Flavopiridol induces cell cycle arrest and apoptosis by interfering with CDK1 signaling pathway and inducing excessive reactive oxygen species in human ovarian granulosa cells

doi:10.21203/rs.3.rs-4167568/v1

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Flavopiridol induces cell cycle arrest and apoptosis by interfering with CDK1 signaling pathway and inducing excessive reactive oxygen species in human ovarian granulosa cells

https://doi.org/10.21203/rs.3.rs-4167568/v1

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In recent years, several clinical trials have been conducted to evaluate the use of flavopiridol to treat a variety of cancers, however, it is not clear whether the use of this drug will affect the female reproductive system. Granulosa cells, one of the important cells that constitute the follicle, play a crucial role in determining the reproductive ability of females. In this study, we observed whether different concentrations of flavopiridol have a toxic effect on the growth of immortalized human ovarian granulosa cells. We showed that flavopiridol had an inhibitory effect on cell proliferation at a level of nanomole concentration. Flavopiridol reduced cell proliferation and induced apoptosis by inducing mitochondrial dysfunction and oxidative stress, as well as increasing BAX/BCL2 and pCDK1 levels. These results suggest that reproductive toxicity should be considered when flavopiridol is used to the clinical work.

Human ovarian granulosa cells

Flavopiridol

Oxidative stress

Apoptosis

Cell cycle arrest

The process of oocyte growth, maturation, and ovulation involves the interaction and signal regulation among multiple cell types. Oocyte affects the function and differentiation of granulosa cells, while the latter provide oocytes with the necessary energy and substances (Da Broi et al., 2018, Albertini, 2011, Albertini et al., 2001). Several substances produced by granulosa cells can be used as nutrients to support oocyte growth and maturation (Albertini et al., 2001, Albertini, 2011, Da Silva-Buttkus et al., 2008), while oocytes can secrete GDF9 and BMP15 to regulate the function of surrounding somatic cells and promote the proliferation of granulosa cells by binding to surface receptors, in which gap junctions play an important role in information transmission and substance transport (Turathum et al., 2021, Richani et al., 2021). The interaction between granulosa cells and oocytes jointly regulates the process of follicular growth, maturation, and ovulation.

Cycle-dependent protein kinase is a serine/threonine (Ser/Thr) protein kinase, among them, CDK can be used as a catalytic subunit, while cyclin is a regulatory subunit. They form different Cyclin-CDK complexes and participate in the regulation of cell cycle and transcriptional activity (Zhang et al., 2021). The CDK1, CDK2, CDK4, and CDK6 are mainly involved in the regulation of cell cycle progression, while CDK8, CDK9, CDK12, CDK13, and CDK19 mainly regulate gene transcription (Grison and Atanasoski, 2020). Flavopiridol is a plant-derived flavonoid, originally extracted from a native plant in India, and now it has been synthesized artificially (Soner et al., 2014). A large number of studies have shown that flavopiridol can be used as an inhibitor of CDK1, CDK2, CDK4, CDK6, CDK7, and CDK9, and it regulates a variety of cell functions including its obvious cell cycle arrest effect (Venkataraman et al., 2006). Previous studies have shown that flavopiridol has a good inhibitory effect on lymphoma, leukemia, thyroid cancer, protoplast cell proliferation, and, its anti-proliferative effect in various cancers is mediated by cell cycle arrest, followed by apoptosis (Zhang et al., 2021). Additionally, in clinical trials, flavopiridol also showed some side effects, such as diarrhea and increased transaminase after administration (Hofmeister et al., 2014, Dispenzieri et al., 2006). Therefore, in this study, we explore whether flavopiridol can also cause side effects or toxicity to the female reproductive system.

In addition to the mitotic (M) phase, the cell cycle includes G1, S, and G2 phases. CDK1 plays a major role in controlling the initiation, progression, and termination of cell cycle (Jin et al., 2002). Inhibition of CDK1 activity may prevent cells from entering mitosis because it is the main protein kinase that drives cells through, and cyclinB-CDK1 is the key component of the M-phase promoting factor, which can promote M phase initiation. Inhibition of cyclinB-CDK1 activity leads to G2/M phase arrest, growth inhibition, and apoptosis (Vairapandi et al., 2002). Cell cycle arrest will lead to cell growth inhibition and apoptosis, mitochondrial dysfunction can also trigger apoptosis and autophagy, destroying intracellular ROS homeostasis and DNA damage. The DNA damage response (DDR) mainly includes repairing the non-homologous terminal junction and homologous sufficient pathway of DNA damage when the cell cycle stops, and will continue until the DNA damage is repaired when the cell cycle stops, otherwise, the pathway of cell death is activated by damaged cells (Chang et al., 2014). One of the important biological signals of toxic reactions is ROS. Cellular metabolic dysfunction can be caused by excessive levels of ROS. It is of great significance for cell survival to stimulate cells to produce antioxidant proteins to resist oxidative damage and inhibit ROS balance and maintain homeostasis.

This study aimed to clarify whether flavopiridol has reproductive toxicity in the female reproductive system. Therefore, immortalized human granulosa cell line SVOG was pretreated with different concentrations of flavopiridol in the culture medium, to observe whether it had toxic effects on SVOG cells, and further explored the mechanism of its toxicity to granulosa cells.

2.1. Cell Culture

The SVOG human ovarian granulosa cell line was created by introducing the SV40 large T antigen into primary HGL cells. This cell line is commonly used by researchers to study follicle function and related molecular mechanisms (Chang et al., 2015, Chang et al., 2016). SVOG cells were cultured in vitro in 12-well plates (5000 cells/well) with 1.5 mL of DMEM medium containing high glucose (SH30022, Sangon) and 5% fetal bovine serum (FBS) (26010074, GIBCO), supplemented with 0.1 mg/ml streptomycin and 100 U/ml penicillin (SV30010, Sangon). The cells were cultured at a constant temperature of 37°C and pH 7.

2.2. Drug treatment and cell viability assay

In the previous studies on the effects of flavopiridol on growth inhibition, cell cycle arrest and apoptosis of CD133+/CD44 + prostate cancer stem cells and mantle cell lymphoma, the concentration ranges of 100–500 nM and 20–100 nm were adopted (Soner et al., 2014, Venkataraman et al., 2006), and based on our previous clinical studies evaluating FP's potential to treat cancer, toxicity tests of FP exposed to cells in vitro summarized IC50 to cancer cells at 100–1000 nM (Sedlacek et al., 1996), so the concentration ranges of 25 nm-500 nm were initially selected in this study. Flavopiridol was purchased from MedChemExpress (MCE; HY-1005). Subsequently, the drug concentration was tested by gradient dilutions (final concentrations of 25 nM-500 nM). SVOG cells were cultured in 96-well plates with medium. After 24 hours, the cells were observed for adhesion. Gradient concentrations of flavopiridol (0 nM, 25 nM, 50 nM, 100 nM, 200 nM, 300 nM, 400 nM or 500 nM) were added to the fresh culture medium for 24 hours, ensuring that the final concentration of DMSO did not exceed 0.1%. The cells per well were incubated with 10 uL of Cell Counting Kit-8 reagent (CCK8, Beyotime, C1086) at 37 ℃ for 1 hour, and cell viability was measured by determining the OD value at 450 nm. The IC50 values were calculated using GraphPad Prism 8.

2.3. Apoptosis detection assay

Cell apoptosis was conducted by using the Apoptosis Assays Kit (Beyotime, C1063) and flow cytometry. The apoptosis rate of SVOG cells was assessed with Annexin V-FITC/PI Apoptosis Detection Kit. SVOG cells were incubated in 6-well plates at a density of 1 × 10⁵ cells per well and treated with flavopiridol at concentrations of 0 nM, 50 nM, or 200 nM for 24 hours. After trypsin digestion, 1 × 10⁵ SVOG cells were collected in each experiment and washed twice with pre-cooled PBS.

SVOG cells were suspended in a binding buffer for 15 minutes and then labelled with AnnexinV-FITC and PI at room temperature and in the dark. Following incubation, a binding buffer was added to the cell mixture in proportion to the instructions (Binding buffer, AnnexinV-FITC, and PI). Green (Annexin V-FITC) and red (PI) fluorescence were detected by flow cytometry. The excitation wavelength was 488 nm. Next, after flavopiridol treatment, 5 µl PI was mixed with 400 µl of binding buffer for staining SVOG cells, and incubated for 25 min in darkness, then mixed with 5ul DAPI and 400ul binding buffer for 10 min under the same conditions. After being washed with PBS thrice, the images with the same setting were collected by laser confocal scanning, and the fluorescence intensity was analyzed.

2.4. Intracellular ROS assessment

A reactive oxygen species assay kit (S0033, Beyotime) was used to determine ROS levels. After flavopiridol treatment (0, 50, and 200 nM), the SVOG cells were loaded with 10 mM DCFH-DA and cultured at 37°C for 25 min in darkness. After washing with PBS three times to reduce background intensity, images were collected using laser confocal scanning with the same settings, and fluorescence intensity was analysed.

2.5. Mitochondrial membrane potential (MMP) detection

Cells were seeded in 6-well plates and treated with 0nM and 50 nM,200 nM flavopiridol, respectively for 24 h. The Mitochondrial membrane potential assay kit (Beyotime, C1056) was used by the manufacturer’s protocol. After washing with PBS three times to reduce background intensity, images were collected using laser confocal scanning with the same settings, and fluorescence intensity was analysed.

2.6. Immunofluorescence

12-well plates (5000 cells/well) were used to culture SVOG cells and treated with flavopiridol(0 nM, 50 nM, and 200 nM)for 24h. A solution of 4% paraformaldehyde was applied to the cells after three washes with PBS. Next, Triton X-100 (9002-93-1, Hyclone) permeates for 25 minutes. After blocking with BSA for 1.5h, the cells were incubated with primary antibody γH2AX (1:800;10856-1-AP, Proteintech) at 4 ℃ overnight. The cells were washed with PBS 2 times, and the secondary antibody (A32740, Invitrogen) was added at room temperature for 1h and with DAPI for another 15 min. After washing with PBS three times to reduce background intensity, images were collected using laser confocal scanning with the same settings, and fluorescence intensity was analysed.

2.7. RNA isolation and quantitative real-time PCR (qRT-PCR)

To isolate total RNA from granulosa cells, we used the EZ-10 Spin Column Total RNA Isolation Kit from TransZol and the Vazyme Master Mix to reverse-transcribe the RNA into cDNA. The cycle and amplification conditions were set according to the instructions. The relative gene expression to GAPDH was calculated according to the 2^−△△Ct method (He et al., 2017). The primers are listed in Supplementary Table S1. We selected a set of 4–6 appropriate housekeeping genes, for normalization and quantification purposes. The raw data of qRT-PCR is listed in Supplementary Materials 1 and 2.

2.8. Western blotting

A previously described method was used to extract total proteins from SVOG cells (Liu et al., 2019). Separation of the protein fraction was achieved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), which was then subsequently blotted onto PVDF membranes and blocked with 5% BSA powder dissolved in 1×TBST at room temperature for 1.5 h. The membrane was incubated with the primary antibody overnight at 4°C. BAX (1:2500, Proteintech, China), BCL2 (1:800, Proteintech, China), pCDK1 (1:2000, Cell Signaling Technology, USA), α-Tubulin(1:1500, Proteintech, China),β-actin (1:2000, Proteintech, China), γH2AX (1:800, Proteintech, China), then was incubated with secondary antibodies Goat anti-rabbit lgG H&L(1:5000, Proteintech, China) and Goat anti-mouse lgG H&L(1:5000, Proteintech, China) at room temperature for 1h. Finally, our chemiluminescence reaction is carried out using a high-signal ECL Western Blotting substrate (Tanon, 180–501, Shanghai, China). Protein bands were obtained by using a luminescent imaging system.

2.9. Statistical analysis

Each trial was repeated at least 3 times. Comparison of control and flavopiridol treatment data was conducted by using GraphPad Prism T-test software (San Diego, CA, USA), Results were shown as mean ± standard deviation (SD). An asterisk means important compared with the control group. Significant differences were analyzed by Turkey’s range test. The results were considered statistically significant P < 0.05(*) and extremely significant P < 0.01(**).

3.1. Flavopiridol inhibits SVOG cell viability and proliferation

First, we investigated whether the flavopiridol (ranging from 25 nM to 500 nM) culture affected the proliferation of SVOG cells. By light microscopy, SVOG cells exposed to flavopiridol are shown in Fig. 1A. With increasing flavopiridol concentrations, the floating SVOG cells were increased while adherent cells were decreased compared with the control (Fig. 1A).

We treated SVOG cells with different concentrations of flavopiridol and performed CCK8 experiments. The results showed that flavopiridol prominently inhibited SVOG cell growth and viability in a dose-dependent manner (Fig. 1B), and the IC50 value of flavopiridol on SVOG cells at 24 h was 50 nM. When the concentration was in the range of 25–100 nM, the viability of SVOG cells was slightly inhibited, and the inhibition became more obvious with the increase of concentration (P < 0.01, Fig. 1B). Treatment concentrations above 300nM/mL caused up to 50% of cell death, consequently, the concentrations at 50 nM and 200 nM were used to explore the effect of flavopiridol on SVOG cells.

3.2. Flavopiridol exposure increases apoptosis of SVOG cells

Cell death can take a variety of forms, one of which is apoptosis. The typical images of flow cytometry detection of cell apoptosis by Annexin V/PI staining after SVOG cells in different concentrations of flavopiridol for 24 h. (Fig. 2A). It can sort cells into necrotic cells (upper left, Annexin V- and PI+), apoptotic cells (lower right, Annexin V+, and PI-), normal cells (lower left, Annexin V- and PI-), and late apoptotic or necrotic cells (upper right, Annexin V+, and PI+). Flow cytometry showed that the percentages of early apoptosis cells (Fig. 2B, P < 0.05) and late apoptosis or necrosis cells (Fig. 2B, P < 0.01) in 200nM flavopiridol treatment group was significantly increased compared with the control group.

Also, DAPI was co-stained with PI to further detect apoptosis and necrosis of SVOG cells (Fig. 2C). The relative fluorescence intensity in 200 nM group were significantly enhanced compared with the control group (Fig. 2D, E, P < 0.01).

In addition, we detected the relative mRNA expression levels of apoptosis-associated genes (BAX/BCL2), and the housekeeping gene was used as the internal control to normalize the results. According to the results, relative mRNA expression was significantly higher in the groups treated with 200nM flavopiridol (Fig. 2F, P < 0.01), and as revealed by Western blotting, the BAX/BCL2 levels in the control group were significantly lower than those in the flavopiridol-treated group (Fig. 2G and H, P < 0.05). In conclusion, these results suggest flavopiridol induces SVOG cell apoptosis.

3.3. Flavopiridol exposure increases ROS levels and damages mitochondria in SVOG cells

We investigated whether flavopiridol affects the level of oxidative stress in SVOG cells. In this experiment, a 12-well plate was incubated with a DCFH-DA probe for 25 minutes in an incubator following treatment with 0 nM, 50 nM, or 200 nM flavopiridol for 24 hours. We assayed the effects of different concentrations of flavopiridol on intracellular ROS levels in SVOG cells using a DCFH-DA probe. We conducted fluorescence intensity analysis of DCFH-DA staining levels using ImageJ software (Fig. 3A). The relative fluorescence intensity in 200 nM group were significantly enhanced compared with the control group (Fig. 3B, P < 0.01). The results suggested that the oxidative stress of SVOG cells increased significantly after flavopiridol treatment.

Next, we explored whether flavopiridol treatment can cause intracellular antioxidant stress response. In the flavopiridol-treated group, qRT-PCR showed remarkably lower levels of expression of genes encoding glutathione peroxidase 1 (GPX1), peroxiredoxin 2 (PRDX2), catalase (CAT) and superoxide dismutase 2 (SOD2) (Fig. 3C, P < 0.01).

In our next work, we showed that the mitochondrial membrane potential of mitochondria is decreased by excessive oxidative stress, which damages their function. JC-1 staining was used to detect the MMP of SVOG cells. Mitochondria with high membrane potential show fluoresce red, and mitochondria with low membrane potential show fluoresce green. The ratio of JC-1 aggregate/monomer in fluorescence of live SVOG cells was recorded to measure mitochondrial membrane potential (Fig. 3D). A significant decrease in SVOG fluorescence ratio was observed in flavopiridol-exposed cells as compared to control cells (Fig. 3F, P < 0.05).

These results revealed that flavopiridol-exposed cells increased oxidative stress and impaired mitochondrial function.

3.4. Flavopiridol exposure increases the DNA damage in SVOG cells

Unrepaired DNA damage can also cause apoptosis, then, by labeling the relative level of expression of γH2AX, it was verified that flavopiridol could induce DNA damage in SVOG cells. (Fig. 4A). In the cells exposed to flavopiridol, we observed a greater relative fluorescence of γH2AX compared to the control culture cells, and the fluorescence signal increased with the increase of flavopiridol concentration. (Fig. 4B, P < 0.05). Western blotting results showed that the expression level of γH2AX protein in the flavopiridol-treated group for 24 h was slightly higher than that in the control group, but there was no significant difference (Figs. 4C and D). This result indicates that flavopiridol could cause DNA damage in SVOG cells.

3.5. Flavopiridol arrests the cell cycle at the G2/M phase in SVOG cells

CDKs, CDK4, CDK6, CDK7, and CDK9 can all be inhibited by flavopiridol, an inhibitor of cyclin-dependent kinases. When CDK1 binds to Cyclin B, CDK1 is activated, and phosphorylated Cyclin B binds CDK1 to initiate cell progression to the M phase.

In order to determine whether flavopiridol exposure affects cell proliferation, we used flow cytometry to monitor the progression of SVOG cells through their cell cycle (Fig. 5A). After culturing for 24h, the percentage of SVOG cells in the G2/M (p < 0.05) phase in the flavopiridol exposure group was significantly increased compared with the control group. Flavopiridol can lead to the arrest of the proliferation of SVOG cells in the G2/M phase, and within a certain concentration range, the higher the concentration of the drug, the stronger the blocking effect (Fig. 5B, P < 0.01). We detected the pCDK1 protein levels after exposure to different concentrations of flavopiridol for 24 h by Western blot. Compared to the control group, flavopiridol treatment significantly increased the level of pCDK1 protein expression (Fig. 5C and D, P < 0.01). After SVOG cells treatment with flavopiridol, the expression of pCDK1 increased, and SVOG was blocked in the G2/M phase, resulting in the increase of the G2/M ratio compared with the control

In recent years, several clinical trials have been conducted on the effects of flavopiridol on the treatment of leukemia, multiple myeloma, sarcoma, and other solid tumors (Pinto et al., 2020, Arguello et al., 1998, Wiernik, 2016, Bose et al., 2017, Hofmeister et al., 2014), but its reproductive toxicity is not known. Granulosa cells constitute an important part of follicles, which determine female reproduction (Srikumar and Padmanabhan, 2016). Studies have shown that somatic follicular cells' health and function reflect oocytes' health and ability, and vice versa. In this study, the human ovarian granulosa cell line (SVOG) was selected to study whether the use of flavopiridol will have a certain effect on the female reproductive system. We demonstrated that flavopiridol could reduce cell proliferation, induce mitochondrial dysfunction and oxidative stress, increase BAX/BCL2 and pCDK1, and induce cell cycle arrest at the G2/M phase in vitro.

Flavopiridol’s anti-tumor effect is usually achieved by blocking cell cycle and inducing apoptosis, and it has a direct inhibitory activity on CDK through competitive inhibition of ATP phosphorylation (Parker et al., 1998). In the treatment of acute myeloid leukemia with flavopiridol, it has been found that it inhibits a variety of CDK and leads to the decrease of MCL1 protein expression, and finally induces cell apoptosis (Boffo et al., 2018, Bose and Grant, 2013). It has also been described in previous literature that flavopiridol is an effective cell cycle pan-inhibitor and may be used as a radiosensitizer for some cancer types, including esophageal and ovarian cancer cell lines. (Kari et al., 2003, Sato et al., 2004). In this study, different concentrations of flavopiridol were added to the culture medium to pretreat SVOG to observe its effect on cell proliferation, cell cycle block, and apoptosis. We first set the concentration range of flavopiridol to 25–500 nm to observe the cell morphology and detect the viability of SVOG cells. Under the inverted microscope, we directly observed the morphological changes, multi-antennae and blackening death of the cells treated with flavopiridol. It had a toxic effect on cells, and with the increase in the concentration of flavopiridol, the number of adherent cells of SVOG cells decreased gradually, and the number of floating cells in the culture medium increased. It is speculated that flavopiridol can inhibit cell proliferation and increase cell death. In this concentration range, CCK8 was used to detect the viability of SVOG cells. The results showed that flavopiridol prominently inhibited SVOG cell growth and viability in a dose-dependent manner, and the IC50 value of flavopiridol on SVOG cells at 24 h was 50 nM. Apoptosis (or programmed cell death) is a physiological event that responds to multiple stimuli. After flow cytometry analysis, we found that the apoptosis rate of SVOG cells treated with flavanol was significantly higher than that of the control group. Also, qRT-PCR indicated that relative mRNA levels of apoptotic genes in the 200nM group were significantly higher, and as a result the BAX/BCL2 level in the control group was significantly lower than that in the flavopiridol-treated group as revealed by Western blotting,. Therefore, these results indicate that flavopiridol could induce apoptosis of SVOG cells.

The production of ROS can cause oxidative eustress and oxidative distress, which is a leading cause of cell apoptosis (Aklima et al., 2021). ROS is a beneficial signal molecule with beneficial physiological functions, and the redox state of cells can be maintained in a steady state through the tight coupling system of antioxidants and antioxidant enzymes (Bhattacharyya et al., 2014), but excessive ROS is harmful and damages normal cell function (Yang et al., 2019, Hardy et al., 2021, Saller et al., 2012, Nickel et al., 2014). The balance of reactive oxygen species is essential for cell survival. There are many self-regulatory mechanisms in cells. When there is excessive oxidative stress, in order to reduce the damage caused by oxidative stress, cells establish an effective antioxidant system to inhibit the excessive accumulation of ROS. (Kroemer et al., 1998, Limon-Pacheco and Gonsebatt, 2009). Mitochondria are the main source of cellular ROS, and they play an important role in follicular developmentCells have various antioxidant defense systems, including glutathione peroxidase (GPX1), superoxide dismutase (SOD), catalase (CAT), and peroxiredoxin-2 (PRDX2) (Bock and Tait, 2020, Lubos et al., 2011). Excessive ROS can cause oxidative stress damage and affect the function of mitochondria (Lee and Song, 2021), decrease the mitochondrial membrane potential and cause DNA damage, and induce cell apoptosis (Limon-Pacheco and Gonsebatt, 2009, Vaccaro et al., 2020). In this study, we also detected the MMP of SVOG using JC-1 staining to evaluate the mitochondrial membrane potential and the damage to the function of SVOG cells. It was found that after flavopiridol treatment, the signal of the green JC-1 monomer was higher than that of the control, while the JC-1 aggregates exhibited lower fluorescence intensity than control, showing the compromised mitochondrial function.

Our results show that the oxidative stress of SVOG cells increased significantly after flavopiridol exposure. We explored whether flavopiridol treatment can cause intracellular antioxidant stress response. According to qRT-PCR results, cells treated with flavopiridol expressed a significantly lower level of some antioxidant genes such as GPX1, PRDX2, CAT, and SOD2. After flavopiridol treatment, the level of oxidative stress and antioxidant stress in SVOG cells was disrupted, and the cells were damaged by oxidative stress.

A complex network of DNA repair and DNA damage signaling pathways known as DNA damage response (DDR) enables cells to repair DNA damage and maintain the genome. (Ciccia and Elledge, 2010). If the cells cannot recover and activate the DNA damage response (DDR), the affected cells will die (Gonfloni, 2010). In this study, we verified the effect of flavopiridol on the DNA damage response of SVOG cells by the relative expression level of marker γH2AX. In comparison with the control group, SVOG cells treated with flavopiridol had a higher γH2AX relative fluorescence signal intensity, and the fluorescence signal increased gradually with the increase of flavopiridol concentration. Western blotting results showed that the expression level of γH2AX protein in the flavopiridol-treated group for 24 h was slightly higher than that in the control group, but there was no significant difference. This result indicates that flavopiridol could cause DNA damage accumulation in SVOG cells.

Cyclin-dependent kinase (CDK), a member of the serine/threonine protein kinase family, is a major cell cycle regulator that binds to cyclins to form cyclin-CDK complexes, which phosphorylates hundreds of substrates and regulates cell cycle progression (Sherr, 1996, Malumbres, 2014, Malumbres and Barbacid, 2001, Zheng, 2022, S, 2022). CDK1, also known as mitotic kinase, can bind to CyclinB1 to form a heterodimer CyclinB1/CDK1 (Malumbres and Barbacid, 2007). When CDK1 binds to CyclinB1, CDK1 is activated, phosphorylates key substrates, and enters the M phase to promote mitosis progression (Salaun et al., 2008, Malumbres and Barbacid, 2007, Schafer, 1998). CDK1/cyclin B complex is a necessary protein kinase for G2/M transition. In this study, the results of cell cycle detection by flow cytometry showed that the ratios of G0/G1 and S phase in the flavopiridol treatment group were lower than in the control group, however, the percentage of SVOG cells in the G2/M phase in the flavopiridol exposure group was significantly increased compared with the control culture. In addition, the flavopiridol-treated groups expressed significantly higher levels of pCDK1 than the control groups, according to Western blot analysis. These results suggest that flavopiridol treatment can increase the expression of pCDK1 and inhibit the activity of CDK1, causing the CyclinB1 / CDK1 complex activity. inhibitionThis subsequently leads to the G2 phase block of SVOG cells, and finally the inhibition of cell proliferation.

In summary, we have shown that flavopiridol, at nanomole concentration, effectively inhibits ovarian granulosa cell proliferation by inducing increased pCDK1 and G2/M arrest. Flavopiridol exposure also increases the level of ROS and oxidative stress, induces mitochondrial dysfunction, increases DNA damage, increased BAX/BCL2, and finally cell apoptosis.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships.

Author Statement

Xiao-Zhen Li: Software, Writing-original draft, Methodology, Software, Data curation, Formal analysis, Visualization; Li-Jia Yang, Wei Song, Xue-Feng Xie: Analyzed the data; Jia-Xin Jiang, Xue Zhang, Chang-Yin Zhou, Ang Li, and Fei Li: Formal analysis, Validation; Shen Yin and Qing-Yuan Sun: resources, manuscript editing; Qing-Yuan Sun: supervision, resources, project administration, funding acquisition, writing - review & editing.

Ethical Statements:

This study was approved by the ethics committee of Guangdong Second Provincial General Hospital (2022-SZ-KY-008-01).

Acknowledgments

The authors thank School of Life Sciences of Qingdao Agricultural University for providing the immortalized human ovarian granulosa cell line.

Funding

This study was supported by the Science and Technology Program of Guangzhou, China (202201020292).

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Flavopiridol induces cell cycle arrest and apoptosis by interfering with CDK1 signaling pathway and inducing excessive reactive oxygen species in human ovarian granulosa cells

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1. Cell Culture

2.2. Drug treatment and cell viability assay

2.3. Apoptosis detection assay

2.4. Intracellular ROS assessment

2.5. Mitochondrial membrane potential (MMP) detection

2.6. Immunofluorescence

2.7. RNA isolation and quantitative real-time PCR (qRT-PCR)

2.8. Western blotting

2.9. Statistical analysis

3. RESULTS

3.1. Flavopiridol inhibits SVOG cell viability and proliferation

3.2. Flavopiridol exposure increases apoptosis of SVOG cells

3.3. Flavopiridol exposure increases ROS levels and damages mitochondria in SVOG cells

3.4. Flavopiridol exposure increases the DNA damage in SVOG cells

3.5. Flavopiridol arrests the cell cycle at the G2/M phase in SVOG cells

4. DISCUSSION

5. Conclusions

Declarations

References

Additional Declarations

Supplementary Files

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