Journal of Biomolecular Research & Therapeutics

Danshen, a Traditional Chinese Medicine isolated from the dried root of Salvia miltiorrhiza Bunge and officially listed in the Chinese Pharmacopoeia, is widely used in Asian countries especially in China for the prevention and treatment of cardiovascular, cerebrovascular and liver diseases including angina pectoris, hyperlipidemia, acute ischemic stroke, hepatic fibrosis and hepatitis [1-3]. By virtue of its versatile abilities such as promoting blood circulation, causing coronary vasodilatation and inhibiting platelet aggregation, Danshen is commonly used as adjuvant therapy in order to promote the efficacies of some therapeutic agents [4-6].

The biological activities and chemical constituents of Danshen have been studied since the early 1930s. Basically, the active ingredients in Danshen are divided into two major groups, hydrophilic phenolic acids and lipophilic tanshinones. The phenolic compounds present in Danshen include salvianolic acid B, lithospermic acid, rosmarinic acid and danshensu, and the major lipophilic tanshinones compose of tanshinone I, tanshinone IIA, tanshinone IIB, cryptotanshinone, and dihydrotanshinone I. Chemical structures of tanshinones are extremely similar, most of the compounds have been purified and identified and further synthesized in the past decades [7,8]. The chemical structures of the four major tanshinones are shown in Figure 1.

Figure 1: The chemical structures of the four major tanshinones.

Up to now, the pharmacological activities of tanshinones particularly tanshinone IIA, which is the most abundant tanshinone found in Danshen, have been investigated extensively. Numerous studies have indicated the potential applications of tanshinones in a broad spectrum of diseases such as atherosclerosis, cardiac arrhythmias, hypertension, obesity, metabolic syndrome and cancer [9,10]. The present review summarizes recent studies about the pharmacological activities and possible mechanisms of action for tanshinones in the treatment of different gastrointestinal and hepatic diseases with emphasis on tanshinone IIA and its anticancer potentials.

Colorectal cancer

Colorectal cancer, one of the most commonly diagnosed cancers in the world, is the third leading cause of cancer-related mortality in developed countries [11]. Recently, several tanshinones have been evaluated for their cytotoxicities in different colorectal cancer cell lines. This section describes their anticancer activities and possible underlying mechanisms of action for this disease.

Tanshinone IIA

As one of the major constituents in Danshen, tanshinone IIA has been evaluated for its anticancer activity in different cancer cell lines including breast, lung, liver, prostate and colorectal cancer cells. Their anticancer mechanisms have been proposed [12]. We take colorectal cancer cell as an example and summarize them both in vitro and in vivo.

In 2008, Su et al. [13] studied the growth inhibition and apoptosis induction by tanshinone IIA in Colo-205 human colon adenocarcinoma cells. In this study tanshinone IIA could inhibit cell proliferation and induce cell apoptosis in a concentration-dependent manner, with an increased percentage of cells in the sub-G1 phase. Moreover, it could up-regulate the expression of p53, p21 and Fas proteins, increase cytosolic cytochrome c level, and induce catalytic activation of caspases, which was confirmed by cleavage of caspase-8 and caspase-3. Finally, they concluded that tanshinone IIA could induce apoptosis in Colo-205 cells through both mitochondrial-mediated intrinsic and Fas-mediated extrinsic caspase cell-death pathways [13].

To further understand the molecular mechanisms of the anti-cancer action of tanshinone IIA on human colon cancer cells, the same group also studied the regulation of erythroblastosis oncogene B (ErbB-2) and tumor necrosis factor-α (TNF-α) protein expression by tanshinone IIA both in vitro and in vivo. Results showed that treatment with tanshinone IIA could lead to down-regulation of ErbB-2 expression and up-regulation of TNF-α expression in Colo-205 cells and xenograft tumors in mice, respectively [14].

Based on these results, Su and his group also investigated whether co-treatment with tanshinone IIA could increase the therapeutic effect of 5-fluorouracil (5-FU), a standard chemotherapeutic agent for colon cancer but with low efficacy in Colo-205 cell xenograft model. They found that the tumor volume was significantly reduced after cotreatment with tanshinone IIA and 5-FU when compared with 5-FU alone. Interestingly, the protein expression of P-glycoprotein (P-gp) and microtubule-associated protein light chain 3 (LC3-II) was downregulated in Colo-205 cell xenograft tumor [15]. Nevertheless, these findings demonstrate the potential application of tanshinone IIA as an adjuvant chemotherapy for colorectal cancer.

In addition, Shan et al. [16] explored the different anticancer actions of tanshinone IIA on tumor invasion and metastasis using human colon carcinoma cell lines HT29 and SW480. They demonstrated that tanshinone IIA could inhibit cell migration and invasion of these colon cancer cells concentration- and time-dependently. Tanshinone IIA also showed inhibitory effect on metastasis of SW480 cells by the tail vein metastatic assay in nude mice. Moreover, the effects of tanshinone IIA were associated with down-regulation of urokinase plasminogen activator (uPA), matrix metalloproteinases (MMP)-2 and MMP-9, and up-regulation of tissue inhibitor of matrix metalloproteinase protein (TIMP)-1 and TIMP-2, and further inhibition of the nuclear factorkappaB (NF-κB) signaling pathway [16].

More recently tanshinone IIA was shown to inhibit angiogenesis in human colorectal cancer. Using the mouse xenograft model developed by the colorectal carcinoma cell line C26, it was found that the tanshinone could reduce tumor weight and volume, inhibit tumor angiogenesis, and down-regulate vascular endothelial growth factor (VEGF) level in a concentration-dependent manner. Furthermore, in a cell line study using transfected HCT116 cells, it was showed that tanshinone IIA could repress cyclooxygenase-2 (COX-2) gene promoter activity and mRNA expression, as well as VEGF expression significantly [17].

Other tanshinones

Currently, the anticancer effects of other tanshinones against colorectal cancer were less investigated, compared with tanshinone IIA. One study demonstrated the growth inhibition and apoptosis induction by tanshinone I in Colo-205 cells. It was found that tanshinone I could also inhibit cell proliferation and induce cell apoptosis in a concentration-dependent manner. There was a G0/G1 arrest in the cell cycle analysis. In this study, tanshinone I up-regulated the protein expression of p53, caspase-3, Bax, and p21. These results suggest that tanshinone I could induce apoptosis in Colo-205 cells through both mitochondrial-mediated intrinsic cell-death pathway and p21-mediated G0/G1 cell cycle arrest [18]. Another recent study found that activating transcription factor (ATF)-3 was involved in 15,16-Dihydrotanshinone I-induced apoptosis in human colorectal cancer cells, and the role of ATF-3 depended on the degree of malignancy [19]. According to the IC₅₀ values showed in Table 1, the growth inhibitory action of tanshinone I was greater than that of tanshinone IIA in Colo-205 cells. However whether or not other tanshinones produce similar or even more potent anti-cancer effect in colon cancer needs further exploration.

Table 1: Growth inhibition by tanshinones in different gastric, colorectal and liver cancer cells following 24 hours of drug exposure.

Other gastrointestinal disorders

In addition to colorectal cancer, the potential application of tanshinones in other gastrointestinal disorders has been studied. Among these disorders gastric cancer, small intestine injury and colitis have been reported. Again tanshinone IIA was widely explored for its action in these different diseases. It inhibited growth and provoked apoptosis in human gastric carcinoma cell line MKN-45 cells in a timeand concentration- dependent manner and arrested cells in the G2/M phase. In this regard the protein expression of p53 was up-regulated and Bcl-2 level was down-regulated by tanshinone IIA [20]. Other group also studied the anticancer effects of tanshinone IIA in gastric cancer both in vitro and in vivo. They reported similar findings in MKN45 and SGC7901 cells [21]. Furthermore, they also demonstrated that tanshinone IIA could induce apoptosis in a mouse xenograft model in vivo [22].

Zhu and his associates explored the protective effects of tanshinone IIA on small intestine injury in rats with sepsis and studied the possible mechanisms for this anti-septic action. Using a rat model of sepsis, which was induced by cecal ligation and puncture for 5 hours, they found that post-treatment with tanshinone IIA could attenuate intestinal injury. The apoptosis and protein levels of NF-κB p65, TNF-α, Bax and interleukin-6 (IL-6) in intestinal epithelial cells were all decreased after treatment [23]. In a separate study the effects of tanshinone IIA on experimental colitis induced by trinitrobenzene sulfonic acid were examined. It was showed that tanshinone IIA could inhibit inflammatory response of colitis and attenuate tissue damage through down-regulation of the pro-inflammatory cytokines and reduction of oxidative stress [24]. Additionally, using healthy newborn piglets, it was demonstrated that tanshinone IIA could increase intestinal hemodynamics without changing the systemic circulation, through an endothelium-derived hyperpolarizing factor vasodilating pathway [25]. The potential applications and mechanisms of action in treating gastric cancer, small intestine injury, and colitis are listed in Table 2.

Table 2: Pharmacological activities and underlying mechanisms of tanshinone IIA in gastrointestinal and hepatic diseases.

Liver cancer

Hepatocellular carcinoma is one of the most common cancers around the world leading to 500,000 to 1 million deaths per year worldwide [26]. Effective treatment is still a big challenge in clinics timedue to poor diagnosis, high mortality and chemotherapy resistance [27]. Nowadays, new strategies other than the current available chemotherapies for liver cancer are under development. In this regard an alternative medicine has been proposed and one of which is the herbal medicine. To this end the application of Danshen in liver cancer is fully anticipated. It has been extensively investigated. This section is focusing on recent studies about the anticancer activities of tanshinones in liver cancer.

Tanshinone IIA: As mentioned earlier, tanshinone IIA has been evaluated for its anticancer activity in a variety of cancer cell lines. Tanshinone IIA decreased cell viability and induced caspase-dependent apoptosis in human liver cancer HepG2 cells. Moreover, tanshinone IIA could induce reactive oxygen species (ROS) generation without activation of p38 mitogen-activated protein kinases (MAPK) [28]. Later on, Lee et al. [29] studied the cytotoxic effects of several tanshinones in P-gp overexpressing HepG2 cells, trying to find out whether they could potentiate the cytotoxicity of doxorubicin, a P-gp substrate, in this drug-resistant cell line. Results showed that tanshinone IIA showed the best synergistic effect with doxorubicin without any influence on doxorubicin efflux from cells [29].

In a separate cell line, tanshinone IIA inhibited human hepatocellular carcinoma J5 cells growth in a concentration- and timedependent manner. In addition, the protein expressions of caspase-3, caspase-12, p53, p21, calreticulin, growth arrest- and DNA damageinducible gene 153 (GADD153) and Bax were increased, but the levels of Cdc25c, Cdc2 and Bcl-2 were decreased by tanshinone IIA treatment. Experimental results also showed that tanshinone IIA could arrest J5 cells in the G2/M phase [30]. Furthermore, tanshinone IIA inhibited cell growth in vivo using SCID mice bearing J5 cells.

To further understand the underlying mechanisms of apoptosis induced by tanshinone IIA, using human hepatoma BEL-7402 cells, Dai et al. [34] found that it could induce apoptosis and arrest cells in the G0/ G1 phase. Intracellular calcium was increased, whereas mitochondrial membrane potential was decreased after treatment with the compound. Additionally, mRNA expression of Bad and metallothionein 1A (MT 1A) was up-regulated. Besides, tanshinone IIA inhibited the enhanced metastasis associated with palliative resection, through promoting VEGFR1/PDGFR-related vascular normalization [35]. It also effectively inhibited invasion and metastasis of hepatocellular carcinoma cells in vitro and in vivo, partly through inhibition on the activity of MMP2 and MMP9 [36]. Furthermore, lipid emulsion of tanshinone IIA for intravenous administration showed anti-cancer activity against human hepatocellular carcinoma cell lines including HepG2, SMMC-7721 and BEL-7404 cells [37]. Different research group also developed novel polylactic acid nanoparticles containing tanshinone IIA which could inhibit the growth of SMMC-7721 cells concentration- and timedue dependently. This novel formulation could prevent tumor growth and increase survival of mice with hepatoma [38]. The anticancer activities of tanshinone microemulsion, composed of phospholipid, ethyl oleate, glycerol and Pluronic F68, was also investigated recently, this drug delivery system also potentiated the anticancer effect of tanshinone in in vitro and in vivo hepatocellular carcinoma models [39]. Taken together, these findings indicate the potential clinical application of tanshinone IIA in the treatment of liver cancer.

Other tanshinones: Although most of the studies on liver cancer were focused on tanshinone IIA, other research groups also evaluated the anti-cancer potential of other tanshinones including tanshinone I, cryptotanshinone and dihydrotanshinone, trying to identify their anti-cancer efficacy and potential clinical application in liver cancer. In human liver cancer HepG2 cells, all the aforementioned tanshinones inhibited cell growth and induced caspase-dependent apoptosis, and dihydrotanshinone showed the most potent effect against cancer growth. Moreover, all three tanshinones induced ROS generation as well, but only dihydrotanshinone could activate p38 MAPK activity [28]. Further study also showed that cryptotanshinone could decrease doxorubicin efflux in P-gp overexpressing HepG2 cells significantly [29]. To further study the pro-apoptotic activity of cryptotanshinone, it was found to be mediated by endoplasmic reticulum (ER) stress, and ROS played an important role in this process. Moreover, Park et al. [40] also found that cryptotanshinone potentiated the anti-cancer effects of several chemotherapeutic agents including TNF-α, cisplatin, etoposide and 5-FU in HepG2 cells. These results also indicate the potential application of cryptotanshinone in adjuvant therapy for liver cancer.

In order to compare the efficacy of different tanshinones, several other tanshinones were evaluated for their anticancer effects, including tanshinone I, cryptotanshinone and dihydrotanshinone. Their IC₅₀ values in HepG2 and Hep3B cells were described in Table 1. These tanshinones showed different efficacies and mechanisms of action from tanshinone IIA. Furthermore in addition to investigating the anticancer actions of tanshinones, some novel formulations of tanshinones were developed in order to improve its bioavailability for a better therapeutic efficacy against different cancers.

Other hepatic diseases

As mentioned in the beginning, Danshen has been used in China and perhaps also in other Asian countries for the prevention and treatment of several liver diseases, in particular liver fibrosis and chronic hepatitis, through promoting hepatic microcirculation [41,42]. Indeed the major components in Danshen, tanshinones are shown to have protective effects against inflammation in the liver and their possible anti-inflammatory mechanisms have been proposed. Using primary rat hepatocytes, Cao et al. [43] demonstrated that tanshinone IIA, an effective antioxidant, could inhibit lipid peroxidation through breaking the chain reactions of peroxidation, scavenging lipid free radicals and decreasing their cytotoxicity in liver cells.

Other study reported that tanshinone I, tanshinone IIA, and cryptotanshinone could protect primary rat hepatocytes against apoptosis induced by bile acid, through inhibiting c-Jun N-terminal kinases (JNK) phosphorylation and intracellular ROS generation in primary rat hepatocytes [44]. Furthermore the same research group also studied the inhibition of lactate dehydrogenase (LDH) leakage, glutathione (GSH) depletion, and lipid peroxidation by tanshinone I, tanshinone IIA, and cryptotanshinone in primary rat hepatocytes. They also found that all three tanshinones showed protective effects on acute and sub-acute liver injury in rats [45].

Kim et al. [46] studied the apoptosis induced by tanshinone I and tanshinone IIA in activated rat hepatic stellate cells, which play a central role in liver fibrosis. Results showed that treatment with tanshinone I could induce typical DNA fragmentation and DNA ladder formation in these cells transformed by simian virus 40 (T-HSC/Cl-6). Furthermore, tanshinone I also activated caspase-3 and subsequent proteolytic cleavage of poly(ADP-ribose) polymerase (PARP), and induced mitochondrial membrane depolarization and release of cytochrome c from mitochondria into the cytosol. In addition to these similar effects produced by tanshinone I, tanshinone IIA induced S phase cell cycle arrest and down-regulated cyclin A, cyclin E and cyclin-dependent kinase 2 (CDK2) levels in these cells [47].

Xu et al. [48] investigated the effects of tanshinone IIA on hepatitis induced by concanavalin A (Con A) in mice. Using C57BL/6 mice, they demonstrated that tanshinone IIA could decrease the release of alanine transaminase into plasma, reduce inflammatory infiltration, and inhibit hepatocyte apoptosis. Additionally, this protective effect was associated with the reduction of important inflammatory mediators through modulating NF-κB and IFN-γ/STAT1 signaling pathways. In a separate study, tanshinone IIA attenuated hepatotoxicity induced by ethanol or lipopolysaccharide. It effectively reversed the up-regulation of CD14, iNOS, and stearoyl-CoA desaturase expression and blocked fat accumulation. Their findings also indicate the potential application of tanshinone IIA in treating alcoholic liver disease through reducing Kupffer cell sensitization and fatty acid level in the liver [49].

As a commonly used Traditional Chinese Medicine, the biological activities of Danshen have been extensively studied in the past decades. As today, numerous studies have indicated the versatile abilities of tanshinones, the lipophilic components present in Danshen, in the prevention of a variety of diseases including acute ischemic stroke, cardiac arrhythmias, atherosclerosis, hypertension, obesity, metabolic syndrome, liver fibrosis, hepatitis and different cancers.

In short this review briefly summarizes recent developments on the pharmacological activities and possible mechanisms of action of tanshinones against different gastrointestinal and hepatic diseases. According to the current findings, tanshinone IIA showed persistent anti-cancer effect against colorectal and liver cancers, as well as protective action against inflammation in liver and GI tract. Extensive studies also reveal its possible underlying mechanisms and potential clinical applications, especially in adjuvant therapy during chemotherapy. However, most of the studies summarized above are focused on tanshinone IIA which is the major component among the different tanshinones in Danshen. There are evidences showing that other analogues of tanshinone IIA may have more potent effects against these diseases with less toxicities in the human body. These compounds should have a greater potential as therapeutic agents for the treatment and perhaps also prevention of GI and hepatic disorders in the future.

Today investigations on the biological activities of Danshen and its clinical applications are still undergoing. Dantonic Dripping Pill, which includes active constitutes of Danshen (Salvia miltiorrhiza) and Sanqi (Panax Notoginseng), is the first Traditional Chinese Medicine approved by U.S. Food and Drug Administration for Phase II and Phase III clinical trials. So far Phase II clinical trials have already been completed in the United States to evaluate its safety and efficacy in patients with chronic stable angina pectoris, and Phase III study is ongoing (see http://clinicaltrials.gov/, No. NCT00797953 and NCT01659580). Thus, Danshen and its active components tanshinones are promising candidates to be developed as novel therapeutic agents for treating a broad spectrum of diseases including those in the gastrointestinal tract and liver.

This work was supported by the Innovation and Technology Support Programme from the Innovation and Technology Commission, Hong Kong. Tao Hu is a recipient of postgraduate student scholarship from The Chinese University of Hong Kong, Hong Kong.