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Involvement of ER stress and reactive oxygen species generation in anti-cancer effect of CKD-516 for lung cancer

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Abstract

Purpose

CKD-516 (Valecobulin), a vascular-disrupting agent, inhibits microtubule elongation. We evaluated the effect of CKD-516 on lung cancer cells and the underlying molecular mechanisms.

Methods

The effects of S516, an active metabolite of CKD-516, were evaluated in HUVECs and three lung cancer cell lines and by a microtubule polymerization assay. Tubulin cross-linking was used to identify the binding site of S516 on tubulin, and Western blotting was performed to identify the intracellular pathways leading to cell death. Subcutaneous lung cancer xenograft models were used to assess the in vivo effect of CKD-516 on tumor growth.

Results

S516 targeted the colchicine binding site on β-tubulin. In lung cancer cells, S516 increased endoplasmic reticulum (ER) stress and induced reactive oxygen species (ROS) generation by mitochondria and the ER. In addition, CKD-516 monotherapy strongly inhibited the growth of lung cancer xenograft tumors and exerted a synergistic effect with carboplatin.

Conclusion

The findings suggest that CKD-516 exerts an anticancer effect in company with inducing ER stress and ROS production via microtubule disruption in lung cancer cells. CKD-516 may thus have therapeutic potential for lung cancer.

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Abbreviations

4-PBA:

4-Phenylbutyric acid

ATCC:

American type culture collection

ATF6:

Activating transcription factor 6

Carboplatin:

Cyclobutane-1,1-dicarboxylate-O,Oʹ

CHOP:

C/EBP homologous protein

Cys:

Cysteine

DAPI:

4ʹ,6-diamidino-2-phenylindole

DMSO:

Dimethyl sulfoxide

DPI:

Diphenyleneiodonium

EBI:

N,Nʹ-ethylene-bis(iodoacetamide)

ER:

Endoplasmic reticulum

FBS:

Fetal bovine serum

Grp78:

Glucose-regulated protein 78

DCFH-DA:

2′,7′-dichlorofluorescin diacetate

HO-1:

Heme oxygenase 1

HRP:

Horseradish peroxidase

HUVEC:

Human umbilical vein endothelial cell

IACUC:

Institutional animal care and use committee

Ire1α:

Inositol-requiring enzyme 1α

LC3B:

Microtubule-associated protein 1 light chain 3

MOI:

Multiplicity of infection

MTCC:

Modern cell & tissue technologies

NAC:

N-acetylcysteine

NADPH:

Nicotinamide adenine dinucleotide phosphate

NSCLC:

Non-small cell lung cancer

NQO1:

NAD(P)H dehydrogenase

PERK:

Protein kinase RNA-like endoplasmic reticulum kinase

RIPA:

Radioimmunoprecipitation assay buffer

SDS-PAGE:

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis

ROS:

Reactive oxygen species

UPR:

Unfolded protein response

VDA:

Vascular disrupting agent

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Acknowledgements

This work was supported by the National Research Foundation of Korea (NRF) Grant (NRF-2017R1A4A1015860 and 2018R1A2B2003590).

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Authors and Affiliations

  1. College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, 08826, Republic of Korea

    Soo Jin Kim, Kyung Hwan Jegal, Ji-Hye Im, Suntae Kim & Keon Wook Kang

  2. CKD Research Institution, Chong Kun Dang Pharmaceutical Corporation, Yongin-si, Gyeonggi-do, 16995, Republic of Korea

    Soo Jin Kim & Gyutae Park

  3. College of Pharmacy, Chungnam National University, Daejeon, 34134, Republic of Korea

    Hye Gwang Jeong

  4. Department of Herbal Formulation, MRC-GHF, College of Korean Medicine, Daegu Haany University, Gyeongsan, Gyeongsangbuk-do, 38610, Republic of Korea

    Il Je Cho

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  1. Soo Jin Kim
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280_2020_4043_MOESM1_ESM.pptx

Supplementary material 1 (PPTX 125 kb) Increase in poly-ubiquitinated proteins by S516 treatment. H460 cells were treated with S516 (10 and 30 nM) in the presence or absence of 10 nM bortezomib (BTZ) for 24 h. The combination of S516 and BTZ led to accumulation of poly-ubiquitinated proteins

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Kim, S.J., Jegal, K.H., Im, JH. et al. Involvement of ER stress and reactive oxygen species generation in anti-cancer effect of CKD-516 for lung cancer. Cancer Chemother Pharmacol 85, 685–697 (2020). https://doi.org/10.1007/s00280-020-04043-x

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  • DOI: https://doi.org/10.1007/s00280-020-04043-x

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