Autophagy inhibition enhances RAD001-induced cytotoxicity in human bladder cancer cells

Ji Fan Lin, Yi Chia Lin, Shan Che Yang, Te Fu Tsai, Hung En Chen, Kuang Yu Chou, Yi-Sheng Huang

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

Background: Mammalian target of rapamycin (mTOR), involved in PI3K/AKT/mTOR pathway, is known to play a central role in regulating the growth of cancer cells. The PI3K/AKT/mTOR pathway enhances tumor survival and proliferation through suppressing autophagy, which sustains energy homeostasis by collecting and recycling cellular components under stress conditions. Conversely, inhibitors of the mTOR pathway such as RAD001 induce autophagy, leading to promotion of tumor survival and limited antitumor efficacy. We thus hypothesized that the use of autophagy inhibitor in combination with mTOR inhibition improves the cytotoxicity of mTOR inhibitors in bladder cancer. Materials and methods: The cytotoxicity of RT4, 5637, HT1376, and T24 human bladder cancer cells treated with RAD001 alone or combined with autophagy inhibitors (3-methyladenine (3-MA), bafilomycin A1 (Baf A1), chloroquine, or hydroxychloroquine) was assessed using the WST-8 cell viability kit. The autophagy status in cells was analyzed by the detection of microtubule-associated light chain 3 form II (LC3-II), using immunofluorescent staining and Western blot. Acidic vesicular organelle (AVO) formation in treated cells was determined by acridine orange vital staining. Inhibition of mTOR pathway by RAD001 was monitored by using a homemade quantitative polymerase chain reaction gene array, while phospho-mTOR was detected using Western blot. Induced apoptosis was determined by measurement of caspase 3/7 activity and DNA fragmentation in cells after treatment. Results: Advanced bladder cancer cells (5637, HT1376, and T24) were more resistant to RAD001 than RT4. Autophagy flux detected by the expression of LC3-II showed RAD001-induced autophagy. AVO formation was detected in cells treated with RAD001 and was inhibited by the addition of 3-MA or Baf A1. Cotreatment of RAD001 with autophagy inhibitors further reduced cell viability and induced apoptosis in bladder cancer cells. Conclusion: Our results indicate that simultaneous inhibition of the mTOR and autophagy pathway significantly enhances apoptosis, and it is suggested to be a new therapeutic paradigm for the treatment of bladder cancer.

Original languageEnglish
Pages (from-to)1501-1513
Number of pages13
JournalDrug Design, Development and Therapy
Volume10
DOIs
Publication statusPublished - Apr 18 2016

Fingerprint

Autophagy
Sirolimus
Urinary Bladder Neoplasms
Apoptosis
Phosphatidylinositol 3-Kinases
Organelles
Cell Survival
Western Blotting
Staining and Labeling
Hydroxychloroquine
Caspase 7
Everolimus
Light
Neoplasms
Acridine Orange
Survival
Chloroquine
Recycling
DNA Fragmentation
Microtubules

Keywords

  • Apoptosis
  • Autophagy
  • Bladder cancer
  • Chloroquine
  • RAD001

ASJC Scopus subject areas

  • Pharmaceutical Science
  • Pharmacology
  • Drug Discovery

Cite this

Lin, J. F., Lin, Y. C., Yang, S. C., Tsai, T. F., Chen, H. E., Chou, K. Y., & Huang, Y-S. (2016). Autophagy inhibition enhances RAD001-induced cytotoxicity in human bladder cancer cells. Drug Design, Development and Therapy, 10, 1501-1513. https://doi.org/10.2147/DDDT.S95900

Autophagy inhibition enhances RAD001-induced cytotoxicity in human bladder cancer cells. / Lin, Ji Fan; Lin, Yi Chia; Yang, Shan Che; Tsai, Te Fu; Chen, Hung En; Chou, Kuang Yu; Huang, Yi-Sheng.

In: Drug Design, Development and Therapy, Vol. 10, 18.04.2016, p. 1501-1513.

Research output: Contribution to journalArticle

Lin, JF, Lin, YC, Yang, SC, Tsai, TF, Chen, HE, Chou, KY & Huang, Y-S 2016, 'Autophagy inhibition enhances RAD001-induced cytotoxicity in human bladder cancer cells', Drug Design, Development and Therapy, vol. 10, pp. 1501-1513. https://doi.org/10.2147/DDDT.S95900
Lin, Ji Fan ; Lin, Yi Chia ; Yang, Shan Che ; Tsai, Te Fu ; Chen, Hung En ; Chou, Kuang Yu ; Huang, Yi-Sheng. / Autophagy inhibition enhances RAD001-induced cytotoxicity in human bladder cancer cells. In: Drug Design, Development and Therapy. 2016 ; Vol. 10. pp. 1501-1513.
@article{a2b3822ab3344b9ea9085e453ae522be,
title = "Autophagy inhibition enhances RAD001-induced cytotoxicity in human bladder cancer cells",
abstract = "Background: Mammalian target of rapamycin (mTOR), involved in PI3K/AKT/mTOR pathway, is known to play a central role in regulating the growth of cancer cells. The PI3K/AKT/mTOR pathway enhances tumor survival and proliferation through suppressing autophagy, which sustains energy homeostasis by collecting and recycling cellular components under stress conditions. Conversely, inhibitors of the mTOR pathway such as RAD001 induce autophagy, leading to promotion of tumor survival and limited antitumor efficacy. We thus hypothesized that the use of autophagy inhibitor in combination with mTOR inhibition improves the cytotoxicity of mTOR inhibitors in bladder cancer. Materials and methods: The cytotoxicity of RT4, 5637, HT1376, and T24 human bladder cancer cells treated with RAD001 alone or combined with autophagy inhibitors (3-methyladenine (3-MA), bafilomycin A1 (Baf A1), chloroquine, or hydroxychloroquine) was assessed using the WST-8 cell viability kit. The autophagy status in cells was analyzed by the detection of microtubule-associated light chain 3 form II (LC3-II), using immunofluorescent staining and Western blot. Acidic vesicular organelle (AVO) formation in treated cells was determined by acridine orange vital staining. Inhibition of mTOR pathway by RAD001 was monitored by using a homemade quantitative polymerase chain reaction gene array, while phospho-mTOR was detected using Western blot. Induced apoptosis was determined by measurement of caspase 3/7 activity and DNA fragmentation in cells after treatment. Results: Advanced bladder cancer cells (5637, HT1376, and T24) were more resistant to RAD001 than RT4. Autophagy flux detected by the expression of LC3-II showed RAD001-induced autophagy. AVO formation was detected in cells treated with RAD001 and was inhibited by the addition of 3-MA or Baf A1. Cotreatment of RAD001 with autophagy inhibitors further reduced cell viability and induced apoptosis in bladder cancer cells. Conclusion: Our results indicate that simultaneous inhibition of the mTOR and autophagy pathway significantly enhances apoptosis, and it is suggested to be a new therapeutic paradigm for the treatment of bladder cancer.",
keywords = "Apoptosis, Autophagy, Bladder cancer, Chloroquine, RAD001",
author = "Lin, {Ji Fan} and Lin, {Yi Chia} and Yang, {Shan Che} and Tsai, {Te Fu} and Chen, {Hung En} and Chou, {Kuang Yu} and Yi-Sheng Huang",
year = "2016",
month = "4",
day = "18",
doi = "10.2147/DDDT.S95900",
language = "English",
volume = "10",
pages = "1501--1513",
journal = "Drug Design, Development and Therapy",
issn = "1177-8881",
publisher = "Dove Medical Press Ltd.",

}

TY - JOUR

T1 - Autophagy inhibition enhances RAD001-induced cytotoxicity in human bladder cancer cells

AU - Lin, Ji Fan

AU - Lin, Yi Chia

AU - Yang, Shan Che

AU - Tsai, Te Fu

AU - Chen, Hung En

AU - Chou, Kuang Yu

AU - Huang, Yi-Sheng

PY - 2016/4/18

Y1 - 2016/4/18

N2 - Background: Mammalian target of rapamycin (mTOR), involved in PI3K/AKT/mTOR pathway, is known to play a central role in regulating the growth of cancer cells. The PI3K/AKT/mTOR pathway enhances tumor survival and proliferation through suppressing autophagy, which sustains energy homeostasis by collecting and recycling cellular components under stress conditions. Conversely, inhibitors of the mTOR pathway such as RAD001 induce autophagy, leading to promotion of tumor survival and limited antitumor efficacy. We thus hypothesized that the use of autophagy inhibitor in combination with mTOR inhibition improves the cytotoxicity of mTOR inhibitors in bladder cancer. Materials and methods: The cytotoxicity of RT4, 5637, HT1376, and T24 human bladder cancer cells treated with RAD001 alone or combined with autophagy inhibitors (3-methyladenine (3-MA), bafilomycin A1 (Baf A1), chloroquine, or hydroxychloroquine) was assessed using the WST-8 cell viability kit. The autophagy status in cells was analyzed by the detection of microtubule-associated light chain 3 form II (LC3-II), using immunofluorescent staining and Western blot. Acidic vesicular organelle (AVO) formation in treated cells was determined by acridine orange vital staining. Inhibition of mTOR pathway by RAD001 was monitored by using a homemade quantitative polymerase chain reaction gene array, while phospho-mTOR was detected using Western blot. Induced apoptosis was determined by measurement of caspase 3/7 activity and DNA fragmentation in cells after treatment. Results: Advanced bladder cancer cells (5637, HT1376, and T24) were more resistant to RAD001 than RT4. Autophagy flux detected by the expression of LC3-II showed RAD001-induced autophagy. AVO formation was detected in cells treated with RAD001 and was inhibited by the addition of 3-MA or Baf A1. Cotreatment of RAD001 with autophagy inhibitors further reduced cell viability and induced apoptosis in bladder cancer cells. Conclusion: Our results indicate that simultaneous inhibition of the mTOR and autophagy pathway significantly enhances apoptosis, and it is suggested to be a new therapeutic paradigm for the treatment of bladder cancer.

AB - Background: Mammalian target of rapamycin (mTOR), involved in PI3K/AKT/mTOR pathway, is known to play a central role in regulating the growth of cancer cells. The PI3K/AKT/mTOR pathway enhances tumor survival and proliferation through suppressing autophagy, which sustains energy homeostasis by collecting and recycling cellular components under stress conditions. Conversely, inhibitors of the mTOR pathway such as RAD001 induce autophagy, leading to promotion of tumor survival and limited antitumor efficacy. We thus hypothesized that the use of autophagy inhibitor in combination with mTOR inhibition improves the cytotoxicity of mTOR inhibitors in bladder cancer. Materials and methods: The cytotoxicity of RT4, 5637, HT1376, and T24 human bladder cancer cells treated with RAD001 alone or combined with autophagy inhibitors (3-methyladenine (3-MA), bafilomycin A1 (Baf A1), chloroquine, or hydroxychloroquine) was assessed using the WST-8 cell viability kit. The autophagy status in cells was analyzed by the detection of microtubule-associated light chain 3 form II (LC3-II), using immunofluorescent staining and Western blot. Acidic vesicular organelle (AVO) formation in treated cells was determined by acridine orange vital staining. Inhibition of mTOR pathway by RAD001 was monitored by using a homemade quantitative polymerase chain reaction gene array, while phospho-mTOR was detected using Western blot. Induced apoptosis was determined by measurement of caspase 3/7 activity and DNA fragmentation in cells after treatment. Results: Advanced bladder cancer cells (5637, HT1376, and T24) were more resistant to RAD001 than RT4. Autophagy flux detected by the expression of LC3-II showed RAD001-induced autophagy. AVO formation was detected in cells treated with RAD001 and was inhibited by the addition of 3-MA or Baf A1. Cotreatment of RAD001 with autophagy inhibitors further reduced cell viability and induced apoptosis in bladder cancer cells. Conclusion: Our results indicate that simultaneous inhibition of the mTOR and autophagy pathway significantly enhances apoptosis, and it is suggested to be a new therapeutic paradigm for the treatment of bladder cancer.

KW - Apoptosis

KW - Autophagy

KW - Bladder cancer

KW - Chloroquine

KW - RAD001

UR - http://www.scopus.com/inward/record.url?scp=84964495085&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84964495085&partnerID=8YFLogxK

U2 - 10.2147/DDDT.S95900

DO - 10.2147/DDDT.S95900

M3 - Article

AN - SCOPUS:84964495085

VL - 10

SP - 1501

EP - 1513

JO - Drug Design, Development and Therapy

JF - Drug Design, Development and Therapy

SN - 1177-8881

ER -