Introduction
Cancer, as a group of genetic-based diseases, is characterized by uncontrolled progression of the cell cycle, invasion, and metastasis. According to 2022 data from the World Health Organization (WHO), one in six deaths worldwide is cancer-related, and this rate is reported to be increasing every year [ 1 ]. While it is generally accepted that cancer incidence increases with age, certain specific types such as carcinoma, sarcoma, leukemia, lymphoma, and melanoma are more commonly observed in childhood and young adulthood [ 2 , 3 ]. Surgical methods, radiation therapy and chemotherapeutic drugs are among the limited number of options generally applied alone or in combination in the treatment of cancer patients [ 4 ]. Agents used in cancer treatment can affect not only tumor cells but also healthy cells and tissues [ 5 , 6 ]. Therefore, it is crucial to evaluate long-term side effects for patients undergoing cancer treatment at a young age. Infertility due to anticancer treatments is defined as a frequently encountered and important complication that negatively affects the quality of life, especially in male patients treated during adolescence or at a young age [ 5 , 7 , 8 ]. Epidemiological data reveal that male individuals have higher morbidity in some types of cancer and that persistent decreases in sperm parameters after treatment are frequently observed [ 9 ]. DNA damage, epigenetic aberrations and chromosomal abnormalities caused by anticancer agents in spermatogonial stem cells lead to irreversible impairment of spermatogenesis [ 10 , 11 ]. This cellular damage can negatively affect not only the integrity of sperm DNA but also embryonic development. Therefore, the long-term effects of cancer treatments can significantly affect not only survival but also the future quality of life of individuals, especially their reproductive health [ 12 ]. Germ cells, which exhibit high activity in terms of replication and DNA synthesis, are more sensitive to DNA damage, oxidative stress, and apoptosis compared to other tissues. Increased oxidative stress and germ cell death in male gonads, particularly after chemotherapy, are among the fundamental mechanisms of permanent reproductive disorders. Anticancer agents cause mutagenic damage by targeting mitotically active spermatogonial stem cells during spermatogenesis, which can lead to the accumulation of unrepaired DNA in the germ line and a decline in sperm quality [ 13 , 14 ]. Selinexor (KPT-330, SLX) is one of the targeted therapeutic agents widely used in cancer 2019 and the European Medicines Agency (EMA) in 2021 [ 15 ]. SLX belongs to the class of Selective Inhibitors of Nuclear Export (SINE), which also includes compounds such as KPT-335, KPT-185, KPT-276, and KPT-251. Among these, SLX has emerged as the most prominent agent in both preclinical and clinical studies due to its potent antitumor activity and favorable oral bioavailability [ 16 ]. The antitumor activity of SLX is primarily mediated through the inhibition of Exportin-1 (XPO1), a key nuclear export protein responsible for transporting numerous cargo molecules—including tumor suppressor proteins, growth regulators, transcription factors, and DNA damage response proteins—from the nucleus to the cytoplasm. XPO1 functions as a transport receptor that mediates the export of proteins and various types of RNA from the nucleus [ 17 – 19 ]. To date, more than 200 XPO1 cargo proteins have been identified, and it is estimated that this number could exceed 1,000 [ 20 ]. This nuclear export mechanism is critically involved in promoting cell survival and proliferation in tumor cells by preventing the nuclear accumulation of pro-apoptotic and cell cycle-inhibitory proteins. By inhibiting XPO1, SLX disrupts this process, leading to the nuclear retention and functional reactivation of key tumor suppressors such as p53, p21, and FOXO [ 21 , 22 ]. As a result, SLX induces apoptosis and causes cell cycle arrest, thereby suppressing tumor progression. Beyond its effects on nuclear export, SLX has also been shown to exert inhibitory activity on DNA damage response pathways at both the transcriptional and post-translational levels. This is particularly significant in targeting tumor cells with genomic instability or deficient DNA repair mechanisms. These multifaceted actions contribute to SLX’s broad-spectrum antitumor efficacy and support its clinical potential across various hematological malignancies and solid tumors [ 18 ]. Although SLX has demonstrated effects on cell proliferation, apoptosis, and DNA damage in various types of cancer, there are no data on its direct effects on male germ cells such as spermatogonia and spermatid cells.
The aim of this study was to investigate the apoptotic, antiproliferative, and antimigratory effects of SLX on spermatogonia (GC1 spg) and spermatid (GC2 spd) cells. Furthermore, it also aimed to investigate its effects on DNA damage and oxidative stress.
Method
Cell culture
Mouse-derived GC1 spg (spermatogonia, ATCC CRL-2053) and GC2 spd (spermatocyte, ATCC CRL-2196) cell lines were maintained in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, USA) and 1% penicillin-streptomycin (100 U/mL penicillin and 100 µg/mL streptomycin; Gibco, USA). Cells were incubated at 37 °C in a humidified atmosphere containing 5% CO₂ until they reached adequate confluency for experimental use [ 23 ].
Cell viability assay
The cytotoxic potential of SLX (Cat# 7760, Tocris) on GC1 and GC2 cell lines was assessed using the MTT assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Cat# AR1156, Boster, CA, USA). Cells were seeded into 96-well plates at a density of 1 × 10⁴ cells per well and allowed to adhere overnight under standard incubation conditions. Subsequently, cells were treated with increasing concentrations of SLX (0, 1.25, 2.5, 5, 10, 20, 40, and 80 µM) for 24 and 48 h. After treatment, 5 mg/mL MTT solution was added to each well, and plates were incubated at 37 °C for 1 h to permit formazan crystal formation. Following incubation, the medium was carefully removed, and the formazan crystals were dissolved in dimethyl sulfoxide (DMSO) (Sigma). Absorbance was measured at 570 nm using a microplate Multiskan GO spectrophotometer (Thermo Scientific). All conditions were tested in triplicate wells, and each experiment was performed three times independently.
In vitro wound healing assay
To assess the impact of SLX on cell migration, a wound healing assay was performed using GC1 and GC2 cell lines. A total of 5 × 10⁴ cells per well were seeded into 24-well plates and incubated overnight to allow monolayer formation. Once confluence was achieved, a straight scratch was introduced into the cell layer using a sterile 200 µL pipette tip. To inhibit proliferative activity, culture medium containing %2 FBS was added only to the control wells, whereas treatment wells were supplemented with 16.70 µM SLX for GC1 and 6.85 µM SLX for GC2. Cell migration into the scratched area was monitored at 0, 12, 24 and 48 h using a (Olympus, CKX-53) at 20X magnification. The captured images were analyzed using (Fiji software, free version). All experiments were conducted in triplicate [ 24 ].
Colony formation assay
To evaluate the long-term proliferative capacity of GC1 and GC2 cells under SLX treatment, a colony formation assay was performed. Cells were seeded into 6-well plates at a density of 1 × 10 3 cells per well, using 2.5 mL of complete DMEM medium supplemented with 10% FBS, 1% Penicillin-Streptomycin, and 1% L-glutamine. Following overnight incubation to allow cell attachment, control wells received only complete medium, while treatment wells were supplemented with 16.70 µM SLX for GC1 and 6.85 µM SLX for GC2. Plates were maintained under standard culture conditions for ten days to support colony development. At the end of the incubation, the cells were fixed with cold methanol (Sigma), then stained with a 0.5% crystal violet solution. Excess dye was removed by rinsing with distilled water, and the plates were air-dried at room temperature. Quantifications of staining intensities were performed using Fiji (ImageJ ver. 2.0), an image processing software [ 25 ].
Immunocytochemistry analysis
GC1 and GC2 cell lines were seeded at a density of 5 × 10⁴ cells per well in 24-well culture plates and incubated for 24 h in a humidified incubator at 37 °C with 5% CO₂ to allow adherence and monolayer formation. Following incubation, the culture medium was removed and replaced with fresh medium containing previously determined IC₅₀ concentrations of SLX. Cells were further incubated under these conditions for 48 h. At the end of the treatment period, cells were fixed with 4% paraformaldehyde (PFA; Cat# 387507, Carla Erba, France) at room temperature for 30 min. Subsequently, cells were washed with PBS and permeabilized using 1% Triton X-100 (Cat# A4025, Biomatic, Canada) for 15 min. After washing, cells were incubated with 3% hydrogen peroxide (H₂O₂; Cat# K40391897, Merck, Darmstadt, Germany) for 10 min to quench endogenous peroxidase activity. Blocking was performed with blocking solution (Cat# TP-125-HL, Thermo, UK) for 10 min at room temperature. Primary antibodies specific to ATM, Bax, BRCA1, and Cas-3 proteins were then applied, and the samples were incubated overnight at 4 °C. Following PBS washes, secondary antibodies conjugated with biotin-streptavidin-peroxidase (Cat#: TP-125-HL, Thermo, UK) were added and incubated for 30 min. The color reaction was carried out for 5 min using DAB chromogen (Cat# ACK125, ScyTek, USA), with the negative control processed without the primary antibody. After staining, the cells were counterstained with Mayer’s hematoxylin (Cat# BS-005, Beslab, Turkey) and rinsed with distilled water. The preparations were mounted using mounting medium (Cat# K002, Diagnostic Biosystem, Holland) and analyzed under a Nikon Eclipse 80i microscope. All experiments were performed in three independent replicates [ 26 ]. Immunocytochemistry staining intensities were evaluated by the Fiji-ImageJ (software, version 2.0.0-beta4) analysis method. In this method, 10 different areas were randomly selected for each group. DAB-positive staining was evidenced by the appearance of brown-colored cells. The obtained optical density (OD) values were evaluated by statistical analysis [ 27 ].
Annexin V-FITC/PI dual staining assay
To evaluate the apoptotic effects of SLX on GC1 and GC2 cell lines, a dual staining flow cytometry analysis was performed using the Annexin V-FITC/PI Apoptosis Detection Kit (E-CK-A211, Elabscience, Houston, USA). Cells were seeded into 6-well plates at a density of 2.5 × 10⁵ cells per well and incubated overnight to allow monolayer formation. Subsequently, SLX was applied at the predetermined IC₅₀ concentrations to each cell line, followed by a 48-hour incubation period. After incubation, cells were prepared and incubated with Annexin V-FITC and propidium iodide (PI) for 20 min. Stained cells were immediately analyzed using a CytoFlex flow cytometer (Beckman Coulter). Data were processed using CytExpert software, and cells were categorized into four populations based on fluorescence signals: viable cells (Annexin V−/PI−), early apoptotic cells (Annexin V+/PI−), late apoptotic cells (Annexin V+/PI+), and necrotic cells (Annexin V−/PI+) [ 28 ].
Statistical analysis
The GraphPad Prism 7 program was used to evaluate the data. First, the Shapiro-Wilk normality test was performed to determine whether the values were normally distributed. Normally distributed data were analyzed with the Student’s t-test. Statistical analyses were calculated using 95% confidence intervals, and p < 0.05 values were considered statistically significant. All experiments were performed in triplicate and repeated three times. Results are presented as mean ± standard deviation (SD).
Results
Selinexor inhibits GC1 and GC2 cell proliferation
The cytotoxic effect of SLX on GC1 and GC2 cell lines was evaluated in a concentration- and time-dependent manner using the MTT cell viability assay. The applied SLX concentrations ranged from 0.625 µM to 40 µM, with incubation periods of 24 and 48 h. According to the obtained results, the IC₅₀ value calculated at 24 h in the GC1 cell line was 29.73 ± 1.59 µM, while this value was recorded as 16.70 ± 0.35 µM at 48 h. In the GC2 cell line, the IC₅₀ value was determined to be 16.21 ± 1.06 µM at 24 h and 6.85 ± 0.35 µM at 48 h (Fig. 1 A and B). These findings revealed that SLX significantly decreased cell viability, and the GC2 cell line exhibited greater sensitivity to the SLX. The effects of SLX on the proliferation capacity of GC1 and GC2 cells were assessed using colony formation assay. Throughout the ten-day observation period, the colony-forming abilities of both cell lines were significantly reduced in SLX-treated groups compared to the control groups. In GC1 cells, the average number of colonies in the control group was 225.00 ± 12.12, whereas this number decreased to 120.33 ± 7.02 in the SLX-treated group (Fig. 1 C and D). Similarly, in GC2 cells, the average colony number decreased from 284.00 ± 10.82 in the control group to 103.00 ± 7.55 following SLX treatment (Fig. 1 E and F). These results demonstrated that SLX exerts a potent inhibitory effect on the clonogenic potential of GC1 and GC2 cells, with a more pronounced effect observed in GC2 cells.
Selinexor shows anti-migratuar effects on GC1 and GC2 cells
The effect of SLX on cell migration was investigated using in vitro wound healing assay in GC1 and GC2 cell lines. Cell migration dynamics were assessed by measuring wound closure percentages at 0, 12, 24, and 48 h post-scratch. In the control group of GC1 cells, wound closure rates were recorded as 72.16 ± 4.34% at 12 h, 93.97 ± 2.94% at 24 h, and 97.39 ± 2.33% at 48 h. In contrast, in SLX-treated GC1 cells, these rates were significantly reduced to 56.75 ± 4.74% ( p = 0.0173), 61.73 ± 10.49% ( p < 0.0001), and 81.50 ± 4.45% ( p = 0.0143), respectively (Fig. 2 A, C). In the GC2 cell line, wound closure percentages in the control group were 23.27 ± 8.74% at 12 h, 44.37 ± 2.67% at 24 h, and 96.05 ± 1.69% at 48 h. However, SLX-treated GC2 cells exhibited wound closure rates of 18.98 ± 7.59% (p = non-significant), 24.23 ± 11.86% ( p = 0.0169), and 66.30 ± 6.20% ( p = 0.0010) at the same time points (Fig. 2 B, D). Collectively, these results indicate that SLX exerts a pronounced anti-migratory effect in both GC1 and GC2 cells by significantly inhibiting wound closure over time, with a more evident suppression observed in the GC2 cell line at 48 h.
Selinexor induces apoptosis on GC1 and GC2 cells
To evaluate the proportion of apoptotic cells, flow cytometry analyses were performed using the Annexin V-FITC/PI double staining method in GC1 and GC2 cell lines. In the GC1 control group, the live cell rate was evaluated at 93.88%, with early and late apoptotic cell rates recorded as 1.24% and 4.30%, respectively. Following SLX treatment, live cells rate decreased to 61.30%, while early apoptotic cells increased to 3.27% and late apoptotic cells rose significantly to 30.22% in the GC1 cell line. Similarly, in the GC2 cell line, the rate of live cells in the control group was 92.36%, the rate of early apoptotic cells was 3.96%, and the rate of late apoptotic cells was 3.41%. After SLX treatment, the live cell rate in GC2 cell line decreased to 43.71%, the late apoptotic cell rate markedly increased to 53.08%, and the early apoptotic cell rate was determined as 2.18% (Fig. 3 A). These findings show that SLX treatment significantly increased the rates of late apoptotic cells in both GC1 and GC2 cell lines, while causing a significant decrease in the rates of live cells.
The effects of SLX treatment on apoptosis-related proteins were assessed in GC1 and GC2 cell lines by analyzing the expression levels of Cas-3 and Bax proteins using immunocytochemistry method. In the GC1 cell line, Cas-3 immunoreactivity was detected as 0.020 ± 0.003 in the control group, while it was found that Cas-3 immunoreactivity increased statistically significantly and was 0.530 ± 0.007 in the SLX treated group ( p < 0.0001). Cas-3 immunoreactivity in the GC2 cell line was found to be 0.052 ± 0.006 in the control group and 0.089 ± 0.005 in the SLX treated group ( p < 0.0001). In both cell lines, Cas-3 staining was observed to be localized in the cytoplasmic regions and showed a significant increase in intensity in the SLX groups. Bax immunoreactivity was found to be 0.030 ± 0.004 in the GC1 control group and 0.079 ± 0.009 in the GC1 SLX treated group ( p < 0.0001). In the GC2 cell line, Bax immunoreactivity was detected as 0.053 ± 0.008 in the control group, while it was found that Bax immunoreactivity increased statistically significantly and was 0.106 ± 0.007 in the SLX treated group ( p < 0.0001) (Fig. 3 B, C). These findings obtained from both cell lines show that SLX application activates the apoptosis process by significantly increasing the expression of Cas-3 and Bax proteins.
Selinexor triggers DNA damage and oxidative stress
The effects of SLX treatment on the expression levels of ATM, BRCA1, and iNOS proteins associated with DNA damage and oxidative stress in GC1 and GC2 cell lines were evaluated by immunocytochemistry. In the GC1 cell line, ATM immunoreactivity was detected as 0.020 ± 0.003 in the control group and elevated to 0.050 ± 0.003 in the SLX treated group ( p < 0.0001). BRCA1 immunoreactivity was found to be 0.085 ± 0.009 in the control group and 0.122 ± 0.003 in the SLX treated group ( p < 0.0001). Additionally, the immunoreactivity of iNOS, a key marker of oxidative stress, was observed to increase from 0.088 ± 0.007 in the control group to 0.106 ± 0.005 in the SLX treated group ( p = 0.0074). In the GC2 cell line, ATM immunoreactivity was found to be 0.041 ± 0.004 in the control group and significantly elevated to 0.103 ± 0.007 in the SLX treated group ( p < 0.0001). The immunoreactivity of BRCA1 was detected as 0.039 ± 0.003 in the control group and 0.092 ± 0.004 in the SLX treated group ( p < 0.0001). iNOS immunoreactivity levels was similarly increased after SLX teratment and found as 0.098 ± 0.002 in the control group and 0.128 ± 0.004 in the SLX treated group ( p < 0.0001) (Fig. 4 A and B). These data indicate that SLX stimulates both DNA damage and nitric oxide-mediated oxidative stress mechanisms.
Discussion
The pivotal role of cellular protein transport mechanisms in the proliferation and survival of cancer cells has been elucidated in recent years. XPO1, a key nuclear transport receptor, mediates the translocation of nuclear export signal (NES)-bearing proteins from the nucleus to the cytoplasm. XPO1 is responsible for exporting several crucial proteins, including tumor suppressors such as TP53, BRCA1/2, and APC. This export process is important for regulating cell proliferation, apoptosis, and maintaining genomic stability. However, aberrant overexpression of XPO1 facilitates the cytoplasmic mislocalization of tumor suppressor proteins, thereby impairing their function and promoting cancer cell survival. SLX, an orally bioavailable small-molecule inhibitor of XPO1, covalently binds to the NES-binding groove of XPO1, effectively retaining NES-carrying tumor suppressor proteins within the nucleus. By restoring the nuclear localization and functional integrity of these proteins, SLX exerts potent anti-tumor effects through mechanisms such as induction of apoptosis, cell cycle arrest, and inhibition of cellular proliferation [ 29 ]. The cytotoxic effects of SLX at varying concentrations were evaluated in acute myeloid leukemia (AML) cells [ 22 ], human T cells [ 30 ], lung cancer, lymphoma cancer, multiple myeloma and breast cancer cells [ 18 ]. In addition, Nair et al. investigated the cytotoxic effects of SLX in liposarcoma, malignant peripheral nerve sheath cells, dedifferential liposarcoma, uterine leiomyosarcoma, osteosarcoma and synovial tumor cells [ 31 ]. When the studies are evaluated, it is seen that SLX has IC₅₀ values ranging from 16 nM to 6.5 µM in different cells. In our study, the antiproliferative and cytotoxic effects of SLX on male reproductive cell lines GC1 and GC2 cells were evaluated. As a result of the MTT assay, the 48-hour IC₅₀ value for the GC1 cell line was determined as 16.73 µM and for the GC2 cell line as 6.85 µM. The anti-proliferative effects of SLX and Eltanexor, one of the SINE compounds like SLX, were examined in hematopoietic stem and progenitor cells (HSPCs). It was reported that Eltanexor decreased colony formation by 75% and SLX by 50% after 48 h of treatment [ 32 ]. Uddin et al. evaluated the effects of SLX, Gemtamycin and Paclitaxel combinations on colony formation in KCI-313 cell line. It is reported that this combination showed a synergistic effect and significantly reduced the number and size of the colonies [ 33 ]. As a result of our colony formation assays, SLX treatment caused a statistically significant decrease in the colony forming capacity of GC1 and GC2 germ cell lines compared to the control groups. SLX has been reported to have inhibitory effects on cell migration as well as antiproliferative effects. It was reported that SLX significantly suppressed cell migration in the tongue cancer cell lines SCC-4 and SCC-25, leading to a significant delay in the wound healing process [ 34 ]. Another study demonstrated that the migratory ability of the human cholangiocellular carcinoma cell line HuCC-T1 was significantly inhibited after SLX treatment [ 35 ]. Similarly in our study, SLX significantly suppressed cell migration in a time-dependent manner compared to the control groups in the GC1 and GC2 cell lines. The migration capacity of male germ cells is critical for the normal spermatogenesis process; spermatogonial stem cells migrate to appropriate microenvironments (niches) in the seminiferous tubules, where they both renew themselves and initiate differentiation programs [ 36 ]. Microenvironmental signals such as Sertoli cells, extracellular matrix components, growth factors, and chemokines guide germ cell positioning, survival, and fate decisions. When these interactions are disrupted, the relationship between spermatogonial cells and the microenvironment weakens, and spermatogenesis may be impaired [ 37 ]. In our study, SLX suppressed cell migration in GC1 and GC2 cell lines, suggesting that the drug may negatively affect germ cell microenvironment interactions and thus fertility.
DNA damage occurs continuously in organisms as a result of cellular processes and environmental factors, and DNA damage response (DDR) mechanisms are activated to repair this damage. Double-strand breaks (DSB) are considered to be one of the most serious types of DNA damage and are detected by phosphorylation at the Ser139 residue of histone H2AX (γ-H2AX) [ 38 , 39 ]. Following DNA damage, the major signal transduction kinases ATM, ATR and DNA-PK initiate the repair process by phosphorylating H2AX [ 40 ]. Gravina et al. investigated the effects of the combination of SLX, an XPO1 inhibitor, and docetaxel (DTX) on prostate cancer cell lines. In the study, it was reported that in both DTX-sensitive and DTX-resistant prostate cancer cell lines, the level of γH2AX, a biomarker of DNA damage, increased following the administration of DTX alone, but completely returned to the pre-treatment basal levels within 24 and 48 h. In contrast, coadministration of DTX and SLX did not restore γH2AX levels and SLX increased γH2AX levels [ 41 ]. Similarly, Yuan et al. showed that γ-H2AX levels were increased when SLX and Venetoclax were administered as monotherapy in non-Hodgkin’s lymphoma (NHL) subtype mantle cell lymphoma (MCL) cells, and this effect was even more pronounced in combination therapy. These findings suggest that both drugs cause DNA damage and have a synergistic effect when used together [ 39 ]. However, Burke et al. reported that ATM protein accumulated in γ-H2AX foci after SLX treatment in fibrosarcoma cells [ 42 ]. In our study, ATM immunoreactivity was found to increase in the nucleus in both cell lines.
XPO1 is overexpressed in various solid tumors and is associated with poor prognosis [ 43 ]. XPO1 plays a critical role in the transport of proteins and cell cycle regulators from the nucleus to the cytoplasm. Inhibition of this protein leads to the accumulation of numerous proteins and RNAs in the nucleus and increased nuclear localization of tumor suppressor proteins (such as p53, p73, BRCA1, pRB, APC/β-catenin and FOXO). This leads to induction of apoptosis in cells [ 18 ]. In the in vitro and in vivo model of uterine leiomyosarcoma, SLX and eribulin combination therapy was reported to reduce the expression of genes associated with epithelial-mesenchymal transition (EMT), angiogenesis and metastasis (ADAM15, HIF1A, FN1 and SOX4), while increasing the expression of tumor suppressor genes such as NF2 and BRCA1 [ 44 ]. In our study, BRCA1 immunoreactivity was significantly increased in the nuclei of GC1 and GC2 healthy male germ cell lines. The obtained data show that SLX causes DNA damage in germ cells.
SLX has been reported to show an anticancer effect by initiating the apoptosis process in cancer cells [ 45 ]. This effect is also supported by experimental studies conducted on different types of cancer. Apoptosis analyses on multiple myeloma cells indicate that SLX-treated cells become more susceptible to apoptosis at both increased doses and longer-term treatments compared to the untreated group [ 46 ]. Similarly, Lin LH et al. reported that SLX effectively induced apoptosis in Kasumi-1 AML cells in a dose-dependent manner, and a significant increase in apoptosis rate was observed with increasing drug concentration. Furthermore, a significant increase in the G0/G1 phase and a significant decrease in the S phase of the cell cycle were detected after treatment. It is stated that SLX suppresses DNA synthesis by stopping the cell cycle in the G0/G1 phase in AML cells and thus inhibits cell proliferation [ 47 ]. In an in vitro study conducted on prostate cancer cell lines, it was reported that as a result of the SLX treatmet al.one, an increase in Bax protein levels and a decrease in Bcl-2 protein were detected, creating a pro-apoptotic environment. In an in vitro study performed in prostate cancer cell lines, it was reported that SLX treatment alone increased Bax protein levels and decreased Bcl-2 protein levels, creating a pro-apoptotic environment. In addition, it is reported that in the combination of SLX and docetaxel, a significant increase in the active form of Cas-3 was observed, the increase in Bax levels became more pronounced, and apoptosis in tumor cells increased significantly [ 41 ]. In another study, Martini et al. reported that SLX significantly activated the apoptosis-related caspase cascade in SUM-159 breast cancer cells and resulted in a six-, nine-, and four-fold increase in the catalytic activities of Cas-9, Cas-3, and Cas-8, respectively. These findings suggest that SLX can initiate cellular death processes by targeting both intrinsic (mitochondrial) and extrinsic apoptosis pathways. In the same study, it was reported that SLX treatment resulted in a decrease in the expression levels of inhibitory apoptosis proteins (IAP family: cIAP1, cIAP2, livin, survivin, XIAP) and anti-apoptotic proteins belonging to the Bcl-2 family (Bcl-2, Bcl-xL), while the levels of pro-apoptotic proteins (Bad, Bax, FADD, Fas, SMAC/DIABLO, p21, p27) increased [ 48 ]. In a study conducted on chronic myeloid leukemia (CML) cell lines, SLX was shown to reduce cell viability by causing depolarization in mitochondrial membrane potential. This effect is associated with a significant increase in the expression levels of the pro-apoptotic genes Bax and BAK1 [ 49 ]. In our study, significant increases in Cas-3 and Bax protein levels were observed in GC1 and GC2 male germ cell lines after SLX treatment. In addition, as a result of the analysis by the Annexin V-FITC/PI double staining method, it was determined that the viable cell ratios decreased in both cell lines after SLX treatment, whereas apoptotic cell ratios increased. After SLX treatment, the percentage of apoptotic cells in GC2 cells was higher than in GC1 cells, whereas the percentage of dead/necrotic cells in GC1 cells was higher than in GC2 cells, suggesting that SLX may activate other cellular death pathways along with apoptosis in GC1 cells.
The effects of SLX on oxidative stress in tumor and healthy cells are very limited in the literature. Zhao et al. reported that mitochondrial membrane potential was significantly decreased in gallbladder cancer (GBC) cells treated with SLX, and SLX induced apoptosis in GBC cells through mitochondria-mediated apoptosis [ 50 ]. Another study reported that mitochondrial membrane potential decreased in ovarian cancer cells exposed to KPT-185, an XPO1 inhibitor. In addition, eIF5A protein, which is an indicator of cellular stress and reactive oxygen species (ROS), is reported to be significantly increased in mitochondria [ 51 , 52 ]. In our study, we detected a significant increase in the immunoreactivity of iNOS protein, an indicator of oxidative stress, in GC1 and GC2 cells after SLX administration. These results suggest that SLX causes mitochondrial dysfunction and oxidative stress in male germ cells, and further studies are needed to better understand these mechanisms.
In conclusion, our study revealed that SLX causes cytotoxic and antiproliferative effects on male germ cells in vitro. SLX treatment significantly decreased colony formation and cell migration in GC1 and GC2 cells. Moreover, increases in Bax, Cas-3, ATM, BRCA1, and iNOS immunoreactivities indicate increased apoptosis, DNA damage, and oxidative stress in cells. However, further studies focusing on DNA damage response, cell cycle regulators, and oxidative stress markers are required for a more comprehensive understanding of the effects of SLX in germ cells. Moreover, it is essential to verify the findings of our study using other methods and techniques. In addition, it is of great importance to investigate the effects of SLX on reproductive organs and fertility under in vivo conditions using animal models in order to evaluate the clinical implications of our findings.
Acknowledgements
The authors thank Gaziantep Islam Science and Technology University for the administrative and technical support provided to this study.
Abbreviations
Acute Myeloid Leukemia
Dimethyl Sulfoxide
European Medicines Agency
Epithelial Mesenchymal Transition
U.S. Food and Drug Administration
Gallbladder Cancer
Mantle Cell Lymphoma
Nuclear Export Signal
Non-Hodgkin’s Lymphoma
Optical Density
Standard Deviation
Sinhibitor of Nuclear Export
Selinexor
World Health Organization
Exportin
Author contributions
Dr. Mustafa Öztatlıcı (MÖ) conceived and designed the research. MÖ, Parmila Rahmat Zada (PRZ) and Rukiye Betül Çolaksel (RBÇ) conducted the experiments. MÖ, Dr. Büşra Şen Halıcıoğlu (BŞH) and Dr. Hülya Öztatlıcı (HÖ) analyzed the data. MÖ and BŞH contributed reagents, materials and analytical tools. Writing—original draft preparation: MÖ, RBÇ and PRZ. Writing—review and editing: MÖ, BŞH and HÖ. All authors read and approved the manuscript. The authors declare that all data were generated in-house and that no paper mill was used.
Funding
This work was supported by Gaziantep Islam Science and Technology University Scientific Researches Funding Commission under grant number of [2025-YLP- TF-0002].
Data availability
No datasets were generated or analysed during the current study.
Declarations
Ethics approval and consent to participate
As this study does not involve a clinical trial or human participation, requirements for ethics approval and informed consent are not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Associated Data
Data Availability Statement
No datasets were generated or analysed during the current study.