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Effect of rapamycin treatment in human seminoma TCam-2 cells through inhibition of G1-S transition

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Abstract

Mammalian target of rapamycin (mTOR) is an important serine/threonine kinase that plays a critical role in several processes including cell cycle, protein synthesis, and energy metabolism. Due to its multiple roles and general dysregulation in cancer, the mTOR pathway is an important target in cancer therapy. However, studies on mTOR activity in seminoma are limited. Therefore, our aim was to investigate the expression of mTOR signaling pathway proteins in the TCam-2 cell line after rapamycin treatment. TCam-2 cells were treated with different concentrations of rapamycin (control (no rapamycin treatment), 4 nM, 20 nM, 100 nM, 500 nM, and 1000 nM rapamycin) for 48 h and 72 h. mTOR, p-mTOR, P70S6K, p-P70S6K, proliferating cell nuclear antigen (PCNA), and caspase-3 expression levels were analyzed by western blot. Apotosis and cell cycle were analyzed by flow cytometry. After 48 h of rapamycin administration, mTOR activity was significantly decreased at 1000 nM (p < 0.05). In addition, P70S6K acitivity significantly decreased in groups at all rapamycin concentrations (***p < 0.001, ****p < 0.0001). After 72 h of rapamycin administration, mTOR pathway activity were significantly decreased at 100, 500, and 1000 nM rapamycin-treated groups (p < 0.05). Moreover, P70S6K expression decreased in all treatment groups (****p < 0.0001). Caspase-3 expression were similar in all groups. While PCNA expression tended to decrease at 48 h in a dose-dependent manner, this decrease was not significant. We detected decreased PCNA expression at 1000 nM rapamycin at 72 h (p < 0.05). The rate of apoptosis increased especially at 1000 nM rapamycin at 72 h (***p < 0.001). On the other hand, according to the results of the cell cycle experiment, G1 phase arrest was detected at all rapamycin doses at 48 and 72 h (***p < 0.001). Our study indicated that 1000 nM rapamycin may inhibit TCam-2 seminoma cells growth by halting cell proliferation through inhibition of G1-S transition. Therefore, we believe that the findings obtained will contribute to the development of new treatment approaches for seminoma patients in the future and in the process of restoring testicular functions and preserving fertility.

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We would like to thank Prof. Dr. Hubert Schorle. TCam-2 cell line obtained from Prof. Dr. Hubert Schorle, Department of Developmental Pathology and Department of Molecular Diagnostics Institute of Pathology, Bonn Medical School, Germany.

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Authors and Affiliations

  1. Department of Histology and Embryology, Yeditepe University Faculty of Medicine, 34755, İstanbul, Turkey

    Tugce Onel, Ecem Yildirim & Aylin Yaba

  2. Department of Physiology, Yeditepe University Faculty of Medicine, 34755, İstanbul, Turkey

    Cihan S. Erdogan

  3. Department of Immunology, Yeditepe University Faculty of Medicine, 34755, İstanbul, Turkey

    Basak Aru & Gulderen Yanikkaya Demirel

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  1. Tugce Onel
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  2. Cihan S. Erdogan
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Contributions

Conceptualization: TO, CSE, and AY; data curation: TO, CSE, BA, EY; formal analysis: TO, CSE, BA, EY, AY; investigation: TO, CSE, BA, EY, AY; methodology: TO, CSE, BA, EY, AY; software: TO, CSE, BA, EY, AY; supervision: AY; validation: TO, CSE; visualization: TO, CSE; roles/writing—original draft: TO, CSE, BA, EY, GYD, AY; writing—review and editing: TO, CSE, BA, EY, GYD, AY.

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Correspondence to Aylin Yaba.

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Our study does not include human and animal experiments. TCam-2 cell line was used for our study. There is no ethics committee requirement.

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Supplementary Information

Supplementary Figure 1

İmages of TCam-2 cells for control and all rapamycin concentrations (4, 20, 100, 500, and 1000 nM) before rapamycin treatment (0h), 48 hours after rapamycin treatment (48h), and 72 hours after rapamycin treatment (72h). (PNG 3997 kb)

High resolution image (TIF 5466 kb)

Supplementary Figure 2

Wound healing Strength Assay of TCam-2 cells for control and all rapamycin concentrations (4, 20, 100, 500 and 1000 nM) before rapamycin treatment (0h), 48 hours after rapamycin treatment (48h), and 72 hours after rapamycin treatment (72h) experimental images. (PNG 3809 kb)

High resolution image (TIF 5413 kb)

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Onel, T., Erdogan, C.S., Aru, B. et al. Effect of rapamycin treatment in human seminoma TCam-2 cells through inhibition of G1-S transition. Naunyn-Schmiedeberg's Arch Pharmacol 396, 1009–1018 (2023). https://doi.org/10.1007/s00210-022-02371-8

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