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

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.


Introductıon
Testicular cancer (TC) is the most common solid organ malignancy in young men and the leading cause of cancerrelated deaths in this population (Ye and Ulbright 2012). TC usually occurs during the reproductive period and can cause infertility (Jacobs and Vaughn 2012). Testicular tumors can be broadly classified germ cell tumors, sex cord-stromal tumors, and mixed germ cell/sex cord-stromal tumors (Ye and Ulbright 2012). Although testicular cancer can arise from any cell type found in the testes, more than 95% of testicular cancers are germ cell tumors. The remaining 5% are sex cord-gonadal stromal tumors orginate from Leydig or Sertoli cells. Among germ cell tumors, testicular seminoma is a germ cell tumor of the testis and the most common testicular tumor, accounting for approximately 45% of all primary testicular tumors (Yaba et al. 2016).
The TCam-2 cell line is the first seminoma-derived cell line that retains most of the characteristic features of seminoma (Young et al. 2011). TCam-2 cells express typical marker genes for primordial germ cells and germ cell neoplasia in situ (GCNIS) (SOX17, PRAME, cKIT, TFAP2C, PRDM1/BLIMP1) and show typical GCNIS/seminoma morphology (large round cells with a large nucleus and clear cytoplasm) (de Jong et al. 2008;Eckert et al. 2008;Nettersheim et al. 2012).
Mammalian target of rapamycin (mTOR) is an important serine/threonine kinase (Zhou and Huang 2010) that plays critical roles in cell cycle, protein synthesis, energy metabolism, cell proliferation, growth, differentiation, and apoptosis (Schmelzle and Hall 2000;Martin and Hall 2005;Wullschleger et al. 2006;Chiang and Abraham 2007;Tsang et al. 2007). mTOR is a downstream factor of PI3K/Akt and positively regulates tumor progression (Sekulić et al. 2000). mTORC1 is one of the two major mTOR complexes and the main downstream effector of PI3K/PTEN/AKT signaling in response to growth factors which phosphorylates ribosomal protein S6 kinase (S6K1) to increase protein synthesis (Dibble and Cantley 2015). Rapamycin is a natural product and non-ATP competitive inhibitor of mTORC1; it exerts its' effects via binding to cytosolic FK binding protein (FKBP12) with high affinity, to interact with mTORC1 and via interfering with mTOR activity or complex assembly (Hausch et al. 2013). Previosly, we have reported mTOR signal pathway immunoreactivity in testicular seminoma, phosphorylated serine 2448 (p-mTOR), P70S6K, and p-P70S6K and hypothesized that mTOR signaling pathway may contribute to the development or progression of testicular cancer (Yaba et al. 2016).
Due to its diverse roles and its general dysregulation in cancer, the mTOR pathway is an important target in cancer therapy. However, studies on mTOR activity in seminoma are limited Wang et al. 2016;Yaba et al. 2016;Chen et al. 2018;Wei et al. 2018). Herein, we analyzed the effect of mTOR inhibition in seminoma-like TCam-2 cells with regard to cell proliferation, migration, cell cycle, and apoptosis.

Wound-healing scratch assay
A total of 5 × 10 5 cells/well were seeded on six-well plates and let attached overnight. After overnight attachment, a straight line was created by scratching with a 100 μL pipette tip. Then, the medium was removed, fresh medium with respective drug concentrations were added, and images were obtained for t = 0 h. The migration/healing was determined for 48 and 72 h by obtaining microscopy images of the same area (Axiovert 135; Carl Zeiss Microscopy). Migration/healing was determined as previously described by using Fiji (Treloar and Simpson 2013), and the percent healing against the control group was determined.

Assesment of apoptosis
Viability, apoptosis, and necrosis were evaluated by Annexin V/Propidium Iodide (PI) staining. For this purpose, cells were seeded into six-well plates as 5 × 10 5 cells per well and incubated for overnight for attachment. Cells were then treated with rapamycin at respective doses for 48 and 72 h. For staining, cells were detached by trypsinization, washed once with Dulbecco's phosphate-buffered saline solution (DPBS, Thermo Fisher Scientific, #14190144) and suspended in 1X ice cold Annexin V Binding Buffer (BioVision Inc., #1006) followed by labelling with 5 μL Annexin V-FITC reagent (Biolegend, #640906) and 1 μL PI solution (Thermo Fisher Scientific, #P3566, diluted to 250 μg/mL in DPBS) by incubating for 10 min under dark conditions at room temperature. Cells were read with DxFLEX flow cytometry system (Beckman Coulter Inc.) and analyzed by CytExpert software (Aru et al. 2020). The experiment was done in triplicate, and 2.5 × 10 4 cells per analysis were acquired at medium flow rate.

Cell cycle assessment
DNA content analysis was performed by using Cell Cycle Kit (Beckman Coulter Inc., #C03551). Cells were seeded as 5 × 10 5 cells/six-well plate and incubated overnight for attachment. After incubating with rapamycin at respective doses for 48 and 72 h, cells were detached by tyripsinization, washed once with DPBS, fixed with ice cold 70% ethanol by adding dropwise followed by incubation at 4 °C for an hour. Tubes were stored at − 20 °C overnight, and ethanol was removed by centrifuging cells at 400 x g for 5 min. Pellet was suspended in 500 μL cell cycle kit reagent, and tubes were incubated at room temperature under dark for 30 min.

Statistical analysis
The statistical analysis was performed using GraphPad Prism 7.0 (GraphPad Software). Data were analyzed by using one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison tests. A p value lower than 0.05 was considered statistically significant.

Dose-dependent inhibition of mTORC1 in TCam-2 cells by rapamycin
Rapamycin exerts its biological activity by inhibiting the mTOR, which is a key regulator of cell growth and survival in many cell types (Zhao et al. 2008). In our study, 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 . After 48 h of rapamycin administration, mTOR activity was significantly decreased at 1000 nM (p < 0.05; Fig. 1a and b and Table 1). In addition, P70S6K acitivity significantly decreased in groups at all rapamycin concentrations (**p < 0.01, ***p < 0.001, ****p < 0.0001; Fig. 1a and c and Table 1). After 72 h of rapamycin administration, mTOR pathway activity were significantly decreased at 100, 500, and 1000 nM rapamycin-treated groups (p < 0.05; Fig. 1a and d and Table 1). Moreover, P70S6K activity decreased at all treatment groups (****p < 0.0001; Fig. 1a and e and Table 1).

Rapamycin does not significantly alter caspase-3 and PCNA protein levels in TCam-2 cells
In order to check apoptosis and proliferation in TCam-2 cells after administration of specific doses of rapamycin, we investigated caspase-3 and PCNA protein expression. Caspase-3 expression did not change at either of the doses nor the time points (Fig. 2a, c and e). While levels of PCNA proteins seemed to decrease in the at 48 h in a dose-dependent manner, this decrease was not significant ( Fig. 2a and b). On the other hand, PCNA expression decreased significantly at 1000 nM rapamycin at 72 h (p < 0.05; Fig. 2a and d and Table 1).

Wound-healing scratch assay in TCam-2 cell line
In order to evaluate TCam-2 cell migration after rapamycin treatment, wound-healing scratch assay were used. 4 nM rapamycin treatment significantly inhibited cell migration after 48 h (p < 0.05; Fig. 3a). On the other hand, rapamycin at doses of 20 and 1000 nM significantly decreased cell migration at 72 h (**p < 0.01; Fig. 3b and Table 1). No significant changes were detected at other doses for 48 h and 72 h (Fig. 3a) (Supplementary Fig. 2).

mTOR inhibition by using rapamycin does not promote apoptosis in the TCam-2 cells
The apoptotic effect of rapamycin mediated mTOR inhibition was evaluated by Annexin V/PI staining on 48 and 72 h. Comparisons between the control and treatment groups revealed no significant change in viability, early and late apoptosis, and necrosis rates at 48 hours (p > 0.05) (Fig. 4a, b, c, d and i). On the other hand, rapamycin treatment increased late apoptosis rates at all concentrations tested significantly (p < 0.05 for 4, 20, 100, and 500 nM; p < 0.001 for 1000 nM) at 72 h ( Fig. 4g and i and Table 1) though no significant changes in viability, early apoptosis, and necrosis rates between doses for this timepoint was observed (p > 0.05; Fig. 4e, f, h and i).

mTOR inhibition leads to G1-phase arrest in TCam-2 cells in a dose-dependent manner
It was previously reported that inhibition of mTOR by physiological cell stressors or pharmacological agents decrease proliferation mainly by leading to cell cycle arrest at G1 phase (Tian et al. 2019). It should be noted that inhibition of mTOR during interphase would be responsible for arrest in G1, and therefore, the interphase expression patterns of p-mTOR and p-P70S6K (Fig. 1a-c) should not be ignored. Consistent with the literature, our results revealed that rapamycin at 1000 nM promoted G1 phase arrest in TCam-2 cells in both 48 and Fig. 1 a Representative immunoblot image for p-mTOR, mTOR, p-P70S6K, P70S6K at 0-1000 nM rapamycin doses at 48 and 72 h. b mTOR and c P70S6K activities at 48 h. d mTOR and e P70S6K activities at 72 h. Statistical analysis is shown in Table 1 72 h significantly compared to the control group (p < 0.001 and p < 0.01, respectively) whereas 4, 20,100, and 500 nM showed a lower rate of rapamycin treatment percentage of cells (approximately 25%) were in G1 (**p < 0.01, ***p < 0.001, ****p < 0.0001; Fig. 5a, b and c and Table 1).

Dıscussıon and conclusıon
The mTOR is frequently deregulated in cancer and activating somatic mutations of mTOR have recently been identified in various types of human cancer and so the mTOR is therapeutically targeted (Murugan 2019). mTOR inhibitors have been commonly used as immunosuppressants, and are currently approved for the treatment of human malignancies (Zheng and Jiang 2015). In this study, we aimed to investigate the effect of inhibition of the mTORC1 signaling pathway in TCam-2 seminoma cells. This study demonstrates that treatment with 1000 nM rapamycin can inhibit proliferation of TCam-2 seminoma cells by downregulating PCNA through mTOR inhibition. In a previous study, analyzed whether mTORC1 and MAPK signaling pathway activities were differentially active in both germ cell tumor (GCT) classes in seminoma and non-seminoma germ cell tumors (Chen et al. 2018). In this study, it was shown that the mTORC1 signaling pathway is activated in non-seminoma germ cell tumors (NSGCT, including embryonal carcinoma, yolk sac tumor, and choriocarcinoma), but not in seminomas (Chen et al. 2018). Seminomas have been shown to negatively regulate mTORC1 activity with high REDD1 levels to maintain an undifferentiated state. On the other hand, members of the EGF and FGF receptor families have been shown to be more highly expressed in NSGCTs and stimulate the signaling of the EGF and FGF2 ligands mTORC1 and MAPK. This suggests that mTORC1 activation in NSGCTs contributes to cell proliferation (Chen et al. 2018). We investigated the activity of mTORC1 in the TCam-2 cell line and propose to show for the first time that the mTOR signaling pathway acts as an oncogene in the TCam-2 seminoma cell line. This is evidenced by its dramatic downregulation in seminomas based on western blot experiments, as well as its positive regulation of cell proliferation and migration. mTOR functions as a key control protein that integrates signals from a variety of environmental factors, and mTOR signalling proteins are essential for cell growth, migration, survival, and development (Chi 2012). To date, we know that mTOR signaling proteins play a role in cell adhesion in the testis and are involved in the regulation of the blood-testis barrier (Zhou and Huang 2011). mTOR signaling proteins express in the testis of adult mice, and mTOR pathway proteins may play a role in proliferation and stimulation of meiotic initiation of spermatogonial stem cells (Moreira et al. 2019). Treatment with mTOR inhibitors, sirolimus and everolimus, had destructive effects on the adult mouse testis and impairs gonadal function (Huyghe et al. 2007). Therefore, we suggested that investigating the role of mTOR signalling inhibition in the TCam-2 cell line might be important to understand underlying mechanism of male reproductive cancers.
Rapamycin is widely used in the clinic as an antiproliferative drug and immunosuppressant; moreover, its analogs have been shown to retard tumor proliferation in clinical trials (Varma and Khandelwal 2007). Currently, more studies are focusing on the effect of rapamycin in tumor therapy (Lin et al. 2018). As an mTOR inhibitor, rapamycin has been reported to block cell cycle progression from G1 to S phase by inhibiting p70 ribosomal protein S6 kinase (p70S6K) (Shafer et al. 2010;Marqués et al. 2015). As a threonine kinase, mTOR phosphorylates S6K1 and 4E-BP1 and promotes transcription of key mRNAs associated with cell cycle progression from G1 to S phase (Marqués et al. 2015). Through its interaction with p70S6K, it is associated with cell growth, proliferation and differentiation by regulating ribosome biogenesis, protein synthesis, cell cycle progression, and metabolism (Shin et al. 2011). mTOR inhibition via rapamycin was reported to inhibit proliferation and migration of hemangiomas and reduce growth of vascular tumors. Consistent with this finding, we showed that the activity of the downstream effector molecule mTOR, P70S6K was significantly reduced upon rapamycin treatment at all concentrations tested in TCam-2 seminoma cells, indicating that mTOR inhibition decreases cell cycle progression and  in turn, proliferation. Moreover, in our study, rapamycininduced mTOR inhibition led to G1 phase-arrest during cell cycle progression, which is also in line with reports showing that mTOR is involved in cell cycle progression in G1 (Ponticelli 2004;Song et al. 2007). PCNA is a nuclear protein expressed in proliferating cells, is required for the maintenance of cell proliferation, and is used as a marker of cell proliferation (Strzalka and Ziemienowicz 2011). AKT/mTOR pathway have shown to inhibit skin cancer proliferation by downregulating PCNA (Amornphimoltham et al. 2008). In prostate cancer, agents such as vicenin-2 reduce the expression of PCNA and cyclin-D1 by inhibiting the EGFR/Akt/mTOR pathway (Singhal et al. 2017). The expression levels of p-AKT/AKT, p-mTOR/mTOR, and p-p70S6K/p70S6K were also significantly downregulated in hemangioma tissues and cells compared with the control group, and the expressions of Ki67 and PCNA were significantly decreased . In another study, syndecan binding protein was reported to inhibit apoptosis of gastric cancer cells and promote the growthof gastric cancer by inducing PCNA expression and inactivating the PI3K/ AKT/mTOR pathway (Qian et al. 2021). In our study, we showed that rapamycin decreases PCNA expression in a dose-dependent manner on 72 h, though a significant decrease was only observed upon treatment with 1000 nM. Yet, according to cell cycle analysis, rapamycin promoted G1-phase arrest in all concentrations tested compared to the control group, indicating other possible mechanisms involving in cell cyce arrest rather than PCNA should be clarified. In short, inhibition of mTOR by rapamycin prevents cells growth, thus division and Fig. 4 TCam-2 cells promotes apoptosis without altering necrosis significantly when treated with 1000 nM rapamycin at 72 h. a-d Bar graphs indicating viability, early apoptosis, late apoptosis, and necrosis. TCam-2 cells was found ineffective in initiating this pathway at 0-1000 nM rapamycin doses at 48 h. e, f, h Bar graphs indicating viability, early apoptosis, and necrosis. TCam-2 cells was found inef-fective in initiating the this pathway at 0, 4, 20, 100, 500, and 1000 nM rapamycin doses at 72 h. g Rapamycin treatment for 72 h significantly increased late apoptosis in all cells, with a greater effect on higher doses (4, 20, 100, 500, and 1000 nM) compared to the control (0 nM). i Representative flow cytometry quadrants. Statistical analysis is shown in Table 1 proliferation. Therefore, we suggest that rapamycin, an mTOR inhibitor, can potentially be used as a chemotherapeutic agent in human seminoma cells.
Caspase-3, an effector caspase along with caspase-6 and caspase-7, is an indicator of apoptotic activity in the cell (Seervi and Xue 2015). Previously, cleaved caspase-3 may be a suitable marker to detect the preapoptotis phase (Belloc et al. 2000) and introduce the triggers the apoptotic damage in leukemia cells (Varghese et al. 2003). In our study, Annexin V/PI staining also indicated that untreated cells had high pre-apoptotic cell population, which is in line with the western blotting results. Moreover, Peng et al. also indicated that TCam-2 cells have active caspase-3 under basal conditions (Peng et al. 2019). As previously reported, high apoptotic activity is associated with the poor prognosis (Gregory and Paterson 2018) and extragonadal tumors are reported to have poor prognosis compared to testiuclar tumors (Suster et al. 1998;Mannuel and Hussain 2010). Briefly, the reason of that high levels of active caspase-3 in parallel with the high rates of early apoptosis may be due to high basal early apoptotic events. In addition, it should be noted that late apoptosis is slightly but significantly increased when treated the cells were with rapamycin, indicating the treatment efficacy. mTOR signaling pathway is oftenly dysregulated in different types of cancers by leading tumor growth and metastasis. Therefore, mTOR signaling has been considered an attractive therapeutic target and rapamycin which is one of the mTORC1 inhibitors has been developed to anticancer therapy. While some of the inhibitors of mTOR signalling pathway have been approved to treatment of human cancers, more mTOR inhibitors are being evaluated in clinical studies. Here, we demonstrate that TCam-2 cells are driven by mTORC1 signal pathway. We suggested that mTOR-targeted therapy of TCam-2 cells with rapamycin will contribute to the development of new treatment approaches for seminoma patients and to the restoration of testicular functions and preservation of fertility in the future.  Table 1