Cinobufagin is a
cardiotoxic
Cardiotoxicity is the occurrence of heart dysfunction as electric or muscle damage, resulting in heart toxicity. The heart becomes weaker and is not as efficient in pumping blood. Cardiotoxicity may be caused by chemotherapy (a usual example is th ...
bufanolide
Bufanolide is a C24 steroid and, indirectly, a parent structure of bufadienolide. Its derivatives was found in ''Bufo'' and ''Scilla'', as an aglycone of cardiac glycosides and is usually toxic
Toxicity is the degree to which a chemical su ...
steroid secreted by the Asiatic
toad
Toad is a common name for certain frogs, especially of the family Bufonidae, that are characterized by dry, leathery skin, short legs, and large bumps covering the parotoid glands.
A distinction between frogs and toads is not made in scient ...
''
Bufo gargarizans
The Asiatic toad or Chusan Island toad (''Bufo gargarizans'') is a species of toad endemic to East Asia. The species was previously classified as ''Bufo bufo gargarizans'', a subspecies of the common toad.
Distribution and habitat
It is comm ...
''. It has similar effects to
digitalis
''Digitalis'' ( or ) is a genus of about 20 species of herbaceous perennial plants, shrubs, and biennials, commonly called foxgloves.
''Digitalis'' is native to Europe, western Asia, and northwestern Africa. The flowers are tubular in shap ...
and is used in
traditional Chinese medicine
Traditional Chinese medicine (TCM) is an alternative medical practice drawn from traditional medicine in China. It has been described as "fraught with pseudoscience", with the majority of its treatments having no logical mechanism of acti ...
.
Isolation and purification
Cinobufagin, as well as other bufadienolides, can be isolated from the traditional Chinese medicine called ChanSu. ChanSu is made from a multitude of chemicals present in
Bufo gargarizans
The Asiatic toad or Chusan Island toad (''Bufo gargarizans'') is a species of toad endemic to East Asia. The species was previously classified as ''Bufo bufo gargarizans'', a subspecies of the common toad.
Distribution and habitat
It is comm ...
secretions. Resibufogenin can be eluted out with silica gel column
chromatography
In chemical analysis, chromatography is a laboratory technique for the separation of a mixture into its components. The mixture is dissolved in a fluid solvent (gas or liquid) called the ''mobile phase'', which carries it through a system ( ...
, using a 5:1 ratio of
cyclohexane to
acetone
Acetone (2-propanone or dimethyl ketone), is an organic compound with the formula . It is the simplest and smallest ketone (). It is a colorless, highly volatile and flammable liquid with a characteristic pungent odour.
Acetone is miscib ...
for the
solvent
A solvent (s) (from the Latin '' solvō'', "loosen, untie, solve") is a substance that dissolves a solute, resulting in a solution. A solvent is usually a liquid but can also be a solid, a gas, or a supercritical fluid. Water is a solvent for ...
in the mobile phase. Subsequently, cinobufagin and bufalin can be separated and purified using an
HPLC column with a 72:28
methanol to
water
Water (chemical formula ) is an Inorganic compound, inorganic, transparent, tasteless, odorless, and Color of water, nearly colorless chemical substance, which is the main constituent of Earth's hydrosphere and the fluids of all known living ...
solvent. Yang et al. confirmed this method of isolation for cinobufagin with
Proton NMR
Proton nuclear magnetic resonance (proton NMR, hydrogen-1 NMR, or 1H NMR) is the application of nuclear magnetic resonance in NMR spectroscopy with respect to hydrogen-1 nuclei within the molecules of a substance, in order to determine the struct ...
.
Clinical significance
Cinobufagin has been shown to have clinical applications in
cancer
Cancer is a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. These contrast with benign tumors, which do not spread. Possible signs and symptoms include a lump, abnormal b ...
treatment as well as immunomodulatory and
analgesic properties.
In human adrenocortical cells, cinobufagin inhibits the secretion of aldosterone and cortisol. Cinobufagin is able to inhibit the expression of the StAR protein as well as bind the transcription factor SF-1, which normally binds to the promoter for the StAR gene. This results in less StAR
protein
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, res ...
product and decreased levels of
aldosterone and
cortisol synthesis. Cinobufagin first binds to a Ca
2+/K
+ plasma membrane
ATPase, subsequently inducing the phosphorylation of
extracellular signal-regulated kinases
In molecular biology, extracellular signal-regulated kinases (ERKs) or classical MAP kinases are widely expressed protein kinase intracellular signalling molecules that are involved in functions including the regulation of meiosis, mitosis, and p ...
(ERK). Phosphorylated ERK then blocks the SF-1
transcription
Transcription refers to the process of converting sounds (voice, music etc.) into letters or musical notes, or producing a copy of something in another medium, including:
Genetics
* Transcription (biology), the copying of DNA into RNA, the fir ...
factor from binding to the
promoter region of the StAR
gene
In biology, the word gene (from , ; "... Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''generation'' or ''birth'' or ''gender'') can have several different meanings. The Mendelian gene is a b ...
.
Thus, cinobufagin plays important roles in regulation of
steroid synthesis and
gene expression. It is speculated that cinobufagin may have therapeutic roles in treatment of
Cushing’s syndrome
Cushing's syndrome is a collection of signs and symptoms due to prolonged exposure to glucocorticoids such as cortisol. Signs and symptoms may include high blood pressure, abdominal obesity but with thin arms and legs, reddish stretch marks, ...
and
heart failure.
Immunology
In vitro
''In vitro'' (meaning in glass, or ''in the glass'') studies are performed with microorganisms, cells, or biological molecules outside their normal biological context. Colloquially called " test-tube experiments", these studies in biology ...
, cinobufagin can stimulate the proliferation of immune cells including splenocytes,
peritoneal
The peritoneum is the serous membrane forming the lining of the abdominal cavity or coelom in amniotes and some invertebrates, such as annelids. It covers most of the intra-abdominal (or coelomic) organs, and is composed of a layer of meso ...
macrophages
Macrophages (abbreviated as M φ, MΦ or MP) ( el, large eaters, from Greek ''μακρός'' (') = large, ''φαγεῖν'' (') = to eat) are a type of white blood cell of the immune system that engulfs and digests pathogens, such as cancer ce ...
,
T helper cells
The T helper cells (Th cells), also known as CD4+ cells or CD4-positive cells, are a type of T cell that play an important role in the adaptive immune system. They aid the activity of other immune cells by releasing cytokines. They are considere ...
and
cytotoxic T cells
A cytotoxic T cell (also known as TC, cytotoxic T lymphocyte, CTL, T-killer cell, cytolytic T cell, CD8+ T-cell or killer T cell) is a T lymphocyte (a type of white blood cell) that kills cancer cells, cells that are infected by intracellular p ...
. Additionally cinobufagin can modulate levels of
cytokines produced by immune cells. Exposure to cinobufagin increases levels of
interferon gamma
Interferon gamma (IFN-γ) is a dimerized soluble cytokine that is the only member of the type II class of interferons. The existence of this interferon, which early in its history was known as immune interferon, was described by E. F. Wheeloc ...
and
tumor necrosis factor alpha
Tumor necrosis factor (TNF, cachexin, or cachectin; formerly known as tumor necrosis factor alpha or TNF-α) is an adipokine and a cytokine. TNF is a member of the TNF superfamily, which consists of various transmembrane proteins with a homolo ...
while decreasing overall levels of
interleukin 4
The interleukin 4 (IL4, IL-4) is a cytokine that induces differentiation of naive helper T cells ( Th0 cells) to Th2 cells. Upon activation by IL-4, Th2 cells subsequently produce additional IL-4 in a positive feedback loop. IL-4 ...
and
interleukin 10
Interleukin 10 (IL-10), also known as human cytokine synthesis inhibitory factor (CSIF), is an anti- inflammatory cytokine. In humans, interleukin 10 is encoded by the ''IL10'' gene. IL-10 signals through a receptor complex consisting of two IL-10 ...
.
Analgesic properties
Cinobufagin has been shown to increase pain threshold levels in mice to thermal and mechanical stimuli. It is thought to trigger increased synthesis of β-END and the up-regulation of the
mu opioid receptor in mouse tumor tissue thereby leading to pain relief. β-END binds the
mu opioid receptor to cause the
analgesic effect.
Interaction with cancer cells and related biochemical pathways
C. elegans
''Caenorhabditis elegans'' () is a free-living transparent nematode about 1 mm in length that lives in temperate soil environments. It is the type species of its genus. The name is a blend of the Greek ''caeno-'' (recent), ''rhabditis'' (r ...
can catabolize cinobufagin into five distinct metabolites, each of which has been shown to have cytotoxic effects to
HeLa
HeLa (; also Hela or hela) is an immortalized cell line used in scientific research. It is the oldest and most commonly used human cell line. The line is derived from cervical cancer cells taken on February 8, 1951, named after Henrietta ...
cancer cells.
Cinobufagin can induce
cell cycle
The cell cycle, or cell-division cycle, is the series of events that take place in a cell that cause it to divide into two daughter cells. These events include the duplication of its DNA (DNA replication) and some of its organelles, and sub ...
arrest at the G2 and M phases as well as induce
apoptosis in
osteosarcoma
An osteosarcoma (OS) or osteogenic sarcoma (OGS) (or simply bone cancer) is a cancerous tumor in a bone. Specifically, it is an aggressive malignant neoplasm that arises from primitive transformed cells of mesenchymal origin (and thus a sarcoma ...
cells. Potentially, cinobufagin could be used to stop proliferation of osteosarcoma cells as well as to induce
apoptosis them. At the protein level, cinobufagin treated osteoscarcoma cells showed an increase in the Bax and cleaved-PARP apoptotic proteins, while inhibiting the
GSK-3
Glycogen synthase kinase 3 (GSK-3) is a serine/threonine protein kinase that mediates the addition of phosphate molecules onto serine and threonine amino acid residues. First discovered in 1980 as a regulatory kinase for its namesake, glycogen ...
β/
NF-κB
Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is a protein complex that controls transcription of DNA, cytokine production and cell survival. NF-κB is found in almost all animal cell types and is involved in cellular ...
signaling pathway.
With regards to the induction of
apoptosis, cinobufagin has been shown to selectively bind K
+/Na
+ ATPases in
canine kidney
The kidneys are two reddish-brown bean-shaped organs found in vertebrates. They are located on the left and right in the retroperitoneal space, and in adult humans are about in length. They receive blood from the paired renal arteries; blo ...
cells to trigger a signaling cascade which leads to
caspase dependent pathways for
apoptosis. It is through the activation of
caspases that Cinobufagin can cause
apoptosis.
References
{{Cardiac glycosides
Vertebrate toxins
Epoxides
Bufanolides
Secondary alcohols
Acetate esters