C6 Glioma Cells Research Articles

Enzyme-Triggered Chemodynamic Therapy via a Peptide-H2 S Donor Conjugate with Complexed Fe2.

Inducing high levels of reactive oxygen species (ROS) inside tumor cells is a cancer therapy method termed chemodynamic therapy (CDT). Relying on delivery of Fenton reaction promoters such as Fe2+ , CDT takes advantage of overproduced ROS in the tumor microenvironment. We developed a peptide-H2 S donor conjugate, complexed with Fe2+ , termed AAN-PTC-Fe2+ . The AAN tripeptide was specifically cleaved by legumain, an enzyme overexpressed in glioma cells, to release carbonyl sulfide (COS). Hydrolysis of COS by carbonic anhydrase formed H2 S, an inhibitor of catalase, an enzyme that detoxifies H2 O2 . Fe2+ and H2 S together increased intracellular ROS levels and decreased viability in C6 glioma cells compared with controls lacking either Fe2+ , the AAN sequence, or the ability to generate H2 S. AAN-PTC-Fe2+ performed better than temezolimide while exhibiting no cytotoxicity toward H9C2 cardiomyocytes. This study provides an H2 S-amplified, enzyme-responsive platform for synergistic cancer treatment.

Enzyme‐Triggered Chemodynamic Therapy via a Peptide‐H2S Donor Conjugate with Complexed Fe2+

AbstractInducing high levels of reactive oxygen species (ROS) inside tumor cells is a cancer therapy method termed chemodynamic therapy (CDT). Relying on delivery of Fenton reaction promoters such as Fe2+, CDT takes advantage of overproduced ROS in the tumor microenvironment. We developed a peptide‐H2S donor conjugate, complexed with Fe2+, termed AAN‐PTC–Fe2+. The AAN tripeptide was specifically cleaved by legumain, an enzyme overexpressed in glioma cells, to release carbonyl sulfide (COS). Hydrolysis of COS by carbonic anhydrase formed H2S, an inhibitor of catalase, an enzyme that detoxifies H2O2. Fe2+ and H2S together increased intracellular ROS levels and decreased viability in C6 glioma cells compared with controls lacking either Fe2+, the AAN sequence, or the ability to generate H2S. AAN‐PTC–Fe2+ performed better than temezolimide while exhibiting no cytotoxicity toward H9C2 cardiomyocytes. This study provides an H2S‐amplified, enzyme‐responsive platform for synergistic cancer treatment.

Age-dependent alteration of microRNAs related to brain cancer in C6 glioma cells and young and old hippocampal rats after exposure to 1,2-Diacetylbenzene

Age-dependent alteration of microRNAs related to brain cancer in C6 glioma cells and young and old hippocampal rats after exposure to 1,2-Diacetylbenzene

Translocator Protein Is Involved in the Induction and Regulation of Mitochondrial Permeability Transition Pore Opening in C6 Glioma Cells

Translocator Protein Is Involved in the Induction and Regulation of Mitochondrial Permeability Transition Pore Opening in C6 Glioma Cells

VEGF affects mitochondrial ROS generation in glioma cells and acts as a radioresistance factor.

Vascular endothelial growth factor (VEGF) is closely related to angiogenesis. Anticancer therapy by inhibiting VEGF signaling is well established. However, the role of VEGF in cell-cell communication during the response to ionizing radiation is not well understood. Here, we examined the role of VEGF on radiosensitivity of cells. The addition of recombinant VEGF (rVEGF) on cultured rat C6 glioma cells showed a radioprotective effects on X-ray irradiation and reduced oxidative stress. These effects were also observed by endogenous VEGF in supernatant of C6 glioma cells. Reduction of oxidative stress by VEGF is suggested to underlie the radioprotective effects. The mechanism of VEGF-induced reduction of oxidative stress was indicated by a decreased oxygen consumption rate (OCR) in mitochondria. However, the number of DNA double-strand breaks (DSB) immediately after irradiation was not reduced by the treatment with VEGF. These results suggest that VEGF plays a role in cell survival after irradiation by controlling the oxidative condition through mitochondrial function that is independent of the efficiency of DSB induction.

Investigation of the effect of Sugammadex on glutamate-induced neurotoxicity in C6 cell line and the roles played by nitric oxide and oxidative stress pathways.

This experiment was intended to evaluate the effect of sugammadex on the cytotoxicity induced by glutamate, involving the nitric oxide and oxidative stress pathways. C6 glioma cells were used in the study. Glutamate was given to cells in the glutamate group for 24 h. Sugammadex at different concentrations was given to cells in the sugammadex group for 24 h. Cells in the sugammadex + glutamate group were pre-treated with sugammadex at various concentrations for 1 h and then exposed to glutamate for 24 h. XTT assay was used to assess the cell viability. The levels of nitric oxide (NO), neuronal nitric oxide synthase (nNOS), total antioxidant (TAS), and total oxidant (TOS) in the cells were calculated using commercial kits. Apoptosis was detected by TUNEL assay. Sugammadex at concentrations of 50 and 100 μg/mL significantly enhanced the cell viability in C6 cells after the cytotoxicity induced by glutamate (p < 0.001). Moreover, sugammadex considerably decreased the levels of nNOS NO and TOS and the number of apoptotic cells and increased the level of TAS (p < 0.001). Sugammadex has protective and antioxidant properties on cytotoxicity and could be an effective supplement for neurodegenerative diseases such as Alzheimer and Parkinson if further research in vivo supports this claim.

Unveiling the Anti-cancer Potential of Bergenia ciliata (haw.) Sternb: A Mechanistic Study on UPR Modulation and ROS Generation

Bergenia ciliata (haw.) Sternb, a well-known medicinal plant, has traditionally been used to treat various ailments, such as diabetes, microbial infections, and kidney stones, owing to its anti-inflammatory and anti-tussive properties. Here we aimed to evaluate the anti-cancer potential of the plant extract and deduce the molecular pathways involved. To investigate this, we used the MTT assay to determine the IC50 values of the methanolic extract of B. ciliata (BcME) in MDA-MB-231 and C6-Glioma cell lines. After treating the cancer cell lines with BcME for 18 hours, we evaluated the UPR signalling markers, including pIRE1, Xbp1, ATF6, eIF2α, ATF4, and CHOP, using Immunoblotting. We also used RT-PCR to determine the mRNA levels of ATF4 and performed enzyme assays using Spectrophotometric techniques to measure ROS levels. Our results indicate that B. ciliata has potent anti-cancer properties due to its ability to modulate UPR and ROS pathways. Specifically, the plant extract effectively repressed the cytoprotective UPR by blocking IRE1-Xbp1 and ATF6 pathways while enhancing the PERK-ATF4-CHOP pathway, which is known to switch UPR towards apoptosis. Furthermore, our study revealed that B. ciliata significantly caused the accumulation of ROS in cancer cells and inhibited the antioxidant enzymes catalase and superoxide dismutase. These results conclusively suggest that BcME works synergistically on both UPR and ROS pathways to promote apoptosis and eliminate cancerous cells and thus serves as a potential primary source for bioactive molecules, selectively targeting cancer cells.

Mathematical modelling of the dynamics of image-informed tumor habitats in a murine model of glioma

Tumors exhibit high molecular, phenotypic, and physiological heterogeneity. In this effort, we employ quantitative magnetic resonance imaging (MRI) data to capture this heterogeneity through imaging-based subregions or “habitats” in a murine model of glioma. We then demonstrate the ability to model and predict the growth of the habitats using coupled ordinary differential equations (ODEs) in the presence and absence of radiotherapy. Female Wistar rats (N = 21) were inoculated intracranially with 106 C6 glioma cells, a subset of which received 20 Gy (N = 5) or 40 Gy (N = 8) of radiation. All rats underwent diffusion-weighted and dynamic contrast-enhanced MRI at up to seven time points. All MRI data at each visit were subsequently clustered using k-means to identify physiological tumor habitats. A family of four models consisting of three coupled ODEs were developed and calibrated to the habitat time series of control and treated rats and evaluated for predictive capability. The Akaike Information Criterion was used for model selection, and the normalized sum-of-square-error (SSE) was used to evaluate goodness-of-fit in model calibration and prediction. Three tumor habitats with significantly different imaging data characteristics (p < 0.05) were identified: high-vascularity high-cellularity, low-vascularity high-cellularity, and low-vascularity low-cellularity. Model selection resulted in a five-parameter model whose predictions of habitat dynamics yielded SSEs that were similar to the SSEs from the calibrated model. It is thus feasible to mathematically describe habitat dynamics in a preclinical model of glioma using biology-based ODEs, showing promise for forecasting heterogeneous tumor behavior.

Swimming Training and Nanoformulation of Traditional Herbal Medicines Recovered Sensory-Motor Impairment by Modulating the Molecular Signaling Pathway in Rat Brain Tumor Models

Background: Glioblastoma (GBM) is one of the most aggressive tumors, health-threatening, and resistant to available treatment approaches for people worldwide. Objectives: This study aimed to evaluate effects of swimming training and a supplement comprising four nanoliposome herbal extracts in a rat model with mid-brain tumor. Methods: In this experimental study, 56-male-Wistar rats (230 ± 20 g) were divided into eight groups (n = 7): Normal group, model group, model + exe group, model + lipo, model + extract, model + lipo-extract, model + extract-exe, model + lipo-extract + exe. Mid-brain tumor model was induced by injection of the C6 glioma cell line (5 × 105 cell suspension) using stereotaxic techniques in the substantia nigra area of rats. Consumption of nanoformulation of herbals extract (100 mg/kg/day), crude extract (100 mg/kg/day), and swimming training (30 min, 3 days/week) were performed for six weeks. P53, Hras, IL-10, and Casp8 were studied by bioinformatics analysis. We assessed the relative expression of genes in the mid-brain tumor via the quantitative Real-Time PCR (qRT-PCR) method. Beam test and sciatic functional index assessed nerve function and motor coordination. Results: In silico analysis highlighted substantial genes with significant differential expression compared to healthy status as biomarkers in pathogenesis conditions. The expression level of the P53 and Hras increased with swimming training and nanoliposomes enriched complex complement. Besides, the expression of the IL-10 and Casp8 decreased with swimming training and nanoliposomes enriched complex complement. Conclusions: Our findings show that swimming training and nanoliposome-enriched combined supplements in rats can possibly be considered effective complementary medicine for motor impairment recovery induced by the brain tumor in the substantia nigra area.

Triterpenoids from the leaves of Abies koreana and their biological activities

Seven undescribed triterpenoids, abikoranes A–G, along with three known triterpenoids were isolated from the leaves of Abies koreana E. H. Wilson. The structures of compounds were elucidated by 1D and 2D NMR, HRMS, ECD, specific rotation, and DP4+ analysis. Abikorane A represents the second example of nor-3,4-seco-17,14-friedo-lanostane triterpenoid. Among the isolates, some compounds showed strong cytotoxic activities against some of four tested cancer cell lines (A549, SK-OV-3, SK-MEL-2, and HCT-116) with the values of IC50 0.89–9.62 μM, inhibited lipopolysaccharide-stimulated nitric oxide production with IC50 values of 11.57–15.16 μM, and exhibited significant nerve growth factor release effect (192.54 ± 12.33%) from C6 glioma cells.

Induction of apoptosis in glioma cells by lycorine via reactive oxygen species generation and regulation of NF-κB pathways.

Glioma is an extremely aggressive primary brain tumor, which is highly resistant to chemotherapy, presenting a therapeutic challenge. Here, we explored the anti-glioma effects and the underlying mechanism of lycorine, an isoquinoline alkaloid isolated from lycoris on glioma cells. We found that lycorine could dose dependently inhibit C6 glioma cell growth and induce cell apoptosis and intracellular reactive oxygen species (ROS) production. The half-maximal inhibitory concentration (IC50) values of lycorine on C6 glioma cells at 48h was 2.85μM. Meanwhile, lycorine treatment caused dysfunction of the NF-κB signal, as demonstrated by the up-regulation of NF-κB inhibitor protein IκB and the downregulation of the NF-κB phosphorylation protein p-p65. The addition of NF-κB inhibitor SC75741 further confirmed the importance of the NF-κB pathway in lycorine-induced cell-growth inhibition. Moreover, lycorine might act synergically with temozolomide (TMZ) to reduce drug resistance by blocking the NF-κB pathway. Our study suggested that lycorine exerts an anti-glioma effect by inducing ROS production and inhibiting NF-κB, which validated that lycorine may be a potential candidate for glioma treatment alone or in combination with TMZ.

DNA Methyltransferase Inhibitors Induce Cerebral Dopamine Neurotrophic Factor Expression in C6 Glioma Cells

DNA Methyltransferase Inhibitors Induce Cerebral Dopamine Neurotrophic Factor Expression in C6 Glioma Cells

Combined Effects of Focused Ultrasound and Photodynamic Treatment for Malignant Brain Tumors Using C6 Glioma Rat Model.

Glioblastoma (GBM) is an intractable disease for which various treatments have been attempted, but with little effect. This study aimed to measure the effect of photodynamic therapy (PDT) and sonodynamic therapy (SDT), which are currently being used to treat brain tumors, as well as sono-photodynamic therapy (SPDT), which is the combination of these two. Four groups of Sprague-Dawley rats were injected with C6 glioma cells in a cortical region and treated with PDT, SDT, and SPDT. Gd-MRI was monitored weekly and 18F-FDG-PET the day before and 1 week after the treatment. The acoustic power used during sonication was 5.5 W/cm² using a 0.5-MHz single-element transducer. The 633-nm laser was illuminated at 100 J/cm². Oxidative stress and apoptosis markers were evaluated 3 days after treatment using immunohistochemistry (IHC): 4-HNE, 8-OhdG, and Caspase-3. A decrease in tumor volume was observed in MRI imaging 12 days after the treatment in the PDT group (p<0.05), but the SDT group showed a slight increase compared to the 5-Ala group. The high expression rates of reactive oxygen species-related factors, such as 8-OhdG (p<0.001) and Caspase-3 (p<0.001), were observed in the SPDT group compared to other groups in IHC. Our findings show that light with sensitizers can inhibit GBM growth, but not ultrasound. Although SPDT did not show the combined effect in MRI, high oxidative stress was observed in IHC. Further studies are needed to investigate the safety parameters to apply ultrasound in GBM.

A data assimilation framework to predict the response of glioma cells to radiation.

We incorporate a practical data assimilation methodology into our previously established experimental-computational framework to predict the heterogeneous response of glioma cells receiving fractionated radiation treatment. Replicates of 9L and C6 glioma cells grown in 96-well plates were irradiated with six different fractionation schemes and imaged via time-resolved microscopy to yield 360- and 286-time courses for the 9L and C6 lines, respectively. These data were used to calibrate a biology-based mathematical model and then make predictions within two different scenarios. For Scenario 1, 70% of the time courses are fit to the model and the resulting parameter values are averaged. These average values, along with the initial cell number, initialize the model to predict the temporal evolution for each test time course (10% of the data). In Scenario 2, the predictions for the test cases are made with model parameters initially assigned from the training data, but then updated with new measurements every 24 hours via four versions of a data assimilation framework. We then compare the predictions made from Scenario 1 and the best version of Scenario 2 to the experimentally measured microscopy measurements using the concordance correlation coefficient (CCC). Across all fractionation schemes, Scenario 1 achieved a CCC value (mean ± standard deviation) of 0.845 ± 0.185 and 0.726 ± 0.195 for the 9L and C6 cell lines, respectively. For the best data assimilation version from Scenario 2 (validated with the last 20% of the data), the CCC values significantly increased to 0.954 ± 0.056 (p = 0.002) and 0.901 ± 0.061 (p = 8.9e-5) for the 9L and C6 cell lines, respectively. Thus, we have developed a data assimilation approach that incorporates an experimental-computational system to accurately predict the in vitro response of glioma cells to fractionated radiation therapy.

Ishige okamurae Attenuates Neuroinflammation and Cognitive Deficits in Mice Intracerebroventricularly Injected with LPS via Regulating TLR-4/MyD88-Dependent Pathways.

Neuroinflammation is one of the critical causes of neuronal loss and cognitive impairment. We aimed to evaluate the anti-neuroinflammatory properties of Ishige okamuae using mice intracerebroventricularly injected with lipopolysaccharides (LPS) and LPS-treated C6 glioma cells. We found that the short- and long-term memory deficits of LPS-injected mice were improved by oral administration of Ishige okamurae extracts (IOE). LPS-induced neuronal loss, increase in amyloid-β plaque, and expression of COX-2 and iNOS were restored by IOE. In addition, LPS-induced activation of Toll-like receptor-4 (TLR-4) and its downstream molecules, such as MyD88, NFκB, and mitogen-activated protein kinases (MAPKs), were significantly attenuated in the brains of mice fed with IOE. We found that pretreatment of IOE to C6 glioma cells ameliorated LPS-induced expression of TLR-4 and its inflammatory cascades, such as MyD88 expression, reactive oxygen species production, MAPKs phosphorylation, and NFκB phosphorylation with consequent downregulation of COX-2, iNOS, proinflammatory cytokines, and nitric oxide expression. Furthermore, IOE (0.2 µg/mL) was found to have equivalent efficacy with 10 μM of MyD88 inhibitor in preventing LPS-induced inflammatory responses in C6 glioma cells. Taken together, these results strongly suggest that IOE could be developed as a promising anti-neuroinflammatory agent which is able to control the TLR-4/MyD88-dependent signaling pathways.

HIF-1α Causes LCMT1/PP2A Deficiency and Mediates Tau Hyperphosphorylation and Cognitive Dysfunction during Chronic Hypoxia.

Chronic hypoxia is a risk factor for Alzheimer's disease (AD), and the neurofibrillary tangle (NFT) formed by hyperphosphorylated tau is one of the two major pathological changes in AD. However, the effect of chronic hypoxia on tau phosphorylation and its mechanism remains unclear. In this study, we investigated the role of HIF-1α (the functional subunit of hypoxia-inducible factor 1) in tau pathology. It was found that in Sprague-Dawley (SD) rats, global hypoxia (10% O2, 6 h per day) for one month induced cognitive impairments. Meanwhile it induced HIF-1α increase, tau hyperphosphorylation, and protein phosphatase 2A (PP2A) deficiency with leucine carboxyl methyltransferase 1(LCMT1, increasing PP2A activity) decrease in the rats' hippocampus. The results were replicated by hypoxic treatment in primary hippocampal neurons and C6/tau cells (rat C6 glioma cells stably expressing human full-length tau441). Conversely, HIF-1α silencing impeded the changes induced by hypoxia, both in primary neurons and SD rats. The result of dual luciferase assay proved that HIF-1α acted as a transcription factor of LCMT1. Unexpectedly, HIF-1α decreased the protein level of LCMT1. Further study uncovered that both overexpression of HIF-1α and hypoxia treatment resulted in a sizable degradation of LCMT1 via the autophagy--lysosomal pathway. Together, our data strongly indicated that chronic hypoxia upregulates HIF-1α, which obviously accelerated LCMT1 degradation, thus counteracting its transcriptional expression. The increase in HIF-1α decreases PP2A activity, finally resulting in tau hyperphosphorylation and cognitive dysfunction. Lowering HIF-1α in chronic hypoxia conditions may be useful in AD prevention.

In Vitro Cytotoxic Effects and Mechanisms of Action of Eleutherine Isolated from Eleutherine plicata Bulb in Rat Glioma C6 Cells.

Gliomas are the most common primary malignant brain tumors in adults, and have a poor prognosis, despite the different types of treatment available. There is growing demand for new therapies to treat this life-threatening tumor. Quinone derivatives from plants have received increased interest as potential anti-glioma drugs, due to their diverse pharmacologic activities, such as inhibiting cell growth, inflammation, tumor invasion, and promoting tumor regression. Previous studies have demonstrated the anti-glioma activity of Eleutherine plicata, which is related to three main naphthoquinone compounds-eleutherine, isoeleutherine, and eleutherol-but their mechanism of action remains elusive. Thus, the aim of this study was to investigate the mechanism of action of eleutherine on rat C6 glioma. In vitro cytotoxicity was evaluated by MTT assay; morphological changes were evaluated by phase-contrast microscopy. Apoptosis was determined by annexin V-FITC-propidium iodide staining, and antiproliferative effects were assessed by wound migration and colony formation assays. Protein kinase B (AKT/pAKT) expression was measured by western blot, and telomerase reverse transcriptase mRNA was measured by quantitative real-time polymerase chain reaction (qRT-PCR). Eleutherine reduced C6 cell proliferation in a dose-dependent manner, suppressed migration and invasion, induced apoptosis, and reduced AKT phosphorylation and telomerase expression. In summary, our results suggest that eleutherine has potential clinical use in treating glioma.

Brain Homogenate of a Rat Model of Alzheimer's Disease Modifies the Secretome of 3D Cultured Periodontal Ligament Stem Cells: A Potential Neuroregenerative Therapy.

Alzheimer's disease (AD) is a progressive neurodegenerative disease leading to neuronal cell death and manifested by cognitive disorders and behavioral impairment. Mesenchymal stem cells (MSCs) are one of the most promising candidates to stimulate neuroregeneration and prevent disease progression. Optimization of MSC culturing protocols is a key strategy to increase the therapeutic potential of the secretome. Here, we investigated the effect of brain homogenate of a rat model of AD (BH-AD) on the enhancement of protein secretion in the secretome of periodontal ligament stem cells (PDLSCs) when cultured in a 3D environment. Moreover, the effect of this modified secretome was examined on neural cells to study the impact of the conditioned medium (CM) on stimulation of regeneration or immunomodulation in AD. PDLSCs were isolated and characterized. Then, the spheroids of PDLSCs were generated in a modified 3D culture plate. PDLSCs-derived CM was prepared in the presence of BH-AD (PDLSCs-HCM) and the absence of it (PDLSCs-CM). The viability of C6 glioma cells was assessed after exposure to different concentrations of both CMs. Then, a proteomic analysis was performed on the CMs. Differentiation into adipocytes and high expression of MSCs markers verified the precise isolation of PDLSCs. The PDLSC spheroids were formed after 7 days of 3D culturing, and their viability was confirmed. The effect of CMs on C6 glioma cell viability showed that both CMs at low concentrations (> 20 mg/mL) had no cytotoxic effect on C6 neural cells. The results showed that PDLSCs-HCM contains higher concentrations of proteins compared to PDLSCs-CM, including Src-homology 2 domain (SH2)-containing PTPs (SHP-1) and muscle glycogen phosphorylase (PYGM) proteins. SHP-1 has a role in nerve regeneration, and PYGM is involved in glycogen metabolism. The modified secretome derived from 3D cultured spheroids of PDLSCs treated by BH-AD as a reservoir of regenerating neural factors can serve as a potential source for AD treatment.

Natural borneol serves as an adjuvant agent to promote the cellular uptake of piperlongumine for improving its antiglioma efficacy

Piperlongumine (PL) can selectively inhibit the proliferation of various cancer cells by increasing reactive oxygen species (ROS) level to cause a redox imbalance in cancer cells rather than in normal cells. However, the clinical application of PL is limited by its poor cellular uptake. Natural borneol (NB) is extracted from the fresh branches and leaves of Cinnamomum camphora (L.) Presl. with the purity of (+)-borneol no less than 96.0%. NB has been often used as an adjuvant agent to promote the cellular uptake of other drugs. This study aims to investigate the effect of NB on the cellular uptake of PL for improving its antiglioma efficacy and underlying mechanism. NB obviously promoted the cellular uptake of PL with a 1.3-fold increase in the maximum peak concentration and an earlier peak time of 30 min in C6 glioma cells. The cellular uptake of PL was enhanced by NB through down-regulating the expression levels of P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2). The combination of NB and PL significantly induced higher levels of ROS, which increased apoptosis and enhanced G2/M cycle arrest of C6 glioma cells, compared to PL alone administration. NB-enhanced antiglioma efficacy of PL without side effects was confirmed in tumor-bearing mice, which was attributed to the improved cellular uptake of PL. The distribution of PL in the tumor tissue of combined group increased 2.39 times than that of PL-treated group. We firstly report NB as an adjuvant agent to improve the antiglioma efficacy of PL in a ROS-dependent manner, which is due to the enhanced cellular uptake of PL by NB though down-regulating the expression levels of ABCB1 and ABCG2. This work provides a new strategy to promote the cellular uptake of PL with great potential for the treatment of glioma.

Glucose Infusion Induced Change in Intracellular pH and Its Relationship with Tumor Glycolysis in a C6 Rat Model of Glioblastoma.

The reliance on glycolytic metabolism is a hallmark of tumor metabolism. Excess acid and protons are produced, leading to an acidic tumor environment. Therefore, we explored the relationship between the tumor glycolytic metabolism and tissue pH by comparing 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) and hyperpolarized [1-13C]pyruvate MR spectroscopy imaging (MRSI) to chemical exchange saturation transfer (CEST) MRI measurements of tumor pH. 106 C6 glioma cells were implanted in the brains of male Wistar rats (N = 11) using stereotactic surgery. A 60-min PET acquisition after a bolus of FDG was performed at 11-13days post implantation, and standardized uptake value (SUV) was calculated. CEST measurements were acquired the following day before and during constant infusion of glucose solution. Tumor intracellular pH (pHi) was evaluated using amine and amide concentration-independent detection (AACID) CEST MRI. The change of pHi (∆pHi) was calculated as the difference between pHi pre- and during glucose infusion. Rats were imaged immediately with hyperpolarized [1-13C]pyruvate MRSI. Regional maps of the ratio of Lac:Pyr were acquired. The correlations between SUV, Lac:Pyr ratio, and ∆pHi were evaluated using Pearson's correlation. A decrease of 0.14 in pHi was found after glucose infusion in tumor region. Significant correlations between tumor glycolysis measurements of Lac:Pyr and ∆pHi within the tumor (ρ = 0.83, P = 0.01) and peritumoral region (ρ = 0.76, P = 0.028) were observed. No significant correlations were found between tumor SUV and ∆pHi within the tumor (ρ = - 0.45, P = 0.17) and peritumor regions (ρ = - 0.6, P = 0.051). AACID detected the changes in pHi induced by glucose infusion. Significant correlations between tumor glycolytic measurement of Lac:Pyr and tumoral and peritumoral pHi and ∆pHi suggest the intrinsic relationship between tumor glycolytic metabolism and the tumor pH environment as well as the peritumor pH environment.