Tanshins and Chemotherapy

Tanshinones, just like berberine down-regulate CYP3A4.1 This enzyme detoxifies (breaks down) chemo agents. This means these products make chemo more effective as well as reduce side effects of the chemo. It increases drug efficacy and gives patients better results.2

Anticancer effects of Tanshinone IIA

The anticancer effects of Tanshinone IIA (Tan IIA) has been confirmed by numerous researches. However, the underlying mechanism remained to be integrated in systematic format. Systems biology embraced the complexity of cancer; therefore, a system study approach was proposed in the present study to explore the anticancer mechanism of Tan IIA based on network pharmacology. Pathways on anticancer effect of Tan IIA were delineated. Five functional units were clarified according to literature: including regulation on apoptosis, proliferation, sustained angiogenesis, autophagic cell death, and cell cycle. The Gene Ontology (GO) analysis confirmed the classification was statistically significant.
The study by Cao et al., (2018) provides a systematic methodology for discovering the coordination of the anticancer pathways regulated by Tan IIA via protein network.3

Mitochondrial homeostasis has been increasingly viewed as a potential target of cancer therapy, and mitochondrial fission is a novel regulator of mitochondrial function and apoptosis. The aim of a study by Jieensinue et al., (2018) was to determine the detailed role of mitochondrial fission in SW837 colorectal cancer cell viability, mobility and proliferation. In addition, the current study also investigated the therapeutic impact of Tanshinone IIA (Tan IIA), a type of anticancer adjuvant drug, on cancer mitochondrial homeostasis.

Altogether, their results identified mitochondrial fission as a new potential target to control cancer viability, mobility and proliferation. Furthermore, our findings demonstrate that Tan IIA is an effective drug to treat colorectal cancer by activating JNK-Mff-mitochondrial fission pathways.4
IL-2-based therapy is a promising tool to treat colorectal cancer, but drug resistance always occurs in clinical practice. Mitochondrial fission is a novel target to modulate cancer development and progression. The aim of a study by Qian et al., (2018) was to explore the effect of IL-2 combined with Tan IIA on SW480 colorectal cancer cell apoptosis in vitro and to determine whether IL-2/Tan IIA co-treatment could reduce SW480 cell viability via activating mitochondrial fission.

They demonstrated that Tan IIA/IL-2 combination therapy controlled INF2-related mitochondrial fission via the Mst1-Hippo pathway. Moreover, Mst1 knockdown abrogated Tan IIA/IL-2-activated mitochondrial fission. Altogether, their results demonstrated that Tan IIA enhances the therapeutic efficiency of IL-2-mediated SW480 colorectal cancer cell apoptosis via promoting INF2-related mitochondrial fission and activating the Mst1-Hippo pathway.5

A study on anticancer activity of tanshinone II A against human breast cancer

Zhang PR, Lü Q. Sichuan Da Xue Xue Bao Yi Xue Ban. 2009 Mar;40(2):245-9.

Objective: To investigate the proliferation inhibition and apoptosis-associated genes expression of both human breast cancer cells with estrogen receptor (ER) positive and negative (MCF-7 and MDA-MB-231) which treated with tanshinone II A, and to elucidate its mechanism of activity.

Methods: Human ER positive breast cancer cells (MCF-7) and ER negative cells (MDA-MB-231) were tested in vitro for cytotoxicity of tanshinone II A with MTT method. The effect of tanshinone II A on DNA synthesis and apoptosis of both human breast cancer cells were evaluated with Brdu incorporation and flow cytometry. Immunohistochemistry were applied to test the P53, CerBb-2 and Bcl-2 protein expression of both cells.

Results: After Tanshinone II A treatment, a dose- and time-dependent decreased proliferation in both MCF-7 and MDA-MB-231 cells were observed (P < 0.05) with a IC50 0.25 microg/mL. A decreased BrdU incorporation and an increased apoptosis in both cells were also observed (P < 0.05 and P < 0.01 respectively). Immunohistochemistry test demonstrated that tanshinone A upregulate P53 expression in both cells and also weakly upregulate the CerBb-2 expression in MCF-7 (P < 0.05), whereas no influence on CerBb-2 expression of MDA-MB-231 and on Bcl-2 expression of both cells were demonstrated (P > 0.05).

Conclusion: This study suggested that tanshione II A could inhibit the proliferation, induce apoptosis of ER-positive breast cancer cell MCF-7 and ER-negative breast cancer cell in vitro. The mechanism may be associated with the inhibition of DNA synthesis, induction of apoptosis, but may not with the expression level of gene p53, cer Bb-2 and bcl-2.

A study on the effect of Tanshinone II A against human breast cancer in vivo

Zhang X, Zhang PR, Chen J, Lü Q. Sichuan Da Xue Xue Bao Yi Xue Ban. 2010 Jan;41(1):62-7.

To confirm Tanshinone II A's (Tan II A) anti-cancer activity on nude mice bearing human breast cancer cells with estrogen receptor (ER) positive and negative and to elucidate the mechanism of its activity in vivo.

Methods: Established the animal model of nude mices bearing human breast cell, both ER positive MCF-7 and ER negative MDA-MB-231, each group was divided into 3 subgroups, respectively by intraperitoneal injection of Tan II A at a dose of 30 mg/kg 4 times/week, by gavage of Tamoxifen at a dose of 1 mg/kg 7 times/ week and by solvent control for 4 weeks. All animals were tested for anti-cancer activity including the weights and the volumes of the tumor, apoptosis index by flow cytometry and expression of p53, bcl-2, cerbB-2 by immunohistochemistry method after the treatment.

Results: In MCF-7 group, there were a 33.64% tumor mass volume reduction and a 32.24% tumor mass weight reduction after Tan II A treatment; in MDA-MB-231 group, a 38.34% tumor mass volume reduction and a 39.82% tumor mass weight reduction were observed in Tan II A subgroups; the differences between Tan II A and Tamoxifen or solvent control were statistically significant in both groups (P < 0.05); increase of apoptosic fiction by flow cytometry examination in Tan II A subgroups in both MCF-7 (48.31% +/- 5.84%) and MDA-MB-231(50.25% +/- 5.03%) groups were observed, there were both significant differences between Tan II A and the other subgroups (P < 0.05). Statistically significant decrease of p53 and bcl-2 expression were observed in Tan lI A between solvent control subgroup in both MCF-7 and MDA-MB-231 groups (P < 0.05) while cerbB-2 had no significant difference with control group (P > 0.05).

Conclusion: Tan lI A can inhibit both breast cancer cell MCF-7 and MDA-MB-231 growth in vivo, which had better anti-cancer effect than Tamoxifen. The mechanism may be associated with the induction of apoptosis, down regulation of the expression level of gene bcl-2 and p53, but may not with the expression level of cerbB-2.

Tanshinone IIA inhibits viral oncogene expression leading to apoptosis and inhibition of cervical cancer.

Munagala R, Aqil F, Jeyabalan J, & Gupta RC. Cancer Letters, vol. 356, no. 2, pp. 536–546, 2015.

Human papilloma virus (HPV) is the well-established etiological factor of cervical cancer. E6 and E7 oncoproteins expressed by HPV are known to inactivate tumor suppressor proteins p53 and pRb, respectively. Tanshinone IIA (Tan IIA) is a diterpenoid naphthoquinone found in the traditional Chinese medicine Danshen (Salvia sp.). Tan IIA has been shown to possess anti-tumor activity against several cancer types.

In this study, we show that Tan IIA potently inhibited proliferation of the human cervical cancer CaSki, SiHa, HeLa and C33a cells. Mechanistically in HPV positive CaSki cells, Tan IIA was found to (i) downregulate expression of HPV E6 and E7 genes and modulate associated proteins E6AP and E2F1, (ii) cause S phase cell cycle arrest, (iii) induce accumulation of p53 and alter expression of p53-dependent targets, (iv) modulate pRb and related proteins, and (v) cause p53-mediated apoptosis by moderating Bcl2, Bax, caspase-3, and PARP cleavage expressions. In vivo, Tan IIA resulted in over 66% reduction in tumor volume of cervical cancer xenograft in athymic nude mice. Tan IIA treated tumor tissues had lower expression of proliferation marker PCNA and changes in apoptosis targets were in agreement with in vitro studies, further confirming reduced proliferation and involvement of multiple targets behind anti-cancer effects.

This is the first demonstration of Tan IIA to possess significant anti-viral activity by repressing HPV oncogenes leading to inhibition of cervical cancer. Together, our data suggest that Tan IIA can be exploited as a potent therapeutic agent for the prevention and treatment of cervical and other HPV-related cancers.

Synergistic antitumor effects of tanshinone II A in combination with cisplatin via apoptosis in the prostate cancer cells

Hou LL, Xu QJ, Hu GQ, Xie SQ. Yao Xue Xue Bao. 2013 May;48(5):675-9.

Treatment with the combination of Chinese herbs and cytotoxic chemotherapies showed a higher survival rate in clinical trials. In this report, the results demonstrated that the tanshinone II A, a key component of Salvia miltiorrhiza bunge, when it is combined with the cytotoxic drug cisplatin showed synergistic antitumor effects on human prostate cancer PC3 cells and LNCaP cells in vitro.

Antiproliferative effects were detected with MTT assay. Cell cycle distribution and apoptosis were detected by flow cytometer. Protein expression was detected by Western blotting. The intracellular concentration of cisplatin was detected by high performance liquid chromatography. The results demonstrated that tanshinone II A significantly enhanced the antiproliferative effects of cisplatin on human prostate cancer PC3 cells and LNCaP cells with the increase of the intracellular concentration of cisplatin.

These effects were correlated with cell cycle arrested at S phase and cell apoptosis. The apoptosis might be achieved through death receptor pathway and mitochondrial pathway. Furthermore, the Bcl-2 family members were also involved in this apoptotic process. Collectively, these results indicated that the combination of tanshinone II A and cisplatin had a better treatment effect in vitro not only on androgen-dependent LNCaP cells but also on androgen-independent PC3 cells.

The Herbal Compound Cryptotanshinone Restores Sensitivity in Cancer Cells that are Resistant to the Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand

Tse AK-W, Chow K-Y, Cao H-H, et al. jbc.M113.483909. doi: 10.1074/jbc.M113.483909

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis and kills cancer cells but not normal cells. However, TRAIL resistance due to low level of TRAIL receptor expression is widely found in cancer cells and hampers its development for cancer treatment. Thus, the agents that can sensitize the tumor cells to TRAIL-mediated apoptosis are urgently needed.

We investigated whether tanshinones, the major bioactive compounds of Salvia miltiorrhiza (Danshen), can up-regulate TRAIL receptor expression. Among the major tanshinones being tested, cryptotanshinone (CT) showed the best ability to induce TRAIL receptor 2 (DR5) expression. We further showed that CT was capable of promoting TRAIL-induced cell death and apoptosis in A375 melanoma cells. CT-induced DR5 induction was not cell type-specific, as DR5 induction was observed in other cancer cell types. DR5 knockdown abolished the enhancing effect of CT on TRAIL responses.

Mechanistically, induction of the DR5 by CT was found to be p53-independent but dependent on the induction of CCAAT/enhancer-binding protein-homologous protein (CHOP). Knockdown of CHOP abolished CT-induced DR5 expression and the associated potentiation of TRAIL-mediated cell death. In addition, CT-induced ROS production preceded up-regulation of CHOP and DR5, and consequent sensitization of cells to TRAIL. Interestingly, CT also converted TRAIL-resistant lung A549 cancer cells into TRAIL-sensitive cells. Taken together, our results indicate that CT can potentiate TRAIL-induced apoptosis through up-regulation of DR5.

Tanshinone IIA and non-small cell lung cancer

The binding mode of tanshinone IIA within the crystal structure of the VEGFR2 protein was evaluated with molecular docking analysis by use of the CDOCKER algorithm in Discovery Studio 2.1. The CCK-8 results showed that tanshinone IIA can significantly inhibit A549 cell proliferation in a dose- and time-dependent manner. The expression of VEGF and VEGFR2 was decreased in Western blot. Finally, molecular docking analysis revealed that tanshinone IIA could be stably docked into the kinase domain of VEGFR2 protein with its unique modes to form H-bonds with Cys917 and π-π stacking interactions with Val848. In conclusion, tanshinone IIA may suppress A549 proliferation, induce apoptosis and cell cycle arrest at the S phase. This drug may suppress angiogenesis by targeting the protein kinase domains of VEGF/VEGFR2.6

Tanshinone IIA Reverses the Malignant Phenotype of SGC7901 Gastric Cancer Cells

Min Xu, Fa-Le Cao, Nai-Yi Li, et al. Asian Pac J Cancer Prev. 2013;14(1):173-7.

Backgrounds: Tanshinone IIA (TIIA), a phenanthrenequinone derivative extracted from Salvia miltiorrhiza BUNGE, has been reported to be a natural anti-cancer agent in a variety of tumor cells. However, the effect of TIIA on gastric cancer cells remains unknown. In the present study, we investigated the influence of TIIA on the malignant phenotype of SGC7901 gastric cancer cells. Methods: Cells cultured in vitro were treated with TIIA (0, 1, 5, 10g/ml) and after incubation for different periods, cell proliferation was measured by MTT method and cell apoptosis and cell cycling were assessed by flow cytometry (FCM). The sensitivity of SGC7901 gastric cancer cells to anticancer chemotherapy was investigated with the MTT method, while cell migration and invasion were examined by wound-healing and transwell assays, respectively.

Results: TIIA (1, 5, 10g/ml) exerted powerful inhibitory effects on cell proliferation (P < 0.05, and P < 0.01), and this effect was time- and dose-dependent. FCM results showed that TIIA induced apoptosis of SGC7901 cells, reduced the number of cells in S phase and increased those in G0/G1 phase. TIIA also significantly increased the sensitivity of SGC7901 gastric cancer cells to ADR and Fu. Moreover, wound-healing and transwell assays showed that TIIA markedly decreased migratory and invasive abilities of SGC7901 cells. Conclusions: TIIA can reverse the malignant phenotype of SGC7901 gastric cancer cells, indicating that it may be a promising therapeutic agent.

Roles of Reactive Oxygen Species in Anticancer Therapy with Salvia miltiorrhiza Bunge

Hung YC, Pan TL & Hu WL. Oxidative Medicine and Cellular Longevity, vol. 2016, Article ID 5293284, 2016.

Cancer is a leading cause of death worldwide. We aim to provide a systematic review about the roles of reactive oxygen species (ROS) in anticancer therapy with Salvia miltiorrhiza Bunge (Danshen). Danshen, including its lipophilic and hydrophilic constituents, is potentially beneficial for treating various cancers. The mechanisms of ROS-related anticancer effects of Danshen vary depending on the specific type of cancer cells involved. Danshen may enhance TNF-????-induced apoptosis, upregulate caspase-3, caspase-8, caspase-9, endoplasmic reticulum stress, P21, P53, Bax/Bcl-2, DR5, and AMP-activated protein kinase, or activate the p38/JNK, mitogen-activated protein kinase, and FasL signaling pathways. Conversely, Danshen may downregulate human telomerase reverse transcriptase mRNA, telomerase, survivin, vascular endothelial growth factor/vascular endothelial growth factor receptor 2, CD31, NF-????B, Erk1/2, matrix metalloproteinases, microtubule assembly, and receptor tyrosine kinases including epidermal growth factor receptors, HER2, and P-glycoprotein and inhibit the PI3K/Akt/mTOR or estrogen receptor signaling pathways. 

Therefore, Danshen may inhibit cancer cells proliferation through antioxidation on tumor initiation and induce apoptosis or autophagy through ROS generation on tumor progression, tumor promotion, and tumor metastasis. Based on the available evidence regarding its anticancer properties, this review provides new insights for further anticancer research or clinical trials with Danshen.

Apoptosis Induced by Tanshinone IIA and Cryptotanshinone Is Mediated by Distinct JAK/STAT3/5 and SHP1/2 Signaling in Chronic Myeloid Leukemia K562 Cells

Jung JH, Kwon T-R, Jeong S-J, et al. Evidence-Based Complementary and Alternative Medicine. Volume 2013 (2013) http://dx.doi.org/10.1155/2013/805639

Though tanshinone IIA and cryptotanshinone possess a variety of biological effects such as anti-inflammatory, antioxidative, antimetabolic, and anticancer effects, the precise molecular targets or pathways responsible for anticancer activities of tanshinone IIA and cryptotanshinone in chronic myeloid leukemia (CML) still remain unclear. In the present study, we investigated the effect of tanshinone IIA and cryptotanshinone on the Janus activated kinase (JAK)/signal transducer and activator of transcription (STAT) signaling during apoptotic process. We found that both tanshinone IIA and cryptotanshinone induced apoptosis by activation of caspase-9/3 and Sub-G1 accumulation in K562 cells. However, they have the distinct JAK/STAT pathway, in which tanshinone IIA inhibits JAK2/STAT5 signaling, whereas cryptotanshinone targets the JAK2/STAT3. In addition, tanshinone IIA enhanced the expression of both SHP-1 and -2, while cryptotanshinone regulated the expression of only SHP-1. Both tanshinone IIA and cryptotanshinone attenuated the expression of bcl-xL, survivin, and cyclin D1. Furthermore, tanshinone IIA augmented synergy with imatinib, a CML chemotherapeutic drug, better than cryptotanshinone in K562 cells.

Overall, our findings suggest that the anticancer activity of tanshinone IIA and cryptotanshinone is mediated by the distinct the JAK/STAT3/5 and SHP1/2 signaling, and tanshinone IIA has the potential for combination therapy with imatinib in K562 CML cells.

Tanshinones and diethyl blechnics with anti-inflammatory and anti-cancer activities from salvia miltiorrhiza bunge (danshen)

Gao H, Sun W, Zhao J et al. Scientific Reports, vol. 6, Article ID 33720, 2016.

Four novel compounds (1–4) as well as fourteen reported compounds (5–18) were isolated and purified from Salvia miltiorrhiza Bunge (Danshen). The structures of novel compounds were determined by 1D and 2D NMR, HRESIMS data, etc. The anti-inflammatory properties of all the compounds on RAW264.7 macrophages and their cytotoxicity on H1299 and Bel-7402 cell lines coupled with a structure-activity relationship (SAR) were investigated. Compound 4 demonstrated the best anti- inflammatory activity and was chosen for further research. Compound 4 greatly suppressed secretion of nitric oxide (NO), tumor necrosis factor (TNF)-α and interleukin-6 (IL-6) in the RAW264.7 macrophages stimulated by LPS. Additionally, the protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) was decreased and the nuclear translocation of NF-κB was attenuated after treatment with compound 4 in vitro. Compound 4 was able to dramatically inhibit LPS-induced activation of JNK1/2 and ERK1/2 and remarkably disrupted the TLR4 dimerization in LPS-induced RAW264.7 macrophages. Thus, the new compound 4 suppressed LPS-induced inflammation partially is due to the blocking TLR4 dimerization. In addition, the anti-cancer activity investigation indicated that most of isolated compounds exhibited cytotoxicity and the SAR analysis showed that the intact D ring was indispensable and unsaturated D ring played vital role.

Opposite Effects of Single-Dose and Multidose Administration of the Ethanol Extract of Danshen on CYP3A in Healthy Volunteers

Qiu F, Jiang J, Ma Ym, et al. Evidence-Based Complementary and Alternative Medicine. Volume 2013 (2013) http://dx.doi.org/10.1155/2013/730734

The aim of this study was to investigate the effect of single- and multidose administration of the ethanol extract of danshen on in vivo CYP3A activity in healthy volunteers. A sequential, open-label, and three-period pharmacokinetic interaction study design was used based on 12 healthy male individuals. The plasma concentrations of midazolam and its metabolite 1-hydroxymidazolam were measured. Treatment with single dose of the extract caused the mean C max of midazolam to increase by 87% compared with control. After 10 days of the danshen extract intake, the mean AUC0-12, C max, and t 1/2 of midazolam were decreased by 79.9%, 66.6%, and 43.8%, respectively. The mean clearance of midazolam was increased by 501.6% compared with control. The in vitro study showed that dihydrotanshinone I in the extract could inhibit CYP3A, while tanshinone IIA and cryptotanshinone could induce CYP3A. In conclusion, a single-dose administration of the danshen extract can inhibit intestinal CYP3A, but multidose administration can induce intestinal and hepatic CYP3A.

CYP enzymes (CYPs) play an important role in detoxification and systemic clearance of xenobiotics. Of >55 human CYP isozymes presently known, CYP3A is considered to be the most important drug-metabolizing enzyme. It participates in metabolism of >60% of all marketed drugs. As the critical role of CYP3A in drug metabolism, inhibition or induction of this enzyme often leads to drug interactions. In recent years, some studies revealed the effect of danshen extract on CYP3A4, especially the danshen extract rich in lipophilic constituents. Kuo et al., 2006 reported that the ethyl acetate extract of danshen could induce CYP3A in C57BL/6J mice.7  By using a reporter gene assay and RT-PCR, Yu et al., 2009 demonstrated that cryptotanshinone and tanshinone IIA in the ethanol extract of danshen could activate the human pregnane and xenobiotic receptor (PXR) and consequently induce the expression of the CYP3A4 gene. It was found in the in vitro study using human liver microsomes that the ethanol extract of danshen had a significant inhibition toward CYP3A4-mediated midazolam metabolism.8

Drugs that are substrates for CYP3A-mediated metabolism are likely to be potential candidates for drug-herb interactions. The duration and dosage of exposure to the ethanol extract of danshen appear to be critical for drug-danshen interactions. An increase in the plasma drug concentration is possible during concomitant administration of the ethanol extract of danshen and prescribed drugs. By contrast, prolonged intake of the danshen extract followed by drug administration may result in subtherapeutic drug concentrations. Although we have shown that the ethanol extract of danshen has the potential to inhibit CYP3A4, particularly after single exposure at high concentrations, the inductive effect predominates with chronic exposure. It is suggested that caution should be taken when co-administrating the danshen extract rich in tanshinones with therapeutic drugs which are substrates for CYP3A4.

Hepatoprotective effect of cryptotanshinone from Salvia miltiorrhiza in d-galactosamine/lipopolysaccharide-induced fulminant hepatic failure

Jin Q, Jiang S, Wu YL, Bai T, Yang Y, Jin X, Lian LH, Nan JX. 2014 Jan 15;21(2):141-7. doi: 10.1016/j.phymed.2013.07.016

Cryptotanshinone from Salvia miltiorrhiza Bunge was investigated for hepatoprotective effects in d-galactosamine (GalN)/lipopolysaccharide (LPS)-induced fulminant hepatic failure. Cryptotanshinone (20 or 40mg/kg) was orally administered 12 and 1h prior to GalN (700mg/kg)/LPS (10μg/kg) injection. The increased mortality and TNF-α levels by GalN/LPS were declined by cryptotanshinone pretreatment. In addition, cryptotanshinone attenuated GalN/LPS-induced apoptosis, characterized by the blockade of caspase-3, -8, and -9 activation, as well as the release of cytochrome c from the mitochondria.

In addition, cryptotanshinone significantly suppressed JNK, ERK and p38 phosphorylation induced by GalN/LPS, and phosphorylation of TAK1 as well. Furthermore, cryptotanshinone significantly inhibited the activation of NF-κB and suppressed the production of proinflammatory cytokines.

These findings suggested that hepatoprotective effect of cryptotanshinone is likely associated with its anti-apoptotic activity and the down-regulation of MAPKs and NF-κB associated at least in part with suppressing TAK1 phosphorylation.

Coexisted components of Salvia miltiorrhiza enhance intestinal absorption of cryptotanshinone via inhibition of the intestinal P-gp.

Dai H, Li X, Li X, Bai L, Li Y, Xue M. Phytomedicine. 2012 Nov 15;19(14):1256-62. doi: 10.1016/j.phymed.2012.08.007. Epub 2012 Oct 5.

Cryptotanshinone, derived from the roots of Salvia miltiorrhiza Bge and Salvia przewalskii Maxim, is the major active component and possesses significant antibacterial, antidermatophytic, antioxidant, anti-inflammatory and anticancer activities. The objective of this study was to investigate the intestinal absorptive characteristics of cryptotanshinone as well as the absorptive behavior influenced by co-administration of the diterpenoid tanshinones and danxingfang using an in vitro everted rat gut sac model. The results showed a good linear correlation between cryptotanshinone of absorption and the incubation time from 10 to 70min. The concentration dependence showed that a non-linear correlation existed between the cryptotanshinone absorption and the concentration at 100μg/ml. Coexisting diterpenoid tanshinones and danxingfang could significantly enhance the absorption of cryptotanshinone. Coexisting diterpenoid tanshinones and danxingfang, which influenced cryptotanshinone's absorption, manifested as similar to that of the P-glycoprotein inhibitor. The underlying mechanism of the improvement of oral bioavailability was proposed that coexisting diterpenoid tanshinones and danxingfang could decrease the efflux transport of cryptotanshinone by P-glycoprotein.

Conclusion. In the present investigation, an in vitro everted rat gut sac system has been developed as a functional technique to study the intestinal transport and absorption of cryptotanshinone and drug interaction. The overall absorptive profile of cryptotanshinone by the rat small intestine can be enhanced by co-incubation of coexisting diterpenoid tanshinones and danxingfang in the gut sac system. This enhancement effect on the intestinal absorption of cryptotanshinone was proved to be one of the major mechanisms on the improvement of cryptotanshinone's bioavailability in vivo. We have verified that the P-gp inhibitors, verapamil, can markedly enhance the intestinal absorption of cryptotanshinone, with a similar pattern as the coexisting diterpenoid tanshinone compounds and danxingfang did. Therefore, it could be concluded that coexisting diterpenoid tanshinones and danxingfang can enhance the intestinal absorption of cryptotanshinone via inhibition of the intestinal P-gp activity like a P-gp inhibitor, and subsequently improve the oral bioavailability of cryptotanshinone.

Cryptotanshinone inhibits angiogenesis in vitro

In the course of screening of angiogenesis inhibitor from natural products, cryptotanshinone (tanshinone) from Salvia miltiorrhiza (dan shen) was isolated as a potent small molecule inhibitor of angiogenesis. Cryptotanshinone inhibits bFGF-induced angiogenesis of BAECs at ten micromolar ranges in vitro without cytotoxicity. These results demonstrate that cryptotanshinone is a new anti-angiogenic agent and double bond at C-15 position of the dihydro-furan ring plays a crucial role in the activity.9

Cryptotanshinone suppresses androgen receptor-mediated growth in androgen dependent and castration resistant prostate cancer cells.

Xu D, Lin TH, Li S, Da J, Wen XQ, Ding J, Chang C, Yeh S. Cancer Lett. 2012 Mar;316(1):11-22. Epub 2011 Oct 10.

Androgen receptor (AR) is the major therapeutic target for the treatment of prostate cancer (PCa). Anti-androgens to reduce or prevent androgens binding to AR are widely used to suppress AR-mediated PCa growth; however, the androgen depletion therapy is only effective for a short period of time. Here we found a natural product/Chinese herbal medicine cryptotanshinone (CTS), with a structure similar to dihydrotestosterone (DHT), can effectively inhibit the DHT-induced AR transactivation and prostate cancer cell growth. Our results indicated that 0.5 μM CTS effectively suppresses the growth of AR-positive PCa cells, but has little effect on AR negative PC-3 cells and non-malignant prostate epithelial cells. Furthermore, our data indicated that CTS could modulate AR transactivation and suppress the DHT-mediated AR target genes (PSA, TMPRSS2, and TMEPA1) expression in both androgen responsive PCa LNCaP cells and castration resistant CWR22rv1 cells. Importantly, CTS selectively inhibits AR without repressing the activities of other nuclear receptors, including ERα, GR, and PR. The mechanistic studies indicate that CTS functions as an AR inhibitor to suppress androgen/AR-mediated cell growth and PSA expression by blocking AR dimerization and the AR-coregulator complex formation. Furthermore, we showed that CTS effectively inhibits CWR22Rv1 cell growth and expressions of AR target genes in the xenograft animal model. The previously un-described mechanisms of CTS may explain how CTS inhibits the growth of PCa cells and help us to establish new therapeutic concepts for the treatment of PCa.

Coexisted components of Salvia miltiorrhiza enhance intestinal absorption of cryptotanshinone via inhibition of the intestinal P-gp

Haixue Dai H, et al. Phytomedicine. 2012 doi:10.1016/j.phymed.2012.08.007

Cryptotanshinone, derived from the roots of Salvia miltiorrhiza Bge and Salvia przewalskii Maxim, is the major active component and possesses significant antibacterial, antidermatophytic, antioxidant, anti-inflammatory and anticancer activities. The objective of this study was to investigate the intestinal absorptive characteristics of cryptotanshinone as well as the absorptive behavior influenced by co-administration of the diterpenoid tanshinones and danxingfang using an in vitro everted rat gut sac model. The results showed a good linear correlation between cryptotanshinone of absorption and the incubation time from 10 to 70min. The concentration dependence showed that a non-linear correlation existed between the cryptotanshinone absorption and the concentration at 100μg/ml. Coexisting diterpenoid tanshinones and danxingfang could significantly enhance the absorption of cryptotanshinone. Coexisting diterpenoid tanshinones and danxingfang, which influenced cryptotanshinone's absorption, manifested as similar to that of the P-glycoprotein inhibitor. The underlying mechanism of the improvement of oral bioavailability was proposed that coexisting diterpenoid tanshinones and danxingfang could decrease the efflux transport of cryptotanshinone by P-glycoprotein.

Cryptotanshinone Reverses Reproductive and Metabolic Disturbances in PCOS Model Rats via Regulating the Expression of CYP17 and AR

Evidence-Based Complementary and Alternative Medicine. Volume 2014 (2014), http://dx.doi.org/10.1155/2014/670743

Objective. To explore the effect of Cryptotanshinone on reversing the reproductive and metabolic disturbances in polycystic ovary syndrome (PCOS) model rats and the possible regulatory mechanisms.

Methods. PCOS model rats were induced by subcutaneous injection of dehydroepiandrosterone (DHEA) and verified by histological screening of vaginal exfoliated cells. After Cryptotanshinone intervention, the rats’ body weight and ovary morphological were observed; the serum biochemical assessments were analyzed by radioimmunoassay (RIA) and key genes and proteins related with anabolism of androgen and insulin were detected by Real-Time PCR and Immunohistochemical (IHC).

Results. The estrous cyclicity of PCOS model rats was significantly recovered by Cryptotanshinone. The body weight, ovarian coefficient, and ovarian morphology had been improved and the serum biochemical indicators including testosterone (T), androstenedione (A2), luteinizing hormone (LH), LH/follicle stimulating hormone (FSH), sexual binding globulin (SHBG), low density cholesterol (LDL-C), fasting insulin (FINS) were reversed after Cryptotanshinone intervention. Specifically, the levels of Cytochrome P450, 17-a hydroxylase/17,20 lyase (CYP17), and androgen receptor (AR) were downregulated significantly.

Conclusions. Our data suggest that Cryptotanshinone could rebalance reproductive and metabolic disturbances in PCOS model rats and could be a potential therapeutic agent for the treatment of PCOS.

Tanshinone ⅡA Protects against Lipopolysaccharides-Induced Endothelial Cell Injury via Rho/Rho Kinase Pathway

Chinese Journal of Integrative Medicine 2014,20(3)

Objective: To test whether tanshinone ⅡA (Tan ⅡA), a highly valued herb derivative to treat vascular diseases in Chinese medicine, could protect endothelial cells from bacterial endotoxin (lipopolysaccharides, LPS)-induced endothelial injury.

Methods: Endothelial cell injury was induced by treating human umbilical vein endothelial cells (HUVECs) with 0.2 μg/mL LPS for 24 h. Y27632 and valsartan were used as positive controls. The effects of tanshinone ⅡA on the LPS-induced cell viability and apoptosis rate of HUVECs were tested by flow cytometry, cell migration by transwell, adhesion by a 96-well plate pre-coated with vitronectin and cytoskeleton reorganization by immunofluorescence assay. Rho/Rho kinase (ROCK) pathway-associated gene and protein expression were examined by microarray assay; quantitative real-time polymerase chain reaction and Western blotting were used to confirm the changes observed by microarray.

Results: Tan ⅡA improved cell viability, suppressed apoptosis and protected cells from LPS-induced reductions in cell migration and adhesion at a comparable magnitude to that of Y27632 and valsartan. Tan ⅡA, Y27632 and valsartan also normalized LPS-induced actomyosin contraction and vinculin protein aggregation. A microarray assay revealed increased levels of fibronectin, integrin A5 (ITG A5), Ras homolog gene family member A (RhoA), myosin light chain phosphatase, phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K, or PIP2 in Western blotting), focal adhesion kinase, vascular endothelial growth factor and vascular endothelial growth factor receptor 2 in the damaged HUVECs, which were attenuated to different degrees by Tan ⅡA, Y27632 and valsartan.

Conclusion: Tan ⅡA exerted a strong protective effect on HUVECs, and the mechanism was caused, at least in part, by a blockade in the Rho/ROCK pathway, presumably through the down-regulation of ITG A5.

The anti-atherosclerotic effect of tanshinone IIA is associated with the inhibition of TNF-α-induced VCAM-1, ICAM-1 and CX3CL1 expression

Chang C-C et al. Phytomedicine. 2013 doi:10.1016/j.phymed.2013.09.012

Tanshinone IIA is one of the major diterpenes in Salvia miltiorrhiza. The inhibitory effect of Tanshinone IIA on atherosclerosis has been reported, but the underlying mechanism is not fully understood. The present study aimed to study the anti-atherosclerosis effect of Tanshinone IIA on the adhesion of monocytes to vascular endothelial cells and related mechanism. Results showed that Tanshinone IIA, at the concentrations without cytotoxic effect, dose-dependently inhibited the adhesion of THP-1 monocytes to the TNF-α-stimulated human vascular endothelial cells. The expressions of cell adhesion molecules including VCAM-1, ICAM-1 and E-selectin were induced by TNF-α in HUVECs at both the mRNA and protein levels. The mRNA and protein expressions of VCAM-1 and ICAM-1, but not E-selectin, were both significantly suppressed by Tanshinone IIA in a dose dependent manner. In addition, the TNF-α-induced mRNA expression of fractalkine/CX3CL1 and the level of soluble fractalkine were both reduced by Tanshinone IIA.

We also found that Tanshinone IIA significantly inhibited TNF-α-induced nuclear translocation of NF-κB which was resulted from the inhibitory effect of Tanshinone IIA on the TNF-α-activated phosphorylation of IKKα, IKKβ, IκB and NF-κB. As one of the major components of Salvia miltiorrhiza, Tanshinone IIA alone exerted more potent effect on inhibiting the adhesion of monocytes to vascular endothelial cells when compared with Salvia miltiorrhiza. All together, these results demonstrate a novel underlying mechanism for the anti-inflammatory effect of Tanshinone IIA by modulating TNF-α-induced expression of VCAM-1, ICAM-1 and fractalkine through inhibition of TNF-α-induced activation of IKK/NF-κB signaling pathway in human vascular endothelial cells.

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