INTRODUCTION:

Onset of cancer is a complicated process, which develops when genetic mutations occur in normal regulatory genes, leading to the transformation of a normal cell to a cancer cell. Alterations in the tumor suppressor and proto-oncogenes force the normal cell to lose its regulatory mechanism, turn tumorigenic, divide uncontrollably and metastasize to other parts of the body ¹. For long, cancer as a disease existed with no known cause or cure. Brain tumors manifest in a variety of ways, including clinical presentation, biological aggressiveness, histological differentiation, and therapeutic response. Infiltrative gliomas are more normal in cerebrum cancers and their separation can be oligodendroglial or astrocytic². The most elevated central nervous system (CNS) tumors include anaplastic astrocytoma and glioblastoma multiforme (GBM).

They can impact anyone at whatever stage throughout everyday life; however, most customarily have an effect on the elderly. After being diagnosed with the most aggressive astrocytoma, GBM (WHO grade IV), the median survival time of it is still 50 weeks³.

The tumor microenvironment is attributed a decisive role in tumor development, enhanced resistance and metastasis. Hypoxia is the sort of factor, that's the end result of an inequity among the delivery and utilization of oxygen⁴. As a result of the widespread proliferation of tumor cells eliminates cells from the vasculature, ensuing in a loss of oxygen and nutrients in the blood in the local area. Hypoxia is a condition in which the tissues are starved of oxygen. Hypoxia presents naturally in physiological conditions (e.g. embryonic development and exercising muscle), as well as in pathophysiological conditions (e.g. myocardial infarction, inflammation and solid tumor formation)⁵. Through conserved hypoxic response mechanisms, mammalian cells can detect a sustained drop in oxygen concentration (hypoxia). These pathways work with variation to hypoxia-induced physiological pressure by managing changes in gene expression, and are likewise basic for some, physiological occasions, including angiogenesis, increasing glucose uptake and its metabolism, evasion of apoptosis and regulation of cell cycle, during tumorigenesis⁶. A family of hypoxia-inducible transcriptional factors (HIFs) lies at the heart of these adaptive pathways. HIF proteins (HIF-1 and HIF-2) are stabilized when the concentration of molecular oxygen (O₂) becomes scarce, leading to the induced expression of downstream target genes that mediate cell adaptation and survival⁷. While these proteins are highly homologus, increasing evidence suggests they have unique transcriptional targets and tissue specific expression. In GBM, HIF-1 regulates a vast array of genes products controlling red blood cell production (erythropoesis), the formation of new blood vessels (angiogenesis), as well as glycolytic enzymes that can produce energy from glucose in the presence of less oxygen, invasion, natural cell death (apoptosis), vascular tone, acidic condition (pH regulation), epithelial homoeostasis and drug resistance⁸. At present, the main mechanism underlying HIF-1 activation in hypoxic and non-hypoxic environment is no completely understood⁹. We still have a very limited understanding of the molecular profiles and the regulatory mechanisms that control tumor cells in hypoxic conditions. The transformation of a low-grade tumor to high grade takes weeks and sometimes months. The expression level and pattern of the genes can be different in high grade GBM compared to the low grade due to the presence of prolonged existence of severe hypoxia in high grade^10,11. Therefore, the present work attempts to identify the expression of hypoxia regulated genes which involved in adaptation and tumor progression including glucose metabolism, apoptosis and survival, and angiogenesis. We have performed gene expression analysis by PCR in glial cells exposed to hypoxia. We observed different expression level of adaptive pathway genes in hypoxic models.

MATERIALS AND METHODS:

Human Glioblastoma cell lines U87MG were utilized in this study. This cell lines are basically used for in-vitro models for glioblastoma research studies. The cultured and hypoxic treated cell were procured from Department of Biotechnology, Jaypee Institute of Information and Technology, Noida. The development of a suitable in-vitro model for hypoxic conditions that resemble hypoxic tumors was developed. The aim of this work is to study and compare the gene expressions in normoxia and hypoxia in human glial cell lines. The variability of cell response especially in terms of HIF-1 expression has also been studied.

RNA Isolation:

Trypsinization is the most common way of isolating adherent cells from the vessel wherein they are being purged utilizing trypsin, a protein-debasing proteolytic specialist. When trypsin is added to a cell culture, it breaks down the proteins that allow cells to adhere to the vessel. Since PBS is isotonic and non-toxic to cells, it has a wide range of applications. It's useful for diluting chemicals. It's used to clean cell-filled containers. PBS can be used as a diluent in ways to dry biomolecules because the water molecules inside it will structure around the substance to be ‘dried' and adhered to a solid surface (for example, a protein). The thin film of water that binds to the material prevents denaturation and other conformational changes. Carbonate buffers can serve the similar purpose, but they are less effective. While assessing protein adsorption in ellipsometry, PBS can be utilized as a kind of perspective range.

Preparation of cDNA:

In 1970, reverse transcriptase was found in a number of retroviruses, including the human immunodeficiency virus (HIV) and the avian myeloblastosis virus (AMV). The Conversion of RNA template molecules into a DNA double helix by providing a very vital tool for molecular biology research through the process of catalyzation of Reverse transcriptase. In combination with the polymerase chain reaction technique known as RT-PCR, they're extensively used to generate complementary DNA (cDNA) libraries from a variety of expressed mRNAs and to evaluate the extent of mRNA production. (1) RNA-structured DNA polymerase, (2) RNase H, and (3) DNA-structured DNA polymerase are the three enzymatic activities of Reverse transcriptase.

Expression of different genes through PCR:

In this study, we studied 4 adaptive genes (VEGF, GLUT-1, EGFR and BNIP-3) which are regulated by HIF-1 under hypoxic conditions.

RESULTS:

Qualitative analysis of RNA on Gel Electrophoresis:

Total RNA was loaded on a denaturing gel, which has shown three bands of tRNA (28s rRNA, 18s rRNA and 5.8s rRNA). We observed a good quality of RNA in all four samples of Normoxia (N), 1 day hypoxia (1DH), 2 days hypoxia (2DH) and 3day hypoxia (3DH) Total RNA was further checked by UV spectrophotometer at A260 nm.

Figure:1 Total RNA was loaded on denaturing agarose gel, three bands of 28s rRNA, 18s rRNA and 5.8s rRNA were observed.

Amplify a given region of c-DNA (region of interest)

The expression level of hypoxia adaptive gens which are regulated by HIF-1 under hypoxic conditions. In this study, we selected HIF-1 (transcription factor) as shown in fig 2, BNIP3 (pro-apoptotic genes) fig 6, Glut-1 (Glucose transporter-1) fig 3, VEGFA (Angiogenesis) and EGFR (Growth factor) from different adaptive pathways under hypoxic condition fig 4 and 5. We also compared the expression level of all the genes with normoxic condition. In hypoxic conditions, we have observed an increased expression level of all adaptive genes as compared to normoxia.

Figure: 2 HIF-1alpha (345bps) was observed in an increasing order under hypoxic condition which is absent in normoxic condition. M: DNA Marker; N: Normoxia; 1DH: 1 day hypoxia; 2DH: 2-day hypoxia; 3DH: 3-day hypoxia.

Figure: 3. Glut-1- (165bps) was observed in an increasing order under hypoxic condition which is very low in normoxic condition. M: DNA Marker; N: Normoxia; 1DH: 1 day hypoxia; 2DH: 2-day hypoxia; 3DH: 3-day hypoxia.

Figure: 4 VEGFA (248bps) was observed in an increasing order under hypoxic condition which is very low in normoxic condition. M: DNA Marker; N: Normoxia; 1DH: 1 day hypoxia; 2DH: 2-day hypoxia; 3DH: 3-day hypoxia.

Figure: 5. EGFR (178bps) was observed in an increasing order under hypoxic condition which is showing low expression in normoxic condition. M: DNA Marker; N: Normoxia; 1DH: 1 day hypoxia; 2DH: 2-day hypoxia; 3DH: 3-day hypoxia.

Figure: 6. BNIP3 (245 bps) was observed in an increasing order under hypoxic condition which is absent in normoxic condition. M: DNA Marker; N: Normoxia; 1DH: 1 day hypoxia; 2DH: 2-day hypoxia; 3DH: 3-day hypoxia.

DISCUSSION:

Glioblastoma multiforme (GBM) is a typical and perhaps the most malignant brain tumor in adults, representing up to half of all essential mind cancers¹². Despite advanced research in the field of tumor biology, the hypoxic tumor microenvironment and behavior is comparatively less well studied. Hypoxia reasons modifications in a tumor's biology and its microenvironment. This could be due to activation of transcription factors and resulting alterations in gene expressions^13-15. Cells exposed to a hypoxic stress must rapidly adapt, otherwise an imbalance in their energy supply/consumption ratio ensues. the hypoxia can affect tumor cells in two ways: as a stress that slows proliferation or causes cell death (apoptosis or necrosis), or as a factor that contributes to malignant development and enhanced the resistance to the radiation therapy and other cancer treatments^16-17. Hypoxia-induced genomic and proteomics alterations inside tumor cells are mostly responsible for increased malignant development and therapeutic resistance. Through molecularly driven cell-cycle arrest, differentiation, programed cell death (apoptosis), or necrosis, hypoxia-induced genomic and proteomic alterations may cause growth standstill or impairment. The hypoxia-induced genomics and proteomics alterations inside tumor cells are mostly responsible for increased malignant development and therapeutic resistance.^18,19. Although these proteomic and genomic changes were mainly studied in short term hypoxia, while development of GBM is a longer time process, takes weeks to months and years to be grown. For low-grade glioma, the survival time is 3-8 years; for glioblastoma 6-12 months²⁰. Glioma malignancy increases with the intensity and duration of hypoxia. Therefore, a substantial study of in vitro models of severe hypoxia is required to gain a better understanding of the mechanisms of tumorigenicity. Knowledge of suitable invitro tumor model is a helpful strategy to identify possible drug targets.

CONCLUSION:

Hypoxia and HIFs play important roles in the initiation, development, and recurrence of GBM, as well as maintaining the phenotypic of glioma cells, as indicated and addressed above. —Future GBM research will concentrate on determining the upstream regulatory mechanisms that control HIF protein levels in glioma cells, as well as the downstream regulatory processes that control GC survival, tumor initiation, and GBM recurrence. Attempting to eliminate GCs by all possible means, such as targeted HIF synthesis to block the HIF1a and HIF2a pathways, destroy their nutrient environment, and destroy their hypoxic / perivascular niches, could lead to new GBM treatments.

CONFLICT OF INTEREST:

The authors declare no potential conflicts of interest with respect to research, authorship, and/or publication of this manuscript.

ACKNOWLWDGEMENT:

We acknowledge all the participants of the study.

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Received on 05.10.2021 Modified on 13.05.2022

Research J. Pharm. and Tech 2023; 16(4):1685-1688.

DOI: 10.52711/0974-360X.2023.00276