Abstract
Background/Aim: Liposarcoma is a type of soft-tissue sarcoma arising from fat tissue. It is relatively common among soft-tissue sarcomas. Chloroquine (CQ), an antimalarial drug, can inhibit autophagy and induce apoptosis in cancer cells. Rapamycin (RAPA) is an inhibitor of mTOR. The combination of RAPA and CQ is a strong inhibitor of autophagy. Previously, we showed that the combination of RAPA and CQ was effective against a de-differentiated liposarcoma patient-derived orthotopic xenograft (PDOX) mouse model. In the present study, we investigated the mechanism of efficacy of the combination of RAPA and CQ to target autophagy in a well-differentiated liposarcoma (WDLS) cell line in vitro. Materials and Methods: The human WDLS cell line 93T449 was used. The WST-8 assay was used to test the cytotoxicity of RAPA and CQ. Western blotting was used to detect microtubule-associated protein light chain 3-II (LC3-II) which is a component of autophagosomes. Immunostaining of LC3-II was also performed for autophagosome analysis. Τhe TUNEL assay was used to detect apoptotic cells, and apoptosis-positive cells were counted in three randomly selected microscopic fields for statistical validation. Results: RAPA alone and CQ alone inhibited the viability of 93T449 cells. The combination of RAPA and CQ inhibited 93T449 cell viability significantly more than either drug alone and increased the number of autophagosomes which led to extensive apoptosis. Conclusion: The combination of RAPA and CQ increased the number of autophagosomes which led to apoptosis in 93T449 WDLS cells, suggesting novel effective treatment for this recalcitrant cancer by targeting autophagy.
Liposarcoma is a soft-tissuesarcoma (STS) and is a recalcitrant disease (1, 2). Well-differentiated liposarcomas (WDLS) can become dedifferentiated after repeated recurrence (3, 4). We previously observed that the combination of the mTOR-inhibitor rapamycin (RAPA) and the anti-malarial drug chloroquine (CQ) had synergistic efficacy in a patient-derived orthotopic xenograft (PDOX) mouse model of dedifferentiated liposarcoma, with a high number of apoptosis-positive cancer cells (5).
Autophagy is inhibited by mTOR inhibitors such as RAPA due to overproduction of autophagosomes, and exacerbated by CQ which prevents autophagosome-lysosome fusion (6). Autophagy is considered an important cellular homeostatic process that coordinates the degradation of proteins and organelles to provide cellular energy (7). Autophagy plays a defensive role in cancer cells to ensure their survival (8). Inhibition of autophagy can lead to accumulation of autophagosomes in cancer cells and apoptosis (9, 10). The microtubule-associated protein light chain 3 (LC3-II) protein is an autophagosome marker (11, 12). Since it is a component of the inner membrane of autophagosomes, the amount of LC3-II is proportional to the number of autophagosomes (10). In the present study, we investigated whether the combination of RAPA and CQ could inhibit proliferation of a WDLS cell line by targeting autophagy and thereby induce apoptosis.
Materials and Methods
Cell lines and cell culture. The human 93T449 cell line, a well-differentiated liposarcoma (American Type Culture Collection, ATCC, Manassas, VA, USA) was cultured in Roswell Park Memorial Institute (RPMI) 1640 medium, supplemented with 10% fetal bovine serum (FBS) and 1 IU/ml penicillin/streptomycin.
Reagents. RAPA was obtained from MedChemExpress (Monmouth Junction, NJ, USA) and dissolved in dimethyl sulfoxide (DMSO). CQ diphosphate (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in phosphate buffered saline (PBS). The concentration of RAPA and CQ stock solution was 100 mM and 10 mM, respectively.
Cell viability assay. 93T449 cell viability was assessed using the WST-8 reagent (Dojindo Laboratories, Kumamoto, Japan). Cells were cultured in 96-well plates (1.5×104cells/well) in RPMI (100 μl/well) and incubated at 37°C overnight. Cells were treated with different concentrations of RAPA ranging from 1.6 μM to 100 μM, or CQ ranging from 1.6 μM to 200 μM, or their combination for 24 h. After the treatment period, 10 μl of WST-8 solution was added to each well and further incubated at 37°C for 1 h. Absorption was measured at 450 nm using a microplate reader (SUNRISE; TECAN, Männedorf, Switzerland). Drug sensitivity curves were obtained using Microsoft Excel for windows (Microsoft, Redmond, WA, USA), and half-maximum-inhibitory-concentration (IC50) values were calculated using ImageJ v. 1.53 (National Institutes of Health, Bethesda, MD, USA). Cell-viability percent was calculated using the following formula: Cell viability (percentage of control) = (sample OD − blank OD)/(control OD − blank OD) ×100.
Western blotting for LC3-II to determine the effects of RAPA and CQ on autophagy. 93T449 cells were seeded in 25 mm3 tissue culture flasks and incubated at 37°C in a humidified atmosphere containing 5% CO2. At approximately 80% confluence, cells were treated for 24 h to determine the efficacy of RAPA (6 μM), CQ (80 μM), and their combination (6 μM + 80 μM), respectively. Following treatment, the cells were lysed in lysis buffer [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1mM EGTA, 1% Triton X-100 (Cell Signaling Technology, Beverly, MA, USA)] on ice for 20 min. The protein samples were separated by 10% sodium dodecyl sulphate polyacrylamide-gel electrophoresis (Wako, Tokyo, Japan) under reducing conditions. The proteins were electrophoretically transferred to nitrocellulose membranes (GE Healthcare Bio-Sciences, Piscataway, NJ, USA) and sequentially incubated with primary and secondary antibodies in iBind solution (iBind Western System, Life Technologies, Carlsbad, CA, USA) for 2.5 hours. Anti-LC3-II mAb-HRP-DirecT and anti-GAPDH as a loading control were purchased from MBL Co. (Nagoya, Japan) and Proteintech (Rosemont, IL, USA), respectively. The anti-LC3-II mAb-HRP-DirecT and anti-GAPDH were used at a dilution of 1:1,000 and 1:50,000, respectively. HPR-conjugated anti-rabbit IgG (Proteintech) was used at a dilution of 1:5,000 as the secondary antibody.
Immunocytochemical staining for LC3-II. To examine the efficacy of RAPA and CQ on autophagy, 93T449 cells were cultured overnight at 6.0×104 cells/well on 8-chamber cell-culture slides (CELLTREAT Scientific Products, Pepperell, MA, USA). The cells were then divided into RAPA alone, CQ alone, and RAPA + CQ groups (2 wells each) and incubated for 24 h. The cells were fixed in 4% paraformaldehyde in phosphate buffer for 30 min. Samples were then sequentially incubated with an anti-LC3-II antibody (MBL, Nagoya, Japan) for 1 hour and with an anti-IgG secondary antibody (Alexa Fluor® 488; code no. A11001; Invitrogen, Carlsbad, CA, USA) for 30 minutes. Samples were then examined under a IX71 microscope (Olympus Corporation, Tokyo, Japan).
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) apoptosis assay. 93T449 cells were grown overnight at 6.0×104 cells/well on 8-chamber cell-culture slides (CELLTREAT Scientific Products) to assess apoptosis induced by RAPA and CQ or their combination. The cells were treated with either RAPA alone, CQ alone, or RAPA + CQ, for 24 hours. The One-step TUNEL In Situ Apoptosis Kit (Green, FITC) (E-CK-A320) (Elabscience, Houston, TX, USA) was used to measure apoptosis. Using an IX71 microscope (Olympus Corporation), fluorescence images were captured at 200x magnification. A total of three fields were chosen at random from each group. Positive cell counts were expressed as mean±SEM.
Statistical analysis. All statistical analyses were carried out using EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan), a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria) (13). Tukey-Kramer HSD was used as the parametric test for between-group comparisons. Error bars show SEM. A p-value ≤0.05 was defined as statistically significant.
Results
Efficacy of RAPA and CQ, alone or in combination, on viability of 93T449 cells. RAPA or CQ alone significantly decreased the viability of 93T449 cells in a dose-dependent manner, compared to the untreated control cells (p=0.0033 and 0.0027, respectively) (Figure 1A). The RAPA + CQ combination treatment further inhibited 93T449 cell viability compared to the control, RAPA alone, and CQ alone groups (p=0.00004, 0.0097, and 0.0077, respectively). There was no significant difference between RAPA alone and CQ alone (p=0.997) (Figure 1B).
A. Viability of the well-differentiated liposarcoma 93T449 cell line as a function of CQ or RAPA concentration (n=3). B. Cell viability of the liposarcoma 93T449 cell line with RAPA, 6 μM; CQ, 80 μM; or their combination. *p<0.05. Error bars indicate standard error of the mean (SEM). CQ, chloroquine; RAPA, rapamycin.
Western blotting of LC3-II to determine the effect of RAPA and CQ or their combination on autophagy in 93T449 cells. In the RAPA+CQ-treated 93T449 cells, LC3-II expression was higher than in the control, RAPA-alone-, and CQ-alone-treated cells, indicating the combination of RAPA and CQ induced over-production of autophagosomes and thereby inhibited autophagy, with CQ having the main effect (Figure 2).
Western blotting of LC3 in 93T449 cells treated with RAPA, CQ and their combination. LC3-II, Microtubule-associated protein light chain 3; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CQ, chloroquine; RAPA, rapamycin.
Immunocytochemical staining for LC3-II to determine the effect of RAPA and CQ or their combination on autophagy in 93T449 cells. Clear cytoplasmic LC3-II-positive puncta were only observed in the RAPA- and CQ combination-treated 93T449 cells, indicating autophagosomes were over-produced in the cytoplasm, and thereby inhibited autophagy (Figure 3).
Immunocytochemical staining of 93T449 cells, treated with RAPA, CQ and their combination, for microtubule-associated protein light chain (LC3-II) shown in green. Nuclei are stained with DAPI (blue). Scale bars: 50 μm. CQ, chloroquine; RAPA, rapamycin.
TUNEL assay to determine the effects of RAPA and CQ or their combination to induce apoptosis in 93T449 cells. CQ combined with RAPA induced more apoptotic TUNEL-positive cells than the untreated control (p=0.015), CQ alone (p=0.005), or RAPA alone (p=0.001) (Figure 4 and Figure 5).
Terminal deoxynucleotidyl transferase dUTP nick- end labeling (TUNEL) assay of 93T449 cells treated with RAPA, CQ and their combination. DAPI (blue) indicates cell nuclei and fluorescein isothiocyanate FITC (green) indicates apoptotic 93T449 cells. Scale bar: 50 μm. CQ, chloroquine; RAPA, rapamycin.
Percentage of apoptotic 93T449 cells (%), treated with RAPA, CQ and their combination, determined by the TUNEL assay. *p<0.05 using the Tukey-Kramer HSD test. Error bars indicate standard error of the mean (SEM). CQ, chloroquine; RAPA, rapamycin.
Discussion
Aberrant activation of mTOR has been reported in liposarcoma (14, 15). mTOR inhibitors have been reported to be effective in soft-tissue sarcomas (14). A phase III trial is investigating the efficacy of the mTOR-inhibitor ridafosfamide on metastatic sarcomas (16).
CQ is an antimalarial drug with a history of development since 1934. Hydroxychloroquine has a variety of effects, including anti-inflammatory effects, and is used clinically (17). CQ is an autophagy inhibitor (18), and has shown anti-cancer efficacy in combination with cancer chemotherapy drugs (19, 20). The efficacy of the combination of mTOR inhibitors and CQ has also been demonstrated in Phase I and II trials on solid tumors, melanoma and renal cell carcinoma (21, 22).
We previously established a patient-derived orthotopic xenograft (PDOX) mouse model of dedifferentiated liposarcoma and showed that the combination of CQ and RAPA was effective in inhibiting tumor growth by inducing apoptosis (5). Excessive accumulation of autophagosomes has been shown to induce apoptosis in cancer cells (9, 10). Shimizu et al. also refer to autophagic cell death to distinguish it from apoptosis. Autophagic cell death is caused by the excessive accumulation of autophagosomes (23).
In the present study, we showed that LC3-II is overexpressed when 93T449 WDLS cells were treated with the combination of RAPA and CQ, indicating over-production of autophagosomes (Figure 2). Immunostaining showed that autophagosomes were highly over produced in the cytoplasm, only when treated with the combination of RAPA and CQ (Figure 3). The overproduction of autophagosomes resulted in a large increase of apoptosis-positive 93T449 cells which were treated with the combination of RAPA and CQ (Figure 4 and Figure 5). The present study demonstrates that the combination of CQ and RAPA induces apoptosis by overaccumulation of autophagosomes in WDLS.
Our previous demonstration of the efficacy of the combination of RAPA and CQ in a PDOX mouse model of de-differentiated liposarcoma (5) and the present study indicate that the combination of RAPA and CQ is effective in liposarcoma. The combination of mTOR inhibitors and autophagy inhibitors can be a new paradigm of treatment for recalcitrant sarcoma.
Acknowledgements
This paper is dedicated to Manabu Nagira and the memory of A.R. Moossa, MD, Sun Lee, MD, Professor Li Jiaxi, Masaki Kitajima, MD, Shigeo Yagi, PhD, Jack Geller, MD, and Joseph R. Bertino, MD and J.A.R, mead respectively. The present study was supported by the Robert M. Hoffman Foundation for Cancer Research.
Footnotes
Conflicts of Interest
The Authors declare that they have no conflicts of interest in relation to this study. AntiCancer Inc. uses PDOX models for contract research.
Authors’ Contributions
N.M. conceived the study, N.M., Y.A. Y. K. and K.O. performed the experiments and J.M. and R.M.H. provided scientific advice. N.M. wrote the paper and R.M.H. revised the paper.
- Received March 28, 2023.
- Revision received May 14, 2023.
- Accepted May 29, 2023.
- Copyright © 2023 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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