Yihong Kaufmann

Effect of dietary glutamine on tumor glutathione levels and apoptosis-related proteins in DMBA-induced breast cancer of rats

Glutamine (GLN) is a non-essential amino acid that is present in nearly every biochemical pathway and is the major intraorgan nitrogen carrier. GLN via glutamate, is one of the precursors for the synthesis of glutathione (GSH), the major endogenous antioxidant in mammalian cells, which protects them from oxidative injury and cell death. Cancer cells have higher GSH levels than the surrounding normal cells, which attributes to a higher rate of cell proliferation and resistance to chemotherapy. Therefore, selective tumor depletion of GSH presents a promising strategy in cancer treatment. Experimental studies have associated decreased GSH levels with inhibition of proliferation and stimulation of apoptosis. Previous results of our laboratory have provided evidence that dietary GLN diminished tumor development in implantable as well as 7,12-dimethylbenz[a]anthracene (DMBA)-induced breast cancer and elevated GSH in the host tissues. In this study we examined the effects of GLN on GSH levels in DMBA-induced mammary tumors and correlated the results with protein and mRNA expression of apoptosis-related proteins Bcl-2, Bax and caspase-3 in tumor cells. The results have shown that GLN supplementation caused a significant decrease in the tumor GSH levels and the ratio GSH/oxidized GSH (GSSG), accompanied by up-regulation of Bax and caspase-3, and down-regulation of Bcl-2. These findings suggest that dietary GLN supplementation suppresses mammary carcinogenesis by activation of apoptosis in tumor cells and this probably is a result of GSH down-regulation.

Tamoxifen and raloxifene suppress the proliferation of estrogen receptor-negative cells through inhibition of glutamine uptake

PurposeModulation of estrogen receptor (ER) plays a central role in selective estrogen receptor modulators (SERMs) molecular mechanism of action, although studies have indicated that additional, non-ER-mediated mechanisms exist. It has been suggested that the induction of oxidative stress by SERM could be one of the non-ER-mediated mechanisms held responsible for their pro-apoptotic role in ER-negative cells. Tumor cells are known for their high requirement of glutamine (Gln) that serves multiple functions within the cells, including nutritional and energy source, as well as one of the precursors for the synthesis of natural antioxidant glutathione (GSH). We hypothesized that one of the mechanisms responsible for ER-independent anti-neoplastic properties of SERMs and also for their adverse side effects could be dependent on the inhibition of Gln uptake.MethodsHuman ER-negative MDA-MB231 breast cancer cells were treated with different doses of Tam and Ral. Gln uptake was monitored by using [3H]Gln assay. The effect of Tam and Ral on Gln transporter ASCT2 expression, glutathione (GSH) levels and cellular proliferation was determined.ResultsTam and Ral inhibited Gln uptake in a dose-dependent manner through inhibition of ASCT2 Gln transporter. This effect of the anti-estrogens was associated with inhibition of GSH production and apoptosis. Treatment of cells with N-acetyl L-cysteine and 17 beta-estradiol 2 reversed the effects of Ral and Tam.ConclusionsOur results indicate that one of the mechanisms of action (and possibly some of the side effects) of TAM and RAL is associated with inhibition of cellular Gln uptake, oxidative stress and induction of apoptosis.

Oral Glutamine Protects against Acute Doxorubicin-Induced Cardiotoxicity of Tumor-Bearing Rats

Doxorubicin (DOX), a widely used anticancer drug, has a dose-dependent cardiotoxicity, attributed mainly to free radical formation. The cardiomyocyte oxidative stress occurs rapidly after DOX treatment, resulting in harmful modifications to proteins, lipids, and DNA. Previous data showed that oral l-glutamine (Gln) prevented cardiac lipid peroxidation and maintained normal cardiac glutathione (GSH) levels in DOX-treated rats. Our aim in this study was to examine the effect of Gln on DOX-induced cardiac oxidative stress in a tumor-bearing host. Female Fisher344 rats with implanted MatBIII mammary tumors were randomized into 2 groups: a Gln group that received l-Gln (1 g.kg(-1).d(-1)) (n = 10) via a Gln-enriched diet and/or gavage with 50% Gln suspension during the whole experiment and a control group that was fed the same diet formulation without Gln and/or were gavaged with water. All rats received a single injection of 12 mg/kg DOX and were killed 3 d later. GSH levels of hearts, livers, tumors, and blood, as well as cardiac histological alterations, lipid peroxidation, peroxinitrite levels, and caspase-3 activation were determined. Cardiac physiologic alterations were assessed by ultrasound imaging before and 3 d after DOX administration. The Gln supplementation resulted in lower cardiac lipid peroxidation and peroxintrite levels and elevated cardiac catalase enzyme activity and GSH compared with the controls, without affecting those of the tumors. DOX-induced alterations of the echocardiographic parameters were significantly reduced in the Gln-supplemented rats. These data indicate that Gln is able to reduce the oxidative damage of cardiomyocytes that occurs soon after DOX administration and thus protects the heart of a tumor-bearing host from DOX-induced cardiomyopathy.

Effect of glutamine on glutathione, IGF-I, and TGF-β1,

Abstract Background. Our previous results have showed that oral glutamine (GLN) supplementation decreased carcinogenesis in 7,12-dimethylbenz[a]antracene (DMBA) breast cancer model. We also have found that GLN raises blood glutathione (GSH) levels in an implantable breast cancer model. The process of tumor growth was accompanied by depressed GSH production and increased levels of insulin-like growth factor-I (IGF-I) and transforming growth factor β1 (TGF-β1). GSH is counter-regulatory to IGF-I. We therefore hypothesized that in DMBA model of breast cancer, the increased GSH levels seen with oral GLN would be associated with lowered levels of IGF-I &TGF-β 1 . Methods. Time-dated pubertal Sprague-Dawley rats were gavaged at time 0 with 1 g/kg/day glutamine (GLN) ( n = 18), isonitrogenous Freamine (FA) ( n = 18), or water (H 2 O) ( n = 18). Rats were further randomized on day 7 to 100 mg/kg DMBA or oil. After 14 days, the animals were sacrificed and blood GSH, IGF-1, TGF-β1, breast tissue, and gut mucosa GSH levels were measured. Results. Oral GLN increased significantly blood, breast tissue, and gut mucosa levels of GSH in both DMBA and control groups in comparison with the control groups not treated with GLN. At the same time, the levels of blood IGF-I and TGF-β1 decreased significantly in both DMBA-treated and control groups. DMBA did not significantly affect any of these levels. Conclusions. Oral GLN increased GSH levels and lowered IGF-I and TGF-β1 in a range that is considered clinically significant. However, the effect of GLN in maintaining normal gut GSH production in the presence of DMBA was much more significant. Inconsistent with our hypothesis, reduction in IGF and TGF-β1 levels did not correlate with DMBA’s effect on gut GSH production.

Conductive interstitial thermal therapy device for surgical margin ablation: In vivo verification of a theoretical model

Purpose: To demonstrate the efficacy and predictability of a new conductive interstitial thermal therapy (CITT) device to ablate surgical margins. Method: The temperature distributions during thermal ablation of CITT were calculated with finite element modelling in a geometrical representation of perfused tissue. The depth of ablation was derived using the Arrhenius and the Sapareto and Dewey (S&D) models for the temperature range of 90 to 150°C. The female pig animal model was used to test the validity of the mathematical model. Breast tissues were ablated to temperatures in the range of 79–170°C, in vivo. Triphenyltetrazolium chloride viability stain was used to delineate viable tissue from ablated regions and the ablation depths were measured using digital imaging. Results: The calculations suggest that the CITT can be used to ablate perfused tissues to a 10–15 mm width within 20 minutes. The measured and calculated depths of ablation were statistically equivalent (99% confidence intervals) within ± 1mm at 170°C. At lower temperatures the equivalence between the model and the observations was within ± 2 mm. Conclusion: The CITT device can reliably and uniformly ablate a 10–15 mm wide region of soft tissue. Thus, it can be used to secure negative margins following the resection of a primary tumor, which could impede local recurrences in the treatment of local diseases such as early staged, non-metastatic, breast cancer.

Dead or alive? Autofluorescence distinguishes heat-fixed from viable cells

Purpose: A proof-of-concept study to evaluate a new autofluorescence method to differentiate necrotic thermally fixed cells from viable tissue following thermal ablation. Methods: A conductive interstitial thermal therapy (CITT) device was used to ablate swine mammary tissue and rabbit VX-2 carcinomas in vivo. The ablated regions and 10-mm margins were resected 24 h following treatment, embedded in HistOmer® and sectioned at 3 mm. The fresh sections were evaluated for gross viability with triphenyl tetrazolium chloride, 1 h post-resection. Representative non-viable and viable areas were then processed and embedded into paraffin, and sectioned at 5 µm. Standard H&E staining and proliferating cell nuclear antigen (PCNA) immunohistochemistry were compared against autofluorescence intensity, at 488-nm wavelength, for cellular viability. Results: Heat-fixed cells in non-viable regions exhibit increased autofluorescence intensity compared to viable tissue (area under receiver operating characteristics (ROC) curve = 0.96; Mann-Whitney P < 0.0001). An autofluorescence intensity-based classification rule achieved 92% sensitivity with 100% specificity for distinguishing non-viable from viable samples. In contrast, PCNA staining did not reliably distinguish heat-fixed, dead cells from viable cells. Conclusions: Examination of H&E-stained sections using autofluorescence intensity-based classification is a reliable and readily available method to accurately identify heat-fixed cells in ablated surgical margins.

Oral glutamine prevents DMBA-induced mammary carcinogenesis via upregulation of glutathione production

OBJECTIVE Glutamine is suggested to participate in glutathione synthesis. Furthermore, there is a positive relation between glutathione level and natural killer (NK) cell activity. Previously we demonstrated that supplemental glutamine inhibited tumor growth in an implantable tumor model and 7,12-dimethylbenz[a]anthracene (DMBA)-induced oral squamous cell carcinoma model; these reductions were associated with enhancing NK cell cytotoxicity, blood glutathione levels, and/or gut glutathione release. Therefore, we hypothesized that oral glutamine might suppress DMBA-induced mammary carcinogenesis by upregulation of glutathione production and/or augmentation of NK cell activity. METHODS Rats were randomized to six groups: DMBA + glutamine, DMBA + Freamine, DMBA + water, oil + glutamine, oil + Freamine, or oil + water. At age 50 d, rats were gavaged with DMBA or sesame oil. Rats also received glutamine, Freamine, or water by gavage from 1 wk before DMBA administration until sacrifice at 1, 2, 4, or 11 wk after DMBA. Tumor appearance, blood, gut mucosa and breast glutamine and/or glutathione concentrations, and NK cell cytotoxicity were measured. The gut extractions, defined as the difference of concentrations across the gut, were calculated. RESULTS Oral glutamine reduced the tumorigenesis of DMBA by 50% in this model. DMBA altered the difference of glutathione concentrations across the gut; however, oral glutamine maintained the normal gut glutathione release. NK cell activities were lower in DMBA groups at early (week 1) and late (week 11) time points, but oral glutamine recovered the NK cell activity only at week 11. In addition, blood, gut, and breast glutathione concentrations were enhanced by glutamine supplementation. CONCLUSION These results indicate that one of the mechanisms of dietary glutamine anticancer action is through upregulating gut glutathione metabolism.

Modulation of p53 and c-myc in DMBA-Induced Mammary Tumors by Oral Glutamine

Abstract: Previous studies established that oral glutamine (GLN) reduced tumor development in implantable and 7,12-dimethylbenz(a)anthracene (DMBA)-induced breast cancer models. This finding was associated with a decrease in tumor glutathione (GSH) levels, while maintaining normal gut, blood, and breast GSH. Alterations in GSH levels contribute to the control of apoptotic and cell cycle-regulating signaling. The aim of this study was to examine the role of dietary GLN on activation of p53 and c-myc, which play critical roles in cancer development and sensitivity to radiation and chemotherapy. Mammary gland carcinomas were induced in rats by DMBA. The rats were gavaged daily with GLN or water (controls), starting 1 wk prior DMBA-application and throughout the duration of the experiment (11 wk after DMBA). Tumor DNA was examined for mutations in p53 exons 5 and 6. Protein and mRNA levels of p53, p21WAF1/CIP1, PTEN, IGF-IR, mdm2, and c-myc in tumors of GLN-supplemented rats were compared with those of the control rats (received water). The sequencing of p53 showed that it was wild type. Increased phosphorylation of p53, as well as higher mRNA and protein levels of p21WAF1/CIP1, PTEN, and mdm2, and lower levels of IGF-IR were detected in tumors of GLN-supplemented rats vs. controls. Both phosphorylated c-myc and c-myc mRNA levels were reduced by GLN. The up-regulation of tumor p53 signaling and down-regulation of c-myc, in addition to previously established inhibition of Akt signaling in DMBA-breast cancer model, suggest that dietary GLN could be a useful approach for increasing the effectiveness of cancer treatment.

Glutamine Affects Glutathione Recycling Enzymes in a DMBA-Induced Breast Cancer Model

Malignancy depletes host glutathione (GSH) levels to increase treatment-related toxicity and increases itself to resist the treatments. Our previous studies have shown that dietary glutamine (GLN) prevented 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumors through enhancing gut GSH release and reducing tumor GSH level. In addition, GSH synthesis, metabolism, and recycling are accomplished in γ -glutamyl cycle. We hypothesized that the GLN prevention might be through a differential regulation of the γ -glutamyl cycle enzymes. Female Sprague-Dawley rats were randomized into DMBA-tumor bearing, DMBA-treated, and control groups subdivided into GLN and water groups. GLN supplementation was given at 1 g/kg/day by gastric gavage. The activities and messenger RNA levels of γ -glutamyl transpeptidase (GTP), γ -glutamylcysteine synthetase (GCS), 5-oxo-L-prolinase (OPase), γ -glutamyl transferase (GTF), and glutaminase (GLNase) were determined in gut mucosa and breast tumor using specific enzyme assays and semiquantitative reverse transcription polymerase chain reaction. GLN upregulated gut GTP, GCS, OPase, and GLNase in DMBA-tumor bearing, DMBA-treated, and/or control rats; however, it downregulated these enzymes in the tumor. The paradoxical effect of GLN on key GSH recycling enzymes in the gut versus tumor suggests that dietary supplemental GLN could be used in the clinical practice to increase the therapeutic index of cancer treatments by protecting normal tissues from, and sensitizing tumor cells to, chemotherapy and radiation-related injury.

Conductive interstitial thermal therapy (CITT) device evaluation in VX2 rabbit model.

We have developed a conductive interstitial thermal therapy (CITT) device to precisely and reliably deliver controlled thermal doses to the surgical margins at the cavity site following tumor resection, intraoperatively. The temperature field created by CITT ablation of a perfused tissue was modeled with a finite element package Femlab™. The modeling suggested that a maximum probe temperature of 120°C and an ablation time of 20 minutes were required to ablate highly perfused tissue such as the VX2 carcinoma. Deployable pins enable faster and more reliable thermal ablation. The model predictions were tested by thermal ablation of VX2 carcinoma tumors implanted in adult New Zealand rabbits. The size of the ablated region was confirmed with a viability stain, triphenyltetrazolium chloride (TTC). Histopathological examination revealed 3 regions in the ablated area: a carbonized region (1–3 mm); a region that contained thermally fixed cells; and an area of coagulated necrosis cells. Cells in the thermally fixed region stained for PCNA (proliferating cell nuclear antigen) and were bounded by the carbonized layer at the cavity wall, and by necrotic cells that exhibit nuclear fragmentation and cell dissociation, 5 to 10 mm away from the CITT probe. Adjacent tissue outside the target region was spared with a clear demarcation between ablated and normal viable tissue. It is suggested that the CITT device can be used, clinically, to inhibit local recurrence by creating negative surgical margins following the resection of a primary tumor in non-metastatic early staged tumors.