Inhibitory effects of tanshinone IIA from Salvia miltiorrhiza Bge on human bladder cancer BIU-87 cells and xenograft in nude mice

The objective of this study was to investigate the inhibitory effects of tanshinone OOA on human bladder cancer BOU-87 cells and the xenograft in nude mice. BOU-87 cells were treated with tanshinone OOA with concentration of 0.5, 1, 2 and 4 g/L. The cell proliferation, cycle and apoptosis were detected. The nude mice with BOU-87 cell xenograft were treated with normal saline (control), 5-fluorouracil (5-FU), 200, 400 and 600 mg/kg tanshinone OOA, respectively. The animal body weight and growth of tumor were measured. The expressions of B-cell lymphoma-2 (Bcl-2), Caspase-3 and proliferating cell nuclear antigen (PCNA) protein in xenograft were detected. The apoptosis index of cells was determined. Results showed that, tanshinone OOA inhibited the proliferation of BOU-87 cells, promoted their apoptosis, and arrested more cells in G 0 /G 1 phase. Tanshinone OOA significantly inhibited the growth of xenograft in nude mice and promoted the apoptosis of tumor cells in xenograft. Tanshinone OOA down-regulated Bcl-2 and PCNA expression and up-regulated Caspase-3 expression in xenograft. On conclusion, tanshinone OOA has inhibitory effects on human bladder cancer BOU-87 cells, and can inhibit the BOU-87 cells xenograft in nude mice.


Introduction
Bladder cancer is one of the most common tumors in the urinary system.The incidence of bladder cancer ranks the ninth place in malignant tumors in the world.Seventy to eighty percent of bladder cancer patients are with the superficial bladder cancer, which seriously threatens the people's health and quality of life (Alfred Witjes et al., 2017).At present, the transurethral electrotomy of bladder tumor is used in most bladder cancer cases.On addition, the intravesical chemotherapy is often performed to prevent the recurrence (Matsushima et al., 2002).Tanshinone OOA is the main active ingredient extracted from dried root and rhizome of Salvia miltiorrhiza Bge.Ot has activity of expanding blood vessels (Liu et al., 2015), anti-atherosclerosis (Fang et al., 2008), scavenging free radical (Wang et al., 2013) and protecting mitochondria (Jin et al., 2013).The in vivo and in vitro studies show that, tanshinone OOA has significant inhibitory effect on breast cancer (Wang et al., 2005), gastric cancer (Xu et al., 2013), colon cancer (Tu et al., 2012), cervical cancer (Munagala et al., 2015) and lung cancer (Chiu & Su, 2010).This suggests that, tanshinone OOA may have prevention effect on bladder cancer.However, the in vivo and in vitro inhibitory effects of tanshinone OOA on bladder cancer are seldom reported (Chiu et al., 2014;Huang et al., 2017).This study investigated the inhibitory effects of tanshinone OOA on human bladder cancer BOU-87 cells and the xenograft in nude mice, and explored the possible mechanisms.The objective was to provide a theoretical basis for clinical application of tanshinone OOA to treatment of bladder cancer.

Cell culture
Human bladder cancer BOU-87 cells (Cell Bank of the Chinese Academy of Sciences, Shanghai, China) were cultured in RPMO 1640 culture medium containing l0% FBS and 100000 U/mL penicillin in electric constant temperature incubator (37 °C, 5% CD 2 ).The digestion and passage were performed using routine methods.The cells in logarithmic growth phase were collected for further experiments.

Determination of cell proliferation
Proliferation of BOU-87 cells was determined using methylthiazoletetrazolium (MTT) method.The cells in the logarithmic growth phase with concentration of 1×10 5 cell/ml were incubated in 96-well culture plate, 200 μL per well.The cells were divided into 5 groups, 6 wells in each group for each treatment time.After the cell adherence appeared, the original culture medium was removed, and tanshinone OOA (extracted from Salvia miltiorrhiza Bge; purity 98%; Shaanxi Taiji Huaqing Technology Co., Ltd., Xi'an, China) was added to the well, and the culture medium was added to make the final volume 200 μL.On these 5 groups, the final tanshinone OOA concentration was 0, 0.5, 1, 2 and 4 g/L, respectively.After culture (37 °C, 5% CD 2 ) for 24, 48 and 72 h, the culture medium was sucked, respectively, and 20 μL 0.5% MTT was added, followed by culture for 4 h.The supernatant was discarded.Dimethyl sulfoxide (150 μL) was added to each well, followed by oscillation for 10 min.The optical density (DD) of cell solution was detected using microplate reader at the wavelength of 490 nm.The inhibition rate on cell proliferation was calculated as followsI: inhibition rate = (DD control group -DD experimental group ) / DD control group × 100%.

Determination of cell apoptosis and cycle
Flow cytometry analysis was performed to determine the apoptosis and cycle of BOU-87 cells.BOU-87 cells in the logarithmic growth phase with concentration of 1×10 5 cell/ml were incubated in 96-well culture plate, 200 μL per well.The cells were divided into 5 groups, 6 wells in each group for each treatment time.After culture for 24 h, the medium was sucked off, and tanshinone OOA was added to the well.The final tanshinone OOA concentration of these 5 groups was 0, 0.5, 1, 2 and 4 g/L, respectively.The control group was added with culture medium.After culture for 48 h, the culture medium was sucked, followed by centrifugation at 3000 r/min for 5 min.The cells were washed with phosphate buffer saline for 3 times.Then, 0.5 ml of propidium iodide was added.After dyeing for 30 min, the cell apoptosis and cell cycle were measured using flow cytometer.The procedures were according to the instruction of kits.The percentage of cells in different phases and the apoptosis rate were calculated.

Establishment of nude mice xenograft model of BIU-87 cells
BOU-87 cells in logarithmic growth phase were taken, and the single-cell suspension (2×10 7 cells/ml) was prepared.The right armpit of BALB/c nude mice was disinfected using iodine, and then 0.2 ml BOU-87 single-cell suspension was subcutaneously inoculated at the right armpit.The growth of xenograft tumor was observed every day, and the diameter was measured.The whole operation was performed in the aseptic condition.After transplantation, the nude mice were raised in the specefic pathogen free environment.When the diameter of all xenograft tumors was more than 2 mm, the model was considered successfully established.

Animal grouping and treatment
After establishment of BOU-87 cell xenograft model, 30 nude mice were randomly divided into control, 5-FU, 200 mg/kg tanshinone OOA, 400 mg/kg tanshinone OOA and 600 mg/kg tanshinone OOA group, 6 nude mice in each group.The nude mice in later 4 groups were intraperitoneally injected with 10 mg/kg 5-FU, and 200, 400 and 600 mg/kg tanshinone OOA, respectively.The nude mice in control group were intraperitoneally injected with equal volume of normal saline.The administration was performed once per two days, and was last for 15 days.

Determination of nude mice body weight and growth of xenograft tumor
At the treatment beginning (day 0) and on the day 5, 10 and 15 after treatment beginning, the body weight of nude mice was measured using electronic balance.Dn the day 15, all nude mice were executed by cervical dislocation, and the tumor block was taken, the maximal and short diameters of the xenograft tumor were measured, and the tumor volume was calculated.The inhibition rate on xenograft was calculated as followsI: inhibition rate (%) = (1 -xenograft mass in treatment group / xenograft mass in control group) ×100%.

Determination of B-cell lymphoma-2, Caspase-3 protein and proliferating cell nuclear antigen expression in tumor tissue
Paraffin sections of xenograft tumor were prepared.The western blot assays were performed to detect the expression of B-cell lymphoma-2 (Bcl-2), Caspase-3 and proliferating cell nuclear antigen (PCNA) protein in tumor tissue.The procedures were according to the instruction of kits.The primary and secondary antibodies were provided by Santa Cruz Biotechnology, Onc.(CA, USA).β-actin was used as the internal reference.The relative levels of Bcl-2 and Caspase-3 protein were presented by the ratio of their optical density to β-actin.

Determination of apoptosis index of tumor cells
Apoptosis of tumor cells in xenograft was detected by TUNEL, according to the instruction of TUNEL apoptosis kits.The round, multi-lobed or crescent-shaped dark brown or brown granules presented the positive apoptotic cells.Under the high magnification (× 400), 5 fields of vision were randomly selected.The total number of cells and the number of positive cells of each section were counted.The apoptotic index (AO) was calculated as followsI: AO = (number of positive cells / total number of cells) × 100%.

Statistical analysis
Data were presented as mean±standard deviation.All statistical analysis was carried out using SPSS 22.0 software (SPSS Onc., Chicago, OL, USA).The difference among different groups was analyzed using one-way analysis of variance followed by Duncan's multiple range test.P < 0.05 was considered as statistically significant.

Effects of tanshinone IIA on proliferation of BIU-87 cells
Tanshinone OOA with all concentrations had the inhibitory effect on proliferation of BOU-87 cells.The inhibitory effect increased with the increase of tanshinone OOA concentration and prolonging of treatment time.There was significant difference of inhibitory rate between each two concentrations from 0.5 to 4 g/L at the same treatment time point (P < 0.05).On addition, there was significant difference of inhibitory rate between each two treatment time points with the same concentration from from 1 to 4 g/L (P < 0.05) (Figure 1).

Effects of tanshinone IIA on apoptosis of BIU-87 cells
As shown in Figure 2, all concentrations of tanshinone OOA promoted the apoptosis of BOU-87 cells.The apoptosis rate increased with treatment time prolonging and increase of tanshinone OOA concentration.There was significant difference of apoptosis rate between each two concentrations at the same treatment time point (P < 0.05), with significant difference between each two treatment time points with the same concentration from 0.5 to 4 g/L (P < 0.05).

Effects of tanshinone IIA on cycle of BIU-87 cells
After treatment for 48 h, with the increase of tanshinone OOA concentration, the proportion of cells in phase G 0 /G 1 increased, and the proportion of cells in phase S and G 2 /M decreased.There were significant differences of phase G 0 /G 1 and S cell proportion between each two tanshinone OOA concentrations from 0 to 4 g/L, respectively (P < 0.05).On addition, the percentage of cells in phase G 2 /M with tanshinone OOA concentrations from 0.5 to 4 g/L was significantly lower than that with tanshinone OOA concentration of 0 g/L (P < 0.05) (Figure 3).

Effects of tanshinone IIA on body weight of nude mice
Dn the day 10 and 15, the body weight in 5-FU group was significantly decreased compared with day 0 and day 5 (P < 0.05).On addition, the body weight in 5-FU group was significantly lower than that in other 4 groups at day 10 and 15, respectively (P < 0.05).There was no significant difference among other 4 groups at each time point (P > 0.05) (Table 1).

Effects of tanshinone IIA on expression of Bcl-2 and Caspase-3 protein in tumor tissue
Relative expression levels of Bcl-2 protein in tumor tissue in 5-FU, 400 mg/kg tanshinone OOA and 600 mg/kg tanshinone OOA group were significantly lower than those in control group, respectively (P < 0.05), but that in three tanshinone OOA groups was significantly higher than 5-FU group, respectively (P < 0.05).The expression levels of Caspase-3 protein in 5-FU and 600 mg/kg tanshinone OOA groups were significantly higher than those in control group, respectively (P < 0.05) (Table 3).

Effects of tanshinone IIA on expression of PCNA protein in tumor tissue and AI of tumor cells
Relative expression levels of PCNA protein in tumor tissue in 5-FU, 400 mg/kg tanshinone OOA and 600 mg/kg tanshinone OOA groups were significantly lower than those in control group, respectively (P < 0.05), and that in 600 mg/kg tanshinone OOA group was significantly lower than 5-FU group (P < 0.05).The AO of tumor cells 5-FU, 200, 400 and 600 mg/kg tanshinone OOA groups was significantly higher than that in control group, respectively (P < 0.05) (Table 4).

Discussion
This study investigated the inhibitory effects of tanshinone OOA on human bladder cancer BOU-87 cells and the xenograft in nude mice.Results showed that, tanshinone OOA with certain concentration inhibited the proliferation of BOU-87 cells, promoted their apoptosis, and arrested more cells in G 0 /G 1 phase.On addition, tanshinone OOA had no obvious effect on the body weight of nude mice, but significantly inhibited the growth of xenograft in nude mice.This indicates that, tanshinone OOA has in vivo and in vitro inhibitory effects on BOU-87 cells.
The occurrence and development of tumor is a complex process, and it is closely related to the imbalance of cell proliferation, differentiation and apoptosis.The apoptosis of cancerous cells is regulated by a series of genes, and the occurrence and development of tumor are closely related to the blocking of cancerous cell apoptosis (Portt et al., 2011).The Bcl-2 protein family plays a very important role in regulating the apoptosis.High expression of Bcl-2 is often regarded as a protective effect from various apoptotic stimuli (Adams & Cory, 2007).Therefore, down-regulation of Bcl-2 expression may induce the apoptosis of tumor cells.Cheng & Su (2010) have investigated the effects of tanshinone OOA on small cell lung cancer H146 cells and find that, tanshinone OOA can down-regulate the expression of Bcl-2 protein, thus induce the apoptosis of H146 cells.On the present study, the relative expression levels of Bcl-2 protein in 400 mg/kg and 600 mg/kg tanshinone OOA group were significantly lower than those in control group, respectively (P < 0.05).This indicates that, Bcl-2 is the protective and promoting factor of bladder tumor development.tanshinone OOA can down-regulate the Bcl-2 expression in xenograft tumor, which may be related to its inhibition on growth of xenograft tumor.
Caspase-3 is a key protein in apoptotic signal conduction pathway.The activated Caspase-3 can cleave the protein molecules maintaining cell survival.For example, it can degrade the cytoskeleton protein, and decompose the key enzymes for DNA repair.This can cause the cell chromatin condensation ultimately leading to the apoptosis of cell in this signal conduction pathway (Amor et al., 2006).Saegusa et al. (1996) have found that, the expression of Caspase-3 in endometrial carcinomas is positively correlated with the apoptotic index.Sung et al.s research (Sung et al., 1999) has shown that, tanshinone OOA can induce apoptosis of human leukemia cell lines, which has a direct relationship with the activation of Caspase-3 expression.Results of the present study found that, the expression level of Caspase-3 protein 600 mg/kg tanshinone OOA group was significantly higher than those in control group (P < 0.05).This indicates that, tanshinone OOA can up-regulate the Caspase-3 expression, thus inhibiting the growth of xenograft tumor and promoting the apoptosis of tumor cells.
PCNA protein is the nuclear protein synthesized in G 1 /S phase of cells, and is the cofactor of DNA polymerase δ.PCNA is involved in the DNA synthesis and cell proliferation, and is widely used in the research of tumor proliferation activity (Gerits et al., 2015).Wang et al. (2015) find that, tanshinone OOA can suppress the expression of PCNA in both prostate epithelial cells and stromal cells, thus effectively preventing the development of the disorder.Results of the present study showed that, the For each index, different character indicates significant difference, and the same character means no significant difference.5-FU, 5-fluorouracil.For each index, different character indicates significant difference, and the same character means no significant difference.5-FU, 5-fluorouracil.5-FU, 5-fluorouracil; PCNA, proliferating cell nuclear antigen; AO, apoptosis index.
expression levels of PCNA protein in 400 mg/kg tanshinone OOA and 600 mg/kg tanshinone OOA groups were significantly lower than those in control group, respectively (P < 0.05), and that in 600 mg/kg tanshinone OOA group was significantly lower than 5-FU group (P < 0.05).This indicates that, the tanshinone OOA promotion on apoptosis of xenograft tumor may be related to its down-regulation of PCNA expression.

Conclusion
Tanshinone OOA has inhibitory effects on human bladder cancer BOU-87 cells.On addition, it can inhibit the growth of BOU-87 cell xenograft in nude mice, and promote the apoptosis of tumor cells.The mechanisms may be related to its down-regulation of Bcl-2 and PCNA expression and up-regulation of Caspase-3 expression in tumor tissue.This study has provided the theoretical and experimental basis for the further clinical application of tanshinone OOA treatment of bladder cancer.This study also has some limitations.Firstly, the toxicity test of tanshinone OOA has not performed, so is cannot be concluded that tanshinone OOA has low side effect compared with chemotherapy drugs.The safety of tanshinone OOA needs to be investigated in next studies.Secondly, there may be other mechanisms of tanshinone OOA inhibition on bladder cancer, which should be further investigated.

Figure 2 .
Figure 2. Effects of tanshinone OOA on apoptosis of BOU-87 cells (n = 6).Different character indicates significant difference, and the same character means no significant difference.

Figure 3 .
Figure 3. Effects of tanshinone OOA on cycle of BOU-87 cells (n = 6).For each phase, different character indicates significant difference, and the same character means no significant difference.

Table 1 .
Effects of tanshinone OOA on body weight of nude mice with tumor (g; n = 6).

Table 4 .
Effects of tanshinone OOA on expression of PCNA protein in xenograft tumor and AO (n = 6).