*3.3. Tanshinone and mTOR*

mTOR is a conserved and universally expressed serine-threonine kinase. In mammals, it is usually assembled with Raptor, rictor, lst8, and msin1 to form two catalytic subunits of different protein complexes (mTORC1 sensitive to rapamycin and mTORC2 insensitive to rapamycin) and participates in a variety of signal pathways in vivo to regulate cell proliferation, autophagy, and apoptosis. Among them, mTORC1 is the most important. mTORC1 not only consists of three core components: mTOR, regulatory-associated protein of mTOR (raptor), and mammalian lethal with SEC13 protein 8 (mlst8), but also contains two inhibitory subunits: 40 kDa proline-rich Akt substrate (PRAS40) and regulatoryassociated protein of mTOR (deptor) [125,126]. Here, we mainly review the regulatory effect of Tanshinone on mTORC1.

In terms of assembly, Tanshinone can not only directly inhibit the expression and phosphorylation of mTOR so that it interferes with the assembly of the mTORC1 subunit [23], but also inhibit the promotion of mTORC1 assembled catalyst and stabilizer (heat shock protein 90) [30,127,128]. In terms of activity, the activity of mTORC1 is regulated by growth factors, cell energy, stress, and nucleotides, and the lysosome is the main site of its activation [126,129]. In fact, the activity of mTORC1 is mainly related to the tuberous sclerosis complex (TSC), which can inactivate Ras homolog enriched in the brain (rheb) (an important activator of mTORC1), thereby inhibiting mTORC1 [130]. Interestingly, the existing literature shows that Tanshinone can inhibit not only the expression of growth factors and their receptors [131] but also the expression of rheb [132]. In addition, the activity of mTORC1 is also regulated by the energy sensor AMP-dependent kinase (AMPK). AMPK can promote the inhibition of rheb by TSC by stimulating the gap activity of TSC, resulting in the down-regulation of mTORC1 [133,134], which means that activating AMPK in cancer cells can affect mTORC1 to a certain extent. Interestingly, Tanshinone has been shown to inhibit the mTORC1-mediated signal pathway by activating the AMPK-TSC2 axis [135]. Sestrins negatively regulate mTORC1 signaling through GATOR2/Rag and its components, SESN1 and SESN2, can directly bind to complex and AMPK, resulting in AMPK activation and autophosphorylation in a p53-dependent manner. This stimulates AMPK-mediated TSC2 phosphorylation to negatively regulate mTORC1 signaling [136,137]. Previous literature shows that Tanshinone induces the expression of BECN1 and SESN2 proteins in a dose-dependent manner and then induces autophagy in osteosarcoma cells [138]. It also regulates the upstream gene p53 of the SESN2 protein. Secondly, the down-regulation of pyruvate dehydrogenase kinase 4 (PDK4) has also been reported to lead to the inactivation of mTORC1 [139], and Tanshinone is known as an inhibitor of PDK4, which means that the inhibition of mTORC1 activity by Tanshinone may be partly due to the inhibition of PDK4 [140]. In addition, mitogen-activated protein kinase (MAPK) downstream of the growth factor receptor can also up-regulate mTORC1 activity. MAPK-related signal molecules can promote the activation of mTORC1 through the phosphorylation of Raptor. For example, MEK1/2 can not only phosphorylate Raptor but also promote Raptor phosphorylation through ERK1/2 and P90 ribosomal S6 kinase (RSK1/2). This increases mTORC1 activity [141,142]. Secondly, RAS and RAF kinases, key molecules in the MAPKmediated signal pathway, also regulate the activation of its upstream signal molecule, PI3K [143,144]. In fact, MAPK and mTOR-mediated signaling pathways have mutual crosstalk in many aspects. They not only receive the activation of various growth factors but also have the same downstream molecules, such as Src kinase, FOXO (forkhead box o), c-myc transcription factor, and various metabolism-related enzymes [143]. This means that inhibition of this pathway can inhibit the activation of mTORC1 and its related pathways to a certain extent. Tanshinone not only directly inhibits the expression and phosphorylation of MAPK but also inhibits the activation and expression of related molecules of its

mediated signal pathway, including ERK, JNK, p38 MAPK, Ras, etc. [45,54,145,146]. It can also indirectly affect MAPK by up-regulating ROS in cancer cells [66].

PI3K is a large class of lipid kinases and one of the most important upstream molecules of mTORC1-mediated related pathways. At present, it is mainly divided into three categories: class I (subdivided into classes IA and IB), class II, and class III, of which class I is the most important [142], and class IA PI3K is a heterodimer that consists of a catalytic subunit (P110α, p110β or P110δ) and a regulatory subunit (p85α/p55α/p50α, p85β, or p55γ). It is activated by various growth factors. After being activated, class IA PI3Ks synthesize lipid secondary messenger phosphatidylinositol 3,4,5 triphosphate (PIP3) from phosphatidylinositol 4,5 diphosphate (PIP2), which recruits protein kinase Akt to the plasma membrane, where it is activated by 3-phosphoinositide dependent kinase 1 (PDK1), Subsequently, activated Akt phosphorylates TSC2 and inhibits the TSC complex, eventually leading to the activation of mTORC1 [142,147]. However, as described above, Tanshinone can inhibit the expression of various growth factors and their receptors. In fact, Tanshinone can weaken or even inhibit the induction of PI3K protein expression and phosphorylation by these factors, which is partly because Tanshinone directly inhibits PI3K protein expression and phosphorylation [148–150]. Secondly, Tanshinone also showed the pharmacological action of inhibiting the phosphorylation and expression of catalytic subunit P110α/γ and subunit p85 (down-regulating the expression of its gene) [38,151–153]. In addition, Tanshinone has the pharmacological effect of promoting the expression of phosphatase and tensin homologue (PTEN), a negative regulatory gene of PI3K [133,154,155], which negatively regulates PI3K Akt mTOR signal transduction by transforming PIP3 back to PIP2 [156] and the deletion of this gene usually occurs with the progression of prostate cancer [157,158]. In addition, the expression and phosphorylation of PDK1 and Akt downstream of PI3K were also inhibited by Tanshinone [148–150,159].

The two most important molecules in mTORC1-mediated cell activity are P70S6 kinase 1 (S6K1) and eIF4E binding protein (4E-BP) 1 [160]. The phosphorylation of S6K1 by mTORC1 leads to increased protein and nucleotide synthesis [125,126,160]. 4ebp is a negative regulator of 5- cap dependent mRNA translation, and mTORC1 induces the separation of 4E-BP1 from eIF4E so as to reduce its inhibition of protein synthesis [125,126,160]. By targeting these two molecules, mTORC1 synthesizes proteins required for cell growth, cell cycle process, and cell metabolism, and then induces tumor growth and progression. Tanshinone also has a regulatory effect on these two molecules. As shown in previous literature, Tanshinone can not only prevent the binding of S6K1 to mTORC1 [161] but also significantly inhibit the expression and phosphorylation of S6K1 and 4E-BP1 [159,162]. It is worth mentioning that Tanshinone also regulates the key downstream molecule PKC of mtorc2 [128]. Protein kinase C (PKC) is considered to be the main downstream molecule of mtorc2. Mtorc2 completes various cell activities by targeting the special type of this molecule [126].

This laboratory evidence shows that Tanshinone can inhibit mTOR-mediated tumor malignant behavior in prostate cancer cells by regulating the expression and phosphorylation of upstream and downstream protein molecules of the mTOR-mediated signaling pathway or by regulating other molecular pathways with obvious crosstalk with mTOR. It is worth noting that although Tanshinone is similar to rapamycin in structure, the regulation of mTOR does not directly inhibit the synthesis of mTOR like rapamycin, which means that Tanshinone has fewer side effects on normal tissue and is safer [163,164]. In short, Tanshinone has a significant inhibitory effect on prostate cancer. its pharmacological mechanism. As previously described, it depends on the regulation of the mTOR-mediated signaling pathway, but the current research is still limited, The regulatory mechanism of some Tanshinones on mTOR has only been confirmed in other tumor cells. Therefore, a large number of studies are still needed to further clarify the effect of Tanshinone on the mTOR-mediated signaling pathway in prostate cancer cells. Furthermore, State3 [27] is another key molecule of Tanshinone that exerts an anti-tumor effect. Although it has been confirmed to be involved in the inhibitory effect of Tanshinone on prostate cancer, the

current research is very lacking and should be given enough attention in the future. We plotted the relevant regulation of Tanshinone on the mTOR pathway in Figure 3

**Figure 3.** Effect of Tanshinone on mTOR. induced by Tanshinone are noted by using →, while the inhibition represented by symbol.

#### **4. Dilemma of Clinical Application of Tanshinone**

Tanshinone, as a secondary metabolite, accumulates mainly in the roots of Salvia miltiorrhiza, but in very low yields. Currently, Tanshinone relies on traditional chemical isolation and purification from Salvia divinorum roots [8]. However, traditional methods are characterized by low efficiency, high energy consumption, and unfriendliness to the environment and plant resources [8,165], and secondly, due to the increasing demand for Tanshinone in the market, wild salvia has been over-harvested and the resources are on the verge of extinction [8,165]. Although artificial domestication of salvia has been cultivated since the 1970s, due to the low yield of secondary metabolites and the long growth period of cultivated plants, the production of Tanshinone from cultivated salvia cannot meet the rapidly growing market demand [17]. Therefore, both the reform of the purification process of Tanshinone and the improvement of yield through modern biotechnology have received great attention. Various in vitro culture systems of Salvia divinorum, including suspension cells, guard tissues, adventitious roots, hairy roots, and new techniques such as the use of endophytic fungi and transgenic plants, have been reported to significantly increase the yield of Tanshinone [18,166]. However, these efforts are still insufficient for the increased demand for Tanshinone in the market, and therefore a new technique with a higher yield is urgently needed to provide the supply.

In recent years, the discovery of key genes for the biosynthesis of pharmaceutical active ingredients and the use of synthetic biology strategies to design and modify microbial strains to produce natural products are considered to be promising resource acquisition methods [167]. It is reported that at present, with Saccharomyces cerevisiae as the chassis cell, a high-yield engineering strain of miltiradiene, an important intermediate of Tanshinone, with a yield of up to 488 mg/L has been constructed through functional module design, and cloned the modifying enzyme gene CYP76AH1 of the Tanshinone biosynthesis pathway, which successfully converted miltiradiene into ferruginol [17,168]. However, due to the limited understanding of the transcription genes of Salvia miltiorrhiza, the further elucidation of Tanshinone biosynthesis has been hindered [167,169]. It is worth mentioning that the formation of the furan ring, which has puzzled scientists for a long time, has been gradually decoded [170]. However, there is still a long way to go. It is still necessary to pay more attention to and explore genes and enzymes related to Tanshinone biosynthesis in the future.

Second, like most natural drugs, Tanshinone also has the characteristics of low water solubility, poor stability, large first-pass elimination, and low bioavailability [17,171], which greatly limits the clinical application of Tanshinone. It is reported that when Cryptotanshinone is administered at a body weight of 100 mg/kg (mg/kg), the bioavailability of oral and intraperitoneal injection in rats is 2.1% and 10.6%, respectively. In fact, most of the studies on Tanshinone have pointed out the nanomolar to lower micromolar after oral administration. The maximum/peak concentration (Cmax) value within the range [17,18] greatly limits the clinical application of Tanshinone. Although the currently prepared new formulations of Tanshinone for injection, such as microemulsion, microspheres, solid dispersion, liposomes, and nanoparticles, can significantly improve the bioavailability of Tanshinone, the complexity of the process, high cost, and low tissue specificity still limit the clinical application of these formulations [166,171]. However, it is worth noting that in previous studies, researchers greatly enhanced the specificity of Tanshinone nanoparticles for prostate cancer tissue by combining them with prostate-specific membrane antigen [60], which provides a way for the development of Tanshinone anti-prostate cancer-related nanoparticles in the future. However, the clinical trials of Tanshinone nanoparticles are still lacking, and the clinical application of Tanshinone nanoparticles is still making little progress. In addition, Tanshinone derivatives by changing the Tanshinone skeleton group also seem to be a promising solution, but in most cases, the anti-tumor effect of Tanshinone derivatives will be reduced or even lost, for example, sodium Tanshinone sulfonate [172,173]. Although previous studies have found some Tanshinone derivatives with significantly increased bioavailability and anti-tumor efficacy [42,54,55], due to the lack of in vivo experiments and related toxicological experiments, the safety of these Tanshinone derivatives is also a worrying problem and is also limited by the lack of raw materials. Therefore, future research should not only explore new Tanshinone derivatives but also focus on the safety of Tanshinone derivatives.

In addition, the safety of Tanshinone is also an aspect of concern for scientists. Although the experiments of Wang et al. confirmed that in both acute and subchronic toxicity studies, no abnormalities of other organs were observed in Sprague Dawley rats treated with Tanshinone injection, except that it caused focal inflammation at the injection site [174], which is consistent with the conclusions drawn from the in vivo experiments of Tanshinone and prostate cancer. In fact, limited studies pointed out that high concentrations of TsIIA and CYT showed serious growth inhibition, developmental malformation, and cardiotoxicity to zebrafish embryos [175,176]. Similarly, high concentrations of TsIIA were also toxic to normal human endothelial cells. It was reported that high concentrations of (25 uM) TsIIA could kill endothelial cells within 24 h [177]. Therefore, future research should focus on the toxic reaction of Tanshinone to normal tissues to confirm the safety and stability of Tanshinone, which is crucial for the development of clinical drugs.

#### **5. Conclusions and Prospects**

It is well understood that blocking the products of a single signal pathway or gene is frequently insufficient to prevent or treat malignant tumors. Tanshinone is expected to be a candidate drug for the treatment of prostate cancer because it can regulate the proliferation, survival, migration, and metabolism of prostate cancer through multiple targets, links, and pathways. Although the current experiments have confirmed the significant antiprostate cancer effect of Tanshinone, there are still some problems with the current research. First, the current research on the anti-prostate cancer effect of Tanshinone is still limited, and most of the studies remain to verify the expression of proteins and genes related to apoptosis, cell cycle, and invasion in prostate cancer cells. In order to further explore the specific molecular mechanism that causes these changes, more research is still needed to explore the specific mechanism of Tanshinone against prostate cancer in the future. In addition, as described above, TsI and DHT have stronger anti-prostate cancer effects. However, current research is mostly focused on TsIIA and CYT. TsI and TsIIA should be given sufficient attention in the future. In addition, Tanshinone can significantly regulate the tumor immune microenvironment, which is more important for prostate cancer with poor immunotherapeutic efficacy. In the future, we should also pay attention to the effect of Tanshinone on the immune microenvironment of prostate cancer.

Although Tanshinone has been widely reported to be beneficial to health, its clinical application is still subject to many restrictions. First, as a secondary metabolite, Tanshinone mainly comes from the root of Salvia miltiorrhiza. Due to the characteristics of the long growth cycle and low yield of cultivated plants, the supply of Tanshinone is difficult to meet the market demand, although the current research on Tanshinone biosynthesis has made good progress. However, there are many key conversion processes that we are not clear about. Hence, it is still necessary to further explore the genes and enzymes related to Tanshinone biosynthesis. Secondly, like most natural drugs, Tanshinone also has the characteristics of low water solubility and low bioavailability. Fortunately, the currently prepared Tanshinone injection microemulsions, microspheres, solid dispersions, liposomes, nanoparticles, and other new dosage forms can greatly improve the bioavailability of Tanshinone. However, it is still subject to the limitations of complex processes, high costs, and low tissue specificity. Therefore, further preclinical and clinical studies are needed to explore new preparations with low cost, simple processes, and high tissue specificity. It was previously reported that Tanshinone nanoparticles developed in combination with prostate-cancer-specific membrane antigen can significantly enhance the tissue specificity of Tanshinone nanoparticles for prostate cancer. Therefore, future research will continue to consider the development of drug nanoparticles in combination with tumor-specific antigens. Secondly, Tanshinone derivatives synthesized by the Tanshinone skeleton should also be a focus of future research. Although the anti-tumor effect of these derivatives is significantly lower than that of Tanshinone in most cases, some Tanshinone derivatives with increased bioavailability and anti-tumor effects have been found in the current research. Secondly, the current research on the toxic reaction of Tanshinone and Tanshinone derivatives to normal tissues is limited and cannot confirm the safety and stability of Tanshinone and its derivatives. Therefore, more attention should be paid to the study on the toxic reaction of Tanshinone and its derivatives to normal tissues in the future to further clarify the safety of Tanshinone and its derivatives. Finally, in order to better guide future research and verify the possibility of Tanshinone as an anti-prostate cancer drug, we conducted a bioinformatics analysis on the four components of Tanshinone to further determine the possible anti-prostate cancer targets of Tanshinone.

First, after determining the structural formulas of four Tanshinone components (TsI, TsIIA, DHTI, and CYT) through Pubchem (https://pubchem.ncbi.nlm.nih.gov (accessed on 10 July 2022)), we screened drug targets using the Swiss target prediction database (http://www.swisstargetprediction.ch/ (accessed on 10 July 2022)) and the traditional Chinese Medicine System Pharmacology database (https://www.tcmsp-e.com/ (accessed on 10 July 2022)), and submitted the collected targets to the UniProt database (https: //www.uniprot.org/ (accessed on 10 July 2022)), limiting the species to "Homo sapiens", converting the protein targets into official gene names, select gene targets with probability greater than 0 in the Swiss target prediction database, and obtain drug target genes: TsI (53), TsIIA (145), DHTI (80), CYT (94) after excluding duplicate genes. Secondly, we searched the Genecards (https://www.genecards.org/ (accessed on 10 July 2022)) and Disgenet databases (https://www.disgenet.org/ (accessed on 10 July 2022)) by using the keyword

"prostate cancer" to obtain disease targets, and obtained 12,555 disease target genes after excluding duplicate targets in the two databases. Then, we input the drug target genes and disease target genes obtained by the above methods into the online Venny 2.1 mapping platform (https://www.bioinformatics.com.cn/ (accessed on 10 July 2022)) to obtain the cross-target genes of "prostate cancer" and "four Tanshinone components" (Figure 3), TsI (49), TsIIA (126), DHTI (68) and CYT (85) were obtained (Figure 4).

**Figure 4.** Venny of four components of Tanshinone ((**a**): Tanshinone I and Prostate Cancer; (**b**): Tanshinone IIA and Prostate Cancer; (**c**): Dihydrotanshinone I and Prostate Cancer; (**d**): Cryptotanshinone and Prostate Cancer).

These cross genes are considered possible targets of Tanshinone against prostate cancer, and we analyzed them through a range of methods. First, we uploaded these genes to the String online database (https://string-db.org/ (accessed on 10 July 2022)) to form a protein-protein interaction map. The species is "human" and the comprehensive score > 0.4 is the critical value for inclusion in the network. We further visualized these results with the help of Cytoscape 3.9.1 (Figure 5), To find the key targets of four Tanshinone components. At the same time, we also carried out the Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. After inputting these gene data into the David data platform (https://david.ncifcrf.gov/tools.jsp (accessed on 10 July 2022)) and setting the species as "Homo species", we further analyzed the enrichment analysis of four Tanshinone components on prostate cancer-related biological processes (BP), cellular components (CC), molecular functions (MF) and signal pathways. For the obtained information, we met the *p*-value < 0.05; sorted according to the number

of genes; selected the top 10 enrichment information of BP, CC, and MF, and the top 20 enrichment information of KEGG; and used the bioinformatics online platform (https: //www.bioinformatics.com.cn/ (accessed on 10 July 2022)) to visualize the analysis results (Figures 6 and 7).

**Figure 5.** Protein network analysis of four Tanshinone components ((**a**): Tanshinone Ir; (**b**): Tanshinone IIA; (**c**): Dihydrotanshinone I; (**d**): Cryptotanshinone).

Finally, our results show that the target of TsIIA is significantly more than the other three components. Among them, cellular tumor antigen p53 (TP53), myc proto-oncogene protein (MYC), transcription factor AP-1 (JUN), Src, Caspase-3 (CASP3), and EGFR play a key role in the anti-prostate cancer process of TsIIA, followed by CYT, which has more targets than the other two components. EGFR, TNF, STAT3, prostaglandin G/H synthase 2 (PTGS2,), Transcription factor p65 (RELA), and other targets are the key targets of CYT, The second is DHTI, STAT3, receptor tyrosine-protein kinase erbB-2 (ERBB2), CASP3, EGFR, PTGS2, etc. are the key targets of DHTI, and finally, TsI, Vascular endothelial growth factor A(VEGFA), EGFR, MAPK14, protein tyrosine phosphatase receptor type C (PTPRC), enhancer of zeste homolog 2 (EZH2), etc. are the key targets. These key targets are essential for various biological processes of prostate cancer, and these components participate in regulating multiple signaling pathways.

**Figure 6.** Go enrichment analysis of four components of Tanshinone((**a**): Tanshinone Ir; (**b**): Tanshinone IIA; (**c**): Dihydrotanshinone I; (**d**): Cryptotanshinone).

Secondly, our results also show that the biological processes of the four Tanshinone components mainly occur in the cell and participate in the activation and binding of a series of cell receptors and cascade downstream signaling pathways. KEGG analysis shows that PI3K Akt signaling pathway, MAPK signaling pathway, Ras signaling pathway, and rap signaling pathway play a key role in the anti-prostate cancer process of Tanshinone, which is consistent with previous studies, These pathways have significant significance for the metastasis, progression, and angiogenesis of prostate cancer. In addition, as mentioned above, Tanshinone has a significant effect on the regulation of immune pathways. we note that the four Tanshinone components have a regulatory effect on the PD-1/PD-L1 signaling pathway, T cell-related pathways, and immune helper cells and related factors mediated signaling pathways. Although these results are not in our screening results, they are statistically different (*p*-value < 0.05). This is consistent with the protein network analysis, which may have more significant significance for immunotherapy-insensitive prostate cancer. In addition, our results also show that Tanshinone has a regulatory effect on microRNAs in prostate cancer cells, and these small molecules are also essential for tumor survival and metastasis. In conclusion, our results and the existing laboratory data show that Tanshinone can inhibit the metastasis, invasion, and progression of prostate cancer through multiple targets and pathways. However, the current research is limited. Therefore, more research should be carried out in the future to further clarify the relevant mechanisms and molecular pathways of Tanshinone against prostate cancer, and more attention should be paid to the bioavailability and toxicological experiments of Tanshinone.

**Figure 7.** KEGG enrichment analysis of four components of Tanshinone ((**a**): Tanshinone Ir; (**b**): Tanshinone IIA; (**c**): Dihydrotanshinone I; (**d**): Cryptotanshinone).

**Author Contributions:** K.T. conceptualized the review, analyzed the data, and helped to write the manuscript. W.L., T.H., S.X., B.C., Y.Y. and W.Z. helped to write the manuscript and prepared the figures. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by the National Natural Science Fund of China (81660263), Science and Technology Fund Project of Guizhou Health Commission (gzwkj2021–211), and Doctoral Fund of Affiliated Hospital of Guiyang Medical College, Guizhou Province, China (C-2012-6).

**Acknowledgments:** Thanks all the research participants of this study. This study was supported by the National Natural Science Fund of China (81660263), Science and Technology Fund Project of Guizhou Health Commission (gzwkj2021-211), and Doctoral Fund of Affiliated Hospital of Guiyang Medical College, Guizhou Province, China (C-2012-6).

**Conflicts of Interest:** The authors declare no conflict of interest.
