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Effects of 2-(2-Chlorophenyl)ethylbiguanide on ERAD Component Expression in HT-29 Cells Under a Serum- and Glucose-Deprived Condition

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Abstract

We recently characterized the cytotoxic action of a novel phenformin derivative, 2-(2-chlorophenyl)ethylbiguanide (2-Cl-Phen), on HT-29 cells under a serum- and glucose-deprived condition and found that 2-Cl-Phen attenuated ATF4 and GRP78, typical downstream targets of the unfolded protein response (UPR), together with c-Myc protein expression in a transcriptional and posttranscriptional manner. In the current study, we focused on the expression of ER-associated protein degradation (ERAD) components after treatment with 2-Cl-Phen under a serum- and glucose-deprived condition. Among nine ER-localizing factors regulating protein quality control within the ER, the amounts of Herp, GRP78, GRP94, and OS9 proteins were significantly downregulated by treatment with 2-Cl-Phen. In particular, replacement of the culture medium with the serum- and glucose-deprived medium induced the expression of Herp protein at the early phase. This increase in Herp protein was accompanied by an increase in its mRNA, and its induction was significantly dampened by 2-Cl-Phen. However, cotreatment with a proteasome inhibitor, MG132, restored Herp expression only to a limited extent. Taken together, these results show that 2-Cl-Phen changed the expression of several ERAD components, especially by transcriptional inhibition of Herp induction by 2-Cl-Phen when it occurred at an early phase, and this finding provides new insights into understanding the mechanisms of 2-Cl-Phen-mediated cytotoxicity.

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Abbreviations

ATF4:

Activating transcription factor 4

ATF6:

Activating transcription factor 6

2-Cl-Phen:

2-(2-Chlorophenyl)ethylbiguanide

CHX:

Cycloheximide

Derlin-1:

Degradation in endoplasmic reticulum protein 1

ER:

Endoplasmic reticulum

ERAD:

ER stress-associated protein degradation

GRP78:

78 kDa glucose-regulated protein

GRP94:

94 kDa glucose-regulated protein

G3PDH:

Glyceraldehyde 3-phosphate dehydrogenase

Herp:

Homocysteine-induced ER protein

Hrd1:

Hydroxymethylglutaryl reductase degradation 1

OS9:

Osteosarcoma amplified 9

PERK:

PKR-like endoplasmic reticulum kinase

PDI:

Protein disulfide isomerase

RT-PCR:

Reverse transcription polymerase chain reaction

SEL1L:

Suppressor/enhancer of lin-12-like

VCP:

Valosin-containing protein

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Funding

This work is partly supported by the Koshiyama Science and Technology Foundation (to Kensuke Okuda), Grant-in-Aid for Challenging Exploratory Research (no. 17K19901 to Kentaro Oh-hashi), and the OGAWA Science and Technology Foundation (to Kentaro Oh-hashi).

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Author notes

  1. Kensuke Okuda

    Present address: Laboratory of Bioorganic & Natural Products Chemistry, Kobe Pharmaceutical University, 4-19-1, Motoyama-kita, Higashinada, Kobe, 658-8558, Japan

Authors and Affiliations

  1. Department of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan

    Kentaro Oh-hashi, Shiori Matsumoto & Yoko Hirata

  2. United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan

    Kentaro Oh-hashi & Yoko Hirata

  3. Laboratory of Pharmaceutical & Medicinal Chemistry, Gifu Pharmaceutical University, 1-25-4, Daigakunishi, Gifu, 501-1196, Japan

    Takayuki Sakai, Kensuke Okuda & Hideko Nagasawa

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  1. Kentaro Oh-hashi
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  2. Shiori Matsumoto
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Correspondence to Kentaro Oh-hashi.

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Supplementary Fig. 1

Effect of tunicamycin on OS9 protein expression in HT-29 cells. HT-29 cells were incubated in serum-containing medium together with or without tunicamycin (Tm, 1 μg/ml) or serum- and glucose-deprived medium for 24 h. The expression levels of the indicated protein were determined as described in the “Materials and methods” section. (PPTX 54.4 kb)

Supplementary Fig. 2

Effect of MG132 and cycloheximide on ATF4 protein expression in HT-29 cells under a serum- and glucose-deprived condition. HT-29 cells were incubated in the serum-containing or serum- and glucose-deprived medium together with 2-Cl-Phen (50 μM), cycloheximide (CHX, 10 μg/ml), MG132 (MG, 10 μM), or vehicle for 12 h. The expression levels of the indicated protein were determined as described in the “Materials and methods” section. Representative results are shown in this figure. (PPTX 180 kb)

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Oh-hashi, K., Matsumoto, S., Sakai, T. et al. Effects of 2-(2-Chlorophenyl)ethylbiguanide on ERAD Component Expression in HT-29 Cells Under a Serum- and Glucose-Deprived Condition. Appl Biochem Biotechnol 188, 1009–1021 (2019). https://doi.org/10.1007/s12010-019-02969-4

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