*1.4. Tanshinone and PCa*

In a recent population-based retrospective study of 40,692 men diagnosed with PCa, the protective effect of salvia miltiorrhiza on PCa was confirmed by a 5–10% increase in survival rates among men who took salvia miltiorrhiza compared with those who did not. This protective effect is positively correlated with the dose and time of salvia miltiorrhiza use [32]. However, unfortunately, there is only one study on clinical evidence of salvia miltiorrhiza and PCa at present, and in this study, the experimenters also did not further verify whether the intake of salvia miltiorrhiza could inhibit the occurrence of castrationresistant PCa, the occurrence of biochemical recurrence of PCa after radical prostatectomy, and the increase in the aggressivity of PCa.

However, there are many in vitro studies on Tanshinone and PCa cell lines. Human PCa cells: LNCaP, PC3, DU145, and 22RV1, which are commonly used as cell models for PCa in vitro experiments, have different characteristics, respectively. For example, LNCaP has functional ARs, so it is sensitive to androgen and secretes prostate-specific antigen (PSA). 22RV1 cells were androgen-sensitive but not androgen-dependent and showed low aggressiveness.PC3 cells do not depend on androgens, are highly invasive, and have a strong potential for metastasis.DU145 cells are also androgen-independent cells with strong proliferation ability but only moderate metastasis ability [33]. In these in vitro experiments, the researchers pretreated PCa cell lines with varying biological characteristics with a specific concentration of Tanshinone and then used various modern molecular biological methods to identify proteins involved in the cell cycle, apoptosis, growth, and metastasis. Finally, they confirmed that Tanshinone in vitro by adjusting the related protein expression and signaling pathway in PCa cells induced the stagnation of the cell cycle and apoptosis, inhibiting metastasis and invasion of tumor cells (Figure 2). We put the current relevant Tanshinone in vitro effect on Pca-related research summarized in Table 1.

**Figure 2.** Effect of Tanshinone on prostate cancer. induced by Tanshinone are noted by using →, while the inhibition represented by symbol.

Similarly, in vivo experiments with Tanshinone against PCa are also under way. In the experiments, the researchers transplanted human PCa cells (LNCaP, PC3, and 22Rv1) subcutaneously or in situ into immunodeficient mice that did not reject human cells to establish animal models. Finally, it was confirmed that Tanshinone could effectively inhibit the growth of tumors in vivo, whether injected orally or subcutaneously/intraperitoneally. It is worth noting that in the current animal experiment, it has not been observed that the

activity of Tanshinone on other organs and tissues, except tumor tissues, has toxic effects. Table 2 summarizes the current state of Tanshinone and PCa in vivo research.

**Table 1.** Tanshinone and PCa in vitro.



**Table 2.** In animal study of Tanshinone and PCa.

*Note*: *Tanshinone I* (*TsI*), *Tanshinone II A* (*TsIIA*), *Tanshinone II B* (*TsIIB*), *Dihydrotanshinone I* (*DHT*), *Cryptotanshinone* (*CYT*), *Tanshinone derivatives* (*TsD*), *Tanshinone IIA derivatives* (*TIIAD*), *TsIIA nanoparticles* (*TsIIAD*), *Nanoparticles containing doxorubicin and Tanshinone* (*NCDT*), *Nanoparticles synthesized from salvia miltiorrhiza extract* (*SMED*).

#### **2. Tanshinone as a Potential Anti-Cancer Agent for PCa**

*2.1. Tanshinone-Induced Stagnation of the PCa Cell Cycle*

A normal cell cycle is essential for homeostasis and normal development of an organism, and dysregulation of this system often leads to uncontrolled cell proliferation, leading to the occurrence of tumors [61]. The progress of the cell cycle depends on the regulation of various cyclins, cell cycle-dependent protein kinases (CDK), and CDK inhibitors. In mammals, the normal progress of the cell cycle is carefully edited with the expression of different cyclin proteins in characteristic cell cycle stages as the center of the molecular mechanism, and they are assembled with specific CDKs to promote the phosphorylation of the retinoblastoma tumor suppressor (Rb) so as to promote the cell cycle to the next stage [62,63]. Therefore, in order to maintain their own biological characteristics (uncontrolled proliferation), tumor cells induce protein expression of cell cycle progression and the genes encoding the cell cycle inhibitors are missing, or there is inactivation of cell cycle regulators such as Rb and P53 [64,65]. Interestingly, current publications suggest that Tanshinone can significantly inhibit PCa proliferation by inducing PCa cell cycle arrest. The mechanism is that Tanshinone can significantly reduce cyclinD1, A, and E (cyclinD1, A, and E) in PCa cells [34,51,54,56], which seems to be partly due to Tanshinone's ability to directly inhibit the phosphorylation of the pro-proliferative signaling pathway STAT3Tyr705 in PCa cells [51]. Tanshinone also has a significant ability to relate to CDK family-relative proteins in PCa cells. Previous studies have shown that Tanshinone can not only inhibit the expression of CDK in PCa cells but also increase the activation and expression of CDK inhibition (P21, P27, p16) by promoting the phosphorylation of Ser15 residues of p53 [34,59]. It can also significantly inhibit the hyperphosphorylation of Rb in PCa cells [54], which seems to be caused to some extent by Tanshinone increasing ROS in PCa cells [35,66]. In addition, the normal of the cell cycle also depends on other factors of the adjustment. These factors are not only for genomic stability and integrity maintenance, which is very important, but they also participate in the spindle and maintain normal mitosis of the structure of the adjustment. It is interesting to note that, according to the existing literature, Tanshinone and its derivatives in PCa cells also show the effect on the regulation of these factors. including: stasis and DNA damage-inducing protein (GADD45A), polo-like kinase 1 (PLK1), and checkpoint-related protein [54].

#### *2.2. Tanshinone-Induced Apoptosis of PCa Cells*

According to reports, tumor progression frequently means apoptosis and proliferation imbalances, which are related to apoptosis signaling molecules and proteins that scientists are concerned about [67,68]. Interestingly, previous studies have shown that Tanshinone induces apoptosis in PCa cells by altering the balance between the expression of pro-apoptotic and anti-apoptotic proteins in the b-cell lymphoma-2 (bcl-2) family [34,35,38,50,51,55–58]. Secondly, caspase containing cysteine is a key enzyme for performing apoptosis, and caspase-3 is a key executor of apoptosis in mammalian cells [68]. Studies have shown that Tanshinone can significantly induce its expression and phosphorylation in PCa. It also significantly increases the activation and expression of caspase-8 or-9 upstream promoters [34,35,38,50,51,55–58]. The regulation of these proteins by Tanshinone is partly due to the inhibition of the pi3k/akt pathway and MAPK pathway and the expression of hypoxia stress factor 1 (HIF-1) in PCa cells [38,45]. In fact, Tanshinone also has a significant effect on the regulation of ROS and LC3II in PCa cells for the process [36]. In addition, Tanshinone induces apoptosis of PCa cells by other mechanisms. As the report goes, Tanshinone activates mitochondrial-dependent apoptosis of PCa cells by inhibiting the expression of mitochondrial protective Bcl-2 family protein Mcl-1 by inducing the cleavage of ADP ribose polymerase (PARP), promoting the release of cytochrome c from the mitochondria to the cytoplasm and reducing mitochondrial membrane potential. which seems to be partly due to the inhibition of pik3/Akt in PCa cells [38]. Similarly, Tanshinone derivatives also have the same pharmacological effects. In the experiments conducted by Wang et al., they found that Tanshinone derivatives induce PCa cell apoptosis by regulating p53, ERK1, BAX, P38, Bcl-2, caspase-8, cleavedcaspase-8, and PARP1, and significantly affect the phosphorylation of ERK1 and P38 in P38 [54]. Second, Tanshinone has been shown to induce ER stress pathway apoptosis in PCa cells by increasing the expression of key proteins of ER stress pathway apoptosis, such as glucose regulatory protein 78 (BIP/GRP78), ER stress sensor (IRE1-) and its downstream target CAAT/enhancer binding protein homologous protein/growth arrest and DNA-damage-inducing gene 153 (gadd153/cho) [48,53]. In addition, Tanshinone has been reported to up-regulate Fas sensitivity of PCa cells and promote Fas (APO1/CD95) mediated apoptosis of PCa cells by inhibiting phosphorylation of Janus kinase (JAK) and p38MAPK [45]. In addition, Tanshinone has been shown to be an effective sensitizer of tumor necrosis factor-associated apoptosis-inducing ligand (TRAIL) to enhance TRAIL-mediated apoptosis, which seems to be related to Tanshinone activation of mir135A-3p mediated up-regulation of death receptor 5 (DR5) in PCa cells [52].

#### *2.3. Tanshinone-Induced Motility Inhibition of PCa Cells*

Metastasis of tumor cells is often one of the main causes of death in patients. Previous studies have shown that prostate cancer can metastasize through a variety of mechanisms. Among these are the secretion of matrix metalloproteinases (MMPs) to dissolve the extracellular matrix, the promotion of angiogenesis, the recruitment of some cytokines and chemokines, and so on [69,70], which pose significant challenges to clinicians' diagnosis and treatment. Fortunately, existing literature shows that Tanshinone can act as an effective inhibitor of the metastasis and invasion of PCa cells. In the experiment of WuCY et al., Tanshinone inhibits the translocation of phosphorylated STAT3 and protein expression of P-STAT3 and Skp2 in PCa cells in a dose-dependent manner, resulting in inhibition of the translation and transcription of RhoA and SNAI1 genes in PCa cells, which results in reduced invasiveness of PCa cells [35]. Interestingly, inhibition of this pathway also has a

significant impact on the ability of PCa cells to recruit macrophages. Inhibition of this pathway will lead to reduced secretion of related cytokines in PCa cells [32], including CCL2, CCL5, interleukin-1 receptor antagonists (il-1ra) and intercellular adhesion molecule-1, which are significantly associated not only with PCa metastasis but also with the ability to recruit macrophages of Pca [71,72]. In addition, literature has shown that Tanshinone has a significant pharmacological effect of inhibiting angiogenesis in PCa cells, which seems to be related to the inhibition of hif-1 expression induction of vascular endothelial growth factor and its receptor in PCa cells by Tanshinone intervention [30,31]. Similarly, Tanshinone derivatives also have significant inhibitory effects on the metastasis and invasion of PCa cells. In the experiment of Wang et al., they found that Tanshinone derivatives can reduce the expression of protein molecules related to metastasis and invasion in PCa cells, including MMP-1, MMP-9, and VEGF-1 [54].
