Abstract
Background/Aim: Fibroblast growth factor 9 (FGF9) is a member of the human FGF family known for its pivotal roles in various biological processes, such as cell proliferation, tissue repair, and male sex determination including testis formation. Cordycepin, a bioactive compound found in Cordyceps sinensis, exhibits potent antitumor effects by triggering apoptosis and/or autophagy pathways. Our research has unveiled that FGF9 promotes proliferation and tumorigenesis in MA-10 mouse Leydig tumor cells, as the phenomena are effectively countered by cordycepin through apoptosis induction. Moreover, we have observed FGF9-mediated stimulation of proliferation and tumorigenesis in TM3 mouse Leydig progenitor cells, prompting an investigation into the potential inhibitory effect of cordycepin on TM3 cell proliferation under FGF9 treatment. Hence, we hypothesized that cordycepin induces cell death via apoptosis and/or autophagy in FGF9-treated TM3 cells. Materials and Methods: TM3 cells were treated with cordycepin and/or FGF9, and the flow cytometry, immunofluorescent plus western blotting assays were used to determine how cordycepin regulated Leydig cell death under FGF9 treatment. Results: Our findings reveal that cordycepin restricts cell viability and colony formation while inducing morphological alterations associated with cell death in FGF9-treated TM3 cells. Surprisingly, cordycepin fails to elicit the expression of key apoptotic markers, suggesting an alternate mechanism of action. Although the expression of certain autophagy-related proteins remains unaltered, a significant up-regulation of LC3-II, indicative of autophagy, is observed in cordycepin-treated TM3 cells under FGF9 influence. Moreover, the inhibition of autophagy by chloroquine reverses cordycepin-induced TM3 cell death, highlighting the crucial role of autophagy in this process. Conclusion: Our study demonstrates that cordycepin activates autophagy to induce cell death in TM3 cells under FGF9 treatment conditions.
The fibroblast growth factor (FGF) family encompasses 23 FGF ligands (1), which exert their actions via highly conserved transmembrane tyrosine kinase receptors (FGFRs; FGFR1, FGFR2, FGFR3, and FGFR4) (2, 3) involved in various biological functions (4). The interaction of FGFs with FGFRs is facilitated by coreceptors known as heparan sulfate proteoglycans (HSPGs), a type of glycoproteins (5). This binding triggers FGFR monomers to dimerize, leading to tyrosine cross-autophosphorylation of the cytoplasmic domain, subsequently activating multiple signal transduction pathways (6). Among the FGF family, fibroblast growth factor 9 (FGF9) stands out for its critical roles in embryonic development (7), tissue repair (8), and male sex determination with testis formation (9, 10). Studies have unveiled its involvement in promoting the survival and migratory capabilities of human lung fibroblasts, pancreatic development, and odontoblast differentiation (11, 12). Furthermore, FGF9 has been shown to stimulate MAPK pathways, PI3K/AKT pathway, focal adhesion kinase, and PLC/PKC pathway, thereby regulating cell proliferation, survival, motility, and development (13, 14).
Cordycepin (3′-deoxyadenosine), an extract from the fungus Cordyceps sinensis, exhibits diverse physiological functions, including anti-oxidative activity, skin cell regeneration, maintenance of stemness of human mesenchymal stem cells, anticancer effects, and steroidogenesis induction (15-17). Numerous studies have demonstrated its ability to induce apoptosis in liver cancer (18), glioma (19), breast cancer (20), and bladder cancer (21). Cordycepin can also stimulate steroidogenesis (22). Additionally, cordycepin has been found to induce cell death via autophagy across various cell types (23, 24).
Autophagy plays a crucial role in many physiological functions, including development (25), lifespan extension (26), and immunity and defense against microbial invasion (27). It is a catabolic process wherein cellular material is enclosed in double-membrane autophagosomes and delivered to lysosomes for degradation (28). During periods of starvation stress, autophagy degrades cytoplasmic materials to produce amino acids and fatty acids that can be utilized for cell survival (29). However, dysregulated autophagy is implicated in various diseases, such as cancer, neurodegeneration, heart, and liver diseases (30, 31). There is crosstalk between autophagy and apoptosis, where inhibition of autophagy may enhance apoptosis or vice versa, depending on cellular responses (32). Both autophagy and apoptosis share common stimuli and signaling pathways, ultimately determining cell fate (33). Multiple ATG proteins, including ATG5 and ATG7, are involved in catalyzing the formation of phosphatidylethanolamine (PE)-conjugated LC3 (LC3-II), essential for autophagosome formation (34). The closure of an elongated phagophore signifies the formation of a mature autophagosome, which then fuses with a lysosome for cargo degradation and nutrient recycling (28).
Our research has elucidated the anticancer effect of cordycepin on FGF9-induced MA-10 mouse Leydig cell tumor growth by modulating the expression of p-ERK1/2, p-Rb, E2F1, cell cycle-related proteins, and FGFR1-4 proteins both in vitro and in vivo (35). Additionally, we have observed that cordycepin induces apoptosis via the caspase cascade, rather than autophagy, to suppress FGF9-induced MA-10 cell tumor growth in vitro and in vivo (36). Moreover, we have discovered that FGF9 promotes TM3 mouse Leydig normal/progenitor cell proliferation and tumorigenesis (37). However, the potential inhibitory effect of cordycepin on TM3 cell proliferation under FGF9 treatment remains undetermined.
In this study, we investigated the effect of cordycepin-induced cell death in TM3 mouse Leydig progenitor cells through apoptosis and/or autophagy under FGF9 treatment. Our findings demonstrate that cordycepin activates autophagy to induce TM3 cell death in the presence of FGF9. Notably, compared to our previous findings (36), the efficacy of cordycepin in inducing TM3 cell death was lower than in MA-10 cells. Thus, in the presence of FGF9, cordycepin stimulates autophagy in TM3 cells and apoptosis in MA-10 cells, respectively, to induce cell death.
Materials and Methods
Chemicals. Human fibroblast growth factor 9 (FGF9) was procured from PeproTech (Rocky Hill, NJ, USA). Ethanol was obtained from PerkinElmer (Boston, MA, USA), and other chemicals including bovine serum albumin (BSA), 30% acrylamide/Bis-acrylamide solution, methyl tetrazolium (MTT), dimethyl sulfoxide (DMSO), sodium chloride (NaCl), potassium chloride (KCl), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), D-glucose, proteinase inhibitor, cordycepin, and sodium hydroxide (NaOH) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Donor horse serum (HS), fetal bovine serum (FBS), Dulbecco’s modified eagle medium nutrient mixture F-12 (D-MEM/F12), and trypsin-EDTA were obtained from Gibco (Grand Island, NY, USA). Sodium chloride (NaCl), Tris base, and potassium chloride (KCl) were sourced from JT Baker (Phillipsburg, NJ, USA), while hydrochloric acid (HCl), acetic acid, and sodium dodecyl sulfate (SDS) were purchased from Merck (Darmstadt, Germany). Tween-20 was acquired from AppliChem (Darmstadt, Germany). Peroxidase AffiniPure goat anti-rabbit IgG (H+L) was procured from Jackson ImmunoResearch Inc. (West Grove, PA, USA), and donkey anti-mouse IgG conjugated with horseradish peroxidase was obtained from PerkinElmer. Monoclonal antibodies against cleaved caspase-3 (1:2,000; #9661) and Atg12 (1:2,000; #4180), and polyclonal antibodies against cleaved caspase-8 (1:1,000; #9429), cleaved caspase-9 (1:1,000; #9509), PARP (1:1,000; #9542), LC3A/B (1:1,000; #4108), and beclin-1 (1:1,000; #3738) were purchased from Cell Signaling (Beverly, MA, USA). The monoclonal antibody against β-actin (1:8,000; #A5441) was obtained from Sigma-Aldrich (St. Louis, MO, USA). The Enhanced chemiluminescence (ECL) detection kit was sourced from Millipore (Billerica, MA, USA), while the Annexin V-FITC apoptosis detection kit was acquired from Strong Biotech (Taipei, Taiwan, ROC).
Cell culture. The TM3 mouse Leydig progenitor cell line was obtained from ATCC (Manassas, VA, USA) and maintained in D-MEM/F12 medium supplemented with 5% horse serum (HS) and 2.5% fetal bovine serum (FBS). All cells were cultured in a humidified incubator at 37°C with 5% CO2.
MTT viability test. For the 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay, a colorimetric method for assessing cell viability, TM3 cells were seeded at a density of 6×103 cells in 96-well plates per well with 200 μl of culture medium. After 20-24 h of incubation for cell seeding, the cells were switched to serum-free medium for 18 h. Following starvation, cells were treated with BSA (vehicle control), 50 ng/ml FGF9, DMSO (vehicle control), and/or different concentrations of cordycepin (25, 50, or 100 μM) in medium containing 1% FBS for 12 and 24 h, respectively. Subsequently, MTT was added to each well at a final concentration of 0.5 mg/ml and incubated in a humidified incubator at 37°C with 5% CO2 for 3 h. After removing the medium, 50 μl of DMSO was added to each well and placed on a shaker to dissolve the crystals for 30 min in the dark. Cell viability was then measured at λ=570 nm using a VersaMax ELISA reader (Molecular Devices, Sunnyvale, CA, USA).
Morphological observation. TM3 cells were seeded in 6-cm culture dishes at concentrations of 5.5×105/ml for 12 h and 4×105/ml for 24 h. After 20-24 h of incubation after cell seeding, the cells were switched to serum-free medium for 18 h. Subsequently, TM3 cells were treated with BSA (vehicle control), 50 ng/ml FGF9, DMSO (vehicle control), and/or different concentrations of cordycepin (25, 50, or 100 μM) for 12 and 24 h, respectively. Cell morphology was then observed using an Olympus CK40 light microscope, and images were recorded with an Olympus DP20 digital camera (Olympus, Tokyo, Japan) (38).
Clonogenic assay. TM3 cells were treated with BSA (vehicle control) or 50 ng/ml FGF9 plus cordycepin (25, 50, or 100 μM) or DMSO (vehicle control). The cells were then trypsinized and 1,000-1,500 cells were replated in 6-cm culture dishes and returned to the incubator for colony development. After 8-14 days, colonies (containing ≥50 cells in each colony) were stained with a 0.5% crystal violet solution. The plating efficiency (PE) was calculated as the ratio of the number of colonies to the number of cells seeded in culture dishes. The surviving fraction (SF) was determined using the formula: SF=plating efficiency (PE) of treated cells/PE of control cells. PE (%) was obtained from (colonies counted/cell plated) ×100. Survival curves were plotted using the mean surviving fraction values (36).
Annexin V/PI double staining assay. TM3 cell suspensions, harvested by trypsinization and washed with 2 ml of culture medium, underwent centrifugation at 1,000 rpm for 3 min at 4°C. Following two washes with cold PBS and subsequent centrifugation, cellular apoptosis was assessed using the FITC Annexin V Apoptosis Detection Kit as per the manufacturer’s instructions (BD Pharmingen™, Franklin Lakes, NJ, USA). The cells were resuspended in 100 μl of 1X binding buffer at a concentration of 1×106 cells/ml in a 1.5 ml Eppendorf tube. Subsequently, 5 μl of FITC Annexin V and 5 μl of PI were added to the cells in the tube, which were then vortexed. After incubation at room temperature in the dark for 15 min, 400 μl of 1X binding buffer was added to each tube, followed by analysis using a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA, USA).
Western blotting assay. TM3 cells, seeded at densities of 5.5×105 cells for 12 h and 4×105 cells for 24 h in 6-cm culture dishes with 2 ml of culture medium, were subjected to treatment. Post-treatment, cells were washed twice with cold PBS, and cell suspensions underwent centrifugation at 1,000 rpm for 3 min at 25°C. Attached cells were lysed with 50-70 μl of lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-Glycerolphosphate and 1 mM Na3VO3) containing proteinase inhibitors (Sigma-Aldrich, St. Louis, MO, USA), followed by centrifugation at 12,000 × g for 12 min at 4°C. The resulting cell suspensions were collected and stored at −80°C. Protein concentrations of cell lysates were determined using the Bio-Rad protein assay dye reagent concentrate (Bio-Rad Laboratories, Hercules, CA, USA). For the Western blot assay, cell lysates containing 25-35 μg of proteins were resolved in a 12% SDS-PAGE gel with standard running buffer (24 mM Tis-HCl, 0.19 M glycine, and 0.5% sodium dodecyl sulfate, pH 8.3) at room temperature and electrophoretically transferred onto a polyvinylidene difluoride (PVDF) membrane on ice. Electrophoresis was performed at 90V, and transfer onto the PVDF membrane was conducted at 375 mA for 90 min. The PVDF membrane with the transferred proteins was incubated in blocking buffer (5% non-fat milk in 0.1% TBST washing buffer) for 1 h and then with primary antibodies for 16-18 h at 4°C. After washing 3 times with TBST, the membranes were incubated with the appropriate dilution of HRP-conjugated secondary antibodies for 1 h. Signaling was visualized using the UVP EC3 BioImaging Systems (UVP, Upland, CA, USA) by the ECL detection kit. Quantitative analysis was performed using the ImageJ program version 1.5 (NIH, Bethesda, MD, USA).
Autophagy detection. For the assessment of autophagy, cells were stained with acridine orange (AO) from Sigma-Aldrich (St. Louis, MO, USA), a dye known to detect acidic vesicular organelles (AVOs) (39). After harvesting and centrifugation, cell suspensions were treated with 1 μg/ml AO and then incubated at room temperature in the dark for 15 min before analysis using a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA, USA). Excitation fluorescence was set at blue (488 nm), with emission fluorescence recorded in green (510-530 nm) and red (>650 nm). Additionally, TM3 cells were pre-treated with the autophagy inhibitor chloroquine (CQ) for 1 h, followed by treatment with 50 ng/ml FGF9 and/or cordycepin (0 and 50 μM). The PrestoBlue assay for cell viability was employed to confirm whether cordycepin activated autophagy in FGF9-treated TM3 cells.
Immunofluorescent staining. TM3 cells were cultured on glass coverslips at 37°C before fixation with ice-cold methanol at −4°C for 30 s. After blocking with a blocking buffer for 1 h, cells were treated with primary antibodies for 24 h at 4°C. Following extensive washing with PBS, cells were incubated with a fluorescein isothiocyanate-secondary antibody (Invitrogen, Carlsbad, CA, USA) for 1 h in the dark. Nuclei were simultaneously stained with 4′,6-diamidino-2-phenylindole (DAPI, 0.1 μg/ml). After further washing, the coverslips were mounted on glass slides in 50% glycerol. Fluorescent cells were observed using an Axio Imager M2 fluorescence microscope (Zeiss, Oberkochen, Germany). Images were processed using the analyze particles tool in ImageJ program version 1.5 (NIH, Bethesda, MD, USA), with the number of LC3 objects/puncta normalized to the number of nuclei per field of view.
PrestoBlue™ cell viability assay. TM3 cells were seeded at a density of 6×103 cells in 96-well plates per well with 200 μl of culture medium. After treatments, the medium was removed, and 10 μl of PrestoBlue Reagent was added to 90 μl of 1% FBS medium directly to the cells. The cells were then incubated for 1.5 h at 37°C in the dark. Cell viability was determined at λ=570 nm, with 600 nm as a reference wavelength (normalized to the 600-nm value) using a VersaMax ELISA reader (Molecular Devices, Sunnyvale, CA, USA). Cell viability data were analyzed according to the PrestoBlue™ Cell Viability Reagent protocol (Thermo Fisher, Waltham, MA, USA).
Statistical analysis. The data are presented as mean±standard error of the mean (SEM) of at least 3 separate experiments. Statistical significance of differences between control and treatment groups was assessed using one-way or two-way ANOVA followed by Tukey multiple comparisons test, conducted with GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA). A significance level of p<0.05 was applied.
Results
Cordycepin decreased cell viability in FGF9-treated TM3 cells. The impact of cordycepin on FGF9-induced cell viability in TM3 cells was initially assessed using MTT assay. Following an 18-h starvation period, TM3 cells were seeded in 96-well plates and treated with cordycepin (0, 25, 50, and 100 μM) alone or in combination with 50 ng/ml FGF9 for 12 and 24 h. At the same time, morphological changes in cells pre- and post-treatment were observed under light microscopy.
Results revealed that cordycepin alone (25, 50, and 100 μM) significantly decreased TM3 cell viability in a dose-dependent manner at both 12 (Figure 1A) and 24 (Figure 1B) h (p<0.05). While treatment with 50 ng/ml FGF9 alone increased cell viability at both time points (p<0.05), the addition of cordycepin (25, 50, and 100 μM) alongside FGF9 also significantly reduced TM3 cell viability in a dose-dependent manner at 12 (Figure 1A) and 24 (Figure 1B) h (p<0.05).
Effects of cordycepin on cell proliferation and morphology on FGF9-treated TM3 cells. MTT assay for the viability of FGF9-treated TM3 cells. After 18 h of serum starvation, TM3 cells were co-treated with or without 50 ng/ml FGF9 and 0, 25, 50, 100 μM cordycepin for 12 (A) and 24 h (B). Examination of morphology and cell growth were observed under light microscopy and cells were co-treated with or without 50 ng/ml FGF9 plus 0, 25, 50, and 100 μM cordycepin for 12 (C) and 24 h (D). All values are represented as the mean±standard error of the mean (SEM), n=3. p-Values were calculated using one-way ANOVA followed by Tukey’s multiple comparisons test. Different letters above the bars represent statistical differences among different treatments; p<0.05. Yellow arrowheads in (C) and (D) indicate apoptotic cells with rounded up phenomena.
Morphologically, TM3 cells exhibited a rounded-up phenotype upon treatment with 25, 50, and 100 μM cordycepin alone at 12 (Figure 1C) and 24 (Figure 1D) h. With the presence of 50 ng/ml FGF9, only 50 and 100 μM cordycepin induced this rounded-up morphology, suggesting that cordycepin decreased TM3 cell viability, potentially through cell death pathways, under FGF9 treatment.
Cordycepin decreased colony formation in FGF9-treated TM3 cells. The colony formation assay, indicative of a single cell’s ability to proliferate into a cell colony, was employed to further evaluate TM3 cell viability and proliferation under cordycepin treatment with or without FGF9. TM3 cells were seeded in 6-cm culture dishes and treated with cordycepin (0, 25, 50, and 100 μM) alone or in combination with 50 ng/ml FGF9 for 8-14 days to allow colony formation.
Data revealed that cordycepin alone (25, 50, and 100 μM) significantly suppressed TM3 cell proliferation in a dose-dependent manner at both 12 (Figure 2A) and 24 h (Figure 2B) (p<0.05). While 50 ng/ml FGF9 alone did not significantly enhance cell proliferation at either time point (p>0.05), its combination with cordycepin (25, 50, and 100 μM) led to a significant reduction in TM3 cell proliferation in a dose-dependent manner (Figure 2A and B) (p<0.05). Notably, 50 ng/ml FGF9 significantly attenuated TM3 cell proliferation under cordycepin treatment at 12 h (Figure 2A) and partially at 24 h (Figure 2B) (p<0.05), suggesting that cordycepin decreased TM3 cell colony-forming ability, potentially through impairing cell proliferation pathways, under FGF9 treatment.
Effects of cordycepin on colony formation on FGF9-treated TM3 cells. After co-treatment with 0 or 50 ng/ml FGF9 plus 0, 25, 50, and 100 of μM cordycepin for 12 and 24 h, TM3 cells (A and B) were trypsinized and collected. 1,500 cells were then replated in 6-cm culture dishes to allow for colony development. After 8-14 days, colonies were stained by 0.5 % crystal violet solution. The number of colonies between different treatments were then counted and were analyzed by two-way ANOVA with Tukey’s multiple comparisons test (C and D). All values are represented as the mean±standard error of the mean (SEM), n=3 for 12 and 24 h. *p<0.05, **p<0.01 and ***p<0.001 vs. control group, ##p<0.01 and ###p<0.001 vs. the FGF9 alone group, $p<0.05 and $$p<0.01 vs. combined group.
Cordycepin did not induce cell death through apoptosis in FGF9-treated TM3 cells. To assess the apoptotic effect of cordycepin on FGF9-treated TM3 cells, cells were treated with cordycepin (0, 25, 50, and 100 μM) alone or in combination with 50 ng/ml FGF9 for 12 and 24 h, and apoptotic phenomena were detected using annexin V/PI double staining assay and analyzed by flow cytometry.
Results indicated that cordycepin alone (25, 50, and 100 μM) did not induce TM3 cell apoptosis at either time point (Figure 3) (p>0.05). Similarly, 50 ng/ml FGF9 alone did not stimulate cell apoptosis (Figure 3) (p>0.05). Furthermore, cordycepin (25, 50, and 100 μM) did not induce TM3 cell apoptosis in the presence of 50 ng/ml FGF9 at both time points (Figure 3) (p>0.05). These findings suggested that cordycepin did not induce TM3 cell apoptosis under FGF9 treatment conditions.
Cordycepin did not induce cell apoptosis in FGF9-treated TM3 cells. After co-treatment with 0 or 50 ng/ml FGF9 plus 0, 25, 50, and 100 μM cordycepin in TM3 cells for 12 (A) and 24 h (B), cells were stained with annexin V and propidium iodide (PI) and analyzed by flow cytometry. All values are represented as the mean±standard error of the mean (SEM), n=3. Two-way ANOVA with Tukey’s multiple comparisons test was used to compare differences between different groups for 12 (C) and 24 h (D). There are no statistical differences between treatments in (C) and (D).
Cordycepin did not induce caspase-dependent cell death in FGF9-treated TM3 cells. The role of the caspase cascade in cordycepin-induced cell death under FGF9 treatment was examined by western blotting. TM3 cells were treated with cordycepin (0, 25, 50, and 100 μM) alone or in combination with 50 ng/ml FGF9 for 12 and 24 h.
Results revealed that cordycepin alone (25, 50, and 100 μM) did not induce activation of caspase cascade Je markers (cleaved caspase-3, 8, 9, and cleaved PARP) compared to control at both time points (Figure 4) (p>0.05). Similarly, 50 ng/ml FGF9 alone did not stimulate caspase cascade activation compared to control at both time points (Figure 4) (p>0.05). Moreover, cordycepin (25, 50, and 100 μM) did not induce caspase cascade activation in the presence of 50 ng/ml FGF9 at both time points (Figure 4) (p>0.05). These results suggest that cordycepin did not stimulate the caspase cascade to induce TM3 cell apoptosis under FGF9 treatment conditions.
Cordycepin did not induce apoptosis-related protein expression on FGF9-treated TM3 cells. Western blot analysis for the expression of cleaved caspase-8, cleaved caspase-9, cleaved caspase-3 and cleaved PARP in TM3 cells co-treated with or without 50 ng/ml FGF9 plus 0, 25, 50, and 100 μM cordycepin for 12 (A) and 24 (B) h. All values are represented as the mean±standard error of the mean (SEM), n=3. Two-way ANOVA with Tukey’s multiple comparisons test was used to compare differences between different groups. P, positive control.
Cordycepin induced cell death through autophagy in FGF9-treated TM3 cells. Given the lack of significant apoptotic events induced by cordycepin under FGF9 treatment (Figure 3 and Figure 4), the mechanisms of how cordycepin regulated FGF9-induced TM3 cell death were investigated. TM3 cells were treated with cordycepin (0, 25, 50, and 100 μM) alone or in combination with 50 ng/ml FGF9 for 12 and 24 h, stained with acridine orange (AO) to detect acidic vesicular organelles (AVOs), and analyzed by flow cytometry.
Results demonstrated that cordycepin significantly stimulated the expression of AVOs in a dose-dependent manner at both time points (Figure 5) (p<0.05). Although 50 ng/ml FGF9 alone did not increase AVO expression significantly (p>0.05), 100 μM cordycepin still significantly stimulated AVO expression at 12 h (Figure 5A) (p<0.05), but not at 24 h (Figure 5B) (p>0.05) in the presence of 50 ng/ml FGF9 in TM3 cells. Additionally, the expression of LC3 puncta, a marker of autophagy, was significantly stimulated by 50 and 100 μM cordycepin alone at 12 h (Figure 6A) and by 100 μM cordycepin alone at 24 h (Figure 6B) (p<0.05). These findings indicate that cordycepin induced autophagy even in the presence of FGF9 in TM3 cell death.
Cordycepin induced autophagy in FGF9-treated TM3 cells. After co-treatment with 0 or 50 ng/ml FGF9 plus 0, 25, 50, and 100 μM cordycepin for 12 and 24 h, TM3 cells were stained with Acridine orange (AO) for acidic vesicular organelles (AVOs) through flow cytometric analysis and analyzed (A and B). Quantification and analysis of histograms upon autophagy cells from flow cytometric analysis are illustrated at 12 (C) and 24 h (D). All values are represented as the mean±standard error of the mean (SEM), n=3. Two-way ANOVA with Tukey’s multiple comparisons test were used to compare differences between different groups. Different letters above bars represent statistical differences among different treatments; p<0.05.
Cordycepin induced autophagy in FGF9-treated TM3 cells. After co-treatment with 0 or 50 ng/ml FGF9 plus 0, 25, 50, and 100 μM cordycepin for 12 and 24 h, LC3 puncta were detected by immunofluorescent staining. Images were analyzed for bright objects using the analyze particles tool in ImageJ (A and B). Histograms of quantification of autophagic cells upon LC3 puncta immunofluorescent staining are illustrated at 12 (A) and 24 h (B). All values are represented as the mean±standard error of the mean (SEM), n=3. Two-way ANOVA with Tukey’s multiple comparisons test were used to compare differences between different groups. Different letters above bars represent statistical differences among different treatments; p<0.05. Scale bars are equal to 10 μm.
Cordycepin induced autophagy protein expression in FGF9-treated TM3 cells. Under stressful conditions, cells initiate autophagy as a protective mechanism (21, 40). However, excessive autophagy can lead to cell death (41, 42). To elucidate the role of autophagy in TM3 cells under the influence of cordycepin and/or FGF9, the expression levels of Atg5-12, beclin-1 and LC3-II were examined by western blotting assay.
Results showed that no significant differences were observed in the expression levels of Atg5-12 (Figure 7) and beclin-1 (Figure 8) at 12 and 24 h among the different treatments with cordycepin (0, 25, 50, and 100 μM) alone or in combination with 50 ng/ml FGF9 (p>0.05).
Cordycepin didn’t induce the expression of Atg5-12 on FGF9-treated TM3 cells. TM3 cells were co-treated with 0 or 50 ng/ml FGF9 plus 0, 25, 50, and 100 μM cordycepin for 12 and 24 h, respectively. Western blot analyses for the expression of Atg5-12 for 12 and 24 h, in TM3 cells were examined (A and B). Results in (C) and (D) for 12- and 24-h treatments are shown as mean±standard error of the mean (SEM) with at least 3 independent experiments. Two-way ANOVA with Tukey’s multiple comparisons tests was used to determine differences among different treatments. There are no statistical differences between treatments in (C) and (D).
Cordycepin did not induce the expression of beclin-1 on FGF9-treated TM3 cells. TM3 cells were co-treated with 0 or 50 ng/ml FGF9 plus 0, 25, 50, and 100 μM cordycepin for 12 and 24 h. Western blot analyses for the expression of beclin-1 for 12 and 24 h in TM3 cells were examined (A and B). Results in (C) and (D) for 12- and 24-h treatments are shown as mean±standard error of the mean (SEM) with at least 3 independent experiments. Two-way ANOVA with Tukey’s multiple comparisons tests was used to determine differences among different treatments. There are no statistical differences between treatments in (C) and (D).
However, treatment with 50 and 100 μM cordycepin alone for 12 h (Figure 9A and C) and 25, 50, and 100 μM cordycepin alone for 24 h (Figure 9B and D) significantly increased the LC3-II/I ratio compared to the control (p<0.05). Notably, at 12 h, 50 ng/ml FGF9 significantly reversed the LC3-II/I ratio compared to the control (Figure 9A and C) (p<0.05), although this reversal was not observed at 24 h (Figure 9B and D) (p>0.05). Moreover, there was no significant difference in the suppression of the LC3-II/I ratio between treatments with cordycepin alone (25, 50, or 100 μM) and cordycepin combined with FGF9 at 12 h (p>0.05). However, at 24 h, 50 ng/ml FGF9 significantly reversed the LC3-II/I ratio in the presence of 25, 50, and 100 μM cordycepin (Figure 9B and D). Notably, even at 24 h, 100 μM cordycepin still significantly increased the LC3-II/I ratio compared to 50 ng/ml FGF9 alone, as well as combinations of 50 ng/ml FGF9 with 25 μM and 50 μM cordycepin, indicating that cordycepin could induce autophagy-mediated TM3 cell death in the presence of FGF9.
Cordycepin induced the expression of LC3-I/II on FGF9-treated TM3 cells. TM3 cells were co-treated with 0 or 50 ng/ml FGF9 plus 0, 25, 50, and 100 μM cordycepin for 12 and 24 h. Western blot analyses for the expression of LC3-I/II for 12 and 24 h in TM3 cells were examined (A and B). Results in (C) and (D) for 12- and 24-h treatments are shown as mean±standard error of the mean (SEM) with at least 3 independent experiments. Two-way ANOVA with Tukey’s multiple comparisons tests were used to determine differences among different treatments. Different alphabetical letters above bars represent statistical differences among different treatments; p<0.05.
To further confirm the role of cordycepin-induced autophagy in FGF9-treated TM3 cells, an autophagy inhibitor, chloroquine (CQ), which blocks the binding of autophagosomes to lysosomes (43), was employed. TM3 cells were pre-treated with CQ (100 μM) for 1 h and then subjected to treatment with FGF9 (0 and 50 ng/ml) with or without cordycepin (50 μM) for 24 h. The data revealed that 50 ng/ml FGF9 significantly enhanced TM3 cell viability, while 50 μM cordycepin decreased TM3 cell viability, both with and without FGF9 (p<0.05) (Figure 10). Interestingly, CQ augmented the inhibitory effect of cordycepin, further decreasing FGF9-induced TM3 cell viability, even in the absence of FGF9 (Figure 10). Additionally, CQ alone reduced TM3 cell viability, albeit with a lesser effect compared to its combination with FGF9 (Figure 10). These findings suggest that autophagy induction plays an essential role in cordycepin-induced TM3 cell death in the presence of FGF9.
Autophagy inhibitor rescued cordycepin-induced cell death on FGF9-treated TM3 cells. TM3 cells (6.5×103 cells per well) were seeded in 96 well plates with 100 μl of culture medium and pretreated with 100 μM CQ (autophagy inhibitor) for 1 h. Then, TM3 cells were co-treated with or without 50 ng/ml FGF9 and 50 μM cordycepin for 24 h. The PrestoBlue assay was then used to assess cell viability. All values are represented as the mean±standard error of the mean (SEM), n=3. Two-way ANOVA with Tukey’s multiple comparisons test was used to compare differences between different groups. Different letters above bars represent statistical differences among different treatments; p<0.05.
Discussion
FGF9 plays pivotal roles in embryonic development, tissue repair and sex determination (1, 7, 9, 10). It is known to enhance the survival and migratory capabilities of various cell types (11, 12, 44). Conversely, cordycepin, a multifaceted compound, exhibits diverse physiological functions (15, 16). Studies have demonstrated its ability to induce apoptosis in tumor cells across different cancers (19, 21, 22) and to trigger cell death via autophagy in various cell types (23, 24).
In the present study, FGF9 stimulated TM3 cell proliferation, while cordycepin suppressed TM3 cell viability in the presence of FGF9 (Figure 1). Interestingly, the reduction in cell viability by cordycepin was less pronounced in TM3 cells (0.7-0.8-fold) compared to MA-10 Leydig tumor cells (0.4-0.6-fold) (36), suggesting a milder impact of cordycepin on TM3 Leydig progenitor cells. Similar observations have been reported with other compounds, such as isobutyrylshikonin, which exhibited greater cytotoxicity in carcinoma tumor cells than in normal cells (45).
The colony formation assay, a standard method for assessing cell survival and proliferation, revealed that cordycepin decreased colony count in TM3 Leydig progenitor cells with or without FGF9 (Figure 2). Once again, the reduction in cell viability by cordycepin was less pronounced in TM3 cells (0.7-0.8-fold) compared to MA-10 cells (0.4-0.5-fold) (36), highlighting its milder impact on TM3 normal Leydig progenitor cells. Notably, a previous study has shown that cordycepin could decrease colony count in esophageal cancer cells (46), further supporting the versatility of cordycepin in affecting cell survival across different cell types.
Apoptosis, a programmed cell death process, typically involves the activation of the caspase cascade (47, 48). Previous studies have demonstrated that cordycepin induces apoptosis in tumor cells through this pathway (38, 47-49). Surprisingly, in our study, cordycepin did not induce apoptotic features in TM3 cells, as observed through PI/Annexin double staining analyzed by flow cytometry (Figure 3). Additionally, there were no stimulatory effects on the caspase cascade, with or without the presence of FGF9 in TM3 cells (Figure 4). Conversely, cordycepin induced apoptosis in MA-10 cells via caspase cascade activation, consistent with previous findings (36).
Autophagy, a cellular process with dual roles in tumor cell growth promotion and inhibition, can lead to a specific mode of cell death under certain circumstances (23, 41, 50). In our study, cordycepin induced cell death in TM3 cells through autophagy, as evidenced by the upregulated expression of LC3, a marker for autophagosomes (51). Notably, there was no significant change in the expression of Atg5-12 and beclin-1, suggesting a selective activation of autophagy-related proteins. These findings align with previous studies indicating that autophagy can function normally even when Atg5 levels are low, and there is no absolute correlation between the performance of p62 and LC3 (51, 52).
In a previous study, cordycepin did not induce autophagy in MA-10 cells, but an autophagy inhibitor enhanced cordycepin-induced cell death (36). This suggests that MA-10 cells may trigger autophagy to protect themselves, with cordycepin possibly inducing apoptosis at higher doses and longer treatment times. Interestingly, in the present study, cordycepin stimulated autophagy to induce cell death in TM3 cells even in the presence of FGF9. This highlights the differential mechanisms of cell death induction by cordycepin between MA-10 Leydig tumor cells and TM3 Leydig progenitor cells.
Conclusion
FGF9 has the potential to significantly promote the proliferation of TM3 mouse Leydig progenitor cells. However, this stimulatory effect of FGF9 can be inhibited by cordycepin, which leads to the death of TM3 cells. Notably, cordycepin induces cell death through the stimulation of autophagy, rather than apoptosis, in the presence of FGF9. These findings suggest that cordycepin should be used with caution in cancer therapy, as it may also harm normal progenitor cells.
Acknowledgements
The Authors are grateful for the support from the Core Research Laboratory, College of Medicine, National Cheng Kung University, Tainan, Taiwan, Republic of China. This work was supported by grants from National Science and Technology Council, Taiwan, Republic of China (CLFHR11304 to SZW, MOST 110-2320-B-039-079 to LCC, and MOST 110-2320-B-006-025 and NSTC 113-2320-B-006-041 to BMH).
Footnotes
↵* These Authors contributed equally to this study.
Conflicts of Interest
The Authors declare no conflicts of interest.
Authors’ Contributions
The present study was designed by SZW, LCC, YYL and BMH. Experiments were performed by SZW and CYC. Raw data authenticity was confirmed by LCC, YYL and BMH. Data were analyzed by SZW and CYC. Results were interpreted by SZW, CYC and LCC. The initial manuscript was drafted by SZW, CYC, YYL and LCC. The manuscript was revised for important intellectual content by SZW, CYC, YYL, LCC and BMH. The final manuscript was read and approved, ensuring accuracy and integrity of the work by all Authors.
- Received June 20, 2024.
- Revision received August 10, 2024.
- Accepted August 17, 2024.
- Copyright © 2024 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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