Tea is the most commonly consumed beverage in the world and it has beneficial effects, including anti-fibrotic, anti-inflammatory, and anticancer effects, caused by polyphenols (Scalbert et al., 2005; Chacko et al., 2010). Catechins are natural polyphenolic compounds and falavan-3-ols belonging to the flavonoid family. They exist in various foods and medicinal plants, such as teas, grapes, cocoa, and apples (Fan et al., 2017). Catechin compounds include (-)- epigallocatechin-3 gallate (EGCG), (-)-epigallocatechin (EGC), epicatechin-3-gallate (ECG) and (p) catechin (Graham, 1992). Catechin and catechin hydrate (CH) are natural flavon-3-ol phytochemicals that are less toxic than other flavonoids such as EGCG (Alshatwi et al., 2014). The beneficial effects of catechins on various diseases have been previously reported. The antioxidant effect of catechins involves direct mechanisms (scavenging reactive oxygen species [ROS] and chelating metal ions) and indirect mechanisms (inducing antioxidant enzymes, inhibiting pro-oxidant enzymes, and producing phase II detoxification and anti-oxidant enzymes) (Bernatoniene and Kopustinskiene, 2018; Sheng et al., 2023). Oxidative stress has been implicated in the progression of various cardiovascular diseases, including hypertension, endothelial dysfunction, atherosclerosis, ischemic heart disease, cardiomyopathy, cardiac hypertrophy, and congestive heart failure (Bhardwaj and Khanna, 2013; Izzo et al., 2021; Sadler et al., 2022). Catechins from green tea decrease blood pressure and the risk of stroke and coronary heart disease (Fujiki et al., 2015). Catechins present in teas inhibit the growth of various cancer cells, including breast, esophageal, prostate, stomach, small intestine, colon, liver, and lung cancer cells (Zhong et al., 2012; Fujiki et al., 2015; Li et al., 2022).

Cisplatin is a commonly used chemotherapeutic drug for the treatment of various cancer types, including ovarian, bladder, lung, and cervical cancers (Dasari and Tchounwou, 2014). Although cisplatin can effectively destroy cancer cells, it also has the potential to cause damage to healthy tissues, including the testicles. Several studies have described the impact of cisplatin on the testes after treatment. Cisplatin can interfere with spermatogenesis via a reduction in sperm count or even complete cessation of sperm production. The extent of damage may vary depending on factors such as the dosage and duration of cisplatin exposure (Cherry et al., 2004; Sherif et al., 2014). In addition, cisplatin affects the germ and Leydig cells in the testis. Disruption of Leydig cell function by cisplatin treatment can lead to decreased serum levels of testosterone, which is the primary male sex hormone important for sperm production (Maines et al., 1990; García et al., 2012). Furthermore, cisplatin treatment can result in abnormal sperm morphology and can affect sperm maturation in the epididymis (Reddy et al., 2016; Razavi et al., 2019). Cisplatin treatment can increase the risk of infertility in male patients owing to its effects on sperm production and testosterone levels.

As mentioned above, cisplatin is widely used chemotherapy drug for treating various types of cancer, however, one of the significant side effects of cisplatin is its potential to induced germ cell death, leading to infertility in cancer patients. This adverse effect limits its use and affects the quality of life of survivors. Catechins, which are natural antioxidants found in green tea, have shown potential in protecting cell from oxidative stress and apoptosis.

Current study, to investigate the protective effects of catechin against the cisplatin-induced death of GC-1 spermatogonia. The success of this study will not only allow for the understanding of the effects of catechin on cisplatin-induced chronic testicular injury, but may also lead to potential clinical applications for catechin to reduce cisplatin-induced testicular toxicity in cisplatin-receiving cancer patients.

1. Cell culture and treatment

GC-1 spg cells were obtained from the Korean Cell Line Bank (KCLB 21715; Seoul, South Korea). The cells were maintained in Dulbecco’s modified Eagle medium (DMEM) with 10% fetal bovine serum and 1% penicillin-streptomycin, in a humidified atmosphere containing 5% CO₂ at 37℃. Catechin hydrate (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in dimethyl sulfoxide (DMSO) to create a 1 M stock solution, which was diluted to the required concentration using cell culture medium before being added to the cells.

2. Cell proliferation assay

To determine the proliferation rate of GC-1 spg cells, an MTT assay was performed using the EZ-Cytox Viability Assay Kit (EZ1000; Daeil Lab Services Co., Seoul, Korea) according to the manufacturer’s instructions. For viability assay, cells were plated in 96-well plates at a density of 3 × 10³ cells/well in complete growth medium. After 24 h, the medium was replaced with fresh medium containing 0-100 μM catechin hydrate or 10 μM cisplatin and incubated for an additional 24 h. Subsequently, a cell viability reagent was added, and the cells were incubated for 60 min. The absorbance was measured at 490 nm using an Epoch spectrophotometer (BioTek, Winooski, VT, USA).

3. Immunostaining

Cells were plated on 18 mm glass coverslips (BD Biosciences, Franklin Lakes, NJ, USA) and exposed to 10 μM cisplatin, 100 μM catechin hydrate, or both cisplatin and catechin hydrate for 24 h. Following this treatment, the cells were fixed with 4% paraformaldehyde for 15 min at 18℃, then permeabilized with 0.1% Triton X-100 in phosphate-buffered saine (PBS) for 10 min at 18℃. The cells were then incubated overnight at 4℃ with a primary antibody against Ki-67 (ab279653; Abcam, Cambridge, UK), followed by a 1-h incubation at room temperature with an Alexa-Fluor-conjugated secondary antibody (goat anti-mouse IgG Alexa Fluo 594; Thermo Fisher Scientific, Waltham, MA, USA). The proliferation index was calculated as the ratio of Ki-67-positive cells to the total number of cells in each field. The CellROX staining method was performed as described previously (Lee and Park, 2023).

4. Flow cytometry analysis

Annexin V-FITC staining was performed using a Cell Death Apoptosis Kit (Thermo Fisher Scientific) following the manufacturer’s instructions. GC-1 spg cells were seeded in six-well plates, treated with catechin and cisplatin for 24 h, harvested, and washed with PBS. Cells were stained with Annexin V-FITC and propidium iodide (PI) in the dark and analyzed using flow cytometry (CytoFLEX; Beckman Coulter, Miami, FL, USA). This procedure has been described previously (Lee and Park, 2023).

5. RNA extraction and quantitative PCR

Total RNA was extracted from the cells using the Qiagen RNeasy Mini Kit (74106; Qiagen, Hilden, Germany) with on-column DNase treatment (79254; Qiagen), following the manufacturer’s instructions. RNA was reverse-transcribed into cDNA using a RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Rockford, IL, USA). Quantitative PCR (qPCR) was conducted using a QuantStudio 1 system (Applied Biosystems, Foster City, CA, USA) with a SYBR Green mixture (Bioneer, Daejeon, Korea). The PCR conditions and analytical methods were as described in our previous study (Lee and Park, 2023). Expression levels were quantified using the 2^-ΔΔCt method, normalized to endogenous Gapdh levels. The primer sequences were as follows: iNOS F: 5’-GTTCTCAGCCCAACAATACAAGA-3’, R: 5’-GTGGACGGGTCGATGTCAC-3’; NK-kb F: 5’-TGGAGTTCGTGACCGCCGCCG-3’, R: 5’-GCTGGCTCTGCCGGGAAGATG-3’; and COX2 F: 5’-CAGCAAATCCTTGCTGTTCC-3’, R: 5’-TGGGCAAAGAATGCAAACATC-3’.

6. Immunoblotting

Cells were harvested and lysed using RIPA lysis buffer (Thermo Scientific) containing protease inhibitors (Roche, Basel, Switzerland). Total protein was quantified using a bicinchoninic acid assay kit (Thermo Fisher Scientific). Thirty micrograms of protein was separated on 4-16% gradient SDS-PAGE gels (Bio-Rad, Hercules, CA, USA) and transferred to PVDF membranes (Bio-Rad). Antibody treatment and image acquisition were performed as previously described (Lee and Park, 2023). The antibodies used are listed in Table 1.

Table 1. List of antibodies used for immunostaining and Western blotting.

Antibody	Company	Catalog number	Dilution
Cleaved-caspase 3	Cell Signaling	#9661	1:2,000
Caspase 3	Cell Signaling	#9662	1:2,000
BAX	Cell Signaling	#2772	1:2,000
iNOS	Cell Signaling	MCA1658	1:2,000
COX-2	Santa Cruz Biotech	sc376861	1:2,000
β-actin	Santa Cruz Biotech	sc47778	1:2,000

7. Statistical analysis

All data are presented as the mean ± standard error for each group, with experiments conducted independently at least three times. Data analysis was performed using the SPSS statistical package (version 25.0; IBM Corp., Somers, NY, USA). Group comparisons were made using a one-way ANOVA, followed by Tukey’s honest significant difference (HSD) test. Statistical significance was set at *p < 0.05 and **p < 0.01. Graphs were created using Sigma Plot 8.0.

1. Catechin hydrate protects against cisplatin-induced germ cell damage

The structure of (+)-catechin hydrate used in this study is shown in Fig. 1A. To identify catechin hydrate in male germ cells, cell proliferation analysis was performed. Type 2 mouse spermatogonia (GC-1 spg cells) were treated with 0-100 μM catechin. The results showed that catechin did not affect the survival of GC-1 spg cells. However, the cells were significantly damaged by cisplatin, and catechin rescued cisplatin-induced GC-1 spg cell death (Fig. 1B). The Ki67 immunocytochemistry analysis results also showed that catechin hydrate prevented the cisplatin-induced reduction in the proliferation of GC-1 spg cells (Fig. 1C and 1D).

Figure 1.Effect of catechin hydrate on cisplatin-induced GC-1 spg cell damage. (A) The chemical structure of catechin hydrate. (B) Cell viability was measured in GC-1 spg cells following treatment with 0-100 μM catechin hydrate, or 10 μM cisplatin with 0-100 μM catechin hydrate, and cultured for 24 h. The graph shows the mean ± SEM of three independent experiments. (C) Immunostaining of Ki67 in GC-1 spg cells. (D) Relative percentage of Ki-67-positive cells shown as a graph with the mean ± SEM of three different experiments. *p < 0.05; **p < 0.01, compared to controls. Scale bar = 50 μm.

2. Pro-apoptotic protein expression levels in GC-1 spg cells after cispatin and catechin treatment

Flow cytometric analysis was performed using the Dead Cell Apoptosis Kit to assess the proportion of apoptotic GC-1 spg cells after exposure to cisplatin and catechin. A distinct increase in the apoptotic rate was observed in cells 24 h after 10 μM cisplatin treatment, but this significantly reduced after treatment with 100 μM catechin hydrate (Fig. 2A and 2B). Western blotting showed that the expression levels of apoptotic proteins, such as cleaved-caspase 3 and BAX were significantly increased by cisplatin; however, catechin treatment reduced their expression levels in GC-1 spg cells (Fig. 2C and 2D).

Figure 2.Catechin hydrate ameliorated cisplatin-induced GC-1 spg apoptosis. (A) Dot plots were used to visualize flow cytometric analysis of apoptosis in the samples treated with combined cisplatin and catechin hydrate. (B) Apoptotic index data summarizing the results, presented as the mean ± SEM from three independent experiments (n = 4). Significant differences compared to the control are indicated by *p < 0.05; **p < 0.01. (C) The protein levels of apoptotic-related factors such as cleaved caspase 3 and BAX. (D) The graph presents the protein level relative to the β-actin level or the level of the inactive form. The data shown as the mean ± SEM with four determinations per condition. **p < 0.01.

3. Antioxidant and anti-inflammatory effects of catechin hydrate in cisplatin-induced GC-1 spg cell damage

To clarify anti-oxidant effects of catechin hydrate, the expression levels of Nrf2, which is a major regulator of ROS production, were determined. Real-time reverse transcription-PCR showed that Nrf2 expression levels were significantly increased by cisplatin treatment, whereas catechin hydrate treatment decreased Nrf2 levels (Fig. 3A). CellROX staining showed that ROS levels were increased by cisplatin and decreased by catechin hydrate (Fig. 3B). Additionally, an investigation of the anti-inflammatory effects of catechin hydrate on GC-1 spg cells showed that the gene expression levels of iNOS and COX2 were significantly increased by cisplatin, but decreased by combined catechin and cisplatin treatment (Fig. 3C). Consistently, COX2 and iNOS proteins were detected at high levels in cisplatin-treated samples, but not in combined cisplatin- and catechin hydrate-treated samples (Fig. 3D).

Figure 3.Antioxidant and anti-inflammatory mechanisms of catechin hydrate in cisplatin-induced GC-1 spg cell damage. (A) Nrf2 mRNA expression in cisplatin and catechin-hydrate-treated GC-spg after cultured 24 h. The graph shows the mean ± SEM in the log 2 scale. (B) CellROX staining in cisplatin- and catechin-hydrate-treated GC-1 sgp cells. CellROX staining intensity was analyzed using Image J software. Data are shown as a graph with the mean ± SEM. *p < 0.05; **p < 0.01. Scale bar = 100 μm. (C) mRNA expression levels of iNOS and COX2 in each sample. The graph shows the mean ± SEM of the mean in the log 2 scale. (D) The protein expression levels of COX2, iNOS, and β-actin in each sample. The graph shows the relative protein levels as the mean ± SEM. COX2 and iNOS expression levels were normalized to β-actin levels. **p < 0.01.

In the present study, we focused on the protective effects of catechin hydrate on male germ cells damaged by cisplatin treatment. Cisplatin is a chemotherapeutic agent widely used to treat various types of cancer, including carcinomas, germ cell tumors, lymphomas, and sarcomas. Its mode of action has been linked to its ability to crosslink with purines in DNA, interfere with DNA repair mechanisms, cause DNA damage, and subsequently induce apoptosis of cancer cells. However, chemotherapy with cisplatin and cisplatin derivatives is hampered by the occurrence of major side effects in a significant percentage of cancer patients, which considerably limits its prolonged utilization (Romani, 2022). Drug resistance and numerous undesirable side effects, including severe kidney problems, allergic reactions, decreased immunity, gastrointestinal disorders, hemorrhage, and hearing loss, especially occur in younger patients (Dasari and Tchounwou, 2014).

Ismail et al. (2023) reported that cisplatin causes damage to testicular tissue and decreases serum testosterone levels, epididymal sperm counts, and oxidant levels. An antioxidant imbalance was detected owing to increased malondialdehyde and reduced glutathione levels in testicular tissue. They treated adipose-derived mesenchymal stem cells, which led to a moderate epididymal stem cell count, adequate antioxidant protection, suitable hormone levels, and enhanced testicular tissue morphology (Ismail et al., 2023). Another study reported that cisplatin-treated rats showed reduced body weight; absolute testis weight; and sperm count, motility, and viability. Additionally, cisplatin treatment increases the incidence of sperm abnormalities. It also decreases serum testosterone levels and mitochondrial membrane potential, while increasing cytochrome C release from the mitochondria into the cytosol. Cisplatin increases the activities of caspase-3 and -9 and the levels of TNF-α, IL-6, and Bax, but decreases Bcl-2 levels. Resveratrol treatment prevents mitochondria-mediated apoptosis and inflammation in rats with cisplatin-induced testicular damage (Aly and Eid, 2020). A previous report described that nanosome-encapsulated honokiol attenuated cisplatin-induced oxidative damage to DNA by suppressing antioxidation enzymes and mitigating endoplasmic reticulum stress through the downregulation of Bip-ATF4-CHOP signaling (Wang et al., 2020). In another study, cisplatin-induced testicular damage was improved through the attenuation of mitochondria-mediated apoptosis, inflammation, and oxidative stress after resveratrol pretreatment (Aly and Eid, 2020).

Green tea catechin, (-)-epigallocatechin-3-O-gallate (EGCG), has gained significant attention as a potent adjuvant for enhancing the antitumor efficacy of cisplatin while mitigating its harmful side effects. In addition, the antioxidant effect of EGCG moieties ensures fail-safe protection against off-target organ toxicity caused by cisplatin-induced oxidative stress (Bae et al., 2017). Sharma and Goyal reported that cadmium chloride (CdCl₂) intoxication resulted in a significant decline in total testicular protein, cholesterol, and alkaline phosphatase levels, whereas a noticeable increase in acid phosphatase and lipid peroxidation levels were seen compared to their levels in the negative control group. Catechin is effective at reducing CdCl₂-induced augmentation of phase I (P450 and CYPB5) and phase II (DT-diaphorase and glutathione-S-transferase) enzymes in the testes. Furthermore, CdCl₂ intoxication attenuates the antioxidant potential of the testes, which is augmented when supplemented with green tea extract (Sharma and Goyal, 2015). A previous study explored the effects of catechins on testosterone secretion by rat testicular Leydig cells. Both in vivo and in vitro experiments were performed. Catechins increased plasma testosterone levels in male rats in vivo. In vitro, low-dose catechin increased gonadotropin-releasing-hormone-stimulated luteinizing hormone release by the anterior pituitary gland and human-chorionic-gonadotropin-stimulated testosterone release by Leydig cells of male rats (Yu et al., 2010). In animal models, epigallocatechin-3-gallate provides testicular protection in cisplatin-challenged rats through its antioxidant, antinutritive, anti-inflammatory, and antiapoptotic effects. Epigallocatechin-3-gallate causes significant increases of serum testosterone levels and testicular antioxidant status, and significant decreases in interleukin-6, interleukin-1β, malondialdehyde, nitric oxide, cytochrome C, and caspase-3 levels and the Bax/Bcl-2 ratio, in testes of cisplatin-treated rats (Fouad et al., 2017). These results are similar to our in vitro results.

The purpose of this study is to investigate whether catechins can protect spermatogonia from the cytotoxic effects of cisplatin. By understanding this protective mechanism, the study aims to provide insights into developing adjust therapies that can mitigate the adverse side effects of cisplatin, thereby improving the overall treatment outcomes and quality of life for cancer patients undergoing chemotherapy.

In conclusion, the present study showed that catechin hydrate treatment prevented apoptotic cell death and reduced oxidative stress- and inflammation-related factors in cisplatin-treated GC-1 spg cells. These results contribute to a potential clinical method for using catechin hydrate to reduce cisplatin-induced toxicity in cisplatin-based cancer therapies. They also provide dietary guidelines for patients undergoing chemotherapy.

None.

Conceptualization, H-J.P.; data curation, H-J.P.; formal analysis, H-J.P.; investigation, H-J.P., H.W.S., W-Y.L., Y-K.H., S.D.L., and S-G.H.; methodology, H-J.P., W-Y.L.; project administration, H-J.P., Y-K.H., S.D.L., and S-G.H.; resources, H-J.P.; supervision, H-J.P.; writing - original draft, W-Y.L., H-J.P.; writing - review & editing, H-J.P.

This study was supported by the Sangji University Research Fund 2023.

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No potential conflict of interest relevant to this article was reported.