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Thioridazine specifically sensitizes drug-resistant cancer cells through highly increase in apoptosis and P-gp inhibition

  • Research Article
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Tumor Biology

An Erratum to this article was published on 14 August 2015

Abstract

This study was designed to identify conditions that induce an increase in the sensitivity of drug-resistant cancer cells compared to sensitive cells. Using cell proliferation assays and microscopic observation, thioridazine (THIO) was found to induce higher sensitization in drug-resistant KBV20C cancer cells compared to sensitive KB parent cells. By studying cleaved PARP, annexin V staining, and Hoechst staining, we found that THIO largely increased apoptosis specifically in KBV20C cells, suggesting that the difference in sensitization between the resistant and sensitive cells can be attributed to the ability of THIO to induce apoptosis. THIO could also inhibit p-glycoprotein (P-gp) activity in the resistant KBV20C cells. These observations suggest that the mechanisms underlying THIO sensitization in resistant KBV20C cells involve both apoptosis and P-gp inhibition. Furthermore, co-treatment with THIO and vinblastine (VIB) induces higher sensitization in KBV20C cells than KB cells. As observed in a single treatment with THIO, the sensitization mechanism induced by the co-treatment also involves both apoptosis and P-gp inhibition. These results suggest that the THIO sensitization mechanism is generally conserved. Our findings may contribute to the development of THIO-based therapies for patients presenting resistance to antimitotic drugs.

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Abbreviations

THIO:

Thioridazine

VIB:

Vinblastine

VER:

Verapamil

P-gp:

P-glycoprotein

DMSO:

Dimethylsulfoxide

FACS:

Fluorescence-activated cell sorting

FBS:

Fetal bovine serum

TCA:

Trichloroacetic acid

C-PARP:

Cleaved poly ADP ribose polymerase

PBS:

Phosphate buffered saline

SDS-PAGE:

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

S.D.:

Standard deviation

RT:

Room temperature

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Acknowledgments

This work was supported by a research grant (NCC1310120) 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

    Ae-Ran Choi, Ju-Hwa Kim & Sungpil Yoon

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  1. Ae-Ran Choi
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  2. Ju-Hwa Kim
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  3. Sungpil Yoon
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Corresponding author

Correspondence to Sungpil Yoon.

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Supplementary Figure 1

Increasing treatment duration allows low concentration of THIO to sensitize KBV20C cells. (A) KB and KBV20C cells were grown on 6-well plates and treated with 5 nM VIB (VIB), 7.5 μM THIO (THIO-7.5), 10 μM THIO (THIO-10), 12.5 μM THIO (THIO-12.5), 15 μM THIO (THIO-15), 17.5 μM THIO (THIO-17.5), 20 μM THIO (THIO-20) or 0.1% DMSO (Con). After 48 h, all cells were observed using an inverted microscope with a 10× objective lens. We performed two independent experiments. (B) KB and KBV20C cells were plated on 96-well plates and grown to 30-40% confluence. The cells were then stimulated for 72 h with 5 nM VIB (VIB), 7.5 μM THIO (THIO-7.5), 10 μM THIO (THIO-10), 12.5 μM THIO (THIO-12.5), 15 μM THIO (THIO-15), 17.5 μM THIO (THIO-17.5), 20 μM THIO (THIO-20) or 0.1% DMSO (Con). A cell proliferation assay was performed as described in Materials and methods. The data are represented by the mean ± S.D. of at least triplicate results in quadruplicates. Statistical analysis was conducted using one-way analysis of variance (ANOVA) followed by multiple-comparison test; * P <0.05 compared to the corresponding control. (GIF 59 kb)

High resolution image (TIFF 299 kb)

Supplementary Figure 2

THIO increases late apoptosis in drug-resistant KBV20C cells. (A) KB and KBV20C were grown on 60 mm-diameter dishes and treated with 5 nM VIB (VIB-5), 10 μM THIO (THIO-10), 12.5 μM THIO (THIO-12.5), 15 μM THIO (THIO-15) or 0.1% DMSO (Con). After 48 h, FACS analysis was performed as described in Materials and methods. (B) KB and KBV20C were grown on 6-well plates and treated with 5 nM VIB (VIB-5), 10 μM THIO (THIO-10), 12.5 μM THIO (THIO-12.5), 15 μM THIO (THIO-15) or 0.1% DMSO (Con). After 24, 48 h, all cells were then stained with Hoechst as described in Materials and methods. The stained cells were subsequently examined using an inverted fluorescence microscope with a 32X objective lens. (GIF 36 kb)

High resolution image (TIFF 486 kb)

Supplementary Figure 3

THIO sensitization is accompanied with an increase in p21 and a decrease in survivin levels. KB and KBV20C were plated on 60 mm-diameter dishes and treated for 24 h with 7.5 μM THIO (THIO-7.5), 10 μM THIO (THIO-10), 5 nM VIB (VIB-5) or 0.1% DMSO (Con). The cells were used for Western blot analysis using antibodies against pH2AX, pChk2, c-IAP2, c-IAP1, Bcl-XL, pRb, Cdk4, pp70S6K, PCNA, p53, p21, Survivin and GAPDH. (GIF 58 kb)

High resolution image (TIFF 225 kb)

Supplementary Figure 4

Apoptosis of VIB-treated resistant KBV20C cells is increased by THIO, compared to KB cells. (A) KB and KBV20C cells were grown on 6-well plates and treated with 5 nM VIB (VIB-5), 2 nM VIB (VIB-2), 5 μM THIO (THIO-5), 10 μM THIO (THIO-10), 15 μM THIO (THIO-15), 2 nM VIB+5 μM THIO (VIB+THIO-5), 2 nM VIB+10 μM THIO (VIB+THIO-10), 2 nM VIB+15 μM THIO (VIB+THIO-15) or 0.1% DMSO (Con). After 48 h, all cells were observed using an inverted microscope with a 10× objective lens. We performed two independent experiments. (B) KB and KBV20C were plated on 60 mm-diameter dishes and treated for 48 h with 5 nM VIB (VIB-5), 10 μM THIO (THIO-10), 5 nM VIB+10 μM THIO (VIB+THIO-10) or 0.1% DMSO (Con). Annexin V analysis was performed as described in the Materials and methods. (GIF 72 kb)

High resolution image (TIFF 408 kb)

Supplementary Figure 5

P-gp inhibition is increased by THIO/VIB co-treatment compared to single treatment with THIO in resistant KBV20C cells. (A) KBV20C cells were plated on 6-well plates and treated for 24 h with 5 μM verapamil (VER-5), 5 μM THIO (THIO-5), 10 μM THIO (THIO-10), 5 nM VIB (VIB-5), 5 nM VIB+5 μM verapamil (VIB-5+VER-5), 5 nM VIB+5 μM THIO (VIB-5+ THIO-5), 5 nM VIB+10 μM THIO (VIB-5+ THIO-10) or 0.1% DMSO (Con). After 24 h, all cells were then stained with Calcein-AM as described in Materials and methods. The stained cells were subsequently examined using FACS analysis. (B) KBV20C cells were grown on 6-well plates and treated for 24 h with 2 nM VIB (VIB-2), 5 μM verapamil (VER-5), 10 μM THIO (THIO-10), 2 nM VIB+10 μM THIO (VIB-2+THIO-10), 2 nM VIB+5 μM verapamil (VIB-2+VER-5) or 0.1% DMSO (Con). After 24 h, all cells were then stained with Calcein-AM as described in Materials and methods. The stained cells were subsequently examined using FACS analysis. (GIF 41 kb)

High resolution image (TIFF 207 kb)

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Choi, AR., Kim, JH. & Yoon, S. Thioridazine specifically sensitizes drug-resistant cancer cells through highly increase in apoptosis and P-gp inhibition. Tumor Biol. 35, 9831–9838 (2014). https://doi.org/10.1007/s13277-014-2278-1

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