Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 May 10:13:904701.
doi: 10.3389/fphar.2022.904701. eCollection 2022.

Antidepressants Fluoxetine Mediates Endoplasmic Reticulum Stress and Autophagy of Non-Small Cell Lung Cancer Cells Through the ATF4-AKT-mTOR Signaling Pathway

Shali Shao  1 , Xibing Zhuang  1 , Lin Zhang  2 , Tiankui Qiao  1
Affiliations

Antidepressants Fluoxetine Mediates Endoplasmic Reticulum Stress and Autophagy of Non-Small Cell Lung Cancer Cells Through the ATF4-AKT-mTOR Signaling Pathway

Shali Shao et al. Front Pharmacol. .

Abstract

Fluoxetine, one of the latest clinical antidepressants, is reported to have the anti-proliferative effect on cancer cells via immune-related pathways. However, the mechanism is still not known. This study mainly focused on the discovery of the molecular basis of the inhibitory effect of fluoxetine in lung cancer. The specific anti-proliferation effect and autophagy induced by fluoxetine on lung cancer cell were shown in CCK8 and immunofluorescence. The RNA sequence hinted that the endoplasmic reticulum (ER) stress-related protein and mTOR pathway were enriched after fluoxetine treatment. Western blot results revealed that the ER stress pathway was activated by fluoxetine, including PERK, ATF4, and CHOP, while the AKT/mTOR pathway was inhibited. In addition, the transfection of ATF4 siRNA further discovered that ER stress participated in the inhibition of AKT/mTOR pathway and the induction of anti-proliferation and autophagy in the fluoxetine-treated cells. More importantly, fluoxetine was demonstrated to play cytotoxic activity in cancer cells without affecting normal cells. Our results showed that fluoxetine triggered the ATF4-AKT-mTOR signaling pathway to induce cell cycle arrest and autophagy restraining cancer cells' growth in lung cancer. This study found fluoxetine unaffected the proliferation of normal lung epithelial cells, providing safe clinical therapeutic strategies for lung cancer patients with depression.

Keywords: ATF4-AKT-mTOR signaling pathway; autophagy; endoplasmic reticulum stress; fluoxetine; non-small cell lung cancer.

PubMed Disclaimer

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Comparison of paroxetine and fluoxetine for anti-proliferation effect in multiple cell lines. (A–E) Lung cancer cells (H460 and A549), normal lung epithelial cells (BEAS-2B), human hepatoma cell line (Huh7), and normal liver cell line (L02) were treated with paroxetine (0, 5, 10, 20, 30, and 40 μM) for 24 h. CCK8 was used to measure cell viability. (F–J) H460, A549, BEAS-2B, Huh7, L02 cells were treated with fluoxetine (0, 5, 10, 20, 30, and 40 μM) for 24 h, CCK8 was used to measure cell viability. Data are presented as mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001.
FIGURE 2
FIGURE 2
Fluoxetine arrested cell cycle at G0/G1. (A) Cells were treated with fluoxetine (0–40 μM) for 24 h, performed PI/FITC staining, and analyzed with flow cytometry. (B) Percentage of apoptosis cells was analyzed. (C) Distribution of the cell cycle was analyzed by ModFit LT 5.0 software and the result of the Western blot of CDK2, p27, and p21. The images were collected from different parts of the same gel. (D) Quantitative analysis of optical band densitometry. Data are presented as mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001.
FIGURE 3
FIGURE 3
Fluoxetine induced autophagy in a dose and time-dependent way (A) Cells were treated with fluoxetine (0–40 μM) for 24 h. Immunofluorescence of LC3 (green) and nuclear (blue) were used to observe autophagy. Scale bars were 50 μm. (B) The Western blot result of p62 and LC3. Cells were treated with fluoxetine (0–40 μM) for 24 h. The images were collected from different parts of the same gel. (C) Western blot result of LC3 in control and fluoxetine groups. Cells were treated with fluoxetine (20 μM) for 3, 6, 12, and 24 h. The images were collected from different parts of the same gel. (D) Quantitative analysis of optical band densitometry. Data are presented as mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001.
FIGURE 4
FIGURE 4
Fluoxetine induced autophagy in a dose and time-dependent way (A) immunofluorescence of LC3 (green) and DAPI (blue) was used to observe the autophagy. Cells were treated with 20 μM fluoxetine (F), 1 mM 3-MA, 40 μM CQ, the combination of fluoxetine plus rapamycin, and fluoxetine plus chloroquine for 24 h. Scale bars 50 μm. (B) Result of the Western blot of LC3 and p62. The treatment of drugs was used as described previously. Quantitative analysis of optical band densitometry was conducted. Data are presented as mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001.
FIGURE 5
FIGURE 5
Fluoxetine had a connection with ER stress and mTOR signaling pathway. (A) Autophagy-related genes were analyzed in a gene set. (B) Top 8 enriched KEGG pathways. (C)Western blot was used to analyze the expression ER stress related proteins, including BIP, PERK, ATF4, and CHOP. The images were collected from different parts of the same gel. (D) Western blot was used to analyze the expression of p-AKT, AKT, p-mTOR, mTOR, p-p70s6k, and p70s6k. The images were collected from different parts of the same gel. Data are presented as mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001.
FIGURE 6
FIGURE 6
AKT/mTOR signaling pathway was regulated through ATF4 in fluoxetine treatment. (A,D) Expression of ATF4 in normal and tumor tissues was detected in the TCGA database. The blue spots on the left were the normal group, and the red spots on the right were the tumor group. [(B,C), (E,F)] Survival analysis comparing high to low expression of ATF4 in lung cancer. (G) Expression of PERK, p-AKT/AKT and p-mTOR/mTOR in lung cancer cells transfected with ATF4 siRNA for 48 h followed by exposure to fluoxetine (20 μM) for another 24 h was tested by Western blot. The images were collected from different parts of the same gel. Data are presented as mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001.
FIGURE 7
FIGURE 7
Fluoxetine-induced anticancer effect through the ATF4-AKT-mTOR signaling pathway (A) the cell viability in lung cancer cells transfected with ATF4 siRNA for 48 h followed by exposure to fluoxetine (20 μM) for another 24 h was assessed by CCK8. Left and right indicate H460 cells and A549 cells, respectively. (B,C) The distribution of the cell cycle in lung cancer cells transfected with ATF4 siRNA for 48 h followed by exposure to fluoxetine (20 μM) for another 24 h was analyzed by flow cytometry. (D) The autophagy in lung cancer cells transfected with ATF4 siRNA for 48 h followed by exposure to fluoxetine (20 μM) for another 24 h was observed by immunofluorescence of LC3 (green). Scale bars were 50 μm. (E) The expression of CDK2, p27, p21, and LC3 in lung cancer cells transfected with ATF4 siRNA for 48 h followed by exposure to fluoxetine (20 μM) for another 24 h was tested by Western blot. Left and right indicate H460 cells and A549 cells, respectively. The images were collected from different parts of the same gel. Data are presented as mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001.
FIGURE 8
FIGURE 8
Fluoxetine exerted anti-tumor effects while not damaging normal cells. (A–B) Distribution of the cell cycle in normal lung epithelial cells treated with fluoxetine (0–40 μM) was analyzed by flow cytometry. (C) Immunofluorescence of LC3 (green) and DAPI (blue) was used to observe the autophagy. Scale bars were 50 μm. (D–E) Expression of p27, p21, p62, and LC3 was tested by Western blot. The images were collected from different parts of the same gel. Data are presented as mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001.
FIGURE 9
FIGURE 9
Fluoxetine induced cell cycle arrest and autophagy by triggering ATF4-AKT-mTOR signaling pathway in lung cancer cells. Briefly, fluoxetine activated the ER stress related proteins and the increased ATF4 inactivated the AKT/mTOR signaling pathway, thus leading to the cell cycle arrest at G0/G1 phase and autophagy.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Akechi T., Okuyama T., Onishi J., Morita T., Furukawa T. A. (2008). Psychotherapy for Depression Among Incurable Cancer Patients. Cochrane Database Syst. Rev. 2008 (2), CD005537. 10.1002/14651858.CD005537.pub2 - DOI - PMC - PubMed
    1. Amin N., Xie S., Tan X., Chen Y., Ren Q., Botchway B. O. A., et al. (2020). Optimized Integration of Fluoxetine and 7, 8-dihydroxyflavone as an Efficient Therapy for Reversing Depressive-like Behavior in Mice during the Perimenopausal Period. Prog. Neuropsychopharmacol. Biol. Psychiatry 101, 109939. 10.1016/j.pnpbp.2020.109939 - DOI - PubMed
    1. Atkins C., Liu Q., Minthorn E., Zhang S. Y., Figueroa D. J., Moss K., et al. (2013). Characterization of a Novel PERK Kinase Inhibitor with Antitumor and Antiangiogenic Activity. Cancer Res. 73, 1993–2002. 10.1158/0008-5472.CAN-12-3109 - DOI - PubMed
    1. B'Chir W., Maurin A. C., Carraro V., Averous J., Jousse C., Muranishi Y., et al. (2013). The eIF2α/ATF4 Pathway Is Essential for Stress-Induced Autophagy Gene Expression. Nucleic Acids Res. 41, 7683–7699. 10.1093/nar/gkt563 - DOI - PMC - PubMed
    1. Bertolotti A., Zhang Y., Hendershot L. M., Harding H. P., Ron D. (2000). Dynamic Interaction of BiP and ER Stress Transducers in the Unfolded-Protein Response. Nat. Cel Biol. 2, 326–332. 10.1038/35014014 - DOI - PubMed