Silibinin and colorectal cancer chemoprevention: a comprehensive review on mechanisms and efficacy

Komal Raina, Sushil Kumar, Deepanshi Dhar, Rajesh Agarwal,?

1Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences;

2University of Colorado Cancer Center, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA.

Silibinin and colorectal cancer chemoprevention: a comprehensive review on mechanisms and efficacy

Komal Raina1,2, Sushil Kumar1, Deepanshi Dhar1, Rajesh Agarwal1,2,?

1Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences;

2University of Colorado Cancer Center, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA.

Globally, the risk of colorectal cancer (CRC) as well as the incidence of mortality associated with CRC is increasing. Thus, it is imperative that we look at alternative approaches involving intake of non-toxic natural dietary/non-dietary agents, for the prevention of CRC. The ultimate goal of this approach is to reduce the incidence of pre-neoplastic adenomatous polyps and prevent their progression to more advanced forms of CRC, and use these natural agents as a safe intervention strategy during the clinical course of this deadly malignancy. Over the years, pre-clinical studies have shown that silibinin (a favonolignan isolated from the seeds of milk thistle,Silybum marianum) has strong preventive and therapeutic effcacy against various epithelial cancers, including CRC. The focus of the present review is to provide a comprehensive tabular summary, categorically for an easy accessibility and referencing, pertaining to the effcacy and associated mechanisms of silibinin against CRC growth and progression.

colorectal cancer, silibinin, cancer chemoprevention, milk thistle

Introduction

Colorectal cancer (CRC) is the second leading cause of cancer-related deaths in the United States men and women combined; statistical estimates for 2015 indicate～132,700 new CRC cases and 49,700 associated deaths[1]. Though several initiatives involving colon screening and therapeutic interventions have impacted the overall CRC management in the developed countries of the world[2], clearly more efforts are still needed to overcome CRC risk, to arrest the disease progression, and to reduce the mortality numbers associated with this malignancy. One such effort that has gained an appreciable momentum over the last three decades, involves the intake of non-toxic natural dietary/non-dietary agents which can be used globally, for the prevention of CRC. In most cases, these agents, derived from fruits and vegetables as well as herbs and other supplements, have been shown to reduce the incidence of pre-neoplastic adenomatous polyps and/or their progression to more advanced forms of CRC (Fig. 1)[3-4]. In the present review, an attempt has been made to tabulate and summarize the beneficial effects of one such natural chemopreventive agent i.e., silibinin, against CRC. Silibinin is a flavonolignan which is isolated from the seeds of milk thistle,Silybum marianum(Fig. 2); it has a long history of human use for liver ailments[5], widely available as a nutraceutical supplement and now clinically used to manage hepatotoxicity in various countries including the United States.Importantly, silibinin is considered as a very promising cancer chemopreventive agent based on completed studies showing its strong efficacy against various epithelial cancers (Fig. 3), including CRC[3-4,6-9]. As elaborated in detail in this review, silibinin has shown significantin vitroandin vivoanti-CRC efficacy in various pre-clinical models of CRC. Inferences from completed in depth studies investigating the cellular and biological mechanisms associated with anti-CRC effects of silibinin are summarized inTables 1–4, which detail the effect of silibinin on various molecules regulating cell cycle, cell survival, autophagy, apoptosis, angiogenesis, and inflammation in its efficacy against colon tumorigenesis.

Fig. 1Chemoprevention by natural dietary or non-toxic nutraceutical agents. Natural dietary agents, such as silibinin, can reduce the incidence of pre-neoplastic lesions by targeting cancer stem cells and proliferating bulk tumor cells to prevent the disease progression to more advanced forms of the malignancy. Silibinin inhibits tumorigenesis, inflammatory responses, and angiogenesis involved in CRC growth and progression. CRC: colorectal cancer.

Fig. 2Milk Thistle. A:Silybum marianum(Milk Thistle) plant, Family:Asteraceae.B: Chemical structure of silibinin - the principal bioactive constituent of milk thistle extract isolated from the dried seeds of milk thistle.

Silibinin eff i cacy against CRC cell lines in cell culture

Underin vitrocell culture conditions, silibinin has been shown to inhibit the growth and proliferation of a wide range of CRC cells (Table 1). Mechanistic studies have attributed these inhibitory effects to cell cycle arrest as well as both caspase-dependent and -independent apoptotic pathways (Table 1). An increase in cyclin-dependent kinase (CDK) inhibitors Kip1/p27 and Cip1/p21, together with a decrease in the kinase activity of CDK2 and CDK4 in G0/G1 arrested CRC cells; while an increase in the kinase activity of CDC2/p34 molecule along with a decrease in protein expression of cell cycle regulators cdc25C, cdc2/p34 and cyclin B1 by silibinin in G2M arrested CRC cells have been reported (Table 1). Furthermore, a decrease in the hyperphosphorylation of Retinoblastoma (Rb) by silibinin has also been reported as one of the mechanisms involved in the early cell cycle arrest induced by silibinin in CRC cells; though total Rb levels are unaffected, a decrease in the protein expression levels of cyclin -D1, -D3, -A and -B1 and CDK-1, -2, -4, and -6 by silibinin has been reported. Thein vitroeffects of silibinin alone or in combination with other molecules are summarized inTable 1[10-17].

Fig. 3The targets of silibinin. Silibinin inhibits various signaling and regulatory pathways in its chemopreventive and therapeutic efficacy against various epithelial cancers.

Other in depth studies have shown that at physiologically relevant dose (100 μmol/L), silibinin causes oxidative stress in CRC cells, which triggers an apoptotic response, and is followed by a chain of events involving strong inhibition of the PI3K-Akt-mTOR pathway, activation of the ERK1/2 pathway, rough endoplasmic reticulum stress (ER), and suppression of protein translation, which results in autophagy-mediated programmed cell death type II. High-resolution nuclear magnetic resonance spectroscopy (1H,13C,31P-NMR) studies have further confirmed that silibinin interferes with essential mitochondrial metabolic pathways, and the events associated with both phospholipid as well as protein synthesis[13]. Furthermore, silibinin has also been found to interfere with the glucose uptake in these cells; overall, these events result in energy restriction in CRC cells and their severe and irreparable injury by silibinin[13]. Studies have also shown that significant apoptotic death of CRC cells occurs when high silibinin doses are used[13,18], which suggests that silibinin harbors the potential to induce both apoptotic and autophagy associated programmed cell death in CRC cells.

With regards to silibinin efficacy against CRC stem cells (CSC), silibinin has also been shown to interfere with the kinetics of CSC pool generation, which in turn, decreases the self-renewal and rectifies the aberrant differentiation of the CSC[19-20]. These results are detailed inTable 2; the potential of silibinin to target CSC is being extensively investigated by our group (Table 2)[21-22]. Ourin vitrostudies showed that silibinin strongly decreases the percentage of colonosphere formation (a stem cell characteristic) of CRC cells and that this effect on CSC is mediatedviablocking of interleukin (IL)-4/6 signaling in CRC cell lines. Silibinin also caused a strong decrease in IL-4/6 induced activation of STAT-3 and NF-kB transcriptional activity, which was associated with decreased mRNA/protein levels of various CSC regulatory molecules, and CSC-associated markers and transcription factors (Table 2). We also found that silibinin significantly reduces the booster signals of macrophages towards CSC, resulting in decreased colonosphere numbers under both normoxic and hypoxic conditions (Agarwal & colleagues 2015, unpublished data,Table 2). These results are highly significant, given the fact that silibinin has shown efficacy against both CSC and bulk cancer cells, and that CSC are now primarily recognized as the essential factors contributing to initiation/progression as well as the relapse of CRC[23-26].

Notably, severalin vitromechanistic studies have used high doses of silibinin (300 μmol/L); the clinical relevance of such high doses warrants caution; this is founded on the results of several preclinical as well as clinical trial studies that underline limiting the silibinin concentration to ～100 μmol/L dose inin vitrostudies based on silibinin concentrations achievable in mouse plasma[27]and colon tissues of CRC patients[28-29]. Specifically, the results of the clinical trial data showed that silibinin feeding in the form of 720 mg/day (formulated with phosphatidylcholine) as 'silipide capsules' for 7 days to CRC patients leads to high bioavailability of silibinin in colonic tissue (121 nmol/g of silibinin traced in colonic tissue which is equivalent to 121 μmol/L silibinin concentration).

Eff i cacy of silibinin against CRC growth and progression in pre-clinical rodent models

Using azoxymethane (AOM) and 1,2-dimethylhydrazine (DMH) as potential carcinogens to induce sporadic CRC in rodent models, various research groups have demonstrated the potential of silibinin to decrease the number of carcinogen-induced aberrant crypt foci (ACF), crypt multiplicity as well as colonic tumors in these colon tumorigenesis animal models (Table 3). To determine the stage specific efficacy of silibinin feeding, different silibinin feeding protocols based on whether silibinin feeding started before the carcinogen exposure (pre-initiation phase), during the carcinogen exposure (initiation phase), after the carcinogen exposure (post-initiation phase), or through the entire period of the study (pre-/-post initiation phase) were carried out; though all silibinin feeding protocols showed efficacy against CRC growth and progression, the percentage of CRC inhibition was stronger in the animals on the pre-/-post-initiation protocol[30-37]. Silibinin has also shown significant efficacy against spontaneous intestinal tumorigenesis inAPCmin/+mice model (Table 3). Both short and long term intervention studies with silibinin have confirmed that total intestinal polyps are significantly reduced in number and size by silibinin feeding[16,38-40]. While a decrease inproliferation index and an increase in apoptotic index were observed in the polyps of silibinin treated mice, the normal crypt-villus regions of the intestine were not affected. Furthermore, our lab has also used a colitis-related AOM/DSS-induced colon tumorigenesis model to assess the role of inflammatory conditions on colon CSC generation and expansion, and their modulation by silibinin[21]. Our results have indicated the protective effect of oral silibinin feeding in this model as evidenced by the absence of large macroadenomas (＞2-3 mm) in the colon. This effect was accompanied by minimal colonic inflammation (decrease in recruitment of inflammatory cells); tissue analysis indicated a decrease in the expression of pro-inflammatory cytokines, and associated transcription factors: STAT-3 and NF-kB levels in the colonic tissues (Table 3). A decrease in transformed stem cell population (for CSC pool expansion) was also identified (dual staining for BMI-1 and CD44) in the colonic tissues. Silibinin feeding has also shown significant preventive and/or therapeutic efficacy against human CRC xenograft tumors in athymic nude mice (Table 4)[11-12,27,41]. These studies indicate that not only has silibinin an inhibitory effect on tumor growth, but this inhibitory effect is sustained even after silibinin withdrawal; furthermore, silibinin has also shown significant efficacy against established xenograft tumors[41].

Conclusion

Fig.4Targeted proteins of silibinin in CRC. The inflammatory milieu of the colorectal cancer stem cells (CSC) niche is an important growth regulator for both CSC and progenitor cell population. Silibinin has the potential to target colon CSC self-renewal and aberrant differentiation, and associated inflammatory niche during CRC inhibition.

The summarized studies in four tables provide adequate evidence highlighting the potential of silibinin intake to significantly impact CRC growth and progression. These results also indicate silibinin potential to interfere with CSC pool expansionviatargeting both CSC, bulk daughter cells and their inflammatory niche, as well as associated signals involved with the survival and multiplication of colon CSC pool (Fig. 4); the various mechanisms involved in these inhibitory effects are: a) silibinin causes cell cycle arrest resulting in decreased proliferation, and induces multiple programmed cell death mechanisms; b) silibinin interferes with cellular metabolism to induce energy restriction like conditions; and c) silibinin inhibits various signaling and regulatory pathways involved in tumorigenesis, inflammatory responses, and angiogenesis.

Fig. 5Stem cells or their progenitors transformed into colorectal cancer stem cells (CSC) are considered as major factors responsible for colorectal cancer (CRC). Novel preventive and therapeutic strategies are needed to reduce CSC number, target their self-renewal capacity or rectify their aberrant differentiation, or interfere with the pro-tumorigenic signals arising in the colon 'niche' that affects CSC population. Silibinin acts as a 'double edged sword'-striking both CRC 'initiators' and the 'initiated' cells.

Given that silibinin has the potential to target the CSC or the "tumor initiating cells", it can be beneficial for use in the early stages of CRC development, as well asin later stages in cancer treatment to eradicate the CSC pool and bulk tumor cells to prevent cancer relapse (Fig. 5). Given the fact that silibinin consumption is exceptionally safe and that it has high bioavailability in colonic tissue of CRC patients, the efficacy evaluation of silibinin in clinical trials is advocated, both as a CRC preventive agent and as an 'adjunct therapy' for its clinical use in CRC cases which are incurable by current therapeutic modalities.

Acknowledgment

This work was supported by NCI R01 grant CA112304.

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?Corresponding author: Rajesh Agarwal, Ph.D, Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, 12850 E. Montview Blvd, C238, Room V20-2117, Aurora, CO 80045, USA. Tel/fax: (303) 24-4055/(303) 724-7266,

? 2016 by the Journal of Biomedical Research. All rights reserved.

E-mail: rajesh.agarwal@ucdenver.edu.

Received 5 August 2015, Accepted 12 October 2015, Epub 20 November 2015

R730.53, Document code: A

The authors reported no confict of interests.

10.7555/JBR.30.20150111