“…S14). This overexpression ratio is in line with previous studies, where tumour cells showed approximately 2- to 10-fold higher expression of ASCT2 compared to their counterparts 7, 18, 19 . Thus, we used these cell lines for the assessment of interaction between the developed polymers and ASCT2 in in vitro studies.Figure 2
In vivo and in vitro expression of ASCT2.…”
Section: Resultssupporting
confidence: 92%
“…Given that the overexpression ratio of ASCT2 between BxPC3 and HEK293 cells (Supplementary Fig. S14 ) is comparable to that in clinical studies 7 , 18 , 19 , the tumour-selective interaction of the polymer as shown in Fig. 3 strongly indicates the potential in vivo activity of our synthesized polymer for tumour-targeting ligand.…”
Increased glutamine uptake toward the elevated glutaminolysis is one of the hallmarks of tumour cells. This aberrant glutamine metabolism has recently attracted considerable attention as a diagnostic and therapeutic target. Herein, we developed glutamine-functionalized polymer to achieve a selective high affinity to tumour cells overexpressing glutaminolysis-related transporter ASCT2. In in vitro study, our developed polymer exhibited faster and higher cellular uptake in tumour cells than that in normal cells. Uptake inhibition study revealed the dominant contribution of ASCT2 to the polymer-cell interaction. Furthermore, the binding affinity of the polymer to tumour cells was estimated to be comparable to that of the potent ligand molecules reported in the literature. In in vivo study, the polymer showed prolonged retention at tumour site after intratumoral injection. This study offers a novel approach for designing tumour cell-binding synthetic polymers through the recognition of dense transporters related to tumour-associated metabolism.
“…S14). This overexpression ratio is in line with previous studies, where tumour cells showed approximately 2- to 10-fold higher expression of ASCT2 compared to their counterparts 7, 18, 19 . Thus, we used these cell lines for the assessment of interaction between the developed polymers and ASCT2 in in vitro studies.Figure 2
In vivo and in vitro expression of ASCT2.…”
Section: Resultssupporting
confidence: 92%
“…Given that the overexpression ratio of ASCT2 between BxPC3 and HEK293 cells (Supplementary Fig. S14 ) is comparable to that in clinical studies 7 , 18 , 19 , the tumour-selective interaction of the polymer as shown in Fig. 3 strongly indicates the potential in vivo activity of our synthesized polymer for tumour-targeting ligand.…”
Increased glutamine uptake toward the elevated glutaminolysis is one of the hallmarks of tumour cells. This aberrant glutamine metabolism has recently attracted considerable attention as a diagnostic and therapeutic target. Herein, we developed glutamine-functionalized polymer to achieve a selective high affinity to tumour cells overexpressing glutaminolysis-related transporter ASCT2. In in vitro study, our developed polymer exhibited faster and higher cellular uptake in tumour cells than that in normal cells. Uptake inhibition study revealed the dominant contribution of ASCT2 to the polymer-cell interaction. Furthermore, the binding affinity of the polymer to tumour cells was estimated to be comparable to that of the potent ligand molecules reported in the literature. In in vivo study, the polymer showed prolonged retention at tumour site after intratumoral injection. This study offers a novel approach for designing tumour cell-binding synthetic polymers through the recognition of dense transporters related to tumour-associated metabolism.
“…High extracellular Gln concentration has been associated with cell transformation [ 17 ], and its metabolism was related to cell survival and tumor growth by maintaining redox balance, bioenergetics, and supporting macromolecular biosynthesis [ 28 , 42 ]. We have previously reported the upregulation of Gln transporters, ASCT2 and LAT1 , in all grades of astrocytoma [ 20 ]. Here, we showed the upregulation of the mitochondrial isoform of GLS [ 39 ], GLSiso2 ( GAC ), in all grades of astrocytoma at gene and protein levels, and a gradual increase of its expression was observed in parallel to the increment of malignancy.…”
Section: Discussionmentioning
confidence: 99%
“…The relative expression levels of genes involved in the glutaminolysis pathway GLS, GLSiso1, GLSiso2, GLS2, GLUD1, GOT1, GOT2, and GPT2 were analyzed by qRT–PCR, using the SYBR Green approach. The expression of ASCT2 and LAT1 genes were previously described by our group [ 20 ]. A geometric mean of three suitable reference genes was used for normalizing the quantitative data: hypoxanthine phosphoribosyltransferase ( HPRT ), glucuronidase beta ( GUSβ ), and TATA box binding protein ( TBP ) [ 34 ].…”
Background
Glioblastoma is the most frequent and high-grade adult malignant central nervous system tumor. The prognosis is still poor despite the use of combined therapy involving maximal surgical resection, radiotherapy, and chemotherapy. Metabolic reprogramming currently is recognized as one of the hallmarks of cancer. Glutamine metabolism through glutaminolysis has been associated with tumor cell maintenance and survival, and with antioxidative stress through glutathione (GSH) synthesis.
Methods
In the present study, we analyzed the glutaminolysis-related gene expression levels in our cohort of 153 astrocytomas of different malignant grades and 22 non-neoplastic brain samples through qRT-PCR. Additionally, we investigated the protein expression profile of the key regulator of glutaminolysis (GLS), glutamate dehydrogenase (GLUD1), and glutamate pyruvate transaminase (GPT2) in these samples. We also investigated the glutathione synthase (GS) protein profile and the GSH levels in different grades of astrocytomas. The differential gene expressions were validated in silico on the TCGA database.
Results
We found an increase of glutaminase isoform 2 gene (GLSiso2) expression in all grades of astrocytoma compared to non-neoplastic brain tissue, with a gradual expression increment in parallel to malignancy. Genes coding for GLUD1 and GPT2 expression levels varied according to the grade of malignancy, being downregulated in glioblastoma, and upregulated in lower grades of astrocytoma (AGII–AGIII). Significant low GLUD1 and GPT2 protein levels were observed in the mesenchymal subtype of GBM.
Conclusions
In glioblastoma, particularly in the mesenchymal subtype, the downregulation of both genes and proteins (GLUD1 and GPT2) increases the source of glutamate for GSH synthesis and enhances tumor cell fitness due to increased antioxidative capacity. In contrast, in lower-grade astrocytoma, mainly in those harboring the IDH1 mutation, the gene expression profile indicates that tumor cells might be sensitized to oxidative stress due to reduced GSH synthesis. The measurement of GLUD1 and GPT2 metabolic substrates, ammonia, and alanine, by noninvasive MR spectroscopy, may potentially allow the identification of IDH1mut AGII and AGIII progression towards secondary GBM.
“…The mTOR pathway is well described as deregulated in CRC, and the availability of AA functions as a regulator of this pathway, since a high AA microenvironmental bioavailability induces mTOR activity and consequent biological processes, such as protein translation [ 36 ]. Some studies report a relationship between LAT1 and ASCT2, with a two-step mechanism of these AAT being able to regulate mTOR pathway [ 37 , 38 , 39 ]. Firstly, ASCT2 regulates the intracellular concentration of glutamine, and in turn LAT1 uses this intracellular glutamine as an efflux substrate, in order to regulate the uptake of extracellular leucine, which will lead to an activation of mTOR signaling and consequent induction of cell growth and proliferation [ 40 , 41 ] ( Figure 1 ).…”
The development and progression of colorectal cancer (CRC) have been associated with genetic and epigenetic alterations and more recently with changes in cell metabolism. Amino acid transporters are key players in tumor development, and it is described that tumor cells upregulate some AA transporters in order to support the increased amino acid (AA) intake to sustain the tumor additional needs for tumor growth and proliferation through the activation of several signaling pathways. LAT1 and ASCT2 are two AA transporters involved in the regulation of the mTOR pathway that has been reported as upregulated in CRC. Some attempts have been made in order to develop therapeutic approaches to target these AA transporters, however none have reached the clinical setting so far. MiRNA-based therapies have been gaining increasing attention from pharmaceutical companies and now several miRNA-based drugs are currently in clinical trials with promising results. In this review we combine a bioinformatic approach with a literature review in order to identify a miRNA profile with the potential to target both LAT1 and ASCT2 with potential to be used as a therapeutic approach against CRC.
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