMC3142764  PMID: 21799661
AMP-activated protein kinase; insulin; mammalian target of rapamycin; mammary carcinogenesis; metformin
6.  Effect of a low fat versus a low carbohydrate weight loss dietary intervention on biomarkers of long term survival in breast cancer patients ('CHOICE'): study protocol 
BMC Cancer  2011;11:287.
Weight loss in overweight or obese breast cancer patients is associated with an improved prognosis for long term survival. However, it is not clear whether the macronutrient composition of the chosen weight loss dietary plan imparts further prognostic benefit. A study protocol is presented for a dietary intervention to investigate the effects of weight loss dietary patterns that vary markedly in fat and carbohydrate contents on biomarkers of exposure to metabolic processes that may promote tumorigenesis and that are predictive of long term survival. The study will also determine how much weight must be lost for biomarkers to change in a favorable direction.
Approximately 370 overweight or obese postmenopausal breast cancer survivors (body mass index: 25.0 to 34.9 kg/m2) will be accrued and assigned to one of two weight loss intervention programs or a non-intervention control group. The dietary intervention is implemented in a free living population to test the two extremes of popular weight loss dietary patterns: a high carbohydrate, low fat diet versus a low carbohydrate, high fat diet. The effects of these dietary patterns on biomarkers for glucose homeostasis, chronic inflammation, cellular oxidation, and steroid sex hormone metabolism will be measured. Participants will attend 3 screening and dietary education visits, and 7 monthly one-on-one dietary counseling and clinical data measurement visits in addition to 5 group visits in the intervention arms. Participants in the control arm will attend two clinical data measurement visits at baseline and 6 months. The primary outcome is high sensitivity C-reactive protein. Secondary outcomes include interleukin-6, tumor necrosis factor-α, insulin-like growth factor-1 (IGF), IGF binding protein-3, 8-isoprostane-F2-alpha, estrone, estradiol, progesterone, sex hormone binding globulin, adiponectin, and leptin.
While clinical data indicate that excess weight for height is associated with poor prognosis for long term survival, little attention is paid to weight control in the clinical management of breast cancer. This study will provide information that can be used to answer important patient questions about the effects of dietary pattern and magnitude of weight loss on long term survival following breast cancer treatment.
Clinical Trial Registration
PMCID: PMC3150342  PMID: 21733177
biomarkers; dietary patterns; low fat; low carbohydrate; weight loss; breast cancer; long term survival
7.  Wheel running, skeletal muscle aerobic capacity and 1-methyl-1-nitrosourea induced mammary carcinogenesis in the rat 
Carcinogenesis  2010;31(7):1279-1283.
Emerging evidence indicates that intrinsic differences and induced changes in aerobic capacity are probably to play a critical role in the development of chronic diseases like cancer. This study was initiated: (i) to determine how citrate synthase activity, which is routinely used as a marker of aerobic capacity and mitochondrial density in skeletal muscle, was affected by voluntary running on either a motorized activity wheel or a non-motorized free wheel and (ii) to investigate the association between aerobic capacity and the carcinogenic response induced in the mammary gland by intraperitoneal injection of 1-methyl-1-nitrosurea. Overall, wheel running reduced cancer incidence (96 versus72%, P = 0.0006) and the number of cancers per animal (2.84 versus 1.78, P < 0.0001) and induced citrate synthase activity (276 versus 353 U/mg, P < 0.0001, sedentary control versus wheel running,respectively). Both motorized and free wheel running increased citrate synthase activity (373 ± 24, 329 ± 11 and 276 ± 9 U/mg protein, P < 0.0001) and reduced the average number of cancers per rat (2.84, 1.96 and 1.63, P < 0.01), sedentary control, free wheel and motorized wheel, respectively. However, regression analyses failed to provide evidence of a significant association between citrate synthase activity and either cancer incidence or cancer multiplicity. Citrate synthase activity is a single measure in a complex pathway that determines aerobic capacity. The multifaceted nature of intrinsic and inducible aerobic capacity limits the usefulness of citrate synthase activity alone in elucidating the relationship between aerobic capacity and the carcinogenic response.
PMCID: PMC2893798  PMID: 20299525
8.  In vivo molecular mediators of cancer growth suppression and apoptosis by selenium in mammary and prostate models: lack of involvement of gadd genes* 
Molecular cancer therapeutics  2009;8(3):682-691.
We used acute selenium (Se) treatments (i.e., daily single oral gavage of 2 mg Se per kg body weight for 3 days) of female Sprague-Dawley rats bearing 1-methyl-1-nitrosourea (MNU)-induced mammary carcinomas to increase the probability of detecting in vivo apoptosis and the associated gene/protein changes in the cancerous epithelial cells. The results show that whereas control carcinomas doubled in volume in 3 days, Se-methylselenocysteine (MSeC) and selenite treatments regressed approximately half of the carcinomas, accompanied by a 3-4 fold increase of morphologically observable apoptosis and ~ 40% inhibition of BrdU index of the cancerous epithelial cells. The mRNA levels of growth arrest-DNA damage inducible (gadd)-34, 45 and 153 genes were, contrary to expectation, not higher in the Se-treated carcinomas than in the gavage or diet restriction control groups. The gadd34 and 153 proteins were localized in the non-epithelial cells, and not induced in the cancer epithelial cells of the Se-treated carcinomas. On the other hand, both Se forms decreased the expression of cyclin D1, and increased levels of P27Kip1 and JNK activation in a majority of the mammary carcinomas. Furthermore, the lack of induction of gadd genes in vivo by methylseleninic acid was confirmed in human prostate xenograft model in athymic nude mice. In summary, these experiments demonstrated the induction of cancer epithelial cell apoptosis and inhibition of cell proliferation by Se in vivo, through potential involvement of Cyclin D1, P27Kip1 and JNK pathways. They cast doubt on the 3 gadd genes as mediators of Se action in vivo.
PMCID: PMC2822709  PMID: 19276161
selenium; mammary cancer; prostate cancer; gadd genes; apoptosis
9.  Collection of Epithelial Cells from Rodent Mammary Gland Via Laser Capture Microdissection Yielding High-Quality RNA Suitable for Microarray Analysis 
Laser capture microdissection (LCM) enables collection of cell populations highly enriched for specific cell types that have the potential of yielding critical information about physiological and pathophysiological processes. One use of cells collected by LCM is for gene expression profiling. Samples intended for transcript analyses should be of the highest quality possible. RNA degradation is an ever-present concern in molecular biological assays, and LCM is no exception. This paper identifies issues related to preparation, collection, and processing in a lipid-rich tissue, rodent mammary gland, in which the epithelial to stromal cell ratio is low and the stromal component is primarily adipocytes, a situation that presents numerous technical challenges for high-quality RNA isolation. Our goal was to improve the procedure so that a greater probe set present call rate would be obtained when isolated RNA was evaluated using Affymetrix microarrays. The results showed that the quality of RNA isolated from epithelial cells of both mammary gland and mammary adenocarcinomas was high with a probe set present call rate of 65% and a high signal-to-noise ratio.
PMCID: PMC3055717  PMID: 21406068
Laser capture microdissection; Mammary epithelial cells; RNA quality
10.  Effect of Nonmotorized Wheel Running on Mammary Carcinogenesis: Circulating Biomarkers, Cellular Processes, and Molecular Mechanisms in Rats 
The objective of this experiment was to identify circulating growth factors, hormones, and cellular and molecular mechanisms that account for the effects of physical activity on mammary carcinogenesis. A total of 120 female Sprague-Dawley rats were injected with 1-methyl-1-nitrosourea (50 mg/kg) and 7 days thereafter were randomized to either a physically active or a sedentary control group. Individually housed rats were given free access to a nonmotorized, computer-controlled activity wheel and running behavior was reinforced by food reward. Rats self-determined their daily intensity and duration of running. Sedentary control rats received the same amount of food as the physically active rats to which they were paired. Physical activity reduced mammary cancer incidence (P = 0.015) and cancer multiplicity (P = 0.01). Physical activity induced changes in plasma insulin, insulin-like growth factor-I, and corticosterone, suggesting that mechanisms regulating glucose homeostasis were affected. Western blot analyses of mammary carcinomas revealed that proteins involved in cell proliferation were reduced (P < 0.001) and those involved in apoptosis via the mitochondrial pathway were elevated (P < 0.001) by physical activity. The hypothesis that these effects were mediated by activation of AMP-activated protein kinase, and down-regulation of protein kinase B, which collectively down-regulate the activity of the mammalian target of rapamycin, was evaluated. Evidence in support of this hypothesis was found in the Western blot analyses of mammary carcinomas, mammary gland, liver, and skeletal muscle. Collectively, these findings provide a rationale for additional studies of energy-sensing pathways in the elucidation of mechanisms that account for the inhibition of carcinogenesis by physical activity.
PMCID: PMC2667869  PMID: 18708381
11.  Dietary Energy Restriction Modulates the Activity of AMPK, Akt, and mTOR in Mammary Carcinomas, Mammary Gland, and Liver1 
Cancer research  2008;68(13):5492-5499.
Dietary energy restriction (DER) inhibits mammary carcinogenesis yet mechanisms accounting for its protective activity have not been fully elucidated. In this study we tested the hypothesis that DER exerts effects on intracellular energy sensing pathways resulting in alterations of phosphorylated proteins that play a key role in the regulation of cancer. Experiments were conducted using the 1-methyl-1-nitrosourea-induced mammary cancer model in which rats were 0, 20, or 40% energy restricted during the post initiation stage of carcinogenesis. Parallel experiments were done in non carcinogen treated rats in which effects of DER at 0, 5, 10, 20, or 40% in liver were investigated. In a DER dose dependent manner, levels of Thr-172 phosphorylated AMP-activated protein kinase (AMPK) increased in mammary carcinomas with a concomitant increase in phosphorylated acetyl-CoA-carboxylase, a direct target of AMPK, the phosphorylation of which is regarded as an indicator of AMPK activity. Levels of phosphorylated mTOR decreased with increasing DER and down regulation of mTOR activity was verified by a decrease in the phosphorylation state of two mTOR’s targets, 70-kDa ribosomal protein S6 kinase (p70S6K) and 4E-binding protein 1 (4E-BP1). Coincident with changes in mTOR phosphorylation, levels of activated protein kinase B (Akt) were also reduced. Similar patterns were observed in mammary gland and in liver of non carcinogen treated rats. This work identifies components of intracellular energy sensing pathways, specifically mTOR, its principal up-stream regulators, AMPK and Akt, and its downstream targets, p70S6K and 4E-BP1 as candidate molecules on which to center mechanistic studies of DER.
PMCID: PMC2587286  PMID: 18593953
dietary energy restriction; AMP-activated protein kinase; mammalian target of rapamycin; protein kinase B (Akt); mammary carcinogenesis

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