The chemical components of SBD
The related chromatograms were shown in Fig. 1.
SBD inhibited cell proliferation and induced cell cycle arrest in PCa cells
The cytotoxicity of SBD on PC-3 and DU145 cell lines was detected by CCK8 assay. Cells were exposed to various concentration of SBD for 24 h (Figure. 2a) and 48 h (Figure. 2b). According to the results of cell viability, SBD showed an obvious cytotoxic effect against PC-3 and DU145 cell lines in a time and concentration dependent manner, with IC50 of 98.44 µg/mL and 118.2 µg/mL, in 24 h respectively, and 56.39 µg/mL and 66.51 µg/mL in 48 h, respectively. SBD had marginally anti-tumour effects on PC-3 than on DU145.
Afterwards, the cytotoxic phenotypes in PC-3 (Figure. 2c) and DU145 cell lines (Figure. 2d) were detected under EdU incorporation staining. The observations demonstrated that SBD concentration-dependently decreased the radio of EdU-positive nuclei in both PCa cell lines.
Immortal proliferation is a crucial feature of tumour and timing of proliferation rely on the transition speed of cell cycle. It has been observed that cell cycle plays an important role in the progression of PCa cells. The effect of SBD on cell cycle of PC-3 (Figure. 2e) and DU145 cell lines (Figure. 2f) was detected. The data demonstrated that most of the cells were arrested in the S and G2 phases after treatment with SBD in PC-3 cells. Except for an obvious increase in S and G2 phases cell population, SBD treatment decreased the G1 phase cell population (p < 0.05). Consistent with PC3 cells, treatment with SBD resulted in an apparent increase in the G2 phase cell population in the DU145 cell (p < 0.05). In addition, the colony formation experiment indicated that SBD suppressed the colony formation of PCa cells (Figure.2g & 2h).
Taken together, the inhibitory effect of SBD on the cell proliferation and colony formation might partially due to the cell cycle arrest at G2/M phase.
The PI3K/AKT signaling pathway participates in SBD-induced PCa cell apoptosis
Increasing apoptosis is the main index to assess the efficacy of antitumor drugs. We then investigate whether SBD has the ability to induce apoptosis the biological function of SBD in apoptosis, the cell apoptosis in SBD - treated PC-3 (Figure. 3a) and DU145 cells (Figure. 3b) was analyzed with flow cytometry. The percentage of apoptosis cells was dramatically increased in PC3 and DU145 cells treated with SBD when compared with their controls (p < 0.05).
Thus, from prior reports, we hypothesized that SBD could constrain the expression of a panel of proteins associated with cellular apoptosis. The Western blot results that SBD treated PCa cells boosted expression of activated Caspase-3, proving that apoptosis was the major mechanism of SBD -induced growth inhibition in PCa cells. After that, we examined that impact of SBD on the Bcl-2 family of proteins involved in apoptosis. There was an increase in the expression of pro-apoptotic Bax protein in both PC3 and DU145 cells treated with SBD. In addition, treatment with SBD for 48 h down-regulated the expression of anti-apoptotic Bcl-2 protein in PCa cells (Figure. 3c).
The abnormal activation of PI3K/AKT signalling pathway is very frequently observed in PCa. Furthermore, SBD significantly inhibited the phosphorylation of AKT and PI3K in PCa cells (Figure. 3d), which are consistent with the roles for PI3K/AKT signalling pathway in the regulation of cell apoptosis. These dates demonstrated that SBD promote PCa cells apoptosis via inactivation of PI3K/AKT signalling pathway.
SBD inhibits migration, invasion, EMT progression and TME-angiogenesis
The metastasis and invasion are closely related to the malignant degree and prognosis of the tumour. According to the wound-healing assay, SBD significantly inhibited tumour cell migration in both PC3 (Figure. 4a) and DU145 cells (Figure. 4b) in a dose-dependent manner. To determine if SBD could supress migration and invasion of PCa cells, transwell assays were employed in PC3 and DU145 cells. As expected, treatment with SBD for 48 h reduced the number of invaded cells remarkably (p < 0.05, Figure. 4c & 4d). These results indicated that SBD decelerate PCa cells migration and invasion.
As reported in previous studies, EMT is inseparably associated with metastasis of tumour. To further study the potential mechanism of SBD on its anti-metastatic effect, we next assessed the expression of EMT-related protein though western blot. As indicated in Figure. 4e, the expression of the epithelial marker E-cadherin and Vimentin was significantly up-regulated by SBD, while the expression of N-cadherin was down-regulated remarkably in the SBD -treated cells compared to the control cells (p < 0.05, Figure. 4e). Collectively, these results showed that SBD can affect cancer cell invasion, migration and EMT-related protein.
Blockade of AKT resulted in inhibition of SBD-mediated cell proliferation and migration
To further determine the involvement of the PI3K/AKT pathway in the inhibition of tumour cell growth by SBD, PC-3 and DU145 cells were pre-treated with 10 µM MK2206 (AKT-specific inhibitor) for 6 h, followed by 48 h of SBD treatment. After 48 h, cell viability was analysed using CCK-8 assay. Of note, co-treatment with SBD had no significant effect on cell viability compared with those treated alone with SBD (50 µg/mL) in PC-3 (Figure. 5a) and DU145 cells (Figure. 5b). In addition, co-treatment with SBD did not further decrease the expression of p-AKT (p > 0.05).
To further investigate if the cell invasion inhibitory effect of SBD is dependent on AKT/PI3K signalling pathway, MK2206 was pre-treated on PCa cells. The results showed that PC-3 (Figure. 5c) and DU145 cells (Figure. 5d) migration had no significant difference when compared with SBD (50 µg/mL) treatment alone (p > 0.05). These results above indicated that SBD inhibited cell viability and invasion in a mechanism dependent on the AKT/PI3K pathway partially.
SBD inhibited EMT via AKT/PI3K pathway signaling
Based on the abovementioned results, SBD may regulate EMT through PI3K/AKT signaling pathway. According to the results of western blot, we found MK2206 reversed effects of SBD on expression of p-AKT, N-cadherin, E-cadherin and Vimentin on PC-3 and DU145 cells (p<0.05)(Figure. 6a & b). Collectively, these results suggested that treatment with SBD may partially inactivate PI3K/AKT signalling pathway, thereby inhibiting the cell proliferation and invasion, as well as preventing PCa cells from EMT progression.
SBD suppressed tumor growth in 22RV1 cell xenograft mice model
Next, to investigate the anti-tumour effect of SBD on the inhibition of PCa in vivo, we established 2RRV1 subcutaneous xenografts. We explored the effects of Low-dose (SBD-L), Medium-dose (SBD -M), and High-dose (SBD -H) of SBD treatment on tumour growth. As shown in Figure. 7a & 7b & 7c, administration of SBD in 22RV1-bearing mice markedly inhibited tumour volume and the tumour weight. Our results showed that there are significant different between the SBD treatment and the control group. H&E and proliferating cell-related antigen Ki67 staining were conducted to explore the growth level of the xenografts. Ki67-positive rate was decreased markedly in the SBD-treated group with medium and high dose (Figure. 7d & 7e), confirmed that subcutaneous tumour proliferation was significantly reduced in the SBD-M and SBD-L treatment group.
SBD inhibits AKT/PI3K and EMT progression in vivo
Based on the results of the in vitro study, western blotting and immunohistochemical (IHC)assays were used to detect the expression levels of EMT-related proteins in tumour tissues. As shown in Figure. 8a, administration with SBD-M and SBD-H reduced the phosphorylation levels of PI3K and AKT remarkably (p < 0.05) in tumour tissues. In addition, according to the IHC staining results, the expression of E-cadherin and Vimentin was significantly up-regulated, while the expression of N-cadherin was down-regulated remarkably in SBD-M and SBD -H groups when compared to the control groups (p < 0.05, Figure. 8b)
SBD blocks tube formation of human umbilical vein endothelial cells
Angiogenesis is a main process in cancer development and plays a vital role in cancer growth and metastasis. The effects of SBD on angiogenesis in vitro was determined by tube formation and migration experiments. We collected the SBD in the supernatant of tumour cells and subject it to HUVECs. There are significance difference between complete media-treated HUVECs and CM from PCa cells-treated HUVECs, whilst CM from SBD-treated PCa cells obviously diminished the ability of tube formation of HUVEC in concentration-dependent manner (Figure 9A). We next investigated whether SBD-mediated inhibition of the tube formation in HUVEC is due to the drug’s cytotoxicity. The viability and survival rate of HUVEC without or with SBD treatment for 48 h did not change significantly, which ruled out the possibility of drug cytotoxicity (p >0.05) (Figure. 9B). It is well known that the PKC activator PMA is able to induce the tube formation of HUVECs cells. When HUVEC were treated with 10 nM PMA, they formed the typical cobblestone morphology characteristic, which was greatly weakened by SBD treatment (Figure. 9C). These results indicated that SBD inhibited angiogenesis by reducing tube formation of vascular endothelial cells rather than apoptosis.