Transcriptional Activation of Human GD3 Synthase (hST8Sia I) Gene In curcumin-Induced Autophagy in A549 Human Lung Carcinoma Cells

Abstract

Curcumin, a natural polyphenolic compound isolated from the plant Curcuma longa, is known to induce autophagy in various cancer cells, including lung cancer. In the present study, we also confirmed by LC3 immunofluorescence and immunoblotting analyses that curcumin triggers autophagy in human lung adenocarcinoma A549 cell line. In parallel with autophagy induction, gene expression of human GD3 synthase (hST8Sia I) responsible for ganglioside GD3 synthesis was markedly elevated in response to curcumin in A549 cells. To investigate transcriptional activation of hST8Sia I associated with autophagy formation in curcumin-treated A549 cells, functional characterization of the 5’-flanking region of the hST8Sia I gene was carried out using luciferase reporter assay system. Deletion analysis demonstrated that the -1146 to -646 region, which includes putative c-Ets-1, CREB, AP-1 and NF-κB binding sites, functions as the curcumin-responsive promoter of hST8Sia I in A549 cells. Site-directed mutagenesis and chromatin immunoprecipitation assay demonstrated that the NF-κB binding site at -731 to -722 was indispensable for the curcumin-induced hST8Sia I gene expression in A549 cells. Moreover, the transcriptional activation of hST8Sia I by curcumin A549 cells was strongly inhibited by compound C, an inhibitor of AMP-activated protein kinase (AMPK). These results suggest that curcumin controls hST8Sia I gene expression via AMPK signal pathway in A549 cells.

Curcumin Downregulates Human GM3 Synthase (hST3Gal V) Gene Expression with Autophagy Induction in Human Colon Carcinoma HCT116 Cells

“…Dulbecco's modified Eagle's medium (DMEM; WelGENE Co., Daegu, Korea) containing 10% (v/v) heat-inactivated fetal bovine serum (FBS) and 1% PSA (WelGENE Co., Daegu, Korea) was used as normal growth medium and cells were cultured at 37°C under 5% CO 2 . Cell viability was determined by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, as described previously [ 11 , 12 ].…”

“…Isolation of total RNA from HCT116 cells and RT-PCR experiment including first cDNA synthesis by reverse transcriptase were performed as previously described [ 11 – 14 ]. The primers used in RT-PCR are follows as: hST3Gal V (413 bp), 5′-CCCTGCCATTCTGGGTACGAC-3′(sense) and 5′-CACGATCAATGCCTCCACTGAGATC-3′(antisense); β -actin (247 bp), 5′-CAAGAGATGGCCACGGCTGCT-3′ (sense) and 5′-TCCTTCTGCATCCTGTCGGCA-3′ (antisense).…”

“…Immunofluorescence staining was conducted as described previously [ 11 ]. Briefly, after fixation and blocking of curcumin-treated cells, cells were reacted with the anti-GM3 monoclonal antibody (mouse IgM, Kappa-chain, clone, GMR6; Seigakagu, Tokyo, Japan) followed by FITC-conjugated goat-anti-mouse IgG/M/A mixture (Sigma; St. Louis, MO, USA).…”

“…Very recently, we have demonstrated for the first time that gene expression of human GD3 synthase (hST8Sia I) catalyzing ganglioside GD3 synthesis was upregulated during autophagy induction by curcumin in human lung adenocarcinoma A549 cells [ 11 ]. On the contrary, however, we found here that gene expression of human GM3 synthase (hST3Gal V) catalyzing ganglioside GM3 synthesis was downregulated during autophagy induction by curcumin in human colon carcinoma HCT116 cells.…”

Versatile role of curcumin and its derivatives in lung cancer therapy

“…Curcumin reduces the malignancy of A549 cells by activation of autophagy by triggering the expression of GD3 synthase (hST8Sia I) gene. On the contrary, AMPK is a sensor of energy and a decreased level of ATP and ATP/AMP ratio is associated with stimulation of autophagy (Krishan, Sahni, Leck, Jansson, & Richardson, 2020; M. Lee et al, 2018; R. D. Ma et al, 2020). Investigation of molecular pathways exhibits that AMPK function as an upstream mediator of hST8Sia I. Curcumin (20, 40, 60 and 80 mM for 12 and 24 hr) upregulates AMPK to induce hST8Sia I‐mediated autophagy (M. Lee et al, 2018).…”