ASCORBATE AND HYPOXIA INHIBIT CELL MIGRATION AND DOWNREGULATE HIF 1-ALPHA AND TWIST GENE EXPRESSION IN BREAST CANCER CELL LINE MCF-7

• Main Author: Septiani Fidarliyan, Garnish
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/66180
• Institution: Institut Teknologi Bandung

Hypoxia at cellular level can induce the particular changes of certain gene expression. Hypoxia is a favorable microenvironment for promoting cell agresivity. Ability of cancer cells to adapt in hypoxia condition is mediated by the activity of hypoxia-inducible transcription factor subunit ? (HIF-1?). HIF-1? stability can support cancer progression such as proliferation, migration, invasion and metastases through the regulation of TWIST expression. The expression of TWIST can be inhibited through HIF-1? suppression by involving hydroxylases. Activation of hydroxylases require oxygen and 2-oxoglutarate as substrate, also Fe2+ and ascorbate as cofactor. In normoxic condition, hydroxylase hydroxylates oxygen-dependent degradation (ODD) domain of HIF-1? subunit that leads to proteosomal degradation. However, hypoxic condition will maintain HIF-1? accumulation due to a decreased hydroxylase activity. The expression of HIF-1? is known to be reduced both transcriptionally and translationally by increasing ascorbate level during hypoxic. This study is aimed to observe the effect of ascorbate concentration and hypoxia that regulate the inhibition of HIF-1? and TWIST. Scratch assay method was used to observe cell migration to obtain wound closure percentage and migration rate (?m/hour). The detection of HIF-1? and TWIST gene expression was carried out by Real-Time PCR method. The results showed that ascorbate and hypoxia can affect the cell migration of MCF-7 cell line. Under normoxic conditions, ascorbate with the concentration of 0.40 mM (IC50) suppressed cell migration significantly within 34 hours with an average migration rate (Vm) relative to control of 1,28 ?m/hour and a percentage of wound area closure of 19,37%. Under hypoxic condition, ascorbate with the concentration of 1,20 mM (IC75) inhibited cell migration significantly within 34 hours with an average Vm of 0,93 ?m/hour and reduced the percentage of wound area up to 14,2%. Analyzing the effect of ascorbate concentration and hypoxia on the migration of MCF-7 breast cancer cell lines was also carried out by obtaining the relative gene expression of HIF-1? and TWIST. Relative expression of target genes under hypoxic was compared with normoxic condition. The results showed that in hypoxic conditions, HIF-1? gene expression was upregulated ±3,73x when MCF-7 was treated with ascorbate concentration of 0,13 mM (IC25) and ±4,11x when MCF-7 was treated with ascorbate concentration of 0,40 mM (IC50). On the other hand, ascorbate with the concentration of 1,20 mM (IC75) can reduce the relative expression of the HIF-1? gene up to ±6,97x under hypoxic condition. Ascorbate concentration of 1,20 mM (IC75) under hypoxic condition downregulated TWIST gene expression up to ±3,25x. In summary, ascorbate and hypoxia can inhibit MCF-7 cell migration and furthermore can significantly decrease HIF-1? and TWIST gene expression.