Table 1. Biological effects of silibinin against human colorectal cancer (CRC) cell lines under in vitro cell culture conditions.

CRC cell lines	In vitro effects of silibinin	Model and methods	Mechanism of action	Research group
HT-29	growth inhibition: dose (50-100 μg/mL) and time (24-72h) dependent. G0/G1 cell cycle arrest (lower/higher doses). G2/M cell cycle arrest (higher dose: 100 μg/mL). no apoptosis at lower doses. apoptotic death (~ 15%) after 48 hours with 100 μg/mL dose. no induction of cellular differentiation.	FACS based cell cycle distribution analysis. annexin V staining for apoptosis, caspase activity assay, and cytochrome c localization analysis. immunoprecipitation based CDK2-and cdc2/p34-associated H1 histone kinase assays. Northern blot hybridization with 32P labeled Kip/p27 and Cip/p21.	mRNA and protein levels of Kip/p27 and Cip/ p21 ↑ protein levels of cdc25c, cdc2/p34, and cyclin B1 ↓ kinase activity of cdc2/p34 ↓ caspase independent apoptosis.	Agarwal et al. 2003[10]
	inhibitory effects of silibinin (100 μmol/L dose) on β-catenin mediated signaling.	TCF-luciferase reporter plasmids based assays.	β-catenin-dependent TCF-4 transcriptional activity ↓	Rajamanickam et al. 2010[39]
	inhibitory effects of silibinin (1-100 μmol/L dose) on CDK4 signaling pathway.	MTT cell viability assays. FACS based Ki67 labeling analysis. immunoblotting for cell cycle regulatorymolecules.	protein levels of CDK-4, and cyclin D1 ↓ hyper phosphorylation of retinoblastoma ↓	Karim et al. 2013[16]
	dose and time dependent growth inhibition.	cell count assays.	Not explored	Akhtar et al. 2014[15]
	apoptosis of HT29 cells via EGR-l-mediated NSAID-activated gene-1 (NAG-1) up-regulation (silibinin: 50-100 μmol/L dose). inhibitor of p38 MAPK (SB203580) attenuated silibinin-induced NAG-1 expression.	p53 wild-type and p53-null cancer cell lines. siRNA and MAPK inhibitors based confirmatory assays.	NAG-1 up-regulation in p53-independent manner. up-regulation of EGR-1 expression. ectopic expression of EGR-1 significantly upregulates NAG-1 promoter activity and NAG-1 protein expression in a dose-dependent manner.	Woo et al. 2014[17]
Fet, Geo, and HCT116	G2/M cell cycle arrest in Fet and Geo cell lines. G1 arrest in HCT116 cells. IC50 in Fet and Geo lines is 75 μg/mL and 40 μg/mL for HCT116 cells at 72 hours. growth inhibitory effects more due to inhibition of cell cycle regulatory molecules than due to apoptosis.	MTT cell viability assays. FACS based cell cycle distribution and apoptosis analysis. immunoblotting for cell cycle regulatorymolecules.	protein levels of Kip/p27 and Cip/p21 ↑ protein levels of Cyclin Bl/Dl and CDK-2 ↓ no effect on Cox-2 levels.	Hogan et al. 2007[14]
	dose (50-200 μmol/L) and time (24-72 hours) dependent growth inhibition. G1 cell cycle arrest (lower/higher doses) as well as G2M arrest with 200 μmol/L. significant apoptotic death at 100-200 μmol/L.	FACS based cell cycle distribution analysis. annexin V staining for apoptosis. immunoblotting for cell cycle regulatory molecules.	protein levels of cleaved caspase -3 and -9, and cleaved PARP ↑ protein levels of Kip/p27 and Cip/p21 ↑ protein levels of Cyclin- D1/-D3/-A/-B1 and CDK-1/-2/-4/-6 ↓ hyper phosphorylation of Retinoblastoma ↓	Kaur et al. 2009[12]
LoVo	anti-angiogenic effect. inhibits the chemotaxis migration of endothelial cells EA.hy.926 towards CRC cells (IC50: 0.66 μmol/L dose). inhibits EA.hy.926 capillary formation (IC50: 2.6 μmol/L dose). ↓ vascular density index in the choriallontoic membrane assay by 20 μmol/L dose.	transwell migration and matrigel based capillary tube formation assay. chicken egg based choriallontoic membrane assay. mRNA levels by RT-PCR analysis.	mRNA levels of VEGFR-l(Flt-l) ↑ ↓ VEGF secretion by LoVo cells (IC50: 131.7 μmol/L dose).	Yang et al. 2003/2005[42-43]
	dose 10−6 mol/L. invasiveness of CRC cells ↓ IL-6 induced proliferation and invasion of LoVo cells ↓	[3H] thymidine incorporation assay. cell invasion assays. EMSA and MMP-2 promoter activity based luciferase assays. confocal microscopy based MMP-2 localization analysis.	↓ MMP-2 promoter activity via attenuation of AP-1 binding activity. MMP-2 expression ↓	Lin et al. 2012[44]
SW480	cell growth inhibition by 50-200 μmol/L dose after 24-72 hours. no death till 72 hours with doses up to 100 μmol/L. only 200 μmol/L dose affects viability at early time points. inhibitory effects on β-catenin mediated signaling.	viable cell count assays. TCF-luciferase reporter plasmids based assays. confocal microscopy based β-catenin localization analysis. immunoblotting analysis for protein expression.	nuclear and cytoplasmic β-catenin levels ↓ expression of β-catenin regulator CDK-8 ↓ β-catenin-dependent TCF-4 transcriptional activity ↓ expression of β-catenin transcriptional targets: c-Myc and cyclin D1 ↓	Kaur et al. 2010[11]
HT-29, LoVo, and SW480	anti-inflammatory effect (50-100 μmol/L) dose. inhibits TNFα-induced NFκB activation. effects independent of COX-2 expression.	immunoblotting analysis for protein expression. EMSA based gel super shift assays.	nuclear levels of p65 and p50 ↓ IκBα protein levels ↑ phospho- IκBα levels ↓ NFκB transcriptional activity ↓	Raina et al. 2013[45]
SW480 and SW620	300 μmol/L dose synergizes with TRAIL to cause apoptotic death. assumed that autophagy plays a cytoprotective role.	DNA fragmentation assays, FACS analysis and caspase inhibitors based confirmatory assays. mitochondrial membrane potential analysis. mRNA levels by RT-PCR analysis. human recombinant DR5/Fc chimera protein based studies.	mRNA and protein levels of death receptors DR4/-5 ↑ both intrinsic and extrinsic apoptotic pathways involved. Mcl-1 and XIAP ↓	Kuantz et al. 2011/2012[18,46]
	300 μmol/L dose synergizes with HD AC inhibitors: (SAHA and trichostatin A) to cause cellular death.	FACS based cell cycle analysis. HDAC and DNMT activity measurement.	DNMT inhibition.	Kuantz et al. 2013[47]
HT-29, LoVo, and SW480	inhibits mitogenic/growth promoting signals induced by IGF-1 and EGF.	IGF-1 and EGF based effects on cell growth and proliferation. FACS based analysis. immunoblotting for protein expression levels.	PI3K-Akt-mTOR pathway ↓ and ERK1/2 pathway ↑ no inhibitory effect on normal human colon NCM 460 cells.	Raina et al. 2013[13]
SW480	oxidative stress and early on slight apoptosis. intense vacuolization of cytoplasm and rough endoplasmic reticulum swelling. events associated with autophagy ↑ long term exposure causes autophagic cell death (100 μmol/L dose) while higher doses (≥ 200 μmol/L dose) cause apoptosis. potential to cause both apoptotic and autophagic cell death.	oxidative stress and mitochondrial membrane potential analysis, and inhibitors based confirmatory assays. transmission electron microscopy, and dynamics of LC3-I and LC3-II tracking. metabolomics study utilizing 13C, 1H, 3IP based NMR spectroscopy.	early on reactive oxygen species generation. PI3K-Akt-mTOR pathway ↓ and ERK1/2 pathway ↑ interference in mitochondrial metabolism, phopspholipid and protein synthesis, and glucose uptake. energy restrictions causing starvation lead to autophagic cell death.	Raina et al. 2013[13]

Abbreviations: CRC, Colorectal cancer; FACS; fluorescence- activated cell sorting; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; RT-PCR, reverse transcription polymerase chain reaction; TCF-4, T-cell factor-4; TRAIL, TNF-related apoptosis-inducing ligand; HDAC, histone deacetylase; DNMT, DNA methyltransferase; SAHA, suberoylanilide hydroxamic acid; IGF-1, insulin-like growth factor-1; EGF, epidermal growth factor; EMSA, electrophoretic mobility shift assay; NMR, nuclear magnetic resonance.