Eriocalyxin B Biological Activity: A Review on Its Mechanism of Action

Abstract

Natural products, a rich source of bioactive chemical compounds, have served humans as a safer drug of choice since times. Eriocalyxin B, an ent-Kaurene diterpenoid, has been extracted from a traditional Chinese herb Isodon eriocalyx. Experimental data support the anticancer and anti-inflammatory activities of EriB. This natural entity exhibits anticancer effects against breast, pancreatic, leukemia, ovarian, lung, bladder, and colorectal cancer. EriB has capability to inhibit the proliferation of cancer cells by prompting apoptosis, arresting cell cycle, and modulating cell signaling pathways. The regulation of signaling pathways in cancerous cells by EriB involves the modulation of various apoptosis-related factors (Bak, Bax, caspases, XIAP, survivin, and Beclin-1), transcriptional factors (nuclear factor kappa B [NF-κB], STAT3, Janus-activated kinase 2, Notch, AP-1, and lκBα), enzymes (cyclooxygenase 2, matrix metalloproteinase 2 [MMP-2], MMP-9, and poly (ADP-ribose) polymerase), cytokines, and protein kinases (mitogen-activated protein kinase and ERK1/2). This review proposes that EriB supplies a novel opportunity for the cure of cancer but supplementary investigations along with preclinical trials are obligatory to effectively figure out its biological and pharmacological applications.

Nature remains a rich source of biologically active chemical entities since times. Natural products and medicinal plants have played an indispensable role in the discovery of drugs as the source of novel pharmacologically active compounds. ¹ Natural products are the compounds isolated from natural sources, eg, plants, animals, as well as microorganisms. ² They are widely used in traditional medicines since millions of years. ³ In the recent years, natural products have gained popularity because of their nontoxicity and efficacy in comparison to synthetic drugs. ⁴

Plants have always been utilized in traditional medicines for the cure of several diseases. Mesopotamian civilization (2600 BC) provides the first evidence of use of plants in traditional medicines enlisting 1000 medicinal products isolated from different plants. “Ebers Papyrus” (1500 BC), Egyptian medicinal history, provides information about 700 plant-derived drugs. Moreover, Chinese Meteria Medica and Indian Ayurvedic system documented the use of herbal drugs. Arabs devised a medicinal system that combined the use of Chinese and Indian medicinal herbs. ⁵ In the recent years, plants and herbal extracts have thoroughly been investigated for the cure of various ailments. ⁶

It is estimated that about one third of all Food and Drug Administration-approved drugs since last 20 years have plant-based origin. ⁷ Plant-based drugs are the preferable choice as compared to the synthetic drugs because of their safety profiles as compared to synthetic drugs. ^8,9 Most of the scientific research on the plants declares that they act as anti-inflammatory, antioxidant, antiproliferative, as well as cytotoxic agents. ¹ Phytochemicals have been documented to inhibit cancer by modulation of multiple deregulated pathways which are pragmatic to cancer progression. ^10,11

Diterpenoids represent a broad class of isoprenoid natural entities, which occur naturally in plants, fungi, marine organisms, and insects. Kauranes are broad-spectrum tetracyclic diterpenes, which explicit numerous biological activities. ¹² The identification and understanding of mechanisms lying behind the pharmacological effects of these compounds will revitalize the targeted therapies toward an upgraded level. ⁴ Eriocalyxin B (EriB) (Figure 1) is a biologically active ent-kaurene diterpenoid purified from Isodon eriocalyx. ^3,13 This compound has been documented to exert anti-inflammatory, ¹⁴ antiproliferative, ¹³ antitumor, ¹⁵ and anti-angiogenic effects. ¹⁶

Figure 1

Chemical structure and biological activities of eriocalyxin B.

Currently, no one has attempted to provide review about the pharmacological potential of EriB. The present article is aimed at reviewing the biological significance and pharmacological effects of EriB. This review will focus on the pharmacological position of EriB as potential drug candidate depicting the areas for farther research. The literature cited in this chapter is accessed via various e-sites including PubMed, Springerlink, Sciencedirect, Scopus, and Researchgate along with other medical journals. Keywords used for this search include “Eriocalyxin B,” “Biological activities of Eriocalyxin B,” “natural products,” “anticancer,” and “anti-tumor.”

Natural Sources of Eriocalyxin B

The discovery of EriB dates back to the isolation of this compound from the leaves of Isodon eriocalyx var. laxiflora in the year 1982. ¹⁷ Isodon, a perennial herb belonging to family Labitate, found in the Southwest of China (Yunnan Province), exhibits numerous pharmacological effects ¹³ such as anti-inflammatory, antibacterial, and antitumor. It is in use as local folk medicine in China and Japan since long. ^17,18 The plants of genus Isodon are used as folklore medicines in different areas of the world because of their potent biological and pharmacological activities. ¹⁹ EriB was discovered to be the most potent apoptosis-inducing compound in the active fraction of Isodon eriocalyx, as indicated by nuclear magnetic resonance analysis of high-speed counter-current chromatography–purified fraction. ²⁰ EriB occurs in nature as a major bioactive constituent of Isodon eriocalyx while the smaller proportions are also found in Isodon japonicus and Isodon trichocarpus. ¹⁸ The highest contents of EriB are found in the dried leaves of the plants. ¹⁷

Biological Activities of Eriocalyxin B

EriB is potentially an effective compound for pain relief, neuroprotection, inflammation, and immunomodulation. ²¹ It has also been known to possess potent anticancer properties ¹³ (Figure 1). Many in vivo and in vitro experiments have been performed to elucidate the molecular mechanisms and medicinal properties of EriB.

Anticancer Activities

Cancer refers to uncontrolled cellular proliferation marked by the dysregulated epigenetics, along with metabolic and signaling defects. ²² It is considered as the second highest mortality-causing disease in the world, which accounts for 8.8 million deaths per year, estimated by World Health Organization in 2015. The most accessible and the most practiced cure for the treatment of this fatal disease is chemotherapy or synthetic drugs. It is a common practice in most of the cases but the adverse effects and the development of resistance against the disease limit the efficacy of chemotherapy, inviting the need for more effective modalities against cancer. ¹ Natural products provide the cheap and worthy incentives for the development of novel drug candidates. ²³ Since the last few years, pharmaceutical industry has been using the medicinal plants as the imperative pool for the discovery of novel drug candidates. ²⁴

EriB and Cell Cycle Arrest

Cell cycle is a series of precisely regulated cellular events that enable a cell to make an exact copy of itself. ²⁵ It is regulated by the molecular events, which prevent the DNA damage, and control and align up the sequence of the cell divisions in an irreversible manner. Cyclins and cyclin-dependent kinases (CDKs) bind to each other to form activated heterodimers for the control of cell cycle. In addition to cyclins and CDKs, various checkpoints are also present in the cells, which monitor the cell cycle progression. ²⁶ The loss of control at cell cycle checkpoints leads to cancer progression. ²⁷ Anticancer compounds have the ability to inhibit the activity of CDKs as well as various enzymes and protein factors that regulate the cell cycle and stabilize the normal function of cells. ^1,26

Various investigations have shown that EriB is a potential anticancer agent, ²⁸ which arrests the cells’ G2/M phase. ^15,23 In human pancreatic cancer cells (CAPAN-2), 48 hours of treatment of EriB mediated G2/M phase arrest in a dose-mediated mode. The molecular mechanism lying behind the cell cycle arrest proposed that EriB activated p53 protein, thus increasing the expression of CDK inhibitor p21. The results suggest that p53/p21/cdk1-cyclinB1 signaling might cause EriB-mediated G2/M cell cycle arrest. It was also concluded that EriB caused an increase in the phosphorylation of ERK1/2 and p38 mitogen-activated protein kinase (MAPK). The p-ERK1/2 and p-p38 MAPK mediate the activation of p53 via the direct phosphorylation of p53, which ultimately leads to cell cycle arrest in pancreatic cancerous cells. ²⁹ The studies conducted on the human bladder cancer cell line, T24 cells, revealed that G2/M phase arrest was one of the underlying mechanisms through which EriB can induce anticancer effects. ²³ Cell cycle arrest was also noticed at G1 phase in human umbilical vein endothelial cells (HUVECs) treated with EriB. This cell cycle arrest was found to be correlated with the reduced expression of cyclin D1 and CDK4. EriB inhibited the expression of cyclin1, CDK4, and pRb levels upregulating levels of p21Waf1 (CDK inhibitor). As a result of these cell cycle events, the progression of cell cycle was inhibited and DNA synthesis was also stopped. ¹⁶

EriB and Angiogenesis

Angiogenesis is the generation of new capillaries from the old ones. It is an essential hallmark of many ailments such as cancer, atherosclerosis, ischemia, and inflammatory diseases. ^30,31 The newly organized network of vascular tissues nourishes the tumor sites in addition to the management of metabolic wastes. This phenomenon enables the amplification, invasion, and ultimately metastasis. ³² The molecular mediators of angiogenesis include growth factors and cytokines mainly such as vascular endothelial growth factor (VEGF), fibroblast growth factor, tumor necrosis factor-α (TNFα), and angiopoietins, cell-matrix interactions (matrix metalloproteinases and related proteases, extracellular matrix, integrins), and intracellular signaling pathways (Rh0 GTPases, protein kinase C, Notch signaling). ³³ Among the various pro-angiogenic factors, VEGF is the key factor regarding angiogenesis, while VEGFR-2 is the most important modulator of VEGF-stimulated signal transduction. It controls the survival of endothelial cells, their malignancy, and migration. ³⁴ Hence, VEGFR-2 has become a recent drug target in anti-cancer therapies. ¹⁶

Plants and nature-derived compounds have played a pivotal role in developing anticancer drugs. The main advantage of the use of nature-derived compounds is their low toxicity and readily available ingestive form. ³⁵ EriB treatment to zebrafish model (10 and 15 µM) and mouse 4T1 breast tumor models inhibited angiogenesis in vivo. It also altered various genes involved in angiogenesis as indicated by transcriptome profiling. It regulates cell adhesion molecules, p53 signaling, ECM-receptor interactions, and protein-protein interaction leading toward the modulation of VEGFR, thus alleviating angiogenesis. EriB treatment (50 and 100 nM) significantly inhibits the VEGF-induced tumor malignancy, cell migration, as well as cell invasion in HUVECs. Mechanism of EriB-mediated antiangiogenic effect proceeds via competitive inhibition of ATP against VEGFR, thus reducing its phosphorylation. Reduced phosphorylation of VEGFR leads to inhibition of cell viability and angiogenesis. Administration of 5 mg/kg EriB to 4TI breast tumor model reduced the vascularization of tumors in vivo. ¹⁶ Treatment of 1 µmol EriB markedly reduced expression levels of both VEGF and VEGFR in SW1116 cells, which responsible for inhibition of angiogenesis in cancer cells. ³⁶

EriB as Apoptosis Inducer

Apoptosis, programmed cell death, is marked by distinct morphological changes and energy-dependent biochemical cascades. ^37,38 It is considered as a critical component of several biological processes such as functioning of immune system, cellular turnover, and embryonic development. ³⁷ There are 2 important pathways of apoptosis: extrinsic and intrinsic pathways. The extrinsic pathway is activated through various death receptors including Fas, TNF, DR3-6, and TRAIL with their corresponding ligands. The activated receptors form a DISC (death-inducing signaling complex) via recruiting FADD (fas-associated death domain) and procaspase-8/-10.

According to a report, EriB alleviated the growth of cells in a dose-mediated mode in pancreatic cancer cell lines with the half-maximal inhibitory concentration (IC₅₀) values of 0.73-1.40 μM.

Pancreatic CAPAN-2 cells displayed maximum sensitivity toward EriB treatment. EriB markedly reduced the expression of Bcl-2 while enhanced the expression of Bak in CAPAN-2 cells ^29,39 (Table 1). In another study, EriB-treated Kasumi-1 leukemia cells showed vacant and swelling mitochondria, inflated cells, and an increase in the number of lysosomes displaying early apoptosis and deterioration in mitochondria. EriB treatment decreased the levels of Bcl-2 and Bcl-XL leading to mitochondrial instability, activation of caspase-3, and cleavage of poly (ADP-ribose) polymerase (PARP). ⁴⁰

Table 1

Molecular Targets of EriB in Various Cancer Types.

Cancer type	Cell line	Treatment conditions		EC50	Molecular targets	Cell cycle arrest	References
		No of cells/well	Treatment time (hours)
Breast	MCF-7	-	24, 72	0.68-0.80 µM	Akt/mTOR/p70S6┴, LC3B-II↑, Beclin-1↑, p62↓, Bcl-2↓	-	22
	MDA-MB-231	-	24, 72	0.35-0.98 µM
Pancreatic	PANC-1	5 × 103	72	3.5 µM	G2/M arrest, Bak↑, Bax↑, p53↑, p-p38MAPK↑, p-ERK↑, Bcl-2↓, Caspase-3/-7/ -8/-9 Act	G2/M	15,29
	SW1990	1 × 104	72	0.73-1.4 µM
	CAPAN-1	1 × 104	72	0.73-1.4 µM
	CAPAN-2	5 × 103	72	1.4 µM
Ovarian	OCSCs	4500	24, 48, 72	0.5-1 µM	XIAP↓, NF-κB┴, Caspases-3/7Act	-	41
	mOCCs	8000	24, 48, 72	0.5-1 µM
Lymphoma	HUT78	-	72	2 µM	Caspase Act, Bcl-2↓, Bcl-xL↓, Bax↑, ERKAct, NF-κB┴	-	13,28,42
	SU-DHL-4	-	72	1.0-5.6 μM
Leukemia	Kasumi-1,	-	-	0.25 µM	Δψm↓, PARP cleavage Bcl-2↓, Bcl-xL↓, caspases-3Act, pERK1/2↓, AP-1↑, NF-κB┴	-	40
	HL-60	-	-	1 µM
	U937	-	-	1 µM
	NB4	-	-	0.5 µM
	NB4/R1	-	-	1 µM
	NB4/R2	-	-	0.5 µM
Colorectal	HCT	-	-	0.34 µM	pJAK2┴, pSTAT3┴, MMP-2↓, MMP-9↓, VEGF↓, VEGFR2↓, PCNA↓	-	36,42
	SW1116	-	6, 12, 24, 48	-
Lung	A549	-	-	0.24 µM	p- STAT3┴	-	42,43
Bladder	T24	-	-	0.087 µg/mL		-	44
Liver	SMMC7721	3-6 × 103 cells	48	0.598 µM		-	45

↑, Upregulation; ↓, downregulation; ┴, inhibition; Akt, activation Act; AP-1, activator protein 1; Bax, Bcl-2-associated x protein; Bcl-2, B-cell lymphoma 2; EriB, eriocalyxin B;ERK, extracellular signal-regulated kinase; JAK2, janus-activated kinase 2; LC3B-II, light chain 3B-II; MAPK, mitogen-activated protein kinase; MMP-2, matrix metalloproteinase-2; MMP-9, matrix metalloproteinase-9; mTOR, mammalian target of rapamycin; NF-κB, Nuclear factor kappa-light-chain-enhancer of activated B cells; OCSC, ovarian cancer stem cell. PARP, poly (ADP-ribose) polymerase; pJAK2, phospho-JAK2; STAT3, signal transducer and activator of transcription 3; VEGF, vascular endothelial growth factor; VEGFR-2, vascular endothelial growth factor receptor 2; XIAP, X-linked inhibitor of apoptosis protein.

EriB and Nuclear Factor Kappa B Pathway

Since after the discovery of nuclear factor kappa B (NF-κB) in 1986, both of the nuclear and cytoplasmic regulations of NF-κB have been well described. ⁴⁶ It is a family of transcriptional factors that becomes activated in various cancers like breast, leukemia, prostate, pancreas, lymphoma, and colon cancers. ⁴⁷ NF-κB regulates inflammatory as well as immune responses, tumorigenesis, metastasis, ⁴⁸ and chemoresistance. ^{49 -51} p65 and p50 are the 2 heterodimers of NF-κB that mainly reside in the cytoplasm by interacting with IκBα. Activation of cells leads toward IκBα phosphorylation via IKK complex, fast ubiquitination, and consequent degeneration of IκBα. This in turn stimulates translocation of p65/p50 complexes in the nucleus, thus leading toward binding of these complexes with NF-κB response components and mediate the level of their downstream genes. ⁵² These aforementioned steps are a subject of matter for the scientists to discover novel drugs that can target various types of cancers and inflammatory disorders involving NF-κB activation. ^{53 -55} Recent reports support the idea of phytochemicals to be used as the modulators of tumor promoting transcription factor NF-κB, ultimately promoting apoptosis in the cancerous cells. ¹ EriB acts as an inhibitor of NF-κB due to the presence of 2 α, β-unsaturated ketones. EriB significantly downregulated the transcription of NF-κB and its downstream targets (cyclooxygenase 2 and iNOS) in hepatocarcinoma cells. It also prohibits the DNA-interacting action of p65 and p50 subunits. ⁵²

In the case of SMMC-7721 cells, p50 protein is identified as a target of EriB. Cysteine 62 (a critical residue of p50) is targeted by EriB, thus blocking interaction between p50 and its response elements. ⁵¹ In ovarian cancer stem cells, the cell death was attributed toward the inhibition of NF-κB, caspases activation, and downregulation of XIAP. Thus, EriB may act as an effective drug candidate for the discovery of many anticancer drugs. ⁴¹

EriB and Janus-Activated Kinase/ STAT3 Pathway

STAT is a family of transcriptional factors that is engaged in the expression of certain genes, responsible for cell proliferation, survival, angiogenesis, and chemoresistance. STAT3, a member of STAT family, serves as a cytoplasmic transcription factor. Its phosphorylation is carried out through certain growth factors or cytokines, which results in the translocation of STAT3 from the surface of cell toward the nucleus. ³⁶ JAK (Janus-activated kinases), interleukin 6, EGRF, and Src family kinases are responsible for the activation of STAT3. When activated, STAT3 leads to cancer progression and inhibition of apoptosis. The studies conducted by Lu et al. (2014) suggested that JAK2/STAT3 signaling pathways can be the target for the cure of human colon cancer. EriB (1 µmol/L) effectively inhibited phosphorylation of JAK2 along with STAT3 in SW1116 cancerous cells. This condition led to the prohibition of cell proliferation, angiogenesis, invasion, and migration. EriB interacts directly with STAT3 without affecting the upstream targets (protein tyrosine kinases or protein tyrosine phosphatase). The experimental evidences indicate that EriB forms covalent bond with STAT3, thus retarding its activation, ultimately leading to apoptosis of cancer cells. ⁴³ Hence, the growth of cancer cells can be suppressed by using specific inhibitors of JAK2 or STAT3 ⁵⁶ (Figure 2).

Figure 2

A diagram representing the cytotoxic effects of EriB against different types of cancers. EriB, eriocalyxin B; IL-6, interleukin 6; JAK2, Janus-activated kinase; MAPK, mitogen-activated protein kinase; mTOR, mammalian target of rapamycin; NF-κB, nuclear factor kappa B; PARP, poly (ADP-ribose) polymerase.

EriB and MAPK Pathway

MAPK is a family of 6 members including ERK, JNK, and p38 MAPK. They play a crucial role in cellular proliferation, invasion, and differentiation. ^57,58 MAPK signaling pathway is responsible for the growth of cancer cells and insensitivity toward anticancer drugs. ⁵⁸ EriB has the ability to induce phosphorylation of p38 MAPK and ERK1/2 in a dose-mediated mode ²⁹ (Figure 3).

Figure 3

Diagram representing the molecular targets of EriB with their possible mechanism of actions. COX-2, cyclooxygenase 2; EriB, eriocalyxin B; IFN, interferon; IL, interleukin; JAK, Janus-activated kinase; MMP, matrix metalloproteinase; TNF, tumor necrosis factor.

Anti-inflammatory Activity

Inflammation characterizes a number of physiological and pathological conditions. ⁵⁹ It can occur due to the response of several factors such as pathogenesis (cardiac infarction, tissue injury, infection), cellular damage, and toxic agents in the environment. Cellular damage leads to the activation of inflammatory pathways like NF-κB, MAPK, and JAK/STAT. ⁶⁰ Inflammation is a common feature of neurodegenerative ailments. In case of Parkinson’s disease, inflammation in substantia nigra happens due to overproduction of inflammatory factors leading to the dysfunction of dopaminergic neurons. ⁶¹ EriB has competency to protect the dopaminergic neurons via prohibiting the production of proinflammatory cytokines and selectively modulating MPP + induced microglia activation via targeting NF-κB. ⁶² EriB inhibits NF-κB activity in a dose-dependent way and reduces inflammation in the damaged tissues. Thus, EriB can be used as a potent drug candidate in the cure of acute and chronic inflammatory ailments. ⁴¹

Synergistic Effects of EriB

Synergy is a phenomenon that refers to the interaction of multiple agents to create a better effect than the effect produced by individual agent. ⁶³ Synergy is gaining importance nowadays and it has replaced the conventional approach of “one drug, one target, one disease” as it proves to give better therapeutic outputs in the diseases with altered physiological conditions and etiology such as AIDS, cancer, and diabetes. Mono-drugs prove to be less effective in such chronic ailments. ³⁰ Recent studies have supported the idea of development of in vitro models to investigate the synergistic effects of EriB with other synthetic drugs such as gemcitabine (Gem). Treatment of PANC-1, CAPAN-2, and CAPAN-1 cells to EriB with Gem markedly augmented the apoptotic cells ratio to 35%, which was 26% when cells were treated alone either with EriB or Gem. Treatment of Gem or EriB alone did not have any significant effect on phosphorylation of PDK1 although EriB alone inhibited the p-Akt levels. However, the combined treatment of Gem and EriB significantly inhibited the phosphorylation of both Akt and PDK1 resulting in enhanced anticancer potential. EriB and Gem both induce the cleavage of PARP and caspase-3, but their combined exposure exhibit enhanced activation of caspases, cleavage of PARP, reduction in the phosphorylation of PDK1 and Akt as well as the phosphorylation of JNK when compared to the cells treated alone either with EriB or Gem. ⁶⁴

In Vivo Studies and Biosafety Profiling

Intravenous administration of EriB in Sprague Dawley mice to study the pharmacokinetic profiling did not show any adverse effects. The concentration used was 2 mg/kg and the samples were collected at regular intervals. After that, the samples were centrifuged and plasma concentration-time curve was plotted. Results demonstrated that EriB does not exert any adverse effects. ⁶⁵ EriB has also been studied in vivo in pancreatic tumor xenograft models in nude mice. The control group was treated with 1% Pluronic F68 in phosphate buffer saline vehicle while the experimental group was treated for 20 consecutive days with EriB at doses of 1.25 and 2.5 mg/kg. It was noticed that tumor growth was inhibited in the experimental group in a dose-dependent manner. It means that the high dosage of EriB (2.5 mg/kg) has more decreased tumor volume as compared to low dose (1.5 mg/kg). Intraperitoneal treatment of EriB at a high dose for 20 days has been shown to possess no drug-like side effects. ²⁹

It is strongly recommended that more in vivo as well as preclinical studies should be performed to examine the safety dosage and efficacy of EriB. Furthermore, the areas such as genotoxicity, nephrotoxicity, hepatotoxicity, and reproductive toxicity should be addressed by the researchers to consider EriB as an effective drug candidate.

Conclusions and Future Perspectives

This review article highlights the status of EriB as an anticancer and anti-inflammatory compound. The data presented in this review support the efficacy of EriB as a potent anticancer drug, which can be used against various cancer types. It has very low IC50 of 0.24 µM as recorded against lung cancer cell line A549. It shows very interesting synergistic effects when combined with other synthetic anticancer drugs such as Gem. Various in vitro and in vivo studies as well as the biosafety profiling of EriB declare it as a nontoxic compound. The use of EriB as a food supplement in various regions of China and Japan attests its nontoxic nature as a traditional medicine. The molecular mechanisms lying behind the biological properties of this compound show that it modulates various cell signaling pathways including NF-κB, JAK, STAT3, NOTCH, and Wntβ pathways. These signaling pathways are dysregulated in many cancers and lead toward uncontrolled proliferation of cells. Moreover, extensive studies are yet obligatory to fully elucidate the mode of action of EriB against various diseases.