Taraxasterol suppresses the proliferation and tumor growth of androgen-independent prostate cancer cells through the FGFR2-PI3K/AKT signaling pathway

Prostate cancer (PCa) is prevalent among older men and difficult to survive after metastasis. It is urgent to find new drugs and treatments. Several studies show that taraxasterol (TAX) has important anti-inflammatory, anti-oxidative and anti-tumor effects. However, the function and mechanisms of TAX in PCa remain unclear. Here, we found that TAX could significantly suppress the viability and growth of androgen-independent PCa cells and down-regulate the expression of c-Myc and cyclin D1 in vitro. Mechanistically, PI3K/AKT signaling pathway was weakened and the expression of FGFR2 was reduced after TAX treatment in androgen-independent PCa cells. Moreover, TAX evidently inhibited the tumor growth in nude mice and the expression of c-Myc, cyclin D1, p-AKT and FGFR2 were down-regulated in xenograft tumor. These results indicate that TAX suppresses the proliferation of androgen-independent PCa cells via inhibiting the activation of PI3K/AKT signaling pathway and the expression of FGFR2, which means TAX may be a novel anti-tumor agent for later PCa treatment.


Results
TAX suppresses the proliferation of androgen-independent PCa cells.To investigate the optimal treatment concentration of TAX in different androgen-independent PCa cells, DU145 and PC3 cells were treated with varying concentrations of TAX for 48 h to determine the viability of individual groups of cells.The results of CCK-8 assay showed that TAX reduced the viability of androgen-independent PCa cells in a dose-dependent manner and the 50% inhibition concentrations (IC 50 s) of TAX were 56 µM for DU145 cells and 30 μM for PC3 cells (Fig. 1a).Compared with the control cells without TAX treatment, the growth of DU145 and PC3 cells treated with TAX at the IC 50 dose was significantly inhibited (Fig. 1b, c).Moreover, according to the colony formation assay, the treatment of TAX markedly reduced the colony size and number of DU145 (The size of colony ≥ 1.0 mm 2 ) and PC3 (The size of colony ≥ 0.5 mm 2 ) cells (Fig. 1d, e).In addition, the expression of c-Myc and cyclin D1 decreased after TAX treatment at both protein and mRNA levels in DU145 and PC3 cells (Fig. 1f-i).These data demonstrated that the TAX could inhibit the proliferation of androgen-independent PCa cells.
TAX attenuates the PI3K/AKT signaling pathway and down-regulates the expression of FGFR2.Next, the mechanism by which TAX suppressed the proliferation of androgen-independent PCa cells was explored.According to the RNA sequencing in DU145 cells treated with or without TAX, in comparison with control cells, 193 genes were up-regulated and 383 genes were down-regulated by TAX (Fig. 2a).KEGG pathway analysis revealed the most obvious changes of pathways after treated with TAX were cancerrelated pathways, among which the change of the PI3K/AKT signaling pathway was the most obvious (Fig. 2b).Differential expression gene analysis showed that there were 16 PI3K/AKT signaling pathway-related differentially expressed genes after treated with TAX (Fig. 2c), while FGFR2 was the most significant one of them (Fig. 2d).Based on these findings, the expression of AKT, p-AKT and FGFR2 at the protein and mRNA level after treated with TAX was verified in DU145 and PC3 cells.The results of western blotting and real-time qPCR demonstrated that TAX reduced the phosphorylation of AKT and down-regulated the expression of FGFR2 in androgen-independent PCa cells (Fig. 2e-h).Moreover, we also examined the mRNA levels of PDGFA and two classical downstream genes (4E-BP1, S6K1) of mTOR and Akt in the PI3K/Akt pathway in DU145 cells.The results showed that the TAX inhibited the expression of PDGFA and S6K1, and upregulated the expression of 4E-BP1 to varying degrees at the mRNA levels in DU145 cells (Fig. 2i).
The inhibitory effect of TAX on androgen-independent PCa cells could be reversed by Recilisib.Recilisib is a radioprotectant, which can activate the activities of AKT and PI3K in cells 32 .DU145 and PC3 cells were treated with individual or combined TAX and Recilisib to investigate whether the Recilisib could reverse the inhibitory effect of TAX on androgen-independent PCa cells.The cell growth curve showed that Recilisib could enhance the proliferation of DU145 and PC3 cells and partially abolish the affection of TAX(Fig.3a, b).Colony formation assay suggested that the co-treatment of TAX and Recilisib increased the colony size and number of DU145 and PC3 cells compared to the cells of individual TAX treatment (Fig. 3c,  d).The results of western blotting and real-time qPCR demonstrated that the expression of c-Myc, cyclin D1, p-AKT and FGFR2 of DU145 and PC3 cells could be up-regulated after Recilisib treatment compared to the cells of individual TAX treatment at protein and mRNA level (Fig. 3e-h).These results support that Recilisib could partially reverse the anti-proliferation effect of TAX on DU145 and PC3 cells, demonstrating that TAX inhibits the proliferation of androgen-independent PCa cells via weakening PI3K/AKT signaling pathway and reducing the expression of FGFR2.

TAX inhibits the tumor growth of androgen-independent PCa cells in vivo.
To verify the above results, a subcutaneous DU145 cells xenograft model in BALB/c nude mice was established to further evaluate the anti-tumor effect of TAX in vivo.DU145 cells were inoculated into male BALB/c nude mice which were immunosuppressed, with or without TAX treatment for 21 days.Compared with the control group, TAX  4a-c).In the tumor xenografts, immunohistochemistry staining demonstrated that TAX treatment decreased the number of Ki67-positive cells and reduced the expression of c-Myc, cyclin D1, p-AKT and FGFR2 (Fig. 4d, e).These results in vivo indicates that the tumor growth of PCa cells can be inhibited by TAX.In addition, we detected the expression of apoptotic related Caspase-3 and cleaved-Caspase-3 in PCa xenograft of DU145.We found that the TAX increased the expression of cleaved-Caspase-3.These results showed that after TAX treatment the apoptosis of androgen-independent PCa increased (Fig. 4d, e).

Discussion
Although the implementation of prostate-specific antigen (PSA) testing could early detect PCa effectively 33 , the mortality of PCa especially metastatic PCa, remains grim now 1,14 .ADT is the most effective initial treatment for metastatic PCa, but almost all patients will eventually progress to CRPC 34 .Patients with CRPC are often resistant to chemotherapeutic agents and the prognosis is poor.To find more effective therapeutic agents of PCa is a major clinical challenge.Many natural products are widely used in clinical treatment based on their remarkable anti-tumor effect on various cancers and less cytotoxic than other chemotherapeutic agents 35 .Our study investigated the inhibition effect of TAX on the proliferation and tumor growth of androgen-independent PCa cells.TAX could be isolated from many types of plants including Taraxacum officinale Wigg 24,25 .In recent years, the anti-tumor function of TAX has gained increased attention.Previous researches demonstrated that TAX could suppress the tumor cells growth in many types of cancer, such as breast carcinoma, colon carcinoma, cervix carcinoma, ovary carcinoma, and gastric cancer via multiple signaling pathway, including EGFR/AKT1 29 and PI3K/AKT signaling pathway 36 to attenuate the progression of cancer [26][27][28][29] .However, there are few studies on the effect of TAX in PCa cells.A study showed that TAX can significantly inhibit the proliferation of PC3 cells 37 , but the mechanism has not yet been revealed.Here, our study showed that TAX could inhibit the proliferation and colony formation of PC3 and DU145 cells, down-regulating the expression of c-Myc and cyclin D1 (Fig. 1).The inhibition of the tumor growth of androgen-independent PCa cells in vivo further proved the anti-proliferation effect of TAX.These results highlight the potential therapeutic value of TAX in the later treatment of PCa.
A lot of evidence demonstrate that PI3K/AKT signaling pathway is abnormal activation in many types of cancer to promote tumorigenesis by regulating nutrient metabolism, cell proliferation, survival, migration, and angiogenesis 16 .Therefore, PI3K/AKT signaling pathway potential inhibitors may be critical for effective treatment of cancers in the clinic.Fibroblast growth factor receptors 2 (FGFR2) is expressed in a variety of cancers, such as oral mucosal, esophageal, gastric, colorectal, pancreatic, pulmonary, breast, endometrial, cervical and prostate cancers, to promote the carcinogenesis and cancer progression, thus it has been considered as a new target for cancer treatment 38 .Moreover, it has been proved that FGFR2 was overexpressed in the PCa epithelial cells associated with poor differentiation 39 .Here, we found that TAX could prominently suppress PI3K/AKT signaling pathway and reduce the expression of FGFR2 in androgen-independent PCa (Fig. 2).This result was also confirmed in vivo by tumor formation of androgen-independent PCa cells in nude mice (Fig. 4).In addition, compared to the cells of individual TAX treatment, the co-treatment of TAX and Recilisib (an agonist of PI3K/AKT signaling) increased the proliferation and colony formation of androgen-independent PCa cells, and up-regulated the expression of c-Myc, cyclin D1, p-AKT and FGFR2 (Fig. 3), which means that the re-activation of PI3K/AKT could partially abolish the anti-proliferation effect of TAX on androgen-independent PCa cells.Our study suggests that a possible mechanism by which TAX represses the proliferation and tumor growth of androgen-independent PCa cells through down-regulating PI3K/AKT signaling pathway and the expression of FGFR2.These results provide new evidence of TAX as a new candidate agent for the later treatment of PCa, giving new support for FGFR2 as a therapeutic target for PCa.
This study only studied the mechanism of TAX inhibits the proliferation of DU145 and PC3 PCa cells.In fact, the two PCa cell lines PC3 and DU145 used in our study were derived from patients after bone and brain metastases and representative of the type-I androgen depletion independent PCa in which AR is not expressed 40,41 .The study of the inhibitory effect and mechanism of TAX on PC3 and DU145 PCa cells is also of great significance for the later treatment of PCa.Moreover, we had tested the effects of TAX in AR-dependent cells lines LNCaP and C4-2B.The results of CCK-8 assay showed that TAX reduced the viability of LNCaP and C4-2B cells, and the expression of c-Myc and cyclin D1 decreased after TAX treatment at both protein and mRNA levels.At the same time, we also found that TAX reduced AR expression in LNCaP and C4-2B cells, while AR signaling is crucial for the proliferation of androgen-dependent prostate cancer cell.Looking at the literature, AR signaling and PI3K-AKT signaling have cross-dialogue, which jointly regulate the proliferation of androgen-dependent prostate cancer cells 15,16,42 .Therefore, for androgen-dependent prostate cancer cells, the mechanism that TAX inhibits their proliferation is more complex.So, we think the mechanism that TAX inhibits the proliferation of androgen-dependent and androgen-independent prostate cancer cells is not the same.This is a very interesting study, we are carrying out experimental work on it.
In summary, our study demonstrated that TAX suppressed the proliferation and tumor growth of androgenindependent PCa cells in vitro and in vivo.Mechanically, TAX could inhibit the PI3K/AKT signaling pathway and reduce the expression of FGFR2 to play the anti-proliferation role in androgen-independent PCa cells.Moreover, our results showed that some other signaling pathways were also affected by TAX.It's interested in further exploring the other effects of TAX on PCa cells and changes of specific signaling pathways and targets in PCa cells under the effect of TAX in future studies.Hopefully, there will be more and more studies about TAX in PCa cells and giving more understanding of the molecular mechanisms of TAX, which may help TAX become a new choice of clinical treatment agent of PCa.

Materials and methods
All experimental protocols were approved by Dali University.All methods were performed in accordance with relevant guidelines and regulations.

Figure 1 .
Figure 1.TAX inhibited the proliferation of PCa cells.(a) TAX reduced the cell viability of PCa in a dosedependent manner.The DU145 and PC3 cells were treated with TAX (0, 5, 10, 20, 40 and 60 μM for DU145 cells; 0, 5, 10, 15, 20 and 40 μM for PC3 cells) for 48 h and the viability in individual groups of cells was determined by CCK-8.(b, c) The proliferation of DU145 and PC3 cells treated by TAX at the IC 50 dose (56 µM for DU145 cells; 30 μM for PC3 cells) for 0-7 d was detected by CCK-8.(d, e) The colony formation ability of DU145 and PC3 cells were detected by crystal violet staining after treated with TAX for two weeks.(f-i) The expression of c-Myc and cyclin D1 in DU145 and PC3 cells after treated with TAX was examined by western blotting and real-time qPCR.The blots of c-Myc, cyclin D1 and GAPDH were cropped from different parts of the same gel.ns not significant; *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 2 .
Figure 2. TAX attenuated the FGFR2-PI3K/AKT signaling pathway in PCa cells.(a) The Volcano Plot showed the number of the altered genes after treated with TAX for 48 h in DU145 cells.(b) The KEGG pathway analysis 43-45 revealed the changes of pathways after treated with TAX.(c-d) The Heatmaps showed the 16 PI3K/ AKT signaling pathway-related differentially expressed genes after treated with TAX and the most significant five genes of them.(e-h) The expression of FGFR2, AKT and p-AKT in DU145 and PC3 cells after treated with TAX for 48 h was examined by western blotting and real-time qPCR.The blots of FGFR2, AKT, p-AKT and GAPDH were cropped from different parts of the same gel.(i) The expression of PDGFA, 4E-BP1, S6K1 in DU145 cells after treated with TAX for 48 h was examined by real-time qPCR.ns not significant; *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 3 .
Figure 3. Recilisib reversed TAX's inhibitory effect on PCa cells.(a, b) The proliferation of DU145 and PC3 cells treated with individual or combined TAX (56 μM for DU145 cells; 30 μM for PC3 cells) and Recilisib (80 µM/L) for 0-7 d was detected by CCK-8.(c, d) The colony formation ability of DU145 and PC3 cells were detected by crystal violet staining after treated with individual or combined TAX and Recilisib for two weeks.(e, h) The expression of c-Myc, cyclin D1, AKT, p-AKT and FGFR2 in DU145 and PC3 cells after treated with individual or combined TAX and Recilisib was examined by western blotting and real-time qPCR.The blots of c-Myc, cyclin D1 and GAPDH were cropped from different parts of the same gel and the blots of FGFR2, AKT, p-AKT and GAPDH were cropped from different parts of another same gel.ns not significant; *p < 0.05; **p < 0.01; ***p < 0.001.