Tanshinone IIA affects the malignant growth of Cholangiocarcinoma cells by inhibiting the PI3K-Akt-mTOR pathway

In the present study, we aimed to find the target of Tanshinone IIA (Tan-IIA) in Cholangiocarcinoma by network pharmacology-based prediction and investigate the possible mechanism through experimental verification. In this study, we combined Tan-IIA-specific and Cholangiocarcinoma-specific targets with protein–protein interactions (PPI) to construct a Tan-IIA targets-Cholangiocarcinoma network, and network pharmacology approach was applied to identify potential targets and mechanisms of Tan-IIA in the treatment of Cholangiocarcinoma. The anti-cancer effects of Tan-IIA were investigated by using subcutaneous tumorigenic model in nude mice and in the human Cholangiocarcinoma cell lines in vitro. Our results showed that Tan-IIA treatment considerably suppressed the proliferation and migration of Cholangiocarcinoma cells while inducing apoptosis of Cholangiocarcinoma cells. Western blot results demonstrated that the expression of PI3K, p-Akt, p-mTOR, and mTOR were inhibited by Tan-IIA. Meanwhile, After treatment with Tan-IIA, the level of Bcl2 was downregulated and cleaved caspase-3 expression increased. Further studies revealed that the anticancer effects of Tan-IIA were severely mitigated by pretreatment with a PI3K agonist. Our research provides a new anticancer strategy and strengthens support for the use of Tan-IIA as an anticancer drug for the treatment of CCA.


Materials and methods
Ethical approval. The   Plate cloning. Both cell lines were cultured in a well plate at a density of 300 cells per well. The cells were gently rotated to disperse the cells evenly. After 6 h, the Cholangiocarcinoma cells were treated with Tan-IIA (0, 5, 10, 20, and 30 µg/mL) and incubated in a cell culture incubator at 37 °C with 5% CO 2 for two weeks. Next, the cells were washed with PBS three times. Subsequently, 4% paraformaldehyde was used to fix the cells for 15 min. The cells were then stained with crystal violet for 10 min. Subsequently, the staining solution was washed off with PBS. The six well plates were then inverted and an overlay of transparency sheet with a grid was performed and the clones was manually counted directly with the naked eye: clone formation rate = (number of clones/number of inoculated cells) × 100%.
Scratch-induced wound healing assay. Plated in six well culture dishes were 4 × 10 5 of Cholangiocarcinoma cells. After 24 h, a 200 μL tip was used to wound confluent cells. The detached cells were washed with PBS three times, and the cells were incubated with Tan-IIA (HuCCT-1: 27 µg/mL, RBE: 49 µg/mL) for 24 h. Cell migration images were recorded using an inverted microscope. The results of the scratch experiment were obtained using the formula wound closure rate = post-healing area/initial wound area. All experiments were repeated three times. Subsequently, the target membranes were incubated with the specific primary antibodies overnight at 4 °C. Membranes were then washed with TBST three times and then diluted HRP-coupled secondary antibodies were added and incubated for 2 h at room temperature. Finally, immunocomplexes were detected using an ECL detection reagent (Millipore, MA, USA). The measurement dates were obtained from three separate experiments. The intensity of each band was determined using ImageJ software.

Transwell invasion assay. Cell invasion was detected in
In vivo experiments. NOD-SCID (NOD CB17-Prkdcscid/NcrCrl, male, 5 weeks of age) mice were provided by Beijing Vital River Lab-oratory Animal Technology Co., Ltd (Beijing, China). All mice were placed in a 12-h light/dark cycle at 25 + −1 °C and 56% humidity with free access to food and water. The initial body weights of these mice ranged from 20 to 23 g. Following subcutaneous injection of 2 × 10 6 HuCCT-1 Cholangiocarcinoma cells into the back of 15 NOD-SCID mice, the mice were divided into three groups: the control group (n = 5), the Tan-IIA (50 mg/kg) treatment group (n = 5), and the Tan-IIA (50 mg/kg) combined with 740y-p (10 mg/kg) treatment group. Tan-IIA was diluted with DMSO: Methanol: Hydroxypropyl-β-cydodextrin (HP-β-CD) = 1: 1: 1. 740y-p was dissolved in the same way. Seven days after the injection of HuCCT-1 Cholangiocarcinoma cells, drugs were injected intraperitoneally into the experimental groups of mice on every other day, and normal saline was given to the control group. Mice were killed at day 21 of inoculation with tumor cells. All mice were executed by dislocation of the cervical vertebrae. Tumor volumes were measured every 3 days before execution.
Statistical analysis. The statistical software GraphPad 7 was used for data analysis. The experimental results listed in the article represent the dates of at least three separate replicate experiments. Dates are shown as mean and standard deviation. Student's t-test was used for differences between two groups. Multiple group comparisons were made using one-way ANOVA. Differences were considered statistically significant at P values of less than 0.05.

Effects of Tan-IIA on Cholangiocarcinoma cells proliferation, migration, colony formation, and invasion.
To explore the effect of Tan-IIA on Cholangiocarcinoma cells, we incubated Cholangiocarcinoma cells at various concentrations (0, 5, 10, 20, and 30 µg/mL) of Tan-IIA for 12, 24, 48, and 72 h. The effect of Tan-IIA on the proliferation of Cholangiocarcinoma cells was then detected by CCK8 (Fig. 1A,B). This indicated that compared with the control group, Tan-IIA inhibited the proliferation of Cholangiocarcinoma cells in a time-and dose-dependent manner (Fig. 1A,B). At the same time, the plate cloning experiment demonstrated that Tan-IIA significantly suppressed Cholangiocarcinoma cells growth compared with the control group ( Fig. 1C-F). Scratch wound assay showed that Tan-IIA treatment significantly suppressed the motility of Cholangiocarcinoma cells, as determined by the migration area ( Fig. 1G-J). We further assessed the role of Tan-IIA in invasion by a transwell assay. The results showed that Tan-IIA significantly decreased the invasion capacity of Cholangiocarcinoma cells (Fig. 1K-N). In addition, the apoptotic rate detected by flow cytometry analysis showed that Tan-IIA could promote the apoptosis of Cholangiocarcinoma cells (Fig. 1O-R). Next, western blot analysis revealed that compared with the control group, the expression of Bcl-2 was significantly decreased. However, the protein level of BAX was not altered by Tan-IIA. In addition, the results showed that the expression of caspase-3 was markedly decreased, while the cleaved caspase-3 was markedly upregulated induced by Tan-IIA ( Fig. 1S-U).
Common targets of Tan-IIA and Cholangiocarcinoma effects. Relevant targets for Cholangiocarcinoma were obtained from OMIM, GeneCards, PharmGKB, and TTD databases, and the target of Tan-IIA was found from the TCMSP database. After cross-analysis, 17 common drug-disease-related targets were identified ( Fig. 2A). The screened targets were then used to construct the PPI network through the string website  www.nature.com/scientificreports/ (Fig. 2B). Using the data tables obtained from the PPI network, the network pharmacology map was entered into Cytoscape software (Fig. 2C). The relationship between the common target genes and the relationship between the genes and Tan-IIA can be clearly seen in the figure. Then, we analyzed the GO and KEGG results of the common targets of Tan-IIA and Cholangiocarcinoma. Based on the 17 common targets of Tan-IIA and Cholangiocarcinoma, we used R software to analyze the top ten terms of the three major categories of BP, CC, and MF enriched by GO (Fig. 2D), and the results of GO analysis showed that the common targets of Tan-IIA and Cholangiocarcinoma were closely related to cell proliferation. Among the top 30 terms in KEGG analysis (Fig. 2E), we found that the PI3K-AKT pathway was more significantly associated with proliferation (Fig. 2F). www.nature.com/scientificreports/ Tan-IIA inhibited activation of PI3K/AKT/mTOR. Western blotting was used to detect the expression of PI3K, Akt, p-Akt, mTOR, and p-mTOR. The results indicated that Tan-IIA inhibited the expression of PI3K, p-Akt, p-mTOR, and mTOR compared to control group in a dose-dependent manner (Fig. 3A-C). Furthermore, pretreatment of Cholangiocarcinoma cells with the 740 y-p (PI3K agonist) abolished the effects of Tan-IIA on the restrain PI3K, p-Akt, mTOR, and p-mTOR in Cholangiocarcinoma cells (Fig. 3D-F).

PI3K agonists abolished the effects of Tan-IIA.
To further demonstrate that Tan-IIA affects the growth of Cholangiocarcinoma cells by suppressing the PI3K/Akt/mTOR signaling pathway, Cholangiocarcinoma cells were pretreated with 740 y-p (PI3K agonist) or without it in the presence of Tan-IIA. These results confirm that PI3K agonists could attenuate the tumor suppressive effect of Tan-IIA. The data showed that the effect of Tan-IIA inhibited cell proliferation, migration, and invasiveness, and promoted apoptosis was reversed by 740 y-p treatment (Fig. 4A-O). The results showed that Tan-IIA also had a significant anti-tumor effect in vivo. However, the anti-tumor effect of Tan-IIA was largely attenuated by 740y-p ( Fig. 5A-C).

Discussion
Tan-IIA was one of the main components of Danshen. Accumulated evidence from preclinical and clinical studies has confirmed that Tan-IIA has good anti-tumor properties 17 . Several studies have shown that it can inhibit the growth of various tumor cell lines, including liver cancer, pancreatic cancer, and colorectal cancer 7,8,18 . Nevertheless, the inhibitory effect of Tan-IIA on CCA cells and its underlying mechanisms are unknown. Consistent with the previous findings, this study showed that Tan-IIA could inhibit malignant growth, migration, and invasion and promote apoptosis of Cholangiocarcinoma cells, providing a new understanding of the role of Tan-IIA in the treatment of CCA. To investigate the mechanism by which Tan-IIA inhibits Cholangiocarcinoma, we analyzed the possible pathways that Tan-IIA inhibited the malignant proliferation of Cholangiocarcinoma cells through network pharmacology. KEGG analysis showed that Tan-IIA affected the expression of the PI3K/Akt pathway in Cholangiocarcinoma cells. Several studies have shown that elevated expression of PI3K-associated proteins is considered a hallmark of cancer 19 . The PI3K/Akt pathway is closely related to cancer progression in many types of human cancers, including lung cancer, stomach cancer, liver cancer, and pancreatic cancer. Previous studies have shown that the PI3K/Akt pathway plays a significant role in inhibiting tumor proliferation, invasion, migration, and apoptosis 20 . It has been reported that the PI3K/Akt signaling pathway is extremely important in CCA development and progression 21 . In the present study, we detected the expression of PI3K/Akt proteins in Cholangiocarcinoma cells. Consistent with KEGG analysis, we observed that Tan-IIA significantly inhibited the levels of PI3K and p-Akt compared with those in the control group in Cholangiocarcinoma cells.
Recent studies have shown that the activation of the PI3K/Akt pathway can activate or inhibit a variety of downstream target proteins, such as mTOR, Bad, Caspase9 and GSK-3. And the PI3K/Akt/mTOR signaling cascade has a major impact on cell proliferation and survival as well as cell cycle regulation 22,23 . In this pathway, PI3K is activated and further activates Akt proteins located on the plasma membrane, which eventually phosphorylate Akt, and then p-Akt activates various regulators downstream of the pathway, including mTOR 24 . mTOR is the main downstream effector of the PI3K/Akt pathway. Activated mTOR is associated with cell proliferation and survival 25 . Phosphorylation of ribosomal protein S6 kinase (S6K) and eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1) can be mediated by activated mTORC1, which leads to the re-release of eukaryotic translation initiation factor 4E (EIF4E). These factors directly lead to protein translation and cell cycle progression. In addition, mTORC2 can act as an activator of Akt, forming a positive feedback loop between Akt and mTOR, further promoting cell proliferation and survival 26 . It is well documented that Tan-IIA plays an anti-tumor role by regulating the PI3K/Akt/mTOR signaling pathway. Tan-IIA can block the Ras/Raf/MEK/ERK and PI3K/ Akt/mTOR pathways, thereby inhibiting the malignant growth of human pancreatic cancer cells 8 . Tan-IIA has also been reported to induce apoptosis and autophagy in acute monocytic leukemia by inhibiting the PI3K/Akt/ mTOR signaling pathway 27 . In the present study, the results revealed that Tan-IIA significantly inhibited the level of PI3K, p-Akt, mTOR and p-mTOR protein in Cholangiocarcinoma cells. Following the addition of the PI3K agonist (740 y-p), the levels of p-Akt, mTOR and p-mTOR increased, and 740 y-p eliminated the inhibitory effect of Tan-IIA on Cholangiocarcinoma cells. These results indicated that Tan-IIA inhibited the proliferation, migration, and invasion of Cholangiocarcinoma cells by inhibiting the PI3K/AKT/mTOR pathway.
Apoptosis is an important manifestation of cell death, and promoting tumor cell apoptosis plays a crucial role in inhibiting the development and progression of tumors 28 . Previous studies have shown that the PI3K/Akt/ mTOR signaling pathway plays an essential role in regulating cell apoptosis 29 . The novel finding in this study is that inhibition of PI3K by 740 y-p eliminated the effect of Tan-IIA on the proapoptosis of Cholangiocarcinoma cells. Our findings indicated that Tan-IIA induced Cholangiocarcinoma cells apoptosis via inhibition of the Akt/ mTOR pathway. In addition, the Bcl-2 family and the caspase family have been reported to participate in the mitochondria-mediated pathway of apoptosis. Caspase-3 belongs to the cysteine-aspartic acid protease (caspase) family, which regulates apoptosis by interacting with caspase-8 and caspase-9 30 . Whereas Bax and Bcl-2 are members of the Bcl-2 gene family, Bcl-2 mainly plays a role in inhibiting apoptosis and can maintain cell survival 31 . Bax, on the other hand, is a pro-apoptotic factor in the Bcl-2 family that can affect the mitochondrial membrane, leading to the release of cytochrome C and the production of reactive oxygen species, in addition, Bax can form www.nature.com/scientificreports/ heterodimer with Bcl-2 and activate endogenous apoptosis. Therefore, Bax is a major regulator involved in the process of apoptosis 32 . Moreover, compared with the expression of Bax and Bcl-2, respectively, the ratio of Bax/ Bcl-2 is more important for apoptosis. In this study, western blotting and flow cytometry assays revealed that  Tan-IIA at concentrations of 0, 10, 20, and 30 µg/mL was added to fresh medium and co-cultured with Cholangiocarcinoma cells for 24 h. Next, the protein expression levels of PI3K, Akt, mTOR, p-Akt, and p-mTOR were measured using western blots.
(D-F) After the addition of 740y-p (10 µg/mL) to the system in which Tan-IIA (24 h IC50 concentration) and Cholangiocarcinoma cells were co-cultured. The inhibitory effect of Tan-IIA on PI3K-Akt-mTOR pathway was diminished under the influence of PI3K agonists. Compared with control, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.  The results demonstrated that caspase3 and Bax/Bcl2 play critical regulatory roles in the progression of Tan-IIA promoted apoptosis in Cholangiocarcinoma cells.

Conclusion
In conclusion, our study revealed the potential anti-tumor effects of Tan-IIA in Cholangiocarcinoma cells.
Further mechanism studies demonstrated that Tan-IIA promoted apoptosis and suppressed malignant growth, invasion, and migration of Cholangiocarcinoma cells through inhibited PI3K/Akt/mTOR signaling pathway. Furthermore, we suggested that Tan-IIA could be a potent agent for the treatment of CCA.