In TCM, the principle of combining herbs for a Chinese herbal formula is monarch, minister, assistant and guide (Jun, Chen, Zuo and Shi) [5, 22]. The monarch herb(s) are the key ingredient in the formula to target the primary cause or syndrome. Minister herb(s) are used to strength the effect of the Monarch herb(s) or address the secondary syndromes. Assistant herb(s) are utilized to reinforce the curative effect of the Monarch or Minister herb(s), or allay the drastic and toxic effect of the Monarch or Minister herb(s). While the Guide herb(s) are applied to harmonize and integrate the effects of other herbs, or direct the formula to act on the target meridian or the offending part of body. However, Guide herb(s) are not indispensable for a formula, based on the specific conditions, Guide herb(s) might be used or not used in a formula.
According to the TCM theories, Actinidia chinensis, Solanum nigrum and Duchesnea indica are used to against the pathogenic factors of damp-heat and toxicity accumulation, and served as Monarch herbs in TLBZT. In addition to traditional herbal efficacy, Actinidia chinensis, Solanum nigrum and Duchesnea indica also have been proved anticancer potential. It has been reported ethanol extracts from Actinidia chinensis may inhibit colon carcinoma LoVo cells and HT-29 cells proliferation, and induce apoptosis in LoVo cells accompanied by Bcl-2/Bax downregulation and Caspase-3 upregulation [23, 24]. Components of Solanum nigrum, such as solamargine, Solanine, polysaccharide and polyphenol-rich extract of Solanum nigrum have demonstrated anticancer effects against various cancer cells [25–28]. Phenolic extract of Duchesnea indica can inhibit cervical and ovarian cancer growth through induction of apoptosis and cell cycle arrest [29, 30].
Atractylodes macrocephala Koidz, Poria cocos and Coix seed are used as Minister herbs to target spleen-deficiency, damp and the loss of appetite induced by long-term use of Monarch herbs. In addition to traditional efficacy of tonifying Pi (spleen), Atractylodes macrocephala Koidz, Poria cocos and Coix seed or their components also have been showed anticancer effects against cancer cells. Atractylodes macrocephala Koidz extract may inhibit S180 tumor growth . Poricotriol A from Poria cocos may induce apoptosis in leukemia HL-60 cells and lung cancer A549 cells . Pachymic acid from Poria cocos may reduce cell proliferation and induced apoptosis through mitochondria dysfunction in prostate cancer cells . Kanglaite injection, a Coix seed extract, has been widely used as anticancer drug in Chinese oncological clinical . In addition, a variety of compounds from Coix seed bran ethanolic extract, such as coixspirolactam D, coixspirolactam E, coixspiroenone, coixspirolactam A, coixspirolactam C, coixlactam, and ficusal, may significantly inhibite breast cancer cells proliferation .
Scutellaria barbata and Mistletoe are Assistant herbs to enhance the effects of Monarch and Minister herbs by their anti-angiogenesis and anticancer effects. It has been reported Scutellaria barbata may inhibit angiogenesis in vitro and in colorectal cancer model via suppression of Hedgehog pathway and VEGF [16, 17]. Scutellaria barbata extract also have been showed cytotoxity effects against human colon cancer cells . Viscum album (Mistletoe) may induce apoptosis in endothelial cells and inhibit angiogenesis [18, 19]. In addition, Mistletoe lectins could inhibit proliferation and induce apoptosis in colon cancer HT-29 cells . TLBZT is an herbal formula fitted with both TCM theories and the principle of anticancer. In present study, we observed TLBZT, alone or in combination with 5-Fu, significantly inhibited CT26 colon carcinoma growth accompanied by apoptosis.
Apoptosis is an evolutionarily conserved cell suicide process that acts to balance mitosis in the development and maintenance of tissue homeostasis for the removal of superfluous, transformed or damaged cells, and has been recognized as a popular target for anticancer therapy [10, 38, 39]. Two major pathways have been identified in the process of apoptosis. In extrinsic death receptor pathway, the death ligands (FasL, TRAIL, etc.) binds to the death receptors (FAS, TRAIL-R, etc.) which recruits adaptor proteins, such as Fas-associated death domain (FADD), to form ligand-receptor-adaptor protein complex (known as the death-inducing signalling complex, DISC), and then activists Caspase-8, followed by Caspase-3 activation and apoptosis. The intrinsic pathway involves the signals to mitochondria which lead to release of cytochrome C from mitochondria. Released Cytochrome C combines Apaf-1 and Caspase-9 to form apoptosome and activates Caspase-9 which in turn activates Caspases-3, causing the cell to undergo apoptosis. As the members of inhibitor of apoptosis proteins (IAPs), XIAP and Survivin are overexpressed in colorectal cancer, and have been recognized as diagnostic markers and therapeutic targets [12, 13, 40]. XIAP and Survivin may inhibit activation of Caspases, downregulation of XIAP and Survivin could sensitize colorectal cancer cell to drug induced apoptosis [41, 42]. In present study, TLBZT alone or in combination with 5-Fu, significantly induced apoptosis in CT26 colon carcinoma, accompanied by Casapse-3, 8 and 9 activation, and downregulation of XIAP and Survivin, suggested casapses activation and downregulation of XIAP and Survivin may contribute to TLBZT and 5-Fu induced apoptosis.
In addition to apoptosis, cell senescence also contributes to cancer therapeutic response, and has been suggested as a cancer treatment target [43–45]. Cell senescence is a state of stable irreversible cell cycle arrest and loss of proliferative capacity. Senescent cell maintains some metabolic activity but no longer proliferates, and exhibits increased SA-β-gal activity at an acidic pH. Positive of SA-β-gal staining at an acidic pH has been identified as biomarker of cell senescence since 1995 . Cell senescence is closely related to the activation of the CDKN2a (p16INK4A)/pRB or CDKN1a (p21WAF-1/Cip1)/pRB signaling pathway [15, 43–45]. The CDK4 and CDK6 inhibitor p16 participates in regulation of RB phosphorylation, induces cell cycle arrest, and contributes to the induction of cell senescence. p21, an important cell cycle regulator, inhibits a variety of cyclin/CDK complexes, resulted in hypophosphorylation or dephosphorylation of RB protein which binds to E2F and prevents it from activating target genes that are essential in the cell cycle, usually leading to cell cycle arrest. It have been reported natural products, such as Ganoderiol F, Antrodia camphorata extract, Liver-Yin tonifying herbs can inhibit cancer cell growth via cell senescence [46–48]. In present study, TLBZT significantly increased SA-β-gal activity accompanied by an increase in p16 and p21, and downregulation of RB phosphorylation, suggested that TLBZT may induce cell senescence in CT26 carcinoma and related to upregulation of p16 and p21 and downregulation of RB phosphorylation.
Angiogenesis, the process of new blood vessel generate from existing vessels, plays a crucial role in tumor growth and metastasis. Angiogenesis has been recognized as an impotent therapeutic target for cancer treatment since it first proposed by Judah Folkman in 1971 . Currently, angiogenesis targeted drugs, such as bevacizumab (monoclonal antibody against VEGF), sorafenib, sunitinib, pazopanib and everolimus have been wildly used in clinical. CD31 or platelet/endothelial cell adhesion molecule-1(PECAM-1) is a widely used marker protein for angiogenesis [20, 21]. VEGF, secreted by cancer cells, vascular endothelial cells or tumor associate macrophages, is a major driver of tumor angiogenesis [50, 51]. By stimulating vascular endothelial cells proliferation, VEGF can trigger angiogenesis and promote tumor growth. In present study, we detected TLBZT significantly inhibited angiogenesis in CT26 colon carcinoma with concomitant downregulation of VEGF, suggested that anti-angiogenesis may contribute to TLBZT mediated anticancer effects. In TLBZT, Actinidia chinensis, Solanum nigrum, Duchesnea indica, Scutellaria barbata[16, 17], and Mistletoe[18, 19] or their ingredients have been demonstrated anti-angiogenesis effects. The components and the precise mechanism responsible for TLBZT induced anti-angiogenesis effects need to be further explored.