Pharmacogenomics and the Yin/Yang actions of ginseng: anti-tumor, angiomodulating and steroid-like activities of ginsenosides

Abstract

In Chinese medicine, ginseng (Panax ginseng C.A. Meyer) has long been used as a general tonic or an adaptogen to promote longevity and enhance bodily functions. It has also been claimed to be effective in combating stress, fatigue, oxidants, cancer and diabetes mellitus. Most of the pharmacological actions of ginseng are attributed to one type of its constituents, namely the ginsenosides. In this review, we focus on the recent advances in the study of ginsenosides on angiogenesis which is related to many pathological conditions including tumor progression and cardiovascular dysfunctions. Angiogenesis in the human body is regulated by two sets of counteracting factors, angiogenic stimulators and inhibitors. The 'Yin and Yang' action of ginseng on angiomodulation was paralleled by the experimental data showing angiogenesis was indeed related to the compositional ratio between ginsenosides Rg1 and Rb1. Rg1 was later found to stimulate angiogenesis through augmenting the production of nitric oxide (NO) and vascular endothelial growth factor (VEGF). Mechanistic studies revealed that such responses were mediated through the PI3K-->Akt pathway. By means of DNA microarray, a group of genes related to cell adhesion, migration and cytoskeleton were found to be up-regulated in endothelial cells. These gene products may interact in a hierarchical cascade pattern to modulate cell architectural dynamics which is concomitant to the observed phenomena in angiogenesis. By contrast, the anti-tumor and anti-angiogenic effects of ginsenosides (e.g. Rg3 and Rh2) have been demonstrated in various models of tumor and endothelial cells, indicating that ginsenosides with opposing activities are present in ginseng. Ginsenosides and Panax ginseng extracts have been shown to exert protective effects on vascular dysfunctions, such as hypertension, atherosclerotic disorders and ischemic injury. Recent work has demonstrates the target molecules of ginsenosides to be a group of nuclear steroid hormone receptors. These lines of evidence support that the interaction between ginsenosides and various nuclear steroid hormone receptors may explain the diverse pharmacological activities of ginseng. These findings may also lead to development of more efficacious ginseng-derived therapeutics for angiogenesis-related diseases.

Figure 1

The chemical structure of ginsenosides [6]. glc = glucosyl (C₆H₁₁O₆^-); rha = rhamnosyl (C₆H₁₁O₅^-); ara = arabinosyl (C₅H₉O₅^-); p = pyran; f = furan.

Figure 2

The process of angiogenesis. A) Sprouting angiogenesis: formation of blood vessels is a multi-step process, which includes (i) reception of angiogenic signals (yellow spot) from the surrounding by endothelial cells (EC); (ii) retraction of pericytes from the abluminal surface of capillary and secretion of protease from activated endothelial cells (aEC) and proteolytic degradation of extracellular membrane (green dash-line); (iii) chemotactic migration of EC under the induction of angiogenic stimulators; (iv) proliferation of EC and formation of lumen/canalisation by fusion of formed vessels with formation of tight junctions; (v) recruitment of pericytes and deposition of new basement membrane and initiation of blood flow. B) Non-sprouting angiogenesis – intussusceptive microvascular growth: it is initiated by (i) protrusion of opposing capillary walls towards the lumen; (ii) perforation of the EC bilayer and formation of many transcapillaries with interstitial core (red arrow); (iii) formation of the vascular tree from intussusceptive pillar formation and pillar fusion and elongation of capillaries (green arrows).

Figure 3

The balance hypothesis of the 'angiogenic switch'. Angiogenesis is tightly controlled by the balance of two sets of counteracting factors – angiogenic activators and inhibitors. The stability of 'angiogenic switch' determines the time of initiation of the subsequent angiogenic process. When there are more angiogenic stimulators than angiogenic inhibitors, as in the case of solid tumors, normal wound healing or female endometrial repair, the 'angiogenic switch' will be turned on and angiogenesis will proceed. Furthermore, during the process, each step is strictly mediated by the balance of different types of angiogenic stimulators or inhibitors. In some pathological cases, the 'angiogenic switch' remains in the 'ON' mode which leads to 'non-stop' formation of new blood vessels and ultimately many physiological disorders and diseases.

Figure 4

A fundamental step in tumorigenesis – angiogenesis. Angiogenesis is a critical step in the pathogenesis of solid tumors. Tumors remain in a dormant state (avascular phase) for a long time (up to several years), in which tumors keep their size within 1 – 2 mm³. When tumor progression starts, tumor cells secrete a large amount of angiogenic factors, mainly VEGF, to the surrounding tissues and blood capillaries. Once tumor angiogenesis is initiated, tumors enter a 'vascular phase' and become more aggressive. These newly formed blood vessels provide tumor cells with oxygen and nutrients for them to grow and for the initiation of metastasis.

Figure 5

Angiogenesis assays. Angiogenesis is a multi-step process, Different types of in vitro, in vivo or ex vivo bioassays have been designed to mimic the various steps of angiogenesis. (A) In vivo Matrigel Plug assay: liquid form Matrigel (500 μl) containing growth factor (e.g. bFGF) and/or ginsenoside is injected into the abdominal region of C57/BL mice subcutaneously. The Matrigel will solidify at 37°C and form a solid 'plug'. After 5 days of incubation, the mice are sacrificed and in vivo angiogenesis including endothelial cell invasion, migration and formation of neovessels can be examined [124]. (B) Ex vivo rat aortic ring sprouting assay: rat aortic fragments one millimeter in length are embedded in Matrigel and cultured in the presence of growth factors (e.g. endothelial cell growth supplements – ECGS) and/or ginsenosides. The extent of endothelial sprouting from the aortic fragment can clearly indicate the angiogenic properties of ginsenosides [124]. (C) In vivo sponge implantation assay: a sterile polyether polyurethane sponge (170 mm³) containing ginsenosides is inserted into the abdominal region of Balb/c mice. After incubation for 15 days, the animals are euthanized and neovascularization is examined as indicated by the growth of vessels in the granuloma tissue [129]. (D) In vitro tube formation assay: endothelial cells are seeded on the Matrigel and subsequently incubated in medium containing growth factors (e.g. VEGF) and/or ginsenosides. Endothelial cells will rearrange and alight into a tubular structure. Angiogenic properties of ginsenosides can be reflected from the number of tubes, branching points or tube area.

Figure 6

Schematic overview of ginsenosides-mediated genomic and non-genomic pathways. Ginsenosides possess a steroid-like skeleton composed of four trans-rings with different degrees of glyco-substitution. They are amphipathic in nature and can exhibit their actions at different cellular locations; such as the plasma membrane, cytosol and nucleus. Through the non-genomic pathway (indicated by red arrows), (i) they can initiate their actions by binding with the transmembrane receptors (e.g. ATPase pump, ion transporters and channels, voltage-gated channels and G-proteins) and subsequently activating the associated downstream signaling cascades. Moreover, they can intercalate into the plasma membrane resulting in an alteration of membrane fluidity and a trigger of a series of cellular responses. (ii) binding with steroid hormone receptors (SHRs) including glucocorticoid receptor (GR), estrogen receptor (ER), progesterone receptor (PR), androgen receptor (AR) and mineralocorticoid receptor (MR) present inside or outside the nucleus by using their hydrophobic backbone is another alternative to trigger downstream cellular responses. Those activated (phosphorylated) SHRs can activate the target molecules through a signaling cascade that brings about various cellular responses. (iii) the ligand-bound SHRs can translocate into the nucleus, where they regulate gene transcription by binding with the specific Response Elements (XRE). This is the so called 'genomic pathway' (indicated by blue arrows). Consequently, the altered gene products can affect the final cellular responses.

Figure 7

Schematic overview of ginsenoside Rg₁-mediated angiogenic action in HUVEC. Ginsenoside Rg₁, which acts as a functional ligand of glucocorticoid receptor (GR) (either cytosol GR or membrane-bound GR-mGR), promotes angiogenesis through both non-genomic and genomic pathways. Through the non-genomic pathway, it increases nitric oxide (NO) production via the PI3K-Akt pathway: GR → phosphatidylinositol-3 kinase (PI3K)/Akt pathway → endothelial nitric oxide synthase (eNOS) pathway. Rg₁ also increases vascular endothelial growth factor (VEGF) production through the GR → PI3K/Akt → GSK3β → β-catenin/TCF pathway. Gene expression profiling data indicated that Rg₁ could increase the expression of a group of genes (e.g. Rho A, RhoB, IQGAP1, LAMA4, CALM2 and Vav2) which are related to cell-cell adhesion, migration and cytoskeletal remodeling.