Glioblastoma multiforme (GBM) is the most dangerous cancers of the brain. Despite availability of various treatment modalities, GBM chemotherapy remains obscure. Cancer metabolism is considered as one of the important factors for the tumor aggressiveness. Glucose is an important energy source for the cellular metabolism and was found to affect the GBM cancer aggressiveness, and chemo-resistance. Studies have found that GBM cancer is driven by epigenetic proteins. HDACs are important epigenetic proteins that regulate the gene expression by chromatin epigenetics changes, and there by involved in gene transcription in cancer cells. In this study, we have evaluated the role of glucose on GBM cancer cells and identified the cell viability effects. Further, the cell-cycle studies have indicated the apoptotic effects of high dose of glucose. Further the histone deacetylase (HDAC) gene expression was examined during increased glucose availability. We have observed a drastic enhancement in HDAC gene expression. Further, the cancer cell metabolism was analysed by studying the gene expression pertaining to mammalian target of rapamycin (mTOR) pathway. Glucose has induced changes in gene expression of class I HDACs and mTOR pathway genes. Furthermore, the study has also identified the microRNA modulatory effect of glucose. The molecular modelling studies have indicated the interaction of glucose with mTOR, Rictor and caspase-3 proteins suggesting the functional regulatory role of glucose on the expression of genes. The caspase-3 (i. e. the effector caspase) studies confirmed the effect of glucose on caspase-3 activity and the effect was enhanced by the treatment with mTOR complex inhibitors. Proteomic study has identifed the involvement of MAPK, Rho kinase, S6 kinase pathways, Bromodomain, histone acetyl transferases during combined treatment of mTOR complex inhibitor and high glucose combination treatment. Thus, the present study has elucidated the role of glucose on GBM cancer proliferation, and molecular modulatory effect mediated by glucose by varying the chromatin epigenetics, and microRNA modulation.
Research Article
High Glucose modulates the cancer cell fate by regulation of mTOR-HDAC-microRNA axis
https://doi.org/10.21203/rs.3.rs-1957231/v1
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U87MG
glucose
HDAC
mTOR
Rictor
GBM
Glucose is one of the most important molecules required for cellular life and its concentration depends on mTOR signaling pathway. It is the master regulator of cellular metabolism which responds to various stimuli and nutrients (Zhuo Mao, and Wizhen Zhang, 2018). Dysregulation of mTOR pathway leads to cancer phenotype, and various other metabolic disorders. mTOR forms two complexes mTOR complex1 (mTORC1), and mTOR complex 2 (mTORC2). During the low availability of glucose, the mTORC1 signaling inhibit various anabolic processes such as ATP production etc. (Leprivier G & Rotblat B, 2020). In general, carbohydrates are composed of sugars such as fructose, and glucose which have only one sugar (monosaccharides) or two sugars (disaccharides). Tumor cells are highly dependent on glucose during Warburg effect. The hyperglycemia is considered one of the key factors involved in cancer cell proliferation, metastasis, and resistance against apoptosis, and response towards anti-cancer drug treatment (Wenjie Li et al., 2019). Like any other cancers, Glioblastoma was also characterized by aberrant glucose metabolism (Marin-Valencia et al., 2012). Thus, low carbohydrate availability, high fat ketogenic diets (KD) supplementation result in decrease in Warburg effect. In addition, elevation of ketone bodies, might not been metabolized due to ineffective mitochondrial OxPhos (Seyfried TN, et al., 2019). It was observed that the nude mice with diabetes mellitus (DM), and high glucose (HG) availability in the blood also responsible for aggressive tumor growth. In addition, several studies have also demonstrated that the high glucose (HG) to promote GBM progression via upregulation of chemo-attractants, as well as growth factor receptors (Zhiyao Bao et al., 2019). Studies by Thomas N Seyfried et al., 2015 have suggested that calorie-restricted ketogenic diet (KD-R) produce anti-angiogenic effects, and apoptotic inducing effects. This indicates high glucose (HG) is a serious malady in GBM. GBM patients undergoing radiation therapy having increased glucose level result in GBM cancer cell proliferation, and shorter survival period (Marciana N. Duma et al., 2019; Derr RL et al., 2009).
mTORC1 has been found to be deregulated during cancer metabolism. (Kim J & Guan KL, 2019). mTORC1 drive the cancer cell growth, and proliferation. During glucose scarcity, mammalian target of Rapamycin complex 1 (mTORC1) is inhibited leading to the inhibition of cell growth (Liu GY et al., 2020, Nat Rev Mol Cell Biol). But role of mTORC2 was not fully elucidated till date. Emerging studies have suggested the role of the mTORC2 in cancer cell metabolism, and chemo-resistance was still under intensive research. These two complexes shared some common protein components: a serine/threonine protein kinase (mTOR), mammalian lethal with sec-13 protein 8 (mLST8) and DEP-domain containing mTOR-interacting protein (DEPTOR). The additional mTORC1 complex proteins include scaffold protein regulatory-associated protein of TOR (Raptor), and Akt substrate protein proline-rich Akt substrate 40 kDa (PRAS40). The mTORC2 core components include scaffold protein rapamycin insensitive companion of mTOR (Rictor), stress-activated protein kinase-interacting protein 1(mSIN1), and protein observed with Rictor 1 and 2 (PROTOR1/2) (J.D. Weber, and D.H. Gutmann, 2012; W.J. Oh, and E. Jacinto, 2011). Surprisingly deletion of the Rictor gene in the hypothalamic cells result in loss of Akt phosphorylation i. e. Akt ser 473, and the level of Protein kinase C α (PKCα) protein level. The genetic depletion of ribosomal S6 kinase 1 (S6K1), cause reduction in adipose tissue mass. In addition, the regulation of mTORC1 by glucose is characterized by abnormal glucose metabolism and was found to be over expressed in various cancers. Currently, the use of mTORC1 inhibitors was limited by chemo-resistance.
Interestingly, histone deacetylase inhibitors (HDACi) were known to inhibit metabolic reprogramming via disrupting glycolysis and by lowering ATP levels (Trang Thi Thu Nguyen, 2020). Studies have indicated that combined inhibition of Tumor necrosis factor receptor-associated protein 1 (TRAP1) by Gamitrinib and, histone deacetylases (HDAC1/HDAC2) resulted in apoptosis in patient derived GBM xenografts GBM model system (Trang T T Nguyen, 2020). In addition, Histone deacetylases (HDACs) were known to promote cancer cell growth by suppressing the expression of p21, p53 genes. Interestingly, recent studies suggest that HDACs role in the regulation of cancer metabolism (Wardell et al., 2009; Chiaradonna et al., 2015).
mTOR inhibitor rapamycin inhibit the uptake of D-glucose by cancer cells as evident by Fluoro deoxy glucose-positron emission tomography (A. Moreau, et al., 2019). Currently, researchers are focusing on inhibition of mTOR protein kinase to reduce glucose driven hypoxia environment (i. e. HIF α), and angiogenesis (Li J et al., 2014). Importantly, histone acetyl transferases (HATs), and histone deacetylases (HDACs) influence the histone acetylation status that regulate chromatin epigenetics and gene expression that are involved in cancer initiation, progression, and chemo-resistance. Several HDAC proteins were involved in the regulation of metabolism. Recent studies have indicated that HDAC inhibitors such as Suberoyl anilide hydroxamic acid (SAHA), and Valproic acid (VPA) decrease the glucose uptake by cancer cells via inhibition on the expression of glucose transporter type 1 (GLUT1), acetyl-COA, and hexokinase activity (HXK1) in leukemia cells (Wardell S.E et al., 2009). During these conditions cancer metabolism was diverted towards amino acid metabolism. Thus, understanding the relation between glucose metabolism and HDAC genes might provide useful information about underlying factors involved in cancer.
2.1 Cell culture
Human malignant glioma cell line (U87 MG) was obtained from NCCS, Pune, INDIA. The cell line was cultured in Dulbecco Modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), and 1mM concentration of antibiotic cocktail solution ( penicillin, streptomycin, and kanamycin).
2.2 MTT assay
To study the influence of glucose on cell viability on GBM cancer cells. The U87MG cell line was seeded at a density of 0.5 x 104 cells / well of 96 well plate was incubated for 48 h. The cell viability was studied by employing MTT dye (Hi-Media). The optical density (OD) at 570 nm was taken using a Multimode varioscan (Thermo-scientific Ltd).
2.3 Cell-cycle analysis
2× 104 U87MG cells were seeded in a 60 mm dish and was allowed to grow for 24 h. Here various combinations glucose, glucose + Rapamycin, glucose + Torin, glucose + 2-DG was added to the cell-culture dish and incubated for 24 h. Furthe these cells were detached with by the addition of Trypsin-EDTA and further these cells were subjected to treatment with RNAse A (1mg/ml RNase A solution) (Sigma) and was incubated at 37°C for 30 min. Cells were stained with 300 µL of DNA staining solution. The DNA contents was measured by flow cytometer (Beckman Coulter, Brea, CA).
2.4 Caspase-3 assay
Caspase-3 activity was analysed in glucose and mTOR complex inhibitors (i. e. Rapamycin and Torin) treated U87MG cell line using a caspase-3 assay kit. Here the U87MG cells were incubated with various concentrations of glucose and the lysates obtained were subjected to caspase-3 based assay according to manufacturer’s recommendations. The optical density was measured at 405 nm.
2.5 Reverse transcription PCR
Total RNA was isolated from U87MG cells by TRizol based method and was further purified (Qiagen). The quantified and equal amount of RNA was subjected to reverse transcribed to cDNA using superscript III kit (Thermo-scientifics). The polymerase chain reaction was conducted by gene specific primers pertained to mTOR pathway, HDAC genes as well as genes involved in microRNA RNA biogenesis. Here GAPDH was used as an internal control. Primers used were listed in (Mekala et al., 2021)
2.6 MicroRNA expression study
Total RNA was isolated from the control and the glucose-treated U87MG cells. The cDNA was synthesized and was subjected to Reverse transcription PCR (RT-PCR) reaction. The manufacturer’s instructions were followed here (i,e. System biosciences 610A) (Ramaiah et al., 2014).
2.7 Structural biology studies and molecular modelling
The three-dimensional structures of mTOR (PDB ID: 5ZCS-Chain A), caspase 3 (PDB ID: 2J32) and, RICTOR (PDB ID: 5ZCS-Chain E) were reteived from Protein Data Bank (PDB) and the three-dimensional structure of glucose (PubChem ID: 5793) was retrieved from PubChem respectively. The excess water molecules, ions and other bound ligands were removed and the protein was prepared for molecular docking. Also, the energy was minimized by Avogadro software for the glucose molecule (ligand) and the ligand was prepared for the docking (Kim, S. et al., 2019; Hanwell MD. et al., 2012). Then the docking was performed in AutoDock Vina with default parameters (Trott, O. et al., 2010). Finally, the protein-ligand docked complex were visualized in pymol and the interactions were evaluated in Ligplot+
2.8 B-factor study
The B-factor, deformability, motion stiffness, variance, covariance map & matrix and atom pair elastic network of glucose with caspase3, RICTOR, mTOR and HDAC8 were predicted with iMODs normal mode analysis (NMA) (Lopez-Blanco JR et al., 2014). The protein-ligand deformation analysis was performed using iMODs server. The B-factor, deformability, motion stiffness, variance, covariance map, and matrix and atom pair elastic network of glucose with caspase-3, RICTOR, mTOR and HDAC8 were predicted with iMODs normal mode analysis (NMA) (Lopez-Blanco JR et al., 2014). The NMA analysis with dihedral coordinates provides motional and vibrational properties of the protein macromolecules and the nodes will calculate the stereochemistry of the macromolecules.
2.9 Proteomics study
U87MG cells were treated with high glucose (50 mM); glucose, and Rapamycin; and glucose and Torin combination. The cell pellet was carried out 10000 rpm at 10 min. The cell pellet was dissolved in 100 µl of 50 mM NH4HCO3 with 1% SDS. Vortex and sonicate at 20 min. Centrifuge at high speed at 10000 rpm for 10 min. The sodium-dodecyl sulphate-polyacrylamide (SDS-PAGE) was conducted. This was followed by tricarboxylic acid (TCA) precipitation. In this experiment 100 µg of the sample was taken for digestion. The sample was diluted with 50mM NH4HCO3 and then treated with 100mM DTT at 95oC for 1h. Further, the sample is then digested with trypsin and incubated overnight at 37˚C. Further vacuum drying was performed. 10µL injection volume was used on BEH C18 UPLC column for separation of peptides. The peptides separated on the column were directed to Waters Synapt G2 Q-TOF instrument for MS and MSMS analysis. The Score is calculated by the Expression Analysis extension of PLGS (ProteinLynx Global Server) Software based on the relevance of the protein present in both the Samples being compared. The values below 0.5 are considered significantly Under-Regulated and Values above 2 are considered Up-Regulated in the Treated Sample.
Statistical analysis
Statistical Analysis was performed using the graph pad software. All variables were tested in three independent experiments. * = p < 0.05, **=p < 0.01 and *** = p < 0.001.
Glucose affects cell viability
The high glucose levels cause tumor malignancy (Jianjun Han et al., 2016). GBM cancer is characterized by an aberrant glucose metabolism, high glycolytic rate, and thus considered as inhibiting glucose metabolism is a novel, potential therapeutic strategy (Cairns et al, 2011). Here we have used U87MG cells were treated with glucose (2.5 mM to 80 mM). We observed till 40 mM concentration the cell viability was enhanced. Concentration above 40 mM resulted in decrease in cell viability (Fig. 1).
Effect of Glucose on Cell-cycle
Glucose transport via cell membranes regulate cellular metabolism and neuronal cells cannot store this glucose. Thus, the continuous supply of glucose form blood is required for neurons for the growth and energy (Benarroch, 2014). We were interested to understand the effect of high concentration of glucose on the cell proliferation and cell-cycle. Flow-cytometry study has indicated an increased apoptosis in GBM cells treated with high glucose (50mM) up to 39%. Interestingly, GBM cells treated with combination of high glucose and mTORC1/C2 inhibitor Torin (i.e. ATP competitive mTOR inhibitor) enhanced apoptosis up to 69%. This indicates high glucose and Torin cause enhanced apoptosis (Fig. 2).
Glucose and HDACs
Like many cancers GBM cells have deregulated epigenetic mechanisms (Was et al., 2019). Various HDACs were found to be overexpressed in several cancer types (Jinwon Seo et al., 2014). Genetic interference of histone deacetylase 1 (HDAC1), and histone deacetylase 2 (HDAC2) lead to synergistic reduction of viability in GBM cancer with concomitant activation of apoptosis (Nguyen TTT et al., 2022). Thus, we were interested to study the expression of class I histone deacetylases and class II histone deacetylases in U87MG cancer cells treated with glucose at various concentrations such as 10 mM, 25 mM, 50 mM. High glucose concentrations in GBM cancer cells result in increased in expression of histone deacetylae-2, 3, 8 (Class I HDAC), and histone deacetylase-4, and histone deacetylase-7 (Class II HDAC) (Fig. 3).
Effect of mTOR inhibitors on glucose induced HDAC gene expression
HDAC gene expression was found to be linked to cancer metabolism and chemo-resistance (Roca et al., 2019; K. Zhang et al., 2012). Thus, we examined the expression of various HDACs such as class I and class II in glucose in combination of Rapamycin and Torin. Results have indicated the expression of histone deacetylases in glucose and mTOR inhibitors. Results have indicated that HDAC-3, and HDAC-8 were decreased in combination of high glucose and Torin. An increase in gene expression of HDAC-7 was increased (Fig. 4). The modelling study indicated that glucose interacted with caspase-3, and mTOR effectively with good binding affinities such as Cas_glucose= -5.2 Kcal/mol; mTOR_glucose= -5.1 Kcal/mol; Rictor_glucose= -4.7 Kcal/mol (Fig. 5; Fig. 6; Table 1, Table 2; Supplementary table 1).
Table-1. Binding energies and interacting residues
|
Protein |
ligand |
Binding energy (Kcal/mol) |
Interacting amino acid residues |
|---|---|---|---|
|
Caspase 3 |
Glucose |
-5.4 |
ARG64, SER120, HIS121, GLU161, CYS163, SER205, TRP206, ARG207 |
|
mTOR |
-5.1 |
ARG2339, HIS2340, PRO2341, HIS2355, PRO2376, PHE2377, ARG2378 |
|
|
RICTOR |
-4.7 |
LEU194, GLC197, ASN198, VAL201, LEU227, THR230, ILE231, LEU234, HIS237 |
Table-2. H bond interactions study on glucose with caspase-3, mTOR, and Rictor
|
Protein name |
Compound name |
Interaction |
Distance (Å) |
|---|---|---|---|
|
Caspase 3 |
Glucose |
NH2 atom in ARG64 with O2 in Glucose |
3.09 |
|
ND1 atom in HIS121 with O3 in Glucose |
2.94 |
||
|
NE2 atom in GLN161 with O2 in Glucose |
3.18 |
||
|
NE atom in ARG207 with O1 in Glucose |
3.08 |
||
|
NH2 atom in ARG207 with O2 in Glucose |
2.96 |
||
|
N atom in ARG207 with O4 in Glucose |
2.98 |
||
|
O atom in ARG207 with O4 in Glucose |
3.28 |
||
|
mTOR |
O atom in ARG2339 with O2 in Glucose |
3.04 |
|
|
O atom in ARG2339 with O3 in Glucose |
2.92 |
||
|
NE2 in HIS2355 with O4 in Glucose |
3.06 |
||
|
O atom in PRO2376 with O5 in Glucose |
2.96 |
||
|
O atom in PRO2376 with O6 in Glucose |
2.82 |
||
|
NE atom in ARG2378 with O3 in Glucose |
3.22 |
||
|
NH2 atom in ARG2378 with O3 in Glucose |
3.01 |
||
|
RICTOR |
O atom of LEU227 with O4 in Glucose |
2.73 |
|
|
O atom of THR230 with O2 in Glucose |
2.86 |
||
|
O atom of LEU234 with O2 in Glucose |
2.93 |
||
|
O atom of HIS237 with O3 in Glucose |
2.86 |
||
|
O atom of HIS237 with O5 in Glucose |
2.91 |
Supplementary Table-1. Interaction between glucose with caspase-3, mTOR, and Rictor. PDB ID's Cas3- 2J32; mTOR- 5ZCS (Chain-A); Rictor- 5ZCS (chain-E). PubChem ID: Glucose- 5793. The binding energies with stable interactions are Cas_glucose= -5.2 Kcal/mol; mTOR_glucose= -5.1 Kcal/mol; Rictor_glucose= -4.7 Kcal/mol
Glucose induced Apoptosis in cancer cells
Glucose is a vital energy source for cancer cell proliferation. Glucose helps in cell growth, and maintains the oncogenic signaling (Adekola K et al., 2012) and in turn oncogene expression regulates the glucose transport. Excessive availability of glucose lead to apoptosis in cancer cells (K. Matsuura et al., 2016). In our study we have conducted apoptotic assay to study the role of effective caspase such as caspase-3 during high glucose exposure to GBM cancer cells. We observed an increase in expression of caspase-3 (Fig. 7)
Glucose and mTOR gene regulation
The serine / threonine kinase mTOR plays a vital role in the phosphoinositide-3-kinases (PI3K) signaling and is involved in cancer cells and immune cells. mTOR complex is divided in to mTORC1 and mTORC2. mTORC1 is involved in proliferation, translation, lipid synthesis, and metabolism. Whereas mTORC2 is insensitive to rapamycin and phosphorylates its substrates such as Akt, SGK1, and PKC isoforms that are involved in cytoskeletal organization, proliferation, and differentiation (Laplante, and Sabatini, 2012). Here we have treated U87MG cells with glucose at 25 mM, and 50 mM concentration followed by Rapamycin and Torin treatment. Results have shown an enhanced upregulation of mTOR, S6K1, Akt, mSIN1, Bcl2 (proto-oncogene). Interestingly, negligible enhancement was observed with the SGK1, Protor, PKCα expression in glucose treated GBM cancer cells (Fig. 8). Interestingly, the combination of glucose with Rapamycin, or Torin has decreased the expression of PKCα, Protor, and mSIN1 genes (Fig. 9)
Effect of Glucose on microRNAs in U87MG cells
Glucose is one of the most important fuels required for the maintenance of several biological functions. Emerging studies have indicated that microRNAs which are 20-24nt in length, bind to 3’-untranslated region (3’-UTR) of messenger RNA which ultimately results in decreasing protein stability (Srikantan S et al., 2012). Studies have indicated that MicroRNAs (miRs) regulate the glucose and lipid metabolism (Paola Mirra et al., 2018). MicroRNAs such as miR-15b, miR-200, miR-143, miR-155, miR-223, let 7a play a crucial role in cancer cell proliferation, apoptosis, metabolism, and monocyte differentiation. Thus, we tried to understand the role of mTORC1 inhibitor (Rapamycin), and mTORC1/C2 inhibitor (Torin) on the expression of microRNAs during high glucose conditions such as 25 mM and 50 mM concentrations. Surprisingly, we found the miRs such as miR-15b, miR-200, miR-223, miR-143, miR-155, let-7a was enhanced at both 25 mM, and 50 mM. This indicates torin has the ability to decrease the cancer cell proliferation by enhancing the tumor suppression, and regulating metabolism (Fig. 10). Furthermore, glucose in combination with mTOR inhibitor has resulted in decrease in Dicer and enhanced Drosha. In addition, enhanced expression of PTEN and decreased expression of p53 (Fig. 11).
`iMODS NMA analysis of the glucose bound caspase 3, Rictor, mTOR
The iMODS NMA analysis of the glucose bound with the caspase3, RICTOR, mTOR, and HDAC8 were analysed Fig. 12A-D. The B-factor correlates with the deformation of protein molecule at all amino acid residues and hinges will be predicted along with the normal mode analysis (NMA) (Bhowmik R et al., 2022). The change can be observed between the NMA and B-factor, it may represent the changes in the protein macromolecule upon to the addition of the external ligand. In general, the lower eigen value shows the better deformation of the protein-ligand complex (Roy S et al., 2021). Eigen values of glucose with caspase3, RICTOR, mTOR and HDAC8 were calculated as 9.631253e-05, 2.831493e-06, 1.098417e-06, and 4.625399e-04 respectively and clearly shows that the protein-ligand deformation occurs rapidly. In all the docked complexes, glucose is correlated with the structural changes in the caspase3, RICTOR, mTOR and HDAC8 and it may modulate the function and activity of these proteins.
Effect of high glucose, Rapamycin, and Torin on GBM cancer cells
Glucose is an essential energy source for the cancer cell growth. Previous studies have identified that glucose treatment has enhanced glycolytic activity and promoted active endometrial cancer cell proliferation via various pathways such as AMP-activated protein kinase (AMPK), AKT/mTOR/S6 and MAPK pathways (Jianjun Han et al., 2016). Thus, we have studied the possible effect of high glucose (50mM) and combination of high glucose (50mM), and Rapamycin, as well as Torin on the cancer cell proteomics. We have identified the data obtained has indicated rapamycin, torin treatment modulated EGFR and mTOR, BRD4 (bromodomain), HDAC, Histone lysine transferase, histone methyl transferase, protein methyl transferase (Fig. 13) (W. Zhou et al., 2012). The control sample were compared with Rapamycin and Torin.
In case high glucose and rapamycin treatment the down-regulated genes such as Bromodomain adjacent to zinc finger domain protein 2B 0.00729913; Protein phosphatase 1 regulatory subunit 3A; Transcription initiation factor TFIID subunit 9 0.160413561; Rho guanine nucleotide exchange factor 26 0.29819726; Cell division cycle 5-like protein 0.326279793, Mitogen-activated protein kinase kinase kinase 0.357006971; Rho GTPase-activating protein 9 0.339595511; Epidermal growth factor receptor 0.394553708, Histone-lysine N-methyltransferase SETD1A 0.436049294; Ribosomal protein S6 kinase alpha-3 0.491644208.
The genes that are upregulated in rapamycin are Cullin-4A 2.013752683, Microtubule-associated protein 4 2.944679677; Histone-lysine N-methyltransferase 21.54190124
In high glucose and torin treated U87MG cells histone lysine N-methyltransferase 0.016739231; LIM domain-containing protein ajuba 0.186373986. Protein kinase C-binding protein 1 0.339595511; Microtubule-associated protein 1S 0.491644208; Guanine nucleotide exchange factor subunit RIC1 0.477113911. Further, the genes that are upregulated torin treated samples Rho guanine nucleotide exchange factor TIAM1 41.33; Mitogen-activated protein kinase kinase kinase 4 2.247907992. Over all the score below 0.5 are down-regulated protein expression and above 2.0 are considered as upregulated genes (Fig. 13A-C).
During cancer initiation, and progression various molecular changes linked with metabolic reprogramming occurs to meet the energy demands, as well as for various biosynthetic needs (Hanahan and Weinberg, 2011). Among various metabolites cancer cells utilize glucose for active cancer cell metabolism, and aggressiveness. The key pathway for glucose metabolism is mechanistic target of rapamycin (mTOR). Mechanistic target of rapamycin (mTOR) is a kinase forms two complexes which are mTORC1 and mTORC2. During low glucose levels the proteins, lipids, and nucleotide synthesis were inhibited and the metabolism will be diverted towards ATP production (Gabriel Leprivier & Barak Rotblat, 2020). Also, during glucose starvation, hexokinase 2 (HK2) binds to mTORC1 leading to inhibition of mTOR signaling and induction of autophagy (Durán, R. V. et al., 2012). In this, we have studied role of high glucose concentrations such as 25 mM, and 50 mM on the various epigenetic factors such as histone deacetylase proteins as well as microRNAs, and mTOR signaling. To initiate, the U87MG GBM cancer cells were treated with various concentrations of glucose 2.5–80 mM and studied the effect of glucose on GBM cancer cell viability. We have observed increased cell viability till 40 mM concentration and above 40 mM concentration the cell loses their cell viability (Fig. 1). Interestingly, high glucose was found to inhibit the number of s-phase cells and thus delay the cell-cycle progression in human umbilical vein endothelial cells (HUVEC) (Lorenzi M et al., 1987). Studies by Moreno Asso et al., 2013 have found that stimulatory glucose levels in pancreatic beta cells inhibit the anti-proliferative genes and ubiquitously expressed genes. Also, metabolic regulation and cell-cycle progression interlink was also identified in cells (Joanna Kaplon et al., 2022). We observed enhanced apoptosis in high glucose condition as well as in combination of glucose and Torin (i. e. mTORC1/C2 inhibitor) treated GBM cancer cells but not in Rapamycin probably, due to activation of Akt. This indicates significant role of mTORC1/C2 complex inhibition on apoptosis rather than mTORC1 alone (Fig. 2).
Cancer metabolism is regulated by various genetic and epigenetic mechanisms. Various genes such as HDAC, histone methyl transferases (HMTs), and non-coding RNAs regulate the GBM cancer gene expression. The key metabolites are glucose, amino acids, and fatty acids that regulate cancer metabolism and chemo-resistance (Vera Miranda-Goncalves et al., 2018). HDACs were classified in to 4 classes. In which class I (HDAC-1,2,3, and 8) and class II (HDAC-4,5,6,7,9,10) were found to be useful drug targets. Several studies have indicated that availability of glucose resulted in enhancement of HDAC genes (Yang J et al., 2017) and various other epigenetic factors (L.M. Villeneuve et al., 2011). Interestingly, very high expression of the class I HDAC, HDAC3 was found to be highly toxic to neurons (Kim D, et al., 2008).
Studies have revealed that HDAC-1, HDAC-2 was highly upregulated during enhanced glucose metabolism via glucose transport mechanism (GLUT) (Suzanne E. Wardell et al., 2009). Drug discovery studies on GBM have found that certain key genes regulate cancer metabolism and chemo-resistance (Zhou and Wahl, 2019). In our results we have observed increased expression of class I HDACs such as HDAC-2, HDAC-3, and HDAC-8 during increased glucose treatment. Similar findings were made in hepatocellular carcinoma (HCC) (J.Yang et al., 2017) (Fig. 3). Surprisingly, treatment of mTORC1 inhibitor (i.e. Rapamycin) as well as mTORC1/C2 inhibitor (i.e. Torin) resulted in decrease in HDAC-3, HDAC-8, HDAC-4 in Torin treated cells during very high glucose concentration (i.e. 50 mM) (Fig. 4). This indicates that high glucose modulate histone deacetylase gene expression and has interlink with mTORC2 dependent pathway. This also confirms the GBM cancer cell metabolism is regulated by involvement of mTORC2 and HDACs.
Mammalian target of rapamycin (mTOR) inhibition was well known to inhibit cancer cell proliferation. First generation mTOR inhibitors include Rapalogs that bind to the F506 binding protein 12 (FBP12). The second generation mTOR inhibitor include mTORC1/2 inhibitors (e.g., Torin1, Torin2, PP242) bind to ATP biding pocket of mTOR kinase domain in both mTORC1/C2. Interestingly, many of these drugs were under intense clinical trials. Presently, third generation mTOR inhibitors such as Rapa-link that cross blood-brain barrier, inhibit GBM cancer growth and proliferation in various GBM cancer model systems (Kostas A. Papavassiliou and Athanasios G. Papavassiliou, 2021). Studies have identified that mTORC2 complex directly activates protein kinase B (Akt). Further, studies have found that compound, CID613034 that specifically inhibit the mTORC2 and Akt is highly useful for future therapy against GBM (Angelica Benavides-Serrato et al., 2017). Herein we were interested to study the role of increased glucose concentration in mTOR pathway genes. We found that mTOR S6K1, Bcl2, Akt, mSIN1, Protor, PKCα expression was increased during enhanced glucose concentrations (Fig. 8). mSIN1, protor, PKCα decreased in high glucose treated GBM cancer cells treated with mTORC1/C2 inhibitor when compared with mTORC1 alone. This indicates mTORC2 has crucial role in glucose induced mTOR pathway genes and cancer metabolism (Fig. 9) (Table 1–2).
Emerging studies have identified that combination of histone deacetylase inhibitor (HDACi) Panobinostat and PI3K /mTOR inhibitor were found to inhibit the GBM cancer cell viability and apoptosis (Wei Meng et al., 2015). In general, mitochondrial permeability is regulated by various factors such as voltage dependent anion-selective channel protein 1 (VDAC-1), B-cell lymphoma 2 (Bcl-2), Hexokinase (HK2), Bcl2 associated X (Bax). Studies have indicated that Hexokinase 2 and Bcl-2 decrease the mitochondrial cell permeability whereas Bax increases the mitochondrial cell permeability. During high glucoseinduced apoptosis the elevated mitochondrial permeabilitymediated release of mitochondrial cytochrome c, and hexokinase 2 (HK2) was observed (Jia Zhang et al., 2019). Thus, we have examined the apoptotic effect caused by increased glucose concentration in terms of caspase-3 expression at 25 mM and 50 mM concentration. Results indicated the enhancement of caspase-3 expression during high glucose condition and mTORC1/C2 inhibitor suggesting that apoptotic inducing nature of high glucose and Torin in GBM cancer cells. In addition, various molecular modelling studies including LigPlot + studies have confirmed the binding affinity and interactions of glucose with caspase-3 protein, mTOR, and Rictor (Fig. 5–7; Supplementary Table-1).
In GBM cancer miR-15b was down-regulated in various glioblastoma (GBM) patients as well as cell line such as U87MG, and U251 cells (Jian Wang et al., 2017) And the expression of insulin-like growth factor receptor 1(IGFR1) was down-regulated. miR-223 is another important microRNA that inhibit the cell proliferation, and migration, and cytokine signaling involving various interleukins such as IL-8, IL-18, IL-1β, and monocyte chemoattractant protein-1 (MCP-1) (Qiuping Ding et al., 2018). Various microRNAs such as miR-15, let-7a, and miR-200 involved in apoptosis as well as miR-223, miR-143, and miR-155 were known to be involved in cancer metabolism. Studies also identified that ectopic expression of miR-143 in GBM cancer cells was known to inhibit the cancer cell proliferation and epithelial to mesenchymal transition and it self-acts as tumor suppressor (Yi Yan et al., 2016; Eunice L. Lozada-Delgado et al., 2018). In addition, miR-143 expression inhibits the hexokinase 2 (HK2) in lung cancer samples (Rong Fang et al., 2012). Further, miR-200b tumor suppressor inhibit LDHA, suppressed glycolysis, and cell proliferation, invasion of GBM cells ( SU Hu et al., 2016). Interestingly, miR-223 is involved in monocyte to macrophage differentiation, and inhibit the HDAC-2. MiR-200b suppresses the cell proliferation, migration, and invasion in GBM cancer cells and acts as tumor suppressor. In addition, miR-16 decrease the malignancy by down-regulate NF-kB1, and MMP-9 and suppress Bcl2 oncogene. Further, nude mice study with miR-16 expression suppress the glioma growth and invasiveness (Tian-Quan Yang et al., 2014). Thus, we have examined various microRNAs in U87MG cells treated with high glucose and Torin combination (Fig. 10). The Drosha, Dicer, p53, and PTEN gene expression during glucose conditions indicated the Drosha upregulation and Dicer down-regulation was observed (Fig. 11). The B-factor study further supported the molecular interactions between caspase-3, mTOR, and Rictor with glucose (Fig. 12). Further proteomics study indicated various pathways and proteins that deregulated during high glucose condition in combination with Rapamycin, and Torin (Fig. 13).
Glucose metabolism play an important role in cancer aggressiveness and chemo-resistance. Several genes such as HDACs, mTOR were found to be modulated by the available glucose concentration. The expression of various HDACs and mTOR genes were inhibited during the treatment of GBM cancer cells with mTOR complex1 inhibitor (Rapamycin) and mTOR complex 1 & 2 inhibitor (Torin) during the high glucose available condition. In the present study also elucidated the interaction of glucose with various proteins such as mTOR, Rictor and caspase-3 was also elucidated. High glucose condition was shown to cause change in microRNA gene expression. Interestingly, proteomic study using high glucose and mTOR inhibitor have shown the modulation of various pathways that are in chromatin epigenetics and cell proliferation. Thus, this study has revealed the functional genomic, and proteomic changes during high glucose and mTOR inhibition.
Declaration statement
The work has not been published and that is not under consideration for publication elsewhere, that its publication is approved by all.
Funding
This work was supported by SERB, Government of INDIA with grant number EMR/2017/001201 and DBT, Govt. of INDIA for funding with grant number BT/PR20836/MED/30/1727/2016. I would like to thank KLEF management for infrastructural support and encouragement.
Author’s contribution
MJR has conceived the idea, planned, guided, and written the manuscript. KRK has performed RT-PCR, miR studies, and other cell-culture based studies. RPS has performed all the molecular modelling studies. VKD involved in few cells culture based studies such as FACS. SMN helped in Proteomics study. RRM, and MNR was involved in modelling studies.
Ethical approval
This article does not contain any studies with human participants or animals performed by any one of the authors.
Declaration of competing interests
The authors have no conflict of interest to declare that are relevant to the content of this article.
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