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Salinomycin, a p-glycoprotein inhibitor, sensitizes radiation-treated cancer cells by increasing DNA damage and inducing G2 arrest

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Summary

Salinomycin (Sal) is potentially useful for the treatment of cancer. The present study examined a novel mechanism of Sal sensitization in cancer cells. Sal sensitized radiation-treated cancer cells by inducing G2 arrest and causing DNA damage. Sal treatment also reduced p21 levels in radiation-treated cells. Considering that Sal sensitizes doxorubicin (DOX)- or etoposide (ETO)-treated cancer cells by causing DNA damage and reducing p21 expression, the results from our study suggest that the mechanism underlying Sal sensitization is conserved in both chemo- and radiation-treated cells. We also tested the ability of Sal to inhibit p-glycoprotein (P-gp), which plays a role in the efflux of anti-cancer drugs to reduce cellular damage. In particular, we compared Sal to verapamil (Ver), a well-known P-gp inhibitor. Sal inhibits P-gp with a different substrate distinct from that of Ver. In addition, Sal sensitized Ver-resistant cells, indicating that this compound is more effective for sensitizing than Ver. Taken together, the results from our study may contribute to the development of Sal-based therapy for cancer patients treated with P-gp-inhibiting drugs or radiation therapy.

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Abbreviations

DOX:

doxorubicin

DMSO:

dimethylsulfoxide

ETO:

etoposide

Sal:

salinomycin

Ver:

verapamil

MDR:

multi-drug resistance

P-gp:

p-glycoprotein

FACS:

fluorescence-activated cell sorting

Rho:

rhodamin123

CFDA:

carboxyfluorescein diacetate

DAPI:

4′-6-diamidino-2-phenylindole

FBS:

fetal bovine serum

BSA:

bovine serum albumin

TCA:

trichloroacetic acid

PBS:

phosphate buffered saline

SDS-PAGE:

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

TUNEL:

terminal transferase dUTP nick end labeling

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Acknowledgements

This work was supported by research grant (NCC0910170) from the National Cancer Center, South Korea.

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Authors and Affiliations

  1. Research Institute, National Cancer Center, 809 Madu 1-dong, Ilsan-gu, Goyang-si, Gyeonggi-do, 411-764, Republic of Korea

    Won Ki Kim, Ju-Hwa Kim, Kyungsil Yoon, Sunshin Kim, Jungsil Ro, Han Sung Kang & Sungpil Yoon

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  1. Won Ki Kim
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Correspondence to Sungpil Yoon.

Additional information

Won Ki Kim and Ju-Hwa Kim contributed equally to this work.

Electronic Supplementary Materials

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Supplementary Fig. 1

Co-treatment with radiation and Sal increases apoptosis. (A-B) Hs578T cells were grown on 60 mm-diameter dishes and then treated with 4 Gy of radiation, 5 μM Sal, 5 μM Sal with 4 Gy of radiation (4 Gy+Sal), or DMSO (Control). After 48 h, Annexin V (A) and TUNEL analyses (B) were performed as described in the “Materials and method” section. (JPEG 16 kb)

High resolution image (TIFF 665 kb)

Supplementary Fig. 2

Quantification of CFDA and Rho staining confirms that Sal and Ver inhibit different P-gp substrates. (A-B) MCF7 cells were grown and treated for 24 h with 40 μM Ver, 5 μM Sal, or DMSO (Con). The cells were then stained with CFDA (A) or Rho (B). The stained cells were subsequently analyzed using a FACSCalibur flow cytometry system as described in the “Materials and method” section. (JPEG 18 kb)

High resolution image (TIFF 804 kb)

Supplementary Fig. 3

Higher Sal concentrations do not contribute to increased Rho staining. (A-B) Hs578T and MCF7 cells were grown and treated for 24 h with 5 μM Sal (Sal-5), 10 μM Sal (Sal-10), 15 μM Sal (Sal-15), or DMSO (Con). The cells were then stained with Rho as described in the “Materials and method” section. The stained cells were subsequently examined using an inverted fluorescence microscope with 10× (A) and 16× (B) objective lenses. (JPEG 31 kb)

High resolution image (TIFF 2379 kb)

Supplementary Fig. 4

Sal sensitizes relatively Ver-resistant cancer cells. (A-B) Hs578T and MCF7 cells were grown and treated for 24 h with 20 μM Ver (Ver-20), 40 μM Ver (Ver-40), 80 μM Ver (Ver-80), 5 μM Sal (Sal-5), or DMSO (Con). They were subsequently observed using an inverted microscope with a 10× objective lens. (JPEG 35 kb)

High resolution image (TIFF 799 kb)

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Kim, W.K., Kim, JH., Yoon, K. et al. Salinomycin, a p-glycoprotein inhibitor, sensitizes radiation-treated cancer cells by increasing DNA damage and inducing G2 arrest. Invest New Drugs 30, 1311–1318 (2012). https://doi.org/10.1007/s10637-011-9685-6

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