Differential effects of energy stress on AMPK phosphorylation and apoptosis in experimental brain tumor and normal brain

<p>Abstract</p> <p>Background</p> <p>AMP-activated protein kinase (AMPK) is a known physiological cellular energy sensor and becomes phosphorylated at Thr-172 in response to changes in cellular ATP levels. Activated AMPK acts as either an inducer or suppressor of apoptosis depending on the severity of energy stress and the presence or absence of certain functional tumor suppressor genes.</p> <p>Results</p> <p>Here we show that energy stress differentially affects AMPK phosphorylation and cell-death in brain tumor tissue and in tissue from contra-lateral normal brain. We compared TSC2 deficient CT-2A mouse astrocytoma cells with syngeneic normal astrocytes that were grown under identical condition <it>in vitro</it>. Energy stress induced by glucose withdrawal or addition of 2-deoxyglucose caused more ATP depletion, AMPK phosphorylation and apoptosis in CT-2A cells than in the normal astrocytes. Under normal energy conditions pharmacological stimulation of AMPK caused apoptosis in CT-2A cells but not in astrocytes. TSC2 siRNA treated astrocytes are hypersensitive to apoptosis induced by energy stress compared to control cells. AMPK phosphorylation and apoptosis were also greater in the CT-2A tumor tissue than in the normal brain tissue following implementation of dietary energy restriction. Inefficient mTOR and TSC2 signaling, downstream of AMPK, is responsible for CT-2A cell-death, while functional LKB1 may protect normal brain cells under energy stress.</p> <p>Conclusion</p> <p>Together these data demonstrates that AMPK phosphorylation induces apoptosis in mouse astrocytoma but may protect normal brain cells from apoptosis under similar energy stress condition. Therefore, using activator of AMPK along with glycolysis inhibitor could be a potential therapeutic approach for TSC2 deficient human malignant astrocytoma.</p

Influence of Caloric Restriction on Constitutive Expression of NF-κB in an Experimental Mouse Astrocytoma

Many of the current standard therapies employed for the management of primary malignant brain cancers are largely viewed as palliative, ultimately because these conventional strategies have been shown, in many instances, to decrease patient quality of life while only offering a modest increase in the length of survival. We propose that caloric restriction (CR) is an alternative metabolic therapy for brain cancer management that will not only improve survival but also reduce the morbidity associated with disease. Although we have shown that CR manages tumor growth and improves survival through multiple molecular and biochemical mechanisms, little information is known about the role that CR plays in modulating inflammation in brain tumor tissue.Phosphorylation and activation of nuclear factor κB (NF-κB) results in the transactivation of many genes including those encoding cycloxygenase-2 (COX-2) and allograft inflammatory factor-1 (AIF-1), both of which are proteins that are primarily expressed by inflammatory and malignant cancer cells. COX-2 has been shown to enhance inflammation and promote tumor cell survival in both in vitro and in vivo studies. In the current report, we demonstrate that the p65 subunit of NF-κB was expressed constitutively in the CT-2A tumor compared with contra-lateral normal brain tissue, and we also show that CR reduces (i) the phosphorylation and degree of transcriptional activation of the NF-κB-dependent genes COX-2 and AIF-1 in tumor tissue, as well as (ii) the expression of proinflammatory markers lying downstream of NF-κB in the CT-2A malignant mouse astrocytoma, [e.g. macrophage inflammatory protein-2 (MIP-2)]. On the whole, our date indicate that the NF-κB inflammatory pathway is constitutively activated in the CT-2A astrocytoma and that CR targets this pathway and inflammation.CR could be effective in reducing malignant brain tumor growth in part by inhibiting inflammation in the primary brain tumor

DAP12 impacts trafficking and surface stability of killer cell immunoglobulin-like receptors on natural killer cells

Killer cell immunoglobulin-like receptors (KIR) aid in the regulation of natural killer (NK) cell activity. In this study, the effect of the interaction between the two domain stimulatory KIR (KIR2DS) and their adapter, DAP12, was investigated beyond the previously defined signaling function. Flow cytometry analysis showed enhanced KIR2DS surface expression on NKL cells when co-transfected with DAP12. Conversely, KIR2DS4 surface expression on primary cells was decreased when the cells were treated with DAP12 specific siRNA. Treatment of the KIR2DS and DAP12 transfected cells with either cycloheximide or brefeldin A repressed KIR2DS surface expression revealing a role for DAP12 in trafficking newly synthesized KIR to the cell surface. Immunoprecipitation of DAP12 revealed an interaction of DAP12 with an immature isoform of KIR2DS indicating the interaction between these proteins initiates early in the maturation process, likely within the endoplasmic reticulum. An internalization assay demonstrated a significant impact of DAP12 on KIR2DS surface stability. Confocal microscopy showed internalized KIR2DS molecules are recruited to lysosomal compartments independent of DAP12 expression. Our results suggest in vivo conditions that adversely affect DAP12 expression will indirectly reduce surface expression and stability of KIR2DS. These effects could significantly impact ligand recognition and strength of signaling through KIR2DS molecules

Influence of CR on NF-κB expression and activation in CT-2A astrocytoma.

<p>Nuclear expression of phosphorylated NF-κB (p65) (A); cytosolic expression of phosphorylated IκB and total lκB (B) as assessed by western blot analysis, DNA promoter binding activity of activated NF-κB in the CT-2A astrocytoma (C-D) as assessed by EMSA. The histograms illustrate the average relative expression of phosphorylated to total protein normalized to the indicated loading control in either nuclear or cytoplasmic extracts of the indicated tissue (A-B). Values are expressed as normalized means ± S.E.M of 4–5 independent tissue samples/group. The asterisks in indicate that the value is significantly different in the CT-2A astrocytoma under AL and CR condition at * P<0.05 (Student <i>t</i>-test). Two representative samples are shown for each tissue type. (<b>C</b>) Evaluation of the extent of DNA proinflammatory gene promoter binging activity by activated NF-κB in nuclear extracts of CT-2A under AL and CR condition. (<b>D</b>) Coned-down view of the DNA promoter biding activity of activated NF-κB in nuclear extracts of the NF-κB in the CT-2A astrocytoma under AL and CR condition. The histogram illustrates the average relative expression of activated NF-κB in the indicated tissue. Values are expressed as normalized means ± S.E.M of 4–5 independent tissue samples/group. The asterisks indicate that the value is significantly different in the CT-2A astrocytoma under AL and CR condition at * P<0.05 (Student <i>t-</i>test). Two representative samples are shown for each tissue type.</p

Expression and Phosphorylation of NF-κB (p65) in CT-2A astrocytoma.

<p>Western blot analysis of (<b>A</b>) Total protein expression of NF-κB (p65) in the whole lysates of tumor tissue and normal brain parenchyma. 1 and 4 µg of protein was loaded for each sample of tumor and brain tissue. (<b>B</b>) Phosphorylated NF-κB (p65) in nuclear extracts of tumor and normal brain tissues. The histogram illustrates the average relative expression of p-NFκB (p65) (S-536) to histone in nuclear extracts of the indicated tissue. Values are expressed as normalized means ± S.E.M of 4–5 independent tissue samples/group for both <b>A</b> and <b>B</b>. The asterisks in indicate that the value is significantly higher in the CT-2A astrocytoma than in contra-lateral normal brain at ** P<0.01 (Student <i>t</i>-test). Two representative samples are shown for each tissue type. (<b>C</b>) Phosphorylation of NF-κB in CT-2A cells and control mouse astrocytes in absence and presence of TNFα (10 ng/ml). (<b>D</b>) NF-κB immunostaining in CT-2A tumor and contra-lateral normal brain tissues. 3 independent mouse brain tumors were analyzed.</p

Influence of CR on tumor growth, body weight, and plasma biomarkers.

<p>Tumors were implanted i.c. on day 0. All tumor-bearing mice were fed <i>ad libitum</i> (AL) for the first 48 hrs after tumor implantation and were then randomly assigned to one of two diet groups that received rodent chow in either unrestricted (AL; n = 4) or calorically restricted (CR; n = 5) amounts. The two groups were matched for body weight (∼28.0 g) prior to the initiation of CR. The CR group received a feeding regimen of 30% less or 70% of food of AL mice consume daily. All mice were sacrificed 14–15 days after tumor implantation, as described above in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018085#s2" target="_blank"><b>Materials and Methods</b></a><b>section</b>. Final weights of the mice were obtained immediately prior to sacrifice (<b>B</b>). The asterisk in indicate that the tumor growth (A), body weight (B), and glucose level (C) is significantly reduced and ketone level (D) is significantly increased in the CR group compared with the AL group at * P<0.05, ** P<0.001 (Student <i>t</i>-test).</p

Influence of CR on inflammatory protein expressions in CT-2A astrocytoma.

<p>Cyclooxygenase-2 (COX-2) (A) and Allograft Inflammatory Factor 1 (AIF-1) (B) in cytosolic extracts of the CT-2A astrocytoma. COX-2 and AIF-1 have both been reported to be downstream proinflammatory gene-product effectors of the activated NF-κB. The histograms illustrate the average relative expression of the indicated protein normalized to β-actin in CT-2A tumors (A-B). Values are expressed as normalized means ± S.E.M of 4–5 independent tissue samples/group. The asterisks indicate that the value is significantly different in the CR tumor than in the AL tumor at * P<0.05, † P<0.001 (Student <i>t</i>-test). Two representative samples are shown for each tissue type.</p