Incaspitolide A extracted from Carpesium cernuum induces apoptosis in vitro via the PI3K/AKT pathway in benign prostatic hyperplasia

Abstract Benign prostatic hyperplasia (BPH) is a common disease that occurs mainly in older men. The pathogenesis of BPH is complex and patients face a prolonged treatment course, and novel drugs with better selectivity and lower toxicity are required. Incaspitolide A (compound TMJ-12) is a germacrane-type sesquiterpenoid compound extracted from the plant Carpesium carnuum. Extracts of C. carnuum are known to exert suppressive effects on BPH-1 cells. In the present study, we investigated the molecular mechanisms underlying the suppressive effect of TMJ-12 specifically on BPH-1 cells. A cytotoxicity assay indicated that TMJ-12 inhibited BPH-1 cell proliferation, while flow cytometry assays showed that TMJ-12 induced G2/M phase cell cycle arrest and the apoptosis of BPH-1 cells. TMJ-12 was also shown to regulate the expression of several apoptosis- and cell cycle-related proteins, namely Bcl-2, Bax, Bad, Caspase-9, Caspase-3, cyclin-dependent kinase 1 (CDK1), Cyclin B1, CDC25C, and c-Myc, among others. Collapse of the mitochondrial membrane potential (ΔΨm) following exposure to TMJ-12 was detected with the JC-1 staining assay. Further investigation revealed that treatment with TMJ-12 inhibited the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway by increasing the expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Taken together, the results suggest that TMJ-12 prevents BPH-1 cell proliferation via the PI3K/AKT pathway by inducing apoptosis and cell cycle arrest.


Introduction
Benign prostatic hyperplasia (BPH) is a non-malignant enlargement of the prostate [1]. It is a common disease among older men, with up to 80% reported to have some degree of prostate enlargement by the age of 50 years [2]. The symptoms of BPH can be obstructive or irritative or may only affect the patient after micturition. Current treatments for BPH include surgery to resect the obstructive prostate tissue and minimize disease progression, or medications such as the 5-alpha reductase inhibitors, epristeride and finasteride, which blocks the conversion of testosterone to DHT, inhibiting prostatic hyperplasia, reducing prostate size, and slowing disease progression [3]. However, since most BPH patients are elderly, most of them may have serious cardiovascular and cerebrovascular complications and cannot be treated with surgery [4]. Many men are plagued with abhorrent side effects such as erectile dysfunction, ejaculatory problem and a decrease in libido. Of note, high post void residuals, dry mouth, constipation, and the risk of dementia limits their long-term use in the elderly [5,6]. While Natural molecule compounds have milder effects and less adverse reactions, so they are suitable for long-term use [7]. BPH can severely affect the health and well being of patients, and novel drugs with better selectivity and lower toxicity are required. Drugs for the treatment of BPH mainly act by inhibiting prostate cell proliferation.
The phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway is crucial to many aspects of cell growth and survival, and the abnormal activation of the PI3K/AKT signaling pathway is associated with the occurrence and development of oncogenic effects [8]. PI3K is a heterodimer consisting of a catalytic subunit (p110) and a regulatory subunit (p85). Upon activation, PI3Ks phosphorylate phosphatidylinositol 4,5-bisphosphate [PtdIns (4,5)P2] to produce PtdIns(3,4,5)P3 [9]. AKT (protein kinase B) is the primary downstream mediator of the effects of PI3K. AKT is activated by phosphorylation at Thr308 by 3-phosphoinositide-dependent protein kinase 1 (PDK1) Downloaded from http://portlandpress.com/bioscirep/article-pdf/doi/10.1042/BSR20210477/914230/bsr-2021-0477.pdf by guest on 09 June 2021 Bioscience Reports. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version is available at https://doi.org/10.1042/BSR20210477 and at Ser473 in the hydrophobic C-terminal domain by PDK2. Abnormal levels of AKT can affect the expression of proteins regulating various cell cycle-dependent kinases, such as cyclin-dependent kinase 1 (CDK1) and CDK2, thereby affecting cell cycle regulation and causing cell cycle arrest [10,11]. Phosphorylated AKT can promote the phosphorylation of mTORC1 directly or indirectly. The mTORC1 protein complex activates the downstream eukaryotic initiation factor 4E-binding protein 1 (4EBP1) and ribosomal p70S6 kinase (p70S6K), which participate in cell cycle regulation and cell apoptosis [12,13]. The PI3K/AKT pathway can inactivate the Caspase-9 and Caspase-3 proteins, and at the same time, affect the binding of Bad and Bcl-2, thereby inhibiting cell apoptosis [14]. The phosphatase and tensin homolog deleted on chromosome 10 (PTEN), which can convert PtdIns (3,4,5) P3 to PtdIns (4,5) P2, is a negative regulator of the PI3K/AKT pathway [15,16].
Carpesium cernuum, which belongs to the genus Carpesium (Asteraceae), is traditionally used to treat fevers, colds, bruises, and inflammatory diseases [17]. The constituents of this plant have been previously investigated and shown to contain several sesquiterpenoids with diverse bioactivities such as cytotoxicity, anti-inflammatory, and antifungal activities [18]. In recent years, sesquiterpenoids have served as a major source of lead compounds for the development of drugs targeting tumor cells [19]. In the early stage, when we checked the literature found that the crude drug Carpeium cernuum extract has the effect of inhibiting the BPH1 cells, but the author did not elaborate on its mechanism [20]. Therefore, our group has isolated and extracted the main component of Carpeium cernuum, incaspitolide A, and conducted cytotoxicity experiments and mechanism research. The compound incaspitolide A (termed TMJ-12 in this study, see Figure 1A) is one such sesquiterpenoid isolated from C. cernuum [21]. The purity analysis of TMJ-12 were described as supplementary data 1. In the present study, we investigated the effect of TMJ-12 on a benign prostatic hyperplasia cell line (BPH-1) and found that it induced apoptosis and cell cycle arrest by activating PI3K/AKT signaling.
After a 24 h incubation, the cells were treated with TMJ-12 at various concentrations (2, 4, 8, 12, or 16 μM) and the cells were then collected within the specified time for further analysis.

Cytotoxicity assay
The cytotoxicity of TMJ-12 toward the BPH-1 cell line was measured with the MTT assay according to a protocol previously described [22]. Briefly, cells were seeded in 96-well plates at a density of 1 × 10 4 cells/well, incubated at 37°C for 24 h, and then treated with TMJ-12  Each experiment was performed in triplicate.

Hoechst 33258 staining
Cells were seeded in a 6-well plate at an initial density of 6 × 10 5

Detection of the mitochondrial membrane potential (ΔΨm)
The mitochondrial membrane potential of the cells was measured using the JC-1 assay (Beyotime). BPH-1 cells were seeded in 6-well plates (3 × 10 5 cells/well) and incubated at 37°C  and densitometry analyses of the western blot bands were performed with ImageJ software.

Molecular docking
The protein structure of PTEN (PDB ID: 1D5R) required for docking was obtained from the PDB database (https://www.rcsb.org/). AutoDockTools 1.5.6 software [23] was used to remove the water molecules, perform hydrogenation, and calculate the charge of the protein. The structure was then saved in the pdbqt format. Subsequently, the Autodock Vina [24] program was used for molecular docking with the parameters set at num_modes = 10, energy_range = 4, and exhaustiveness = 100.

Statistical analysis
All data are expressed as the mean ± standard deviation (SD) of at least three independent experiments. Each experiment was repeated in triplicate. Statistical analyses were performed with Student's t-test or one-way analysis of variance. Significance levels were defined as follows: not significant (ns), *P < 0.05; **P < 0.01; ***P < 0.001.

Results
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Effect of TMJ-12 on BPH-1 cell viability
To investigate the effect of TMJ-12 on the viability of the BPH-1 cells, the MTT assay was performed on BPH-1 cells treated with increasing concentrations of TMJ-12 (0-16 μM) for 24, 48, or 72 h ( Figure 1B). TMJ-12 was found to reduce BPH-1 cell viability in both a dose-dependent and time-dependent manner. The IC50 of TMJ-12 toward the BPH-1 cells was 8.02 μM after 72 h of incubation ( Figure 1C). TMJ-12 treatment was also observed to induce noticeable morphological changes in these cells (Supplementary Figure 1). The colony formation assay was used to further assess the effects of TMJ-12 on BPH-1 cell proliferation ( Figure 1D, E). Increasing concentrations of TMJ-12 (0-16 μM) were found to reduce the rate of colony formation of the BPH-1 cells in a dose-dependent manner. To understand the underlying molecular events, we examined numerous cell cycle regulators.

TMJ-12 induces G2/M phase cell cycle arrest of BPH-1 cells
Western blot analyses revealed that the cell cycle family proteins CDK1, CyclinB1, CyclinD1, c-Myc, and the CDC25C were consistently declined in treated with TMJ-12 cells ( Figure 2B). One of most improtent protein was CDC25C, which was critical for the G2 checkpoint. While the P27 kip , P21 cip and P53 were substantially elevated. In addition, proliferating cell nuclear antigen (PCNA), a downstream marker of proliferation, was remarkably downregulated in TMJ-12 treated cells ( Figure 2C). Thus, our result showed that effect of TMJ-12 inhibited BPH-1 cell viability was related to cell cycle arrest.

TMJ-12 induces BPH-1 cell apoptosis via the mitochondrial apoptotic pathway
Apoptosis is a form of programmed cell death and mitochondrial changes are considered to be a hallmark of apoptosis [25]. Here, double-staining with annexin V-FITC and PI was employed  (Figure 4A, B).
The ratio of Bax/Bcl-2 was markedly increased in the TMJ-12-treated cells, and the level of cytochrome C in the mitochondria of these cells also increased.

TMJ-12 inhibits activation of the PI3K/AKT signaling pathway
Several studies have demonstrated that the PI3K/AKT signaling pathway is closely associated with the cell cycle and plays a critical role in controlling cell survival/death. Abnormal activation of the PI3K pathway results in disturbances in the control of cell growth and survival, which can contribute toward cells acquiring a competitive growth advantage, metastatic competence, and, frequently, therapeutic resistance [26].Thus, we next investigated whether TMJ-12 activated the PI3K/AKT pathway.
Western blot analysis of BPH-1 cells indicated that treatment with TMJ-12 reduced the expression levels of PI3K (subunit p110α) and its downstream regulator PDK1, as well as the phosphorylation of AKT at Ser473 (Figure 5A, B). Diminished levels of phosphorylation at Ser2448 of the mTOR protein was also observed. Further investigation indicated that TMJ-12 treatment inhibited the expression of EIF4E, the phosphorylation target of mTOR, whereas the expression of PTEN increased. These data together imply that TMJ-12 may inhibit AKT through the activation of PTEN and the inhibition of PDK1 and PI3K. The expression and phosphorylation levels of MAPK/P38 and SAPK/JNK, as components of the side pathway of PI3K/AKT signaling [27], were also determined, as these play crucial roles in regulating apoptosis ( Figure 5C, D). The results showed that the phosphorylation levels of MAPK/P38 decreased while the protein levels of SAPK/JNK increased in a time-dependent manner.
Finally, unbiased blind docking was used to predict the region involved in binding between TMJ-12 and PTEN. For this purpose, all known DNA-binding domain structures of PTEN as identified by X-ray crystallography (PDB ID: 1D5R) were used. The docking of TMJ-12 with ID5R showed a low binding energy of −9.23 kJ/mol ( Figure 6A, B). Both compounds formed similar van der waals interactions with the residuals including Tyr176, Pro169, Arg173, Tyr180, and Tyr177, and the docking results indicated that TMJ-12 can hydrogen bond to Arg173 of PTEN.
Also, we used Oroxin B which is the activator of PTEN as a positive control [28] (Figure 6 C, D).
In comparison to Oroxin B, TMJ-12 formed more hydrogen bonds with PTEN. These data indicated that TMJ-12 enhanced the activity of PTEN. Thus, the docking analysis findings suggest that TMJ-12 can directly bind with PTEN and may thereby inhibit the PI3K/AKT signaling pathway.

Discussion
Natural products have been a rich source of compounds for drug discovery, and natural products continue to enter clinical trials as anticancer and antimicrobial agents [29]. Natural products exhibit diverse structures that correspond to the diversity of their biological functions and binding partners [30]. Our previous study found that TMJ-12, a germacrane sesquiterpenoid extracted from Carpesium cernuum, inhibits the proliferation of the cervical cancer cell line, HeLa, and the liver cancer cell line, HepG2 [31]. In the present study, TMJ-12 was tested on the benign prostatic hyperplasia cell line, BPH-1.
In this study, we focused on the inhibitory effect of TMJ-12 on the proliferation of BPH-1 cells and explored the mechanisms involved. Both the MTT assay and colony formation assay demonstrated that TMJ-12 has a strong inhibitory effect on the proliferation of BPH-1 cells in a time-and dose-dependent manner. Further, flow cytometry assays showed that TMJ-12 can block the growth of BPH-1 cells in the G2/M phase and promote BPH-1 cell apoptosis. Previous studies have demonstrated that cell apoptosis occurs mainly through the intrinsic mitochondrial pathway and the extrinsic death receptor pathway. The mitochondrial pathway of apoptosis is known to depend on the activation of caspases, which ultimately results in cell death characterized by cellular shrinkage, chromatin condensation, and membrane blebbing [32]. This pathway is triggered by cellular stress or developmental cues that cause mitochondrial damage, leading to the release of Cytochrome C, apoptosis-inducing factor, and procaspase-9 into the cytosol and the formation of a complex termed the apoptosome [33,34]. Our data showed that TMJ-12 likely induces mitochondria-mediated apoptosis in BPH-1 cells, as treatment with TMJ-12 resulted in reduced mitochondrial membrane potential, increased Bax/Bcl-2 expression ratio, increased level of Cytochrome C released into the cytosol, activation of Caspase-9 (an initiator caspase) such that it could enter the cell and form a complex termed the apoptosome, followed by the release of Caspase-3 and cleavage of PARP.
Many anti-tumor agents inhibit cell replication by blocking the cell cycle at specific checkpoints. CDC25C is one of the key proteins regulating cell entry to the G2/M phase and providing positive feedback to regulate cell mitosis, and CDC25C is known to be activated by CDK1 and Cyclin B1 [35,36]. Our study showed a significant decrease in CDC25C, p-CDC25C, CDK2, Cyclin B1, and c-Myc, along with an increase in P53, P21 Cip1 , and P27 Kip1 in BPH-1 cells when treated with TMJ-12. These findings indicated that TMJ-12 may block the growth of BPH-1 cells in the G2/M phase.
The effect of TMJ-12 on the PI3K/AKT signaling pathway was also investigated. The PI3K pathway regulates various cellular processes, such as proliferation, growth, and apoptosis. In this study, we observed that TMJ-12 induced the expression of PTEN and inhibited PI3K/AKT phosphorylation, suggesting that TMJ-12 may be involved in cell cycle arrest and apoptosis ( Figure 7). To further investigate whether TMJ-12 affects the PI3K/AKT pathway by regulating PTEN, molecular docking was applied and TMJ-12 was found to exhibit strong affinity toward PTEN in silico.
In conclusion, the present study demonstrated that TMJ-12, which is a compound extracted from Carpesium cernuum, offers broad-spectrum antitumor activity against the benign prostatic hyperplasia cell line BPH-1. TMJ-12 was shown to promote BPH-1 cell apoptosis and cell cycle arrest at G2/M. In addition, several key factors along the PI3K/AKT pathway were found to Downloaded from http://portlandpress.com/bioscirep/article-pdf/doi/10.1042/BSR20210477/914230/bsr-2021-0477.pdf by guest on 09 June 2021 exhibit significantly altered expression following TMJ-12 treatment, which would have resulted in the increased rates of cell apoptosis observed. Overall, our findings suggest that TMJ-12 may be a promising lead compound for the treatment of benign prostatic hyperplasia.  and G2 phases are shown on the right as the means ± SD, n = 3. (B, C) BPH-1 cells were exposed to TMJ-12 (8 μM or 16 μM) for 24 h, and the protein expression levels of core factors associated with cell cycle progression were measured by western blotting. GAPDH was used as the loading control. Data represent the mean ± SD, n = 3. *P < 0.05, **P < 0.01 vs. the control group; ns, not significant.   Data represent the mean ± SD, n = 3. *P < 0.05, **P < 0.01 vs. the control group.