Mechanisms of abemaciclib, a CDK4/6 inhibitor, induced apoptotic cell death in prostate cancer cells in vitro

Highlights • ABE treatment may be a promising treatment strategy in especially mCRPC.• ABE treatment significantly inhibited PC cell growth through apoptosis, depolarization of mitochondria membrane potential, the generation of ROS 2. ABE treatment induced depolarization of mitochondria membrane potential.• ABE caused many cytoplasmic vacuoles in PC cells and these effects were more profound in LNCaP cells than PC-3 cells.


Introduction
Prostate cancer (PC) is the second leading cause of cancer-associated death among men within an estimated approximately 248.530 new cases in the United States for 2021 and the second rate of incidence worldwide [1,2]. The 5-year survival rate is almost 100% in patients with local or regional PC. However, this survival rate is 30% for metastatic PC (mPC) patients when spread to other parts of the body. For PC patients, the androgen receptor (AR) deprivation therapy (ADT) is the primary therapy due to oncogenic features of AR causing cell growth, proliferation and metastasis. Not surprisingly, most PC patients will acquire resistance to ADT with a recurrence of 3-4 years and defined as castration-resistant prostate cancer (CRPC). Different FDA-approved therapies (docetaxel, abiraterone, enzalutamide, cabazitaxel, radioisotope treatment and Poly (ADP-ribose) polymerase inhibitors) have been used for the treatment of metastatic CRPC patients in the clinic. Unfortunately, the median survival rate of CRPC patients is nearly 35 months and therefore new treatment strategies are urgently needed for improving the overall survival rate and quality of life [3][4][5][6].
Cyclin-dependent kinases (CDK) 4/6 inhibitors (CDK4/6i) have been gained attention in recent years due to promising efficacy in the treatment of hormone receptor (HR) positive breast cancer patients and increased median progression-free survival (mPFS) and overall survival. Estrogen receptors (ER) and androgen receptors (AR) regulate the cell cycle process from G1 to S phase transition through regulatory proteins. After induction of growth factor, Cyclin D binds to CDK4/CDK6 and phosphorylates retinoblastoma (Rb). Following hyperphosphorylation of Rb, E2 transcription factor (E2F) is activated and provides the cells into the S phase for DNA replication. Therefore, three CDK4/6i (palbociclib, abemaciclib (ABE) and ribociclib) are FDA approved and inhibit the activation of cyclin D-CDK4/6 complex in the treatment of breast cancer [7][8][9]. In PC, AR activates different signaling pathways including Src, Ras/Raf, protein kinase C, AKT/PI3K and mitogen-activated protein kinase (MAPK) pathways causing cell proliferation. The aberrant activation of cyclin D is mediated by particularly the upregulation of MAPK and Akt/mTOR signaling pathways. The amplification of cyclin D is detected in nearly 4.7% of mCRPC patients. The use of ADT affects the transcription of genes regulating the cell cycle and eventually leads to androgen-independent castration resistance in patients [10][11][12]. Therefore, the use of CDK4/6i could be a new therapeutic modality for the inhibition of AR signaling pathways. On the other hand, the significant toxicity of the first generation of CDK4/6i can delay the clinical applicability of these inhibitors in PC treatment.
Some studies have been investigated the therapeutic potential of CDK4/6i in PC [13][14][15]. In one preclinical study, palbociclib inhibits the proliferation of HSPC and CRPC cells, in vitro and in vivo. Furthermore, Stice et al. (2017) state that newly described CDK4/6 inhibitors (G1T28 and G1T38) as an alternative to taxane can potentially decrease hormone-sensitive and resistant prostate cancer cell proliferation, including wild-type AR (VCaP), AR-V7 (22Rv1 and LNCaP), and the activity of these inhibitors depends on Rb status, in vitro and in vivo [13]. Additionally, five phases Ib/II clinical trials (NCT02059213, NCT02905318, NCT02555189, NCT02494921, NCT03706365) are currently in the treatment of mCRPC or mHSPC, RB-positive patients. In these trials, the potential combination of palbociclib, abemaciclib, ribociclib with ADT, enzalutamide, docetaxel, and abiraterone has been investigated in patients [14,15]. In this context, further preclinical and clinical studies are warranted in the treatment of PC patients to identify the appropriate patient population according to genomic alterations and the best synergistic combination to overcome resistance.
Here, we, for the first time, investigated the inhibition of CDK4/6-Cyclin D complex by ABE in both hormone-sensitive PC and resistant mCRPC cell growth, in vitro. In addition, the underlying molecular mechanisms of ABE-induced cell death in PC cancer cell lines were identified at the molecular level.

Cell viability assay
The PC cells were cultured in 96-well plates (2 × 10 4 cells/well) and treated with different concentrations (0.25, 0.5, 1, 1.5 and 2 µM) of ABE (Biovision, San Francisco, CA, USA for 24 h. ABE was dissolved in DMSO and stored at − 20 • C. PC-3 and LNCaP cells were incubated in a growth medium without ABE, as a negative control. Cell viability percentages were measured by WST-1 assay according to the manufacturer instructions (Biovision, San Francisco, CA, USA). The absorbance value was measured using an ELISA reader at 450 nm (Allsheng, China). The WST-1 assay was used to determine the most effective treatment dose or doses for further analysis.

Annexin V analysis
To explore the rate of apoptotic cell death induced by ABE, Annexin V analysis was conducted. PC cells were cultured in 6-well plates (5 × 10 5 cells/well) and treated with 1 and 2 µM ABE, selected as the most effective doses according to the WST-1 results. The cells were washed twice with cold phosphate buffer solution (PBS, Gibco, San Francisco, CA, USA) and stained with Annexin V-Dead Cell Assay kit (Millipore, Germany). After staining, the cells were examined with Muse Cell Analyzer (Millipore, Germany).

Acridine orange/propidium iodide (AO/PI) dual staining
To determine the effect of ABE on the morphological changes of PC cells, AO/PI dual staining was performed. The PC cells were cultured in 6-well plates (5 × 10 5 cells/well) and treated with 1 and 2 µM ABE. After treatment, the cells were fixed with 4% paraformaldehyde (PFA) then stained by AO and PI. The stained cells were captured with EVOS FL Cell Imaging System (Thermo Fisher Scientific, Waltham, MA, USA).

Mitochondrial membrane potential assay
To evaluate the changes in mitochondrial transmembrane potential, PC cells were cultured in 6-well plates (5 × 10 5 cells/well) and treated with 1 and 2 µM ABE. After treatment, the cells were collected with centrifugation and washed twice with PBS. The collected pellet was incubated with Muse MitoPotential Kit (Millipore, Germany) for 25 min at 37 • C. After staining, the cells were analyzed with Muse Cell Analyzer (Millipore, Germany).

Intracellular reactive oxygen species (ROS) determination assay
To determine the ROS level induced by ABE, oxidative stress assay was performed. The PC cells were cultured in 6-well plates (5 × 10 5 cells/well) and treated with 1 and 2 µM ABE. After treatment, the cells were collected with centrifugation and incubated with Muse Oxidative Stress Assay Buffer (Millipore, Germany) for 30 min at 37 • C. After incubation, the cells were analyzed with Muse Cell Analyzer (Millipore, Germany).

Western blot analysis
To further analyze the protein expression level of pRB and CCDN1 genes, protein isolation and western blot analysis were conducted. Total protein was isolated with RIPA lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) and concentrations of isolated proteins were measured with Qubit 4 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). For blotting, isolated protein lysates were separated at 12% SDS-PAGE and transferred to the nitrocellulose membranes (Bio-Rad, San Diego, CA). After separation, the membranes were blocked in nonfat milk in tris-buffered saline with Tween 20 (TBS-T). After blocking, Anti-p-Rb (sc-377,539), Anti-CCDN1 (sc-8396) and Anti-β-Actin (sc-47,778) (Santa Cruz Biotechnology, CA, USA) primary antibodies were used for detection and β-Actin was used as an internal control. After primary antibody incubation, the membranes were washed and incubated with horseradish peroxidase-conjugated anti-mouse IgG secondary antibody (Bio-Rad, San Diego, CA). Protein detection was conducted using chemiluminescence detection (Bio-Rad, San Diego, CA) and chemiluminescent bands were captured and analyzed with the detection system (Syngene, USA).

Apoptotic protein antibody array analysis
To determine the effects of ABE on the expression levels of apoptosis related proteins in PC cells, a Human Apoptosis Array (Raybiotech, GA, USA) was used. The PC cells were cultured in 6-well plates (5 × 10 5 cells/well) and treated with 2 µM ABE for 24 h. After treatment, the proteins were extracted from the collected pellet and apoptosis array protocol was performed according to the manufacturer instructions. Finally, protein detection was conducted using a chemiluminescence detection system (Syngene, USA).

Statistical analysis
Obtained results were statistically analyzed by using SPSS 22.0 (SPSS Inc., Chicago, IL, USA) and presented as means ± standard deviation (SD). Significant differences were determined using One-way analysis of variance (ANOVA) followed by Tukey's test. The web-based statistical analysis software (https://www.qiagen.com/tr/shop/genes-and-pathw ays/ data-analysis-center-overview-page/other-real-time-pcrprobes-orprimers-data-analysis-center/) was used to determine the relative expression of the selected genes. p<0.05 was considered statistically significant.

Effects of ABE on the cell viability and apoptosis in PC cells
To evaluate the viability of PC cells following treatment with 0.1, 0.25, 0.5, 1 and 2 µM ABE for 24 and 48 h, WST-1 analysis was conducted ( Fig. 1A and B). A dose-dependent significant decrease was detected in both PC cells for 24 and 48 h (p<0.05; Fig. 1). We determined that ABE could considerably suppress PC-3 cell viability at doses of 1 and 2 μM (56.2 ± 0.2% and 35.1 ± 0.1%, respectively) for 24 h (p<0.01, Fig. 1A). Additionally, LNCaP cell viability followed by 1 and 2 μM ABE treatment showed a dose-dependent significant decrease (59.5 To investigate the ABE-induced cell death, we conducted the Annexin-V and dead cell assay ( Fig. 1C and D). Our results showed that 1 and 2 μM of ABE treatment markedly increased the percentage of early apoptotic cells (62.3 ± 0.6% and 75.5 ± 2.2%, respectively) in PC-3 cells compared to the control (p<0.01). On the other hand, ABE treatment significantly increased both the percentage of early and late apoptotic cells in a dose-dependent manner in LNCaP cells (p<0.01, Fig. 1D). In 2 μM of ABE treated group, the percentage of the early and late apoptotic cells increased from 4.03 ± 0.1% and 1.21 ± 0.1% to 40.6 ± 1.5% and 11.3 ± 2.6%, respectively, in LNCaP cells (Fig. 1C and D). Thus, particularly 2 μM ABE treatment could result in significant apoptotic cell death in both PC cells.

Effects of ABE on cell cycle arrest and cell morphology in PC cells
We conducted the cell cycle assay for determining the inhibitory effects of ABE on CCDN1/CDK4/6 complex formation (Fig. 2). Our results indicated that ABE treatment could result in G0/G1 arrest for 24 h in both PC cells. The accumulation of PC-3 cells in the G0/G1 phase significantly increased from 54.4 ± 0.4% to 61.5 ± 0.3% and 58.5 ± 0.4% at 1 and 2 μM of ABE, respectively ( Fig. 2A-B). However, there was no significant difference (from 57.5 ± 0.4% to 57.4 ± 0.4% and 56.5 ± 0.5%) in LNCaP cells at G0/G1 phase following treatments with 1 and 2 μM of ABE, respectively (Fig. 2B).
To further reveal the ABE-induced apoptotic or necrotic cell death, AO/PI dual staining was performed (Fig. 2C). The results demonstrated that ABE treatment exhibited apoptotic morphological changes in PC cells. Following administration of 1 and 2 μM of ABE for 24 h, rounded cells, the formation of nuclear blebbing, and some DNA fragmentation were observed closely related to induction of apoptosis in especially PC-3 cells dose-dependently (Fig. 2C). Furthermore, particularly large cytoplasmic vacuoles were detected in both PC cells following both 1 and 2 μM ABE. However, these vacuoles were more obvious in LNCaP than PC-3 cells at particularly 1 μM ABE.

Effects of ABE on the mitochondrial membrane potential of PC cells
To further evaluate the ABE-induced apoptosis, we conducted the Muse MitoPotential assay (Fig. 3). After treatment with 1 and 2 μM ABE for 24 h, a total depolarized live cell percentage increased (32.1 ± 0.8% and 83.0 ± 0.1%, respectively) in a dose-dependent manner due to the reduction of the mitochondrial membrane potential in PC-3 cells (Fig. 3A-B). In addition, the percentage of total depolarized live cells increased followed by 1 and 2 μM ABE treatment (18.4 ± 0.4% and 64.1 ± 0.2%, respectively) in LNCaP cells. Thus, ABE treatment could result in a change in the mitochondrial membrane potential in PC cells and PC-3 cells were more sensitive to ABE treatment than LNCaP cells.

Effects of ABE on the intracellular level of ROS production in PC cells
Excess ROS levels in cells resulting from oxidative stress, can lead to the activation of cell death processes such as apoptosis. To confirm and support the ABE-induced apoptotic cell death, we performed the ROS determination assay (Fig. 4). After treatment with 1 and 2 μM ABE for 24 h, the intracellular ROS positive cell percentage significantly increased (from 4.7 ± 0.2% to 12.7 ± 0.3% and 39.7 ± 0.6%, respectively) in PC-3 cells (p<0.01, Fig. 4A and B). Furthermore, the intracellular ROS positive cell percentage was 17.9 ± 0.7%, followed by 2 μM ABE treatment compared with control (5.1 ± 0.4%) in LNCaP cells ( Fig. 4A and B). Thus, ABE treatment caused an increase in the intracellular level of ROS in PC cells and the induction of ROS by ABE was higher in PC-3 cells than LNCaP cells inconsistent with the mitochondrial membrane potential assay results.

Effects of ABE on the alteration of gene and protein expression of PC cells
To evaluate the potential mechanism of ABE-induced apoptotic cell death, the mRNA and protein expression levels of pro-apoptotic and anti-apoptotic genes were analyzed by RT-PCR and apoptosis antibody array (Fig. 5A-C). In Fig. 5A, the Bax, Bcl-2, caspase-3 and caspase-8 expression levels considerably increased to 2.1-, 5.6-, 1.8-, and 2.9-fold, respectively at 2 μM ABE in PC-3 cells, whereas only the expression of caspase-8 level was significantly up-regulated following 2 μM ABE treatment in LNCaP cells compared with control (Fig. 5A). To further confirm our findings, 43 apoptosis-related proteins were analyzed. We found that the effects of ABE on apoptosis-related proteins were more profound in PC-3 cells than LNCaP cells. ABE treatment resulted in the upregulation of pro-apoptotic proteins including Bid, Bim, Caspase-3 and Caspase-8 expressions and cell cycle regulatory proteins (p53/ p21/p27) and high temperature requirement protein A2 (HTRA2) in particularly PC-3 cells compared to the control group. (Fig. 5B and C). Additionally, the expression level of several inhibitors of apoptosis proteins (cIAP-2, XIAP, HSP60 and survivin) was down-regulated in ABE treated PC cells.
Additionally, we also explored the potential mechanism of ABEinduced G0/G1 arrest, the mRNA and protein expression levels of CCDN1 (Cyclin D) and Rb1 were analyzed by RT-PCR and western blot analysis ( Fig. 5D and E). According to our results, ABE treatment suppressed the mRNA and protein levels of CCDN1 in PC cells. Furthermore, the mRNA level of Rb1 was significantly up-regulated by 1.6-and 2.0fold at 1 and 2 μM ABE, respectively in PC-3 cells (p<0.001). In Fig. 5E, the expression level phospho Rb (pRb) protein was downregulated by ABE in a dose-dependent manner in PC cells due to G0/ G1 arrest induction.

Discussion
The inhibition of hyper-activated Cyclin D/CDK4/6 complex by CDK4/6i has been shown promising results in the treatment of hormonesensitive breast cancer patients. Due to the similar action of AR and ER in cell growth, we focus on the effects of ABE as a CDK4/6i on PC. Here, for the first time, we compared the efficacy of ABE in AR-PC-3 mCRPC and AR+ mutant LNCaP PC cells. Our preliminary findings showed that ABE potentially suppressed the proliferation of PC cells through apoptosis. However, the response of PC cells to ABE was different. The anti-cancer effects of ABE could be influenced by AR status and molecular features.
The limited number of preclinical studies have addressed the therapeutic potential of CDK4/6i in PC [13][14][15]. In these studies, palbociclib and two new CDK4/6i, G1T28 and G1T38, effectively suppress the proliferation of PC cell lines [wild-type AR (VCaP), AR-V7 (22Rv1 and LNCaP)] and this effect is only mediated by Rb status rather than AR expression. However, we found that ABE was more effective in PC-3 cells than LNCaP cells in this study. The effects of AR on Cyclin D are regulated by the activation of MAPK, Akt and mammalian target of rapamycin (mTOR). Furthermore, the over-expression of androgens can result in the down-regulation of CDK inhibitors (p21, p27 and p16). These changes lead to the inactivation of Rb and transition from G1 to S phase in PC. Therefore, ADT treatment promotes the transition from AR-sensitive PC cells to AR-mutated PC cells through the aberrant activation of these pathways. At this stage, CDK4/6i could be an alternative therapeutic strategy to overcome resistance regulating by aberrant expression of AR [10,11,[16][17][18][19]. On the other hand, palbociclib treatment exerts limited efficacy in hormone-sensitive and CRPC cells in vitro, in vivo and ex vivo, in the previous studies [11,20]. These results could be associated with some genomic alterations including the loss of Rb, higher expression of p16INKa and the amplification of cycle E1/E2 or E2F leading to CDK4/6i resistance [11,21]. Among three CDK4/6i, ABE exerts a higher selectivity for inhibiting the CDK4/cyclin D complex than palbociclib and ribociclib [22]. In our study, ABE exhibited significant anti-cancer activity in AR-mCRPC cells. Therefore, further studies should focus on the effects of new generation CDK4/6i on AR+ wild-type, AR mutant and AR-PC cells.
Furthermore, ABE treatment caused particularly early apoptosis in PC-3 cells through a significant decrease in the mitochondrial membrane potential, increased ROS production, and the over-expression of proapoptotic genes. Interestingly, the inhibition of the cell cycle by ABE would be expected to more promote G0/G1 arrest in PC cells. Cdk inhibitors (flavopiridol, UCN-01) lead to G1 arrest by inhibiting Cdk2 and p53-independent induction of p21Waf1/Cip1 and p27Kip1 in cells expressing wild-type Rb [23][24][25]. Cdk2 /Cyclin E plays a crucial role in the initiation of DNA replication [26]. Additionally, p21 and p27 activation inhibits G1/S phase transition when the cells undergo senescence [27]. Furthermore, our findings showed that the expression of cyclin D level was up-regulated upon treatment with ABE in PC cells despite of the down-regulation of protein level. The mechanism of ABE-induced cyclin D gene expression could be associated with an inactive Cdk4/Cyclin D complex, thereby protecting cyclin D from turnover as in the study of Comstock et al. (2013). Therefore, further investigation is needed to explore the molecular mechanism of the cell cycle following treatment with ABE in PC cells.
CDK4/6i induce senescence or apoptosis in cancer cells [28][29][30][31][32][33][34]. In preclinical studies, the efficacy of ABE in different types of cancer (pancreatic ductal adenocarcinomas (PDAC), breast cancer, lung cancer, cervical cancer) has been evaluated [29][30][31][32][33][34]. The study of Dhir et al. (2019) states that 0.5 µM ABE treatment during 3 days can results in the over-expression caspase-3, the induction of early apoptosis and G1 arrest through the down-regulation of p-Rb in PDAC cells. Furthermore, the levels of senescence-associated secretion (SASP) markers are significantly increased supported by β-galactosidase staining after 7 days ABE treatment (29). The inhibition of CDK4/6 causes elevated levels of ROS for senescence induction (28). However, ABE-induced senescence is not mediated by ROS generation in PDAC cells in that study [29]. In the study of Liu et al. (2021), ABE inhibits the proliferation of HPV-negative cervical cancer cells and induces apoptosis via the repression of CDK4/6-Rb-E2F and mTOR pathways (30). In breast cancer, ABE treatment significantly inhibits the proliferation of both hormone-sensitive and triple negative breast cancer (TNBC) through the induction of apoptosis and senescence in vitro and in vivo [33,34]. Furthermore, the inhibition of CDK4 induces translocation of RelA and suppresses NFkB-driven transcription prior to apoptosis in SW480 colon cancer cells [31]. On the other hand, CDK4 inhibitor causes TRAIL-mediated apoptosis through the down-regulation of Survivin in pancreatic cancer cells and suppresses NFkB activation via degradation of IkB [32]. In our study, ABE treatment caused a significant increase in the rate of particularly early apoptosis through the suppression of CDK4/6/Cyclin D complex, the over-expression of caspase-3 level, pro-apoptotic (Bid, Bim) and cell cycle regulatory proteins (p53/p21/p27) and the down-regulation of cIAP-2, XIAP, HSP60 and survivin in PC-3 cells. Therefore, further investigations are required to elucidate the role of senescence and the contribution of apoptosis-associated signaling pathways into ABE induced cell death in PC cells.
On the other hand, the mRNA levels of caspase-3 and caspase-8 were not correlated with the protein expression according to apoptosis antibody array in PC cells as well as Bcl-2 level in PC-3 cells. In the literature, the regulatory process including post-transcriptional, protein synthesis, translational modifications and protein degradation as well as the spatial and temporal variations of mRNAs can affect the relationship between coding transcript and protein levels. Therefore, the dynamic relationship between transcript levels and protein abundance should be better clarified by understanding genotype-phenotype relationships through transcriptomic and proteomic analysis [35][36][37].
Finally, our preliminary results indicated that ABE caused many cytoplasmic vacuoles in PC cells and these effects were more profound in LNCaP cells than PC-3 cells. The study of Hino et al. (2020) states that ABE induces atypical cell death in A549 human non-small cell lung cancer cells and multiple cytoplasmic vacuoles are derived from lysosomes but are not associated with autophagy [38]. Additionally, ABE treatment does not induce apoptosis and/or necrosis in A549 cells compared to previous studies. In our previous study, we observed the same vacuoles in MDA-MB-231 triple negative breast cancer cells upon treatment with ABE and we found that these vacuoles did not derive from autophagy. Therefore, lysosomal vacuolation could be related to the cytocidal effect of ABE as in the study of Hino et al. (2020). However, further studies should be performed to identify the interaction of apoptosis and atypical cell death with ABE treatment and the underlying molecular mechanism of these death types in different cancer types.
Our results demonstrated for the first time that ABE treatment significantly inhibited PC cell growth through apoptosis, depolarization of mitochondria membrane potential, the generation of ROS and the over-expression of pro-apoptotic and caspase protein levels. Therefore, further preclinical and clinical investigations are warranted to reveal the potential clinical use of ABE for the treatment of PC. In addition, the combination therapy of ABE with chemotherapy drugs may offer a promising advantage, particularly mCRPC therapy. Thus, further investigations will provide additional insight into the clinical use of CDK4/6i for the treatment of both hormone-sensitive and metastatic castration resistant PC.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.