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

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-α-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-κB, 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.


Introduction
Cancer is a leading cause of mortality throughout the world. In addition to conventional therapies such as surgery, chemotherapy, and radiotherapy, traditional Chinese medicine and other complementary or alternative therapies may be necessary for cancer patients [1]. Salvia miltiorrhiza Bunge (Danshen) has been used widely for the treatment of various diseases [2][3][4][5][6][7] including cancers [8][9][10][11][12] for thousands of years within the China community. Danshen, a Chinese herbal medicine, contains two major groups of chemicals [12][13][14][15]. The first group includes lipophilic compounds such as tanshinone I, tanshinone IIA, acetyltanshinone IIA, cryptotanshinone, isocryptotanshinone, dihydrotanshinone,  (Tables 1 and 2). This systematic review provides an appraisal of the roles of reactive oxygen species (ROS) in cancer biology and anticancer therapy with Danshen. Based on the evidence demonstrating anticancer properties of Danshen and the roles of ROS in cancer biology, this review summarizes current data regarding the ROS-related anticancer effects of Danshen components and brings new insights for further anticancer research or clinical trials with this traditional Chinese herb.

Methods
Keyword searches were done using the combined terms "reactive oxygen species and cancer and Danshen" or "reactive oxygen species and cancer and Salvia miltiorrhiza". These searches were done using the Medicine, PubMed, EMBASE, Cochrane library, CINAHL, and Scopus databases. The contents of the identified articles were summarized and the current review focused on the ROS-related anticancer Tanshinone IIA [20] Prostate cancer cells Induces apoptosis and autophagy that depends on intracellular ROS production Tanshinone IIA [21] Gastric cancer cells Suppresses cell growth by blocking glucose metabolism Tanshinone IIA [22] Human non-small cell lung cancer A549 cells Decreases VEGF/VEGFR2 expression and induces apoptosis and cell cycle arrest at the S phase Tanshinone IIA [23] Human oral cancer KB cells Induces apoptosis through the mitochondria-dependent pathway in which there is a loss of the mitochondrial membrane potential and activation of caspase-3 and caspase-9 Tanshinone IIA [24] Human colon cancer cells UDP-glucuronosyltransferase 1A compromises the intracellular accumulation and resultant apoptotic effect of tanshinone IIA Tanshinone IIA [25] Cervical cancer CaSki cells Inhibits cell growth by activating ER stress pathways and promoting caspase cascades with concomitant upregulation of p38 and JNK phosphorylation and signaling Tanshinone IIA [26] Human hepatoma J5 cells Increases Bax and caspase-3 and decreases CD31 expression Tanshinone IIA [27] Non-small cell lung cancer H596 cells Activates ROS-triggered, p53-independent, and caspase-dependent mitochondrial apoptotic cell death pathway Tanshinone IIA [28] 786-O human renal cell carcinoma cells Induces apoptosis by activating p53 expression and subsequently upregulating p21 and Bax Tanshinone IIA [29] Leukemia U937 cells Induces apoptosis by activating PXR, which suppresses the activity of NF-B Tanshinone IIA [30] human non-small lung cancer A549 cells Induces apoptosis by increasing ROS and the ratio of Bax/Bcl-2 and then decreasing the mitochondrial membrane potential, which leads to cytochrome c release Tanshinone IIA [31] Small cell lung cancer H146 cells Inhibits cell growth by upregulating the Bax/Bcl-2 ratio and decreasing the mitochondrial membrane potential Tanshinone IIA [32] Cervical cancer HeLa cells Inhibits cell growth by interfering with the process of microtubule assembly, leading to G2/M phase arrest and subsequent apoptosis Acetyltanshinone IIA [33] Breast cancer Induces G1/S phase arrest and apoptosis by downregulating the receptor tyrosine kinases EGFR/HER2 and activating AMP-activated protein kinase Acetyltanshinone IIA [34] Breast cancer cells Induces ROS generation and Bax translocation to mitochondria, resulting in mitochondrial damage, cytochrome c release, caspase-3 activation, and apoptotic cell death Cryptotanshinone [35] Breast cancer cells Suppresses estrogen receptor signaling 4 Oxidative Medicine and Cellular Longevity

Role of ROS in Cancer
Carcinogenesis is a progressive process from normal to cancerous cells. Reactive oxygen species (ROS) are closely related to carcinogenesis and play an important role in cancer. Previous studies have shown that ROS may be involved in multistep tumorigenesis including tumor initiation and transformation, tumor progression, tumor promotion, tumor angiogenesis, and tumor metastasis [56][57][58]. ROS are generated by both mitochondria and NADPH oxidases. Oxidative stress results from the generation of free radicals such as the superoxide anion, perhydroxyl radical, hydroxyl radical, and nitric oxide, as well as other nonradical but reactive species such as hydrogen peroxide, singlet oxygen, hypochlorous acid, and peroxynitrite [56,57]. Mitochondria in malignant cells are characterized by the overproduction of ROS and differ structurally and functionally from those in normal cells [59]. A major source of ROS is oxidative metabolism in the mitochondria of eukaryotic cells. In normal cells, low-level concentrations of ROS, related to mitochondrial electron transport activity, are required for many cellular processes and signal transduction. Cancer cells generate more ROS as compared to normal cells. The increased generation of ROS in cancer cells may alter mitochondrial metabolism [59,60] and disturb cellular signaling pathways [61,62] that are mediated through the transcription factors NF-B and STAT3, hypoxia-inducible factor-1 , kinases, growth factors, cytokines, and other enzymes [63].
ROS can induce cellular DNA damage and DNA methylation [64] resulting in mutations, which causes healthy cells to transform into malignant cells. Some cancer cells overexpress the ROS-producing NADPH oxidases and ROSremoving antioxidant enzymes. Conversely, there is also evidence showing that excess ROS can result in cancer cell death through autophagy [65,66] and/or apoptosis [62,67]. Cancer cells may be more sensitive than normal cells to the overproduction of ROS. Thus, increasing oxidative stress by generating ROS exogenously may be selective for cancer cells without affecting normal cells [68,69].

Lipophilic Components of Danshen
Tanshinone I, tanshinone IIA, acetyltanshinone IIA, cryptotanshinone, isocryptotanshinone, dihydrotanshinone, and miltirone are the main lipid-soluble potential anticancer constituents of Danshen. These compounds have shown anticancer activity (Table 1) with remarkable dose-and timedependent inhibitory effects on the viability on prostate, lung, breast, leukemia, gastric, oral, colon, cervical, hepatoma, renal, melanoma, rhabdomyosarcoma, and neuroblastoma cancer cells. These effects, in terms of ROS, are described in more detail for each cell type in the following sections.

ROS-Related Anticancer Effects of Tanshinones on Lung
Cancer Cells. Tanshinones inhibited the proliferation of 95D lung cancer cells by increasing caspase-3 activity and inducing apoptosis and prosurvival autophagy [16], through the increased generation of intracellular ROS. Tanshinone IIA decreased vascular endothelial growth factor/vascular  endothelial growth factor receptor 2 (VEGF/VEGFR2) expression and induced apoptosis with cell cycle arrest at the S phase in human non-small cell lung cancer A549 cells [22]. These researchers noted that tanshinone IIA activated a ROS-induced, P53-independent [27], and caspasedependent mitochondrial apoptotic cell death pathway that was characterized by an increased ratio of Bax to Bclx1 or Bax to Bcl-2, decreased mitochondrial membrane potential [30], caspase activation, PARP-1 cleavage, and cytochrome c release in A549 cells and small cell lung cancer H146 cells [31]. Cryptotanshinone also induced ROS-mediated prodeath autophagy through JNK signaling [37].

ROS-Related Anticancer Effects of Tanshinones on Breast
Cancer Cells. Tanshinone I downregulated the PI3K/Akt/ mTOR signaling pathway, induced cell cycle arrest, and inhibited the proliferation of breast cancer MCF-7 and MDA-MB-453 cells [18]. Acetyltanshinone IIA induced G1/S phase arrest and apoptosis with downregulation of receptor tyrosine kinases such as epidermal growth factor receptor (EGFR)/ HER2 and activated AMP-activated protein kinase (AMPK) [33]. Acetyltanshinone IIA also induced ROS generation and Bax translocation to mitochondria resulting in mitochondrial damage, cytochrome c release, caspase-3 activation, and apoptotic cell death in HER2 positive breast cancer cells [34].

ROS-Related Anticancer Effects of Tanshinones on
Leukemia Cells. Some researchers found that tanshinone I activated caspase-3 and decreased human telomerase reverse transcriptase (hTERT) mRNA expression and telomerase activity, as well as downregulating survivin expression, in monocytic leukemia U937 THP-1 and SHI 1 cells [19]. Another study reported that tanshinone IIA induced apoptosis through the activation of PXR, which suppressed NF-B activity in leukemia U937 cells [29]. Cryptotanshinone inhibited cellular movement and induced G2/M phase arrest in acute lymphoblastic leukemia cells [36]. Another study revealed that cryptotanshinone enhanced TNF--induced apoptosis through the ROS-dependent activation of caspase-8 and p38 in chronic myeloid leukemia KBM-5 cells [44]. 15,16-Dihydrotanshinone I induced apoptosis through activation of the JNK and FasL signaling pathways in human HL-60 leukemia cells [48]. Miltirone induced G2/M cell cycle arrest and apoptosis in acute lymphoblastic leukemia cells [50].

ROS-Related Anticancer Effects of Tanshinones on Oral,
Gastric, and Colon Cancer Cells. One previous study reported that tanshinone IIA induced apoptosis through the mitochondria-dependent pathway with the loss of mitochondrial membrane potential and the activation of caspase-9 and caspase-3 in human oral cancer KB cells [23]. Another study that examined the effects of tanshinone IIA reported that it suppressed cell growth by blocking glucose metabolism in gastric cancer cells [21]. Another article revealed that UDP-glucuronosyltransferase 1A compromised the apoptotic effects of tanshinone IIA by reducing its intracellular exposure and switching the NAD(P)H: quinine oxidoreductase 1-triggered redox cycle to metabolic elimination [24]. Other research noted that dihydrotanshinone I induced caspase and ROS-dependent apoptosis and autophagy in colon cancer cells [47].

ROS-Related Anticancer Effects of Tanshinones on Cervical Cancer Cells.
Our previous studies showed that tanshinone IIA had anticancer effects on typical cervical HeLa and advanced cervical CaSki cancer cells. Tanshinone IIA induced apoptosis by interfering with the microtubule assembly process, leading to G2/M phase arrest and subsequent apoptosis in HeLa cells [32]. It also appeared to inhibit cell growth through activating the ER stress pathway and promoting caspase cascades with concomitant upregulation of the phosphorylation of the p38 and JNK-Bax-caspase-3/9 signaling pathways ( Figure 3) in CaSki cells [25].

ROS-Related Anticancer Effects of Tanshinones on Renal
Carcinoma Cells, Melanoma, Neuroblastoma, and Rhabdomyosarcoma Cells. Previous research noted that tanshinone IIA induced apoptosis in renal carcinoma cells by activating p53 expression and subsequently inducing the upregulation of p21 and Bax [28].
The other cryptotanshinone would restore the sensitivity of A375 melanoma cells that were resistant to TRAIL by upregulating the expression of death receptor 5 (DR5) [39]. It also could inhibit sodium nitroprusside-induced apoptosis by an antioxidant effect and by regulating NF-B and the MAPK pathway in Neuro-2a cells [41]. Cryptotanshinone was reported to induce ROS, then activate P38/JNK, and inhibit  Erk1/2 in rhabdomyosarcoma cells. These effects then led to caspase-independent cell death in these cells [40].

ROS-Related Anticancer Effects of Hydrophilic Components Found in Danshen
Polyphenols, as dietary antioxidants, are most abundant in fruits, vegetables, and cereals [70,71]. Numerous clinical studies, as well as in vitro and in vivo experiments, have strongly supported the ability of polyphenols to reduce the risk of many cancers. Some antioxidant polyphenols can downregulate TNF and might be useful as mitochondrially targeted anticancer drugs [72][73][74]. Salvianolic acids A and B are the main water-soluble polyphenolic derivatives found in Danshen. Similar to other natural polyphenols, they have potential anticancer effects ( Table 2). Salvianolic acid A elevated ROS levels, downregulated P-glycoprotein, and triggered apoptosis by increasing caspase-3 activity and upregulating Bax expression, while downregulating Bcl-2 expression and disrupting the mitochondrial membrane potential in multidrug resistance MCF-7 human breast cancer cells [51]. Other research showed that salvianolic acids A and B had antioxidant and antiapoptotic properties that were involved in protecting SH-SY5Y human neuroblastoma cells against 1-methyl-4-phenylpyridinium ion-induced mitochondrial dysfunction. This dysfunction was characterized by loss of the mitochondrial membrane potential, condensation of nuclei, cytochrome c release, and increases in the Bax/Bcl-2 ratio [52,54]. Salvianolic acid B prevented 6-hydroxydopamine-induced apoptosis in SH-SY5Y cells by reducing the increase of caspase-3 activity and the translocation of cytochrome c into the cytosol from 8 Oxidative Medicine and Cellular Longevity mitochondria [55]. Another study revealed that salvianolic acid B induced apoptotic cell death in human glioma U87 cells through p53 and the phosphorylation and activation of p38 MAPK to increase ROS generation [53].

Conclusion
Danshen may be a potential complementary or alternative therapy for various cancer patients. We found the potential utility of this natural product, or its active constituents including lipophilic compounds such as tanshinone I, tanshinone IIA, acetyltanshinone IIA, cryptotanshinone, isocryptotanshinone, dihydrotanshinone, 15,16-dihydrotanshinone I, miltirone, and hydrophilic phenolic acids such as salvianolic acids A and B (Figure 1). The ROS-related anticancer effects of the lipophilic and hydrophilic constituents isolated from Danshen vary, depending on the specific type of cancer cells (Tables 1 and 2). Overall, Danshen can suppress cell proliferation through antioxidation on tumor initiation and induce apoptosis ( Figure 3) or autophagy through ROS generation on tumor progression, tumor promotion, and tumor metastasis. Some components of Danshen may enhance TNF--induced apoptosis and upregulate caspase-3, caspase-8, caspase-9, ER stress, P21, P53, Bax/Bcl-2, DR5, and AMPK and activate the p38/JNK, MAPK, or FasL signaling pathways. Conversely, these compounds can downregulate hTERT mRNA, telomerase, survivin, VEGF/VEGFR2, CD31, NF-B, Erk1/2, MMPs, microtubule assembly, tyrosine kinases such as EGFR/HER2 and P-glycoprotein and inhibit the PI3K/Akt/mTOR or estrogen receptor signaling pathways ( Figure 2). Combined, these effects inhibit cancer cell proliferation by arresting cell cycle progression, inducing cancer cell apoptosis and/or autophagy, and exerting antiangiogenic and antimetastatic effects. However, in accordance with laboratory evidences obtained in vitro and in vivo, rigorous human studies are needed to demonstrate the anticancer effects of Danshen. Future well-designed clinical studies, such as randomized controlled clinical trials, will be necessary to confirm the efficacy of Danshen as an anticancer agent in human patients.