Molecular Mechanisms of Casticin Action : an Update on its Antitumor Functions

All over the ‘realm of History’, human beings relied on natural products as a primary source of medicine to cure many diseases and plants are vital source of novel natural medicines (Mukherjee et al., 2010). Natural medicines have been proven to be a central source of narrative agents with a pharmaceutical potential (Ji et al., 2009). Herbal medicine has held and still holds an important position in primary health care in China and western countries as a fertile source of novel lead molecules and constituted a pharmaceutical potential as part of modern drug discovery (Christen and Cuendet, 2012). One potential source of novel anticancer agents is natural plant products (Cragg and Newman, 2005). Flavonoids constitute a large family of the phytochemicals, including flavanols, flavones, flavonols, flavanones, anthocyanidins, proanthocyanidins and isoflavones (Leibowitz and Yu, 2010). The major sources of flavonoids are fruit and vegetables. This class of phytochemicals possesses various biological functions such as anti-cancer, anti-proliferative, antioxidant, pro-apoptotic, anti-inflammatory, and neuroprotective activities (You et al., 1998; Manosroi et al., 2005; Jiang et al., 2013; Zhu et al., 2013; Tan et al., 2014).


Introduction
All over the 'realm of History', human beings relied on natural products as a primary source of medicine to cure many diseases and plants are vital source of novel natural medicines (Mukherjee et al., 2010).Natural medicines have been proven to be a central source of narrative agents with a pharmaceutical potential (Ji et al., 2009).Herbal medicine has held and still holds an important position in primary health care in China and western countries as a fertile source of novel lead molecules and constituted a pharmaceutical potential as part of modern drug discovery (Christen and Cuendet, 2012).One potential source of novel anticancer agents is natural plant products (Cragg and Newman, 2005).Flavonoids constitute a large family of the phytochemicals, including flavanols, flavones, flavonols, flavanones, anthocyanidins, proanthocyanidins and isoflavones (Leibowitz and Yu, 2010).The major sources of flavonoids are fruit and vegetables.This class of phytochemicals possesses various biological functions such as anti-cancer, anti-proliferative, antioxidant, pro-apoptotic, anti-inflammatory, and neuroprotective activities (You et al., 1998;Manosroi et al., 2005;Jiang et al., 2013;Zhu et al., 2013;Tan et al., 2014).
In addition, recent studies also reported that casticin can enhance efficiency in combination with chemotherapeutics drugs (Xia et al., 2013).This review article is an attempt to cover recent information available on the development of biological and pharmacological potential of casticin in the scientific literature compiled from databases such as PubMed, SpringerLink, ScienceDirect, Oncology and MEDLINE, further to provide comprehensive evidence insight into its natural sources, anticancer properties and mechanisms of action of this drug, which may provide a new perspective of casticin as a anti-neoplastic drug candidate for future cancer therapeutics.

Natural sources of casticin
Accumulated data indicate that casticin (Figure 1) has been isolated from many plants species, using ultra-high performance liquid chromatography diode array detector (UHPLC-DAD) in an ODS column under a mixed solvent system of acetonitrile and water, and microemulsion electrokinetic chromatography (MEEKC), structure was elucidated on the basis of NMR analysis and the compound was dissolved in dimethylsulfoxide (DMSO) to demonstrate the activities of casticin on various model system of different human diseases (Haidara et al., 2006;Hogner et al., 2013).
Further casticin, being a flavonoid natural compound is located in fruits, vegetables, nuts, seeds, herbs, spices, stems, and flowers (Jiang and Morgan, 2004;Miyahisa et al., 2006).It has also shown a variety of pharmacological properties of therapeutic interest such as anti-inflammatory and anticancer activities (Manthey et al., 2001;Touil et al., 2009).The summary of plants containing casticin, parts used, and biological/pharmacological activities, is shown in Table 1.
On the basis of previous in vitro, in vivo and epidemiological studies, it has demonstrated that casticin have a great anticancer potential by targeting various signaling pathways related to the initiation, progression and metastasis of cancer.It appears that casticin hold great promise for cancer chemoprevention and treatment through anti-proliferation, blockage of the cell cycle, induction of apoptosis, inhibition of angiogenesis and elimination of drug resistance.This review summarizes the emerging data concerning bioactive compound with multidirectional mechanisms of action including caspasemediated pathway and regulation of apoptosis-related proteins.
Casticin's inhibitory effects on cell proliferation (Song et al., 2010) and induction of apoptotic cell death of human cervical cancer HeLa cells (Zeng et al., 2012) are primarily mediated by mitochondrial dependent ROS generation and activation of caspase -3 and -9 (Chen et al., 2011a).Apoptotic cells characteristics such as translocation of phosphatidylserine (PS) from internal cell surface to external cell surface, in the early stage of apoptosis (Jiang et al., 2013).Further, after activation of a cascade of various caspases, caspase-3, and PARP are cleaved and activated, followed by DNA fragmentation, nuclear fragmentation, the appearance of apoptotic bodies and cellular shrinkage are considered essential features of apoptosis (Shen et al., 2009).In the late stage of apoptosis, major DNA with formation of typical DNA ladder of 180 -220 bp can be seen (Collins et al., 1997).

Targeting cancer cells by mitochondria-mediated apoptosis
Disruption of mitochondrial integrity is an important component of the apoptosis execution machinery.It is also one of the early events leading to apoptosis, which contain pro-apoptotic proteins such as cytochrome c.Extensive studies have revealed a rapid release of cytochrome c from the mitochondria to the cytoplasm triggered by casticin and activation of its signaling in activation of mitochondrial signaling in a ROS-dependent manner in HeLa cells (Zeng et al., 2012).It has no significant effect on Bcl-2 expression but caused decreases in Bcl-XL and XIAP (Elmore, 2007b;Chen et al., 2011a).Further, apoptosis can be initiated through two alternative signaling pathways: the death receptor-mediated extrinsic apoptotic pathway and the mitochondrial-mediated intrinsic apoptotic pathway (Kok et al., 2005;Reuter et al., 2008).It is increasingly becoming apparent that the mitochondria play critical roles in the regulation of various apoptotic processes leading to cell death (Birt et al., 2001), including drug-induced apoptosis (Cory and Adams, 2002).
It is considered that the mitochondrial, death pathway is controlled by members of the Bcl-2 family (Brunelle and Letai, 2009;Leibowitz and Yu, 2010), which play a central regulatory role to decide the fate of the cells via the interaction between pro-and anti-apoptotic members.Casticin triggers mitochondrial permeability transition (Bradham et al., 1998;Reed et al., 1998;Yang and Cortopassi, 1998a;Yang and Cortopassi, 1998b;Antonsson and Martinou, 2000), which is required for the complete release of cytochrome c.Casticin resulting in production of ROS in a caspase-dependent through reduction of the ratio Bcl-2/Bax and thus favor apoptotic pathways; that are mean Bax, a pro-apoptotic protein in the Bcl-2 family, are up-regulated and this induction of Bax, release cytochrome c in the cytosol, caspase-3 activation and PARP cleavage.In addition, the research carried out by (Chen et al., 2011a) reports that the downregulation of XIAP likely reflects an increase in protein degradation and concluded casticin-induced apoptosis of human cervical cancer cells via the mitochondrial death pathway.

Targeting cancer cells by ROS-mediated apoptosis
ROS, active, transitory and oxygenic compounds are known mediators of intracellular signaling of cascades, including H 2 O 2 , O 2 , and hydroxyl radicals, are metabolites of biochemical processes in the body.In the genesis, ROS is the result of disordered mitochondria function and metabolite augmentation, and there may be ways to regulate ROS selectively in cancer cells (Kim et al., 2010).It is an integrated system to clear ROS in the body to maintain balance.Oxidation of cell membrane phospholipids, enzymes and DNA (Lin et al., 2007a;Appierto et al., 2009) by excessive generation of ROS can induce oxidative stress, alter the function of signal transduction pathways, platelet aggregation, immune control, and the regulation of cell growth, and in some cases can also cause necrosis or apoptosis (Chen and Chan, 2009;Wei et al., 2010).Moreover, casticin generates ROS in human cervical cancer cells and places special emphasis that NAC suppressed the apoptosis of HeLa cells by casticin which indicated that its apoptotic effect is dependent on ROS generation (Chen et al., 2011a).

Targeting cancer cells by caspase-mediated apoptosis
Caspases play important role in apoptosis via triggering of the death receptors and mitochondrial pathways to emit various pro-apoptotic signals to accomplish the programmed cell death (Nunez et al., 1998;Thornberry and Lazebnik, 1998).For the overall functional aspect of caspases, the activation of the caspase cascade requires both initiator caspases, such as caspase-8, and -9, and effector caspases, such as caspase-3.It is generally recognized that there are two major apoptotic pathways: one involves death signals transduced through death receptors, and the other relies on a signal from the mitochondria (Nunez et al., 1998;Thornberry and Lazebnik, 1998;Woo et al., 2003;Li et al., 2005).
Several studies reveal that both pathways are involved in an ordered activation of a set of caspases, which in turn cleave cellular substrates leading to the morphological and biochemical changes of apoptosis (Woo et al., 2003;Yang et al., 2010).The dissipation of ∆Ψm, rapid release of cytochrome c from the mitochondria to the cytosol, activated caspase-9, -8 and -3 and DNA fragmentation are triggered by casticin (Chen et al., 2011a).Furthermore, the presence of the inhibitors such as z-VAD-FMK for caspase-8 and z-LEHD-FMK for caspase-9 attenuated the DOI:http://dx.doi.org/10.7314/APJCP.2014.15.21.9049Molecular Mechanisms of Casticin Action: an Update on its Antitumor Functions apoptosis induced by casticin in human cervical cancer PLC-PRF-5 cells (Yang et al., 2011).
The chemotherapeutic agents cause the dissipation of ΔΨm, along with cytochrome c release from the mitochondria and the subsequent activation of caspase-9 through binding to the protein Apaf-1 mediates apoptosis (Li et al., 1997;Thornberry and Lazebnik, 1998).Casticin is an effective apoptosis-inducing agent in human hepatocellular carcinoma (HCC) cells, which acts through depleting intracellular GSH content and up-regulating DR5, and subsequent activation of caspase-3, -8 and -9.It has been shown that that casticin can inhibit the growth of HCC cells independent of p53 status and thus can be suggested as a good candidate for additional evaluation as a cancer therapeutic agent for human HCC as well as other types of cancer (Yang et al., 2011).

Targeting cancer cells by regulating apoptosis related proteins
p53: The cancer suppressor p53, considered as a guardian of the genome, is an important factor affects the cell response to drug effects on growth inhibition and apoptosis induction (O'Connor et al., 1997;Pirollo et al., 2000).It has also been demonstrated that casticin induced apoptotic cell death in p53 mutant or null breast cancer cell lines (Haidara et al., 2006) and in p53 mutated human cervical cancer HeLa cells (Csupor-Loffler et al., 2009;Chen et al., 2011a).Many studies have been carried out to supports the notion that cells with wild-type p53 exhibit increased sensitivity to radiation or chemotherapeutic agents and revealed that cells with mutant p53 sequence tends to exhibit less growth inhibition in the screen than the wild-type p53 cell lines when treated with the majority of clinically used anticancer agents including DNA cross-linking agents, anti-metabolites, and topoisomerase I and II inhibitors (O'Connor et al., 1997).Whereas, cells lacking wild-type p53 expression still undergoes apoptosis but need a relatively high doses of radiation or chemotherapeutic drugs (Bae et al., 1996).
Casticin acts in a p53-independant manner with regards to its interaction with tubulin, cell cycle arrest in G2/M, p21 induction, Cdk1 activity inhibition, cyclin A down-regulation and finally induction of apoptotic death (Hofseth et al., 2004).As a multi-tasking and multi-directional agent in different cells, it is important for the suppression of tumor formation.The suppressing mechanism of casticin for malignant tumors occurs through c-Myc in p53 mutated Hs578T cells (Song et al., 2010).Furthermore, striking apoptosis was also confirmed in human glioma cells, accompanied by the up-regulation of caspase-3, p53 and pro-apoptotic protein Bax.These effects were absent when the caspase inhibitor z-VAD-   fmk or p53 inhibitor PFTα were applied, suggesting that casticin could trigger cell apoptosis in a caspase-3 and p53-dependent manner (Liu et al., 2013).Accumulated data support that casticin can induces in p53-dependent and -independent manner in various cancer cells.Further studies are required to confirm these effects on p53 signaling pathways.NF-κB: Detail study of literature validated that casticin may act in part by affecting NF-κB signaling pathway (Gillet et al., 2004;Nam, 2006).The expression of the inflammatory mediators is regulated by NF-κB (Ghosh et al., 1998).It has been described to inhibit NF-κB along with many other flavonoids known as NF-κB inhibitors (Gillet et al., 2004;Nam, 2006).NF-κB plays critical role in wide variety of physiological and pathological processes, such as regulating immune response, cell proliferation and apoptosis.They go on to say that a number of proteins in case (NF-κB1 and NF-κB2, each with two alternatively spliced forms, and REL-A, REL-B and c-REL) can form dimers, which are able to bind specific DNA motifs in the promoters of target genes (Brasier, 2006;Gilmore, 2006;Nam, 2006).These heterodimers can activate the transcription of about 200 target genes (Perkins, 2007).Inactive NF-κB1 or NF-κB2 proteins are complexes with IκBα (inhibitory κB) proteins in the cytosol and the phosphorylation of IκBα by IκBα kinase (IKK) leads to IκB degradation and translocation of NF-κB1 and NF-κB2 into the nucleus (Kobayakawa et al., 2004).
Further, the ROS-mediated NF-κB pathway is required for activation of endothelial cell adhesion molecules (Chen et al., 2003).Casticin significantly downregulated vascular inflammation, through inhibition of ROS-NF-κB pathway in vascular endothelial HUVEC cells (Lee et al., 2012).In clinical point view, NF-κB activation is involved in many chronic disease conditions, mainly in the development of atherosclerosis (Kanters et al., 2003).Nuclear NF-κB, p65 translocation and phosphorylation of IκB-α lead to the activation of specific target genes including VCAM-1, ICAM-1, and E-selectin (Liang et al., 2004).Furthermore, the expression of eotaxin, RANTES, VCAM-1, ICAN-1 and activation of eosinophilic inflammation involved in the pathogenesis of asthma is known to be mediated by the NF-kB signaling cascade (Wong et al., 2002;Kuldo et al., 2005;Zerfaoui et al., 2008;Li et al., 2009).
PI3K-Akt: Phosphatidylinositol 3-kinase/Akt signaling pathway is implicated to be one of the most important pathways for cell survival and inhibition of apoptosis (Carnero et al., 2008).It has been demonstrated that Akt can regulate a number of cellular processes, such as cell proliferation and cell growth (Klippel et al., 1996).Inhibition of phosphate-Akt (pAkt) will induce acute myeloid leukemia apoptosis (Papa et al., 2008).Inactivating Akt is a key mechanism for apoptosis induced by various anti-leukemia drugs (Lee et al., 2005;Loges et al., 2006).The PI3K/Akt signaling pathway can override G2/M cell cycle arrest induced by anti-cancer agents (Lee et al., 2005).Casticin inhibited PI3K/Akt signaling pathway in K562 cells and PI3K/Akt inhibitor enhanced casticin-induced cell death (Shen et al., 2009).Another complementary element ERK and PI3K/Akt signal pathway are two important signal pathway associated with cell survival (Xia et al., 1995;Kennedy et al., 1997).

Casticin and its synergistic activity with other chemotherapeutic drugs
Casticin is a multi-targeting molecule that enhances TRAIL-induced apoptosis and triggers G2/M growth arrest through the downregulation of cell survival proteins and the upregulation of DR5 receptors through actions on the ROS-ER stress-CHOP pathway (Zhou et al., 2013b).It is also shown that casticin potentiates TRAIL-induced apoptosis through downregulation of cell survival proteins and induction of DR5 mediated by ROS (Tang et al., 2013).In other hand, a direct effect of casticin on cyclin-A could also be involved in Cdk1 inhibition.The same author also reported that the anti-apoptotic protein Bcl-2 is down regulated, leading to apoptotic cell death (Haidara et al., 2006).

Conclusions and future perspectives
Casticin, naturally occurring compound, has been shown a good pharmacological potentially promising therapeutic effect including anti-inflammatory and antitumor effects.Casticin is located in fruits, vegetables, seeds, herbs, stems, roots, wood, and flowers of the many plants.The previous in vitro and in vivo studies demonstrated the potential applications of casticin to inhibit the growth of several human cancers by targeting cancer cells through a number of parameter including ROS, and capase-mediated apoptosis or by regulating apoptosis related proteins such as NF-κB, p53, and PI3K-Akt.Furthermore, casticin has synergistic activity with other chemotherapeutic drugs such as TRAIL, which enhance to induce apoptosis and triggers G2/M growth arrest through the downregulation of cell survival proteins and the upregulation of DR5 receptors through ROS-ER stress-CHOP pathway (Tang et al., 2013;Zhou et al., 2013b).
Having regard to the foregoing investigations, this review suggests that casticin may represent a novel therapeutic agent for the treatment of human cancers.This review elaborates the current understanding of the chemopreventive effects of casticin through its multiple molecular pathways and highlights its therapeutic value in the treatment and prevention of a wide range of cancers.To support our remarks of the anti-cancer potential of casticin, additional studies and preclinical trials are required to determine its specific intracellular sites of action and derivative targets in order to fully understand the mechanisms of its antitumor activity to validate this compound as medicinal agent in the prevention and treatment of various diseases including cancer.

Figure 1 .
Figure 1.Chemical Structure and Natural Sources of Casticin