Molecular and Cellular Biochemistry

, Volume 419, Issue 1–2, pp 29–40

Elucidation of a novel phenformin derivative on glucose-deprived stress responses in HT-29 cells

  • Kentaro Oh-hashi
  • Nao Irie
  • Takayuki Sakai
  • Kensuke Okuda
  • Hideko Nagasawa
  • Yoko Hirata
  • Kazutoshi Kiuchi
Article
  • 293 Downloads

Abstract

Recently, we developed a variety of phenformin derivatives as selective antitumor agents. Based on previous findings, this study evaluated a promising compound, 2-(2-chlorophenyl)ethylbiguanide (2-Cl-Phen), on the basis of stress responses in the human colon cancer cell line HT-29 under a serum- and glucose-deprived condition. 2-Cl-Phen triggered morphological changes such as shrinkage and plasma membrane disintegration, as well as a decrease in mitochondrial activity and an increase in LDH leakage. To understand intracellular issues relating to 2-Cl-Phen, this study focused on the expression levels of ER stress-inducible genes and several oncogenic genes. Serum and glucose deprivation significantly induced a variety of ER stress-inducible genes, but a 12-h treatment of 2-Cl-Phen down-regulated expression of several ER stress-related genes, with the exception of GADD153. Interestingly, the expression levels of ATF6α, GRP78, MANF, and CRELD2 mRNA were almost completely decreased by 2-Cl-Phen. This study also observed that a 24-h treatment of 2-Cl-Phen attenuated the expression levels of GRP78, GADD153, and c-Myc protein. The decrease in c-Myc protein occurred before the fluctuation of GRP78 protein, while the expression of c-Myc mRNA showed little change with cotreatment of serum and glucose deprivation with 2-Cl-Phen. To further understand the 2-Cl-Phen-induced down-regulation of ATF6-related genes, this study investigated the stability of ATF6α and GRP78 proteins using NanoLuc-tagged constructs. The expression levels of NanoLuc-tagged ATF6α and GRP78 were significantly down-regulated by 2-Cl-Phen in the presence or absence of the translation inhibitor cycloheximide. Taken together, our novel phenformin derivative 2-Cl-Phen has the unique characteristic of diminishing tumor adaptive responses, especially the expression of ATF6-related genes, as well as that of c-Myc protein, in a transcriptional and posttranscriptional manner under a serum- and glucose-deprived condition. Further characterization of cytotoxic mechanisms related to phenformin derivatives may give new insights into developing additional promising anticancer agents.

Keywords

ATF6 c-Myc ER stress GRP78 Phenformin 

Abbreviations

ATF6

Activating transcription factor 6

CRELD2

Cysteine-rich with EGF-like domains 2

ER

Endoplasmic reticulum

GADD153

Growth arrest and DNA damage-inducible protein 153

GRP78

78 kDa glucose-regulated protein

GRP94

94 kDa glucose-regulated protein

G3PDH

Glyceraldehyde 3-phosphate dehydrogenase

MANF

Mesencephalic astrocyte-derived neurotrophic factor

XBP1

X-box binding protein 1

Supplementary material

11010_2016_2747_MOESM1_ESM.ppt (10 mb)
Supplementary material 1 (PPT 10203 kb) Supplementary Fig. 1 Effect of 2-Cl-Phen treatment on proliferation and damage in HT-29 cells cultured with the serum-containing or serum- and glucose-deprived medium. HT-29 cells in the serum-containing or serum- and glucose-deprived medium were treated with 2-Cl-Phen (50 μM) or vehicle for the indicated days. The arrows indicate the typical damaged cells
11010_2016_2747_MOESM2_ESM.pptx (8.2 mb)
Supplementary material 2 (PPTX 8425 kb) Supplementary Fig. 2 Expression of EGFP and NanoLuc-tagged ATF6α in COS7 cells A) The schematic structures of EGFP and NanoLuc-tagged ATF6α used in this study. Each construct has a Flag epitope (F) at the N-terminus. EGFP or NanoLuc (NL) gene was inserted between Flag tag and the N-terminus of full-length (FL) or nuclear localized (N) ATF6α. B) Forty-eight hours after transfection of the indicated construct into COS7 cells in a 6-well plate, fluorescent images were obtained by fluorescent microscopy (OLYMPUS). The scale bar is 100 μm. C) Forty-eight hours after transfection of the indicated construct into COS7 cells in a 6-well plate, the cells were lysed, and NanoLuc activity was measured using an aliquot of the lysate as described in the Materials and Methods section
11010_2016_2747_MOESM3_ESM.pptx (8.4 mb)
Supplementary material 3 (PPTX 8575 kb) Supplementary Fig. 3 Expression of EGFP and NanoLuc-tagged GRP78 in COS7 cells A) The schematic structures of EGFP and NanoLuc (NL)-tagged GRP78 (SP-EGFP-GRP78 and SP-NL-GRP78) used in this study. SP indicates a signal peptide sequence at the N-terminus. B) Forty-eight hours after transfection of the indicated construct into COS7 cells in a 6-well plate, fluorescent images were obtained by fluorescent microscopy. The scale bar is 100 μm. C) Forty-eight hours after transfection of the indicated construct into COS7 cells in a 6-well plate, the cells were lysed, and NanoLuc activity was measured using an aliquot of the lysate as described in the Materials and Methods section

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Kentaro Oh-hashi
    • 1
    • 2
  • Nao Irie
    • 1
  • Takayuki Sakai
    • 3
  • Kensuke Okuda
    • 4
  • Hideko Nagasawa
    • 3
  • Yoko Hirata
    • 1
    • 2
  • Kazutoshi Kiuchi
    • 1
    • 2
  1. 1.Department of Chemistry and Biomolecular Science, Faculty of EngineeringGifu UniversityGifuJapan
  2. 2.United Graduate School of Drug Discovery and Medical Information SciencesGifu UniversityGifuJapan
  3. 3.Laboratory of Pharmaceutical and Medicinal ChemistryGifu Pharmaceutical UniversityGifuJapan
  4. 4.Laboratory of Bioorganic and Natural Products ChemistryKobe Pharmaceutical UniversityKobeJapan

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