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: RAD001; apoptosis; autophagy; bladder cancer; chloroquine.

Figure 1

RAD001 decreased cell viability in human bladder cancer cells. Notes: (A) Cell viability of bladder cancer cells treated with various concentration of RAD001 for 24 hours. Cells were seed in 96-well plates for 24 hours before the addition of indicated concentration of RAD001. The cell viability was then detected at 24 hours of treatment using WST-8 cell viability kit. Four bladder cancer cell lines were used: RT4 (Grade I TCC; wild-type p53), 5637 (Grade II TCC; mutant p53), HT1376 (Grade III TCC; mutant p53), and T24 (Grade III TCC; mutant p53). (B) Cell viability of RT4, 5637, HT1376, and T24 cells upon RAD001 treatment for 24, 48, and 72 hours. Cell viabilities of these cells treated with 0–5 µM RAD001 as described in (A). The values are shown as the mean ± SD of three independent experiments; *P<0.05. Abbreviations: SD, standard deviation; TCC, transitional cell carcinoma.

Figure 2

Inhibition of mTOR-related genes and mTORC1 downstream effectors by RAD001. Notes: qPCR detection of mTOR-related genes, including (A) mTOR complexes, (B) mTORC1-positive regulation, and (C) mTORC2-positive regulation in T24 cells treated with 1 or 5 µM RAD001. Cells were seeded 24 hours before RAD001 treatment. Total RNA samples were extracted at 24 hours posttreatment, reverse transcribed, and subjected to the detection of genes involved in mTOR signaling. The values are shown as the mean ± SD of three independent experiments; *P<0.05. (D) Protein samples were isolated at indicated time points then subjected to the detection of p-4EBP1 and p-RPS6 by Western blot. Abbreviations: mTOR, mammalian target of rapamycin; SD, standard deviation; p-4EBP1, phospho-4EBP1; p-RPS6, phospho-RPS6; mTORC1, mTOR complex 1; mTORC2, mTOR complex 2; qPCR, quantitative polymerase chain reaction; mRNA, messenger RNA.

Figure 3

RAD001 induces autophagy in human bladder cancer cells. Notes: (A) Detection of AVO formation by AO vital staining in RT4, 5637, and T24 cells treated with 1 µM RAD001 with or without 2 hours pretreatment of 200 nM Baf A1. Representative photos from three independent experiments with similar results are shown. Scale bar: 50 µm. (B) Quantitative detection of AVO formation was determined using flow cytometry. Treated cells were collected immediately after AO staining and the percentage of red-fluorescent-positive cells compared to control was measured using flow cytometry. Data are from three independent experiments with similar results and are presented as mean ± SD; *P<0.05. (C) Detection of LC3-II processing and (D) autophagic flux was detected by monitoring the expression level of p62 and LC3-II in cells treated with 0–5 µM RAD001 for 24 hours and cells treated with 1 µM RAD001 with or without the cotreatment of 200 nM Baf A1, respectively. The relative band intensities of p62 and LC3-II were quantitated by denstometric scanning and the relative expression levels are presented as the fold of control cells (lower panels in C and D). The statistical calculation from blots of three independent experiments is shown. The results are presented as the mean ± SD; *P<0.05. Abbreviations: AVO, acidic vesicular organelle; AO, acridine orange; SD, standard deviation; Baf A1, bafilomycin A1.

Figure 3

RAD001 induces autophagy in human bladder cancer cells. Notes: (A) Detection of AVO formation by AO vital staining in RT4, 5637, and T24 cells treated with 1 µM RAD001 with or without 2 hours pretreatment of 200 nM Baf A1. Representative photos from three independent experiments with similar results are shown. Scale bar: 50 µm. (B) Quantitative detection of AVO formation was determined using flow cytometry. Treated cells were collected immediately after AO staining and the percentage of red-fluorescent-positive cells compared to control was measured using flow cytometry. Data are from three independent experiments with similar results and are presented as mean ± SD; *P<0.05. (C) Detection of LC3-II processing and (D) autophagic flux was detected by monitoring the expression level of p62 and LC3-II in cells treated with 0–5 µM RAD001 for 24 hours and cells treated with 1 µM RAD001 with or without the cotreatment of 200 nM Baf A1, respectively. The relative band intensities of p62 and LC3-II were quantitated by denstometric scanning and the relative expression levels are presented as the fold of control cells (lower panels in C and D). The statistical calculation from blots of three independent experiments is shown. The results are presented as the mean ± SD; *P<0.05. Abbreviations: AVO, acidic vesicular organelle; AO, acridine orange; SD, standard deviation; Baf A1, bafilomycin A1.

Figure 4

Inhibition of autophagy increases RAD001-induced cytotoxicity. Notes: Cell viability of (A) RT4, (B) 5637, and (C) T24 cells treated with 1 or 5 µM RAD001 with or without the cotreatment of 1 or 3 mM 3-MA, 100 or 200 nM Baf A1, or 25 or 50 µM CQ for 72 hours. Cell viability was detected using WST-8 reagent, and the values are shown as the mean ± SD of three independent experiments; *P<0.05. #P<0.05 when compared to RAD001 treatment group. Abbreviations: Baf A1, bafilomycin A1; CQ, chloroquine; SD, standard deviation; 3-MA, 3-methyladenine.

Figure 5

Coordinate inhibition of autophagy and mTOR enhances apoptosis in human bladder cancer cells. Notes: Caspase 3/7 activity was detected in RT4, 5637, and T24 cells treated with 1 µM RAD001 with or without the cotreatment of 3 mM 3-MA or 200 nM Baf A1 for 24 hours. The data from three independent experiments are shown. The results are presented as the mean ± SD; *P<0.05. Abbreviations: mTOR, mammalian target of rapamycin; SD, standard deviation; Baf A1, bafilomycin A1; 3-MA, 3-methyladenine.