Background
The worldwide frequency of colorectal cancer (CRC) is third among the cancer rate of relapse in males and fourth among females [
1,
2]. Additionally, colonic adenocarcinoma accounts for 37–45% of all metastatic ovarian tumors. While there is a decline in the death rates for colorectal cancers from 1990 till date. Even with advancement in screening and surgical treatment, no guaranteed therapy has been revealed for metastatic cancer and the half decade endurance rate is disappointedly little (about 9%). Such astonishing incompetence of regular anti-cancer therapies has been authorized to the survival of relatively infrequent, exceedingly drug-resistant, inert or slow propagating cells with stem-like properties: cancer stem cells (CSCs). Existing studies reflects tumors as multifarious varied organ-like systems with a hierarchical cellular organization, rather than merely as collections of homogeneous tumor cells [
3,
4]. CRC rises through the gradual accumulation of alterations in oncogenes and tumor suppressor genes. The accumulation of alterations usually arise because of cumulative effects of epigenetic variations and multiple genetic mutations involving genes that control cell development and segregation [
5].
Natural polyphenols have materialized as a novel approach to defend cells against oxidative stress. Besides anticarcinogenic activities, every polyphenol have a potent antioxidant activity, thus declining the jeopardy of cancer [
6].
Most of the polyphenols have shown chemoprotective, antiproliferative, antioxidative and estrogenic/antiestrogenic activity besides tempting cell-cycle arrest or apoptosis and the detoxification of enzymes. Also, they control the immune system and fluctuations in cellular signaling [
7]. Particularly, tannins, flavonoids, gallic acid, anthocyanins, resvertrol, proanthocyanidins, epigallocatechin-3-gallate, and various plant extracts have shown defensive activity in various cancer models [
8,
9]. Dihydroquercetin (Taxifolin 3, 5, 7, 3′,4′-pentahydroxy-flavanone) is an operative flavonoid, abundantly found in olive oil, grapes, in citrus fruits and onions [
10]. Taxifolin (TAX), a prevalent bioactive component of foods and herbs, showed a tremendous variety of pharmacological and biochemical consequences, including hepatoprotective, anti-diabetic, cardioprotective, antitumor, neuroprotective effects, and anti-inflammatory effects, and played a wonderful role in the preclusion of Alzheimer’s disease [
10]. Also, TAX has an effective anti-oxidant activity which boosts apoptosis encouraged by an assortment of anti-cancer agents and is available in the market under a trade name of Venorutons (semisynthetic form) [
11‐
13]. Recently it was reported that TAX encompasses Nrf2-dependent pathway to encouraged the expression of phase II antioxidant and detoxifying enzymes and exert a critical defensive role against DNA oxidative damage [
14]. Prominently, TAX appreciably has shown accelerated expression of heme oxygenase − 1 (HO-1) by inducing expression of Nrf2 in cytoplasm and nuclear translocation [
14]. The therapeutic promise of dihydroquercetin in chief inflammatory disease states like cancer was recently evaluated [
11]. Particularly, it was revealed that dihydroquercetin can proceed as a scavenger of myeloperoxidase (MPO)-derived RNS [
11]. Interestingly, dihydroquercetin decreases BSO-induced injury to dermal fibroblasts [
15]. In addition, a most current organized study, highlighted that dihydroquercetin was capable to downregulate the collagenase I (MMP-1) in UVB-treated skin cells [
11]. Furthermore, TAX showed inhibitory action against the excess production of ROS and the oxidative enzymes, thus reorganizing cerebral the ischemia–reperfusion injury [
16].
FH535 is considered as twofold small molecule inhibitor of β-catenin/TCF/LEF and peroxisome proliferator-activated receptors (PPARs) [
17]. Earlier studies revealed that FH535 hampers the growth of colon, lung, breast, hepatocellular cancer cells [
18], and suppresses angiogenesis and pancreatic cancer xenograft growth [
19].
In spite of numerous observations signifying the cytotoxicity of TAX in numerous cancer cell lines, the mechanism of action of the TAX during tumor deterioration is chiefly unclear. Hence, in the present study, we explored the anticancer effect of TAX in a systematic manner, using in vitro and in vivo model structures. We found that TAX induces numerous fold advanced levels of cytotoxicity in cancer cells. Further, we showed growth impediment and induction of apoptosis in colorectal cancer cells induced by polyphenolic compound TAX, Down-regulation of Wnt/ β-catenin signal transduction pathway, Anti-proliferative effect of treatment of TAX, β-catenin Inhibitor (FH535) in HCT116 and HT29 cells, Flow cytometric analysis of colorectal cancer cells after TAX treatment for apoptosis and cell cycle, Inhibition of colony formation in HCT and HT29 cells after treatment with TAX and Alteration in CTNNB1 protein level after TAX treatment. Thus our data indicate that TAX could be developed further as a potential anti-cancer agent, both in conventional and combination therapy.
Methods
Ethical declaration
Athymic nude mice studies were performed according to the Institutional principles for the concern and use of animals and the experimental protocol was approved (BAS#0256) by the ethical board of Quaid-i-Azam University, Islamabad, Pakistan and Committee dealing animal care and use, college of Pharmacy, King Saud University, Kingdom of Saudi Arabia. Before starting experiment on human colorectal cancer cell lines HCT116 and HT29 (ATCC® CCL-247 ™ and ATCC® HTB-38 ™ respectively) purchased in July 2017 from American Type Culture Collection (MD, USA), ethical approval was taken from ethics committee of preclinical studies, college of pharmacy, King Saud University, KSA.
Cell culture
Two human colorectal cancer cell lines HCT116 and HT29 were grown in a 5% CO2 atmosphere at 37 °C in medium containing DMEM medium 1640 (GIBCO), 10% fetal bovine serum and 1% penicillin/streptomycin. Taxifolin (TAX) and β- catenin inhibitor (FH535) suspended in DMSO was applied for cell treatment. Cells with 70% confluency were induced with TAX and β- catenin inhibitor at 10-100 μM for 48 h in cell culture medium and the dilution of DMSO applied for each treatment was 0.1% (V/V).
MMT assay/viability assay/ cell proliferation assay
To check the effect of TAX and β- catenin inhibitor (FH535) on the viability of HCT116 and HT29 cell lines, 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazoliumbromide (MMT) protocol was carried. 10 × 10
3 cells /well were plated in 1 ml of culture medium consisting of 10-100 μM dilution of TAX and FH535 in 96-well plate. Cells were incubated for 48 h. at 37 °C, 200 μl of 3–4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide (5-7 mg/ml PBS) was supplemented to each well and set aside for two hours, 200 μl of DMSO were added to each plate which were then spinned (1800×g for 5 min at 4 °C). The readings at 540 nm wavelength were noted on a microplate reader (Elx 800). Impact of TAX and FH535 on inhibition of growth was calculated as % cell viability as DMSO-administrated cells were considered as control. Absorbance numbers of media containing wells were subtracted from test sample values.
$$ \mathbf{Cell}\ \mathbf{viability}=\left[\frac{\mathbf{Absorbance}\ \mathbf{of}\ \mathbf{Sample}\hbox{-} \mathbf{Absorbance}\ \mathbf{of}\ \mathbf{Blank}}{\mathbf{Absorbance}\ \mathbf{of}\ \mathbf{DMSO}\hbox{-} \mathbf{Absorbance}\ \mathbf{of}\ \mathbf{Blank}}\right]\mathbf{X} \mathbf{100} $$
Clonogenic assay
HCT116 cells and HT29 cells were collected subsequently to treatments with active TAX for 48 h. Cells were suspended in fresh medium, cell number was determined, and 500 cells (HCT116) or 1 × 10
3 cells (HT29) were plated into cell culture dishes (35 mm) in triplicate. Following 7 days in culture, 0.5% crystal violet was used for staining colonies [
20]. Cell colonies were count up by using a cubic colony counter (AO scientific) under dark field. The amount of cells in every colony was resoluted by phase contrast microscopy. The colony sizes were calculated on images using Adobe Photoshop software. Data was signified as colony number in TAX group relative to expressed as mean ± SEM of three individual tests.
Cell cycle analysis
HCT116 and HT29 cells were treated with TAX (40-60 μM:48 h.) in complete medium were trypsinized and fixed in 1% paraformaldehyde: 1× PBS and washed with cold PBS twice and spinned. Chilled 70% ethanol was used to suspend cell pellet and incubated overnight, then the cells were spinned for 4-5 min at 800–1000 rpm. To remove ethanol, pellet was washed with chilled PBS twice then finally cells are labeled with FITC and propidium iodide (PI) using the Apo-Direct Kit (BD Pharmagen, CA). Data interpretation was done with a FACScan (Becton Dickinson, NJ). About 1 × 104 cells per sample were harvested. Histograms of DNA were interpreted with ModiFitLT software (verily Software House, ME, USA).
Protein extraction and Immuno-blotting analysis
SDS-PAGE and western blot investigations were executed by earlier described protocol with minor amendments [
21]. Subsequent to 24 h and 48 h of administration with TAX at necessary amounts, HCT116 and HT29 cells lyses was carried out in RIPA buffer augmented with freshly added protease and phosphatase inhibitor cocktail 1:100 (Santa Cruz, CA) and concentration of protein was anticipated by Bradford assay [
22]. To resolve 40-60 μg of protein, 8–12% poly acrylamide gels were used in immune-blotting. Transferred on to a nitrocellulose membrane, with candidate monoclonal primary antibodies, and perceived by super signal west Pico, Dura or Femto Chemiluminescence Reagent (Thermo scientific, USA). Protein bands were quantified by measuring band density using Image J software. The densities of the bands (normalized to actin) relative to that of the untreated control (designated as 1.00) were presented as mean ± SEM of three individual experiments.
Gene expression analysis
Whole RNA was extracted from the cells using a Genei RNA purification kit (Ambion, USA). The RNA extracted was then subjected to first stand complimentary deoxy ribonucleotide (cDNA) synthesis using RevertAid Premium First Strand cDNA Synthesis Kit (Fermentas, Germany). RT- PCR was executed, containing 25 μl total reaction mixture. The reaction contains: SYBR® GreenER™ qPCR SuperMix Universal 12.5 μl, Forward primer (pF) 0.3 μl, Reverse primer (pF) 0.3 μl, cDNA 6 μl, Taq Polymerase 0.5 μl, Nuclease free water 5.4 μl. Thermal cycling conditions were: 4 min at 94 °C for template DNA denaturation with 35 cycles of amplification each consisting of 3 steps: 35 s at 94 °C for DNA denaturation into single strands; 35 s at 60 °C for primers to hybridize or “anneal” and one minute at 72 °C for extension and final 10 min at 72 °C. PCR was carried out in Smartcycler (Cepheid, Germany). Five standards were run in triplicate tubes in Smartcycler for real time PCR. The gene expression levels were determined using 2-ΔΔCT. Designed Primer sequences used were;
CTNNB1 Non-template: 5′-TGTGAATCCCAAGTACCAGTGT-3′.
CTNNB1 Template: 5′- CGTCAGACAAGGAGAAACATT-3′.
β-Actin Non-Template: 5′- CCTCTTCCTCAATCTCGCTC-3′.
β-Actin Template: 5′- GCTCAATGTCAAGGCAGGAG-3′.
Imunofluorescence microscopy
HCT116 and HT29 colorectal cancer cells were cultured in a two chamber tissue culture glass slides and were administrated with 40 μM of TAX at 75% confluence for 24 h. Once the chamber was removed, Phosphate buffer was used to rinse the slides, 2% paraformaldehyde was used to fix the cells and permeablized in methanol. Slides were rinsed with phosphate buffer and 2% serum was used as blocking agent. Primary antibody was incubated overnight. Then incubation with appropriate fluorophore tagged secondary antibody. For mounting antifade 4,6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI) (Invitrogen NY) was used to apply and hematoxylin for counter staining. Analysis was done by using Bio-Rad Radiance system (2100 MP Rainbow) for imaging. The apoptotic and necrotic cells were identified by the Annexin-V-fluos staining Kit (Roche, Switzerland) according to the kit’s procedure. Fluorescence was measured by confocal microscopy (Zeiss 410). Annexin V and propidium iodide was used to stain the cells. The unstained cells in a chosen field were calculated to determine the level of necrosis as well as apoptosis.
In vivo tumor xenograft model
Athymic male mice were acquired from King Faisal Hospital and research center, Riyadh, KSA, were homed under contamination free environment (12 h clock), nourished with a sterilized food adlibitum. HCT116 cells were selected for evaluating the in vivo impact of TAX and β-catenin inhibitor (FH535), as they generate fast tumors in mice. Cells were harvested, suspended in complete RPMI media 1640. Tumor xenografts HCT116 cells in mice were established by injecting cells (1 × 106) subcutaneously mixed with matrigel (Collaborative Biomedical Products, Bedford, MA) in a equal ratio.
Thirty mice were categorized into three groups.
Group1: Served as Control Group, consisting six mice, DMSO was given intra-peritoneally (i.p).
Group2: Divided into two subgroups; Group 2a and 2b consisting of six animals each. Received TAX (15 and 25 mg/kg) intra-peritoneally (i.p) respectively, twice weekly.
Group3: Divided into two subgroups; Group 4a and 4b consisting of six animals each. Received FH535 (15 and 25 mg/kg) intra-peritoneally (i.p) respectively, twice weekly.
During the experiment, food, body weight of animals, and water expenditure were documented twice a week. Digital caliper was used to measure the Tumor volume and calculated using the formula:
$$ L1\times L2\times H\times 0.5238 $$
Where, H = height of the tumor, L1 and L2 = long diameter and short diameter respectively.
Tumor sizes were recorded two times in a week. At the end of the experiment when tumor volume reached to ~1210mm3 animals were sacrificed by CO2 inhalation was used as anesthesia. Tumors were resected, weighed and frozen at -80 °C for subsequent western blotting, RNA extraction and immunohistochemistry.
Immunohistochemistry
Hematoxylin and eosin were used for staining the tumor tissue section for morphological visualization. Furthermore tissues were fixed in 10% formalin. Deparaffinization was done with with EZ Prep (Ventana, Arizona, USA) at 70 °C, heat pretreated in Cell Conditioning 1 (CC1; Ventana, Arizona, USA) using “standard cell conditioning” for antigen reclamation at 90 °C. Overnight incubation with antibodies against β-catenin, cyclinA and cleaved caspase3 (diluted 1:75). Then incubation with ultraview universal HRP multimer (secondary antibody), diamminnobenzidine/ DAB (DAKO, CA) staining and counter staining with hematoxylin. After mounting in DPX (distyrene, a plasticizer and xylene), the sections were covered with cover slips. Immunostained tissue sections were assessed at magnification of 100× and 200× (Olympus BX51 light Microscope and DP72 Olympus Digital Camera, Olympus America Inc., Center Valley, PA, USA).
Data analysis
Densitometry of western blot images was performed using an image analysis software (Image J 1.41). Data of in vitro assays was analyzed GraphPad Prism 5 software to determined IC50 values. Level of significance between different treatments groups relative to control were estimated by one way analysis of variance followed by Tukey’s multiple comparison test. Comparison between more than one parameter was accomplished using two way analysis of variance (ANOVA) followed by Bonferroni multiple comparison test. p < 0.05 was considered statistically significant. Where required correlation analysis was done and R values were calculated.
Discussion
The assorted molecular outlines of CRC and the necessity to categorize patients which could efficiently take clinical benefit from combined chemotherapies ignited the categorization of the mechanisms accountable for sensitivity and confrontation to treatments. The common of sporadic types of CRC are distinguished by deregulation of Wnt/β-Catenin signaling which results in amplified transcriptional activity of the β-Catenin. Regardless of the complexities in to dissect the association of Wnt/β-Catenin pathway in the initiation and development of CRC, the study of these mechanisms is now rising as a potent platform to recognize budding objectives of incursion for CRC cure.
Countless natural compounds have been recommended to operate as anti-cancer agents or lessen the side effects of additional anti-cancer therapeutic drugs by mounting the therapeutic effects of the vital anti-cancer agent or act as “enhancers” [
23,
24].
Taxifolin, a flavanonol plagiaristic of flavonoids, is copious in foods and sages [
25]. It displays an extensive range of bioactivities, amongst which the antioxidant activity is relatively distinctive [
26]. Numerous studies have confirmed TAX demonstrates advantageous chemopreventive activity on colon carcinogenesis, but the specific mode of action by which it obtains its anti-carcinogenic effect remains veiled [
27] . Studies also revealed the therapeutic behavior of TAX confined on in vivo and in silico-mediated tendency of Nrf2, Wnt/ β-catenin, and inflammatory signaling pathways [
28]. Also studies illustrated that TAX defends RPE cells against oxidative strain by hindering the H2O2-induced decline in the intracellular generation of ROS, cell viability and cell apoptosis. The effective mechanism emerges to encompass the stimulation of NRF2 and the upregulation of the phase II antioxidant enzyme system.
Cellular proliferation significant in tumor formation may arise due to amendment in cell cycle regulation [
29,
30]. A chief principal cause of cancer progression is accredited to immediate and uninhibited proliferation results to succession and upsurge of tissue accumulation. Results of MMT assay quantified that TAX are specific in their action and competent against cell cancer cell lines from diverse origin. Cell growth in both HCT116 and HT29 cells is altered by TAX. Administration with TAX resulted in arrest of cellular propagation in a dose dependent aaproach, amplification in hammering of cell viability was observed with increase in the concentration of dose.
Apoptosis is a dominant event to suitable explicit components of cells while shunning inflammatory effect usually escorting necrosis, thus no disparaging effect to the adjacent healthy cells will occur meanwhile the cells undergoes apoptosis. Thus apoptosis is an event ratifying chemotherapy efficiency, believed as an endpoint of anticancer drug therapy [
31,
32].
The in vitro study revealed that the N-terminal of p21 contains 1–82 amino acids, is crucial for the reticence of cyclin–CDK, and involvement with cyclin A, cyclin E, or CDK2 in an uncoupled form or an intricate form [
33]. Observations illustrated that cyclin E allies with p21 via the Cy motif (17). Also studies signify that the Cy-deleted mutant-p21 precipitate the cyclin A–CDK2 and p21 administrated with anti-Cy motif antibody co-precipitated with cyclin A–CDK2. The communication of the p21-allied CDK inhibitor, p27, and cyclin A is mediated by an RNLFG sequence in p27 that communicates with a hydrophobic groove on the surface of cyclin A [
34]. In the present study, assessment of apoptosis tempted in HCT116 and HT29 cells illustrated that TAX are very efficient inducers of apoptosis in concentration dependent approach. Results of our study showed decreased expression of Cyclin B1, D1, E and A, Cdk-2, 4 and 6 and accompany with an upsurge in expression of cdk inhibitors p21 and p27 in TAX administrated cells. Furthermore TAX colorectal cancer cells exhibited arrest in the G2 phase of the cell cycle. These findings are significant since cell cycle regulation is a central goal for anticipation against colorectal cancer. Zhang et al. [
35], illustrated that TAX boosts the anti-proliferation and apoptotic effects of Andrographolide and suggested that TAX might function as an enhancer in andro-induced cell cycle arrest and cell death of DU145 cells, but our study revealed that TAX alone tempted cell-cycle arrest and cell death of HCT116 and HT29 cells.
PARP, a typical caspase substrate, is a crucial competitor of DNA repair against ecological strain and in the continuation of cell viability. Cleavage of PARP is considered as a trademark of apoptosis [
36]. PARP cleavage results concurrently with cleavage of procaspase 3, 7, and 9 in a dose-dependent approach, demonstrating that TAX induced apoptosis in HCT116 and HT29 cells is arbitrated in the passage of an intrinsic apoptosis pathway. Numerous studies have revealed that PARP-1 is overexpressed in different human malignancies [
37‐
39]. In addition, studies revealed that PARP-1 has a part in colon cancer growth [
40,
41], as its expression was considerably increased in colon cancer and was associated with tumor size and histopathology [
41]. Above results strengthened our results showing increased expression of PARP in TAX administrated cells as compared to control.
Bcl-2 family associates are the chief watchdogs of apoptosis. Their overexpression constantly hinders apoptosis due to cytotoxic injuries by abolition of free radicals, deterrence of mitochondrial canal formation, and the discharge of c cytochrome [
42,
43]. In the anti-apoptotic subfamily, Bcl-2 and Bcl-xL, showed decreased expression in TAX administrated cells in concentration dependent approach. In contrast, the expression of Bax and Bak, pro-apoptotic proteins, showed increased expression in TAX treated HCT116 and HT29 cells as compared to control cells. Our results proposed that TAX mediated upsurge in the expression of Bax and decreased regulation of Bcl2 expression may be a promising direction through which TAX induce apoptosis in colorectal cancer. This signifies its pleiotropic influence on the apoptotic signaling pathway.
The canonical Wnt (or Wnt/ β-catenin) signaling pathway is crucial in progression, tissue renewal and stem cell continuance. In addition boosted signaling via this pathway is allied with the expansion and succession of various cancers [
44]. Initiation of the pathway is allied to hypophosphorylation, cytosolic alleviation and growth of β-catenin in the nucleus pursued by intensified candidate gene expression [
45,
46]. Increased cytosolic β-catenin levels and consequent nuclear import authorize its union with transcription players of the Tcf/Lef family that organize the expression of genes, including cyclin D1, vegf, and survivin, all of which promote to cancer progression [
47‐
50]. In this study AKT and β-catenin showed down regulated expression by administration of TAX in HCT116 and HT29 colorectal cancer cells as compared to control cells. Immunofluorescence staining of HCT116 and HT29 cells demonstrated decline in p-AKT expression and β-catenin at dose of 40 μM of TAX treated as compared to control. Significant Alexa fluor staining of p-AKT (cytoplasm) of both cell lines (green fluorescence) were pragmatic in control, while the expression of p-AKT as specified by staining was prominently decreased in TAX administrated cells. Further our findings illustrated that TAX induction resulted in apoptosis through inhibition of β-catenin and p-AKT. While FH535 β-catenin inhibitor baskets AKT phosphorylation which inturn decrease β-catenin protein expression. TAX administration to β-catenin inhibitor induced cells supporting amplified decrease of β-catenin expression, sustaining the fact that these alterations are mediated by protein kinase B (AKT). Our results support the involvement of AKT in encouraging the transcriptional activity of β-catenin.
AKT is the most crucial downstream effector of CK2 and phosphatidylinositol 3 kinase (PI3-K). Phosphorylated AKT encourages cell endurance through hampering pro-apoptotic proteins [
51]. The commencement of PI3K/Akt can trigger the canonical Wnt signaling through the phosphorylation of GSK-3β by the phosphorylated Akt1/2, jamming the configuration of β-catenin destroying complex (28). Therefore, the upregulation of PTEN may hamper the canonical Wnt signaling by encouraging the deprivation of β-catenin. While our results confirmed that administration of TAX to HCT116 and HT29 colon cells reduces the phosphorylation of PI3K, AKT and decreased the protein expression of β-catenin. The recent study revealed that it may not result from the reduced phosphorylation of GSK-3β by PTEN/PI3K/Akt signaling. As it is prone to mutation of β-catenin in HCT116 cells, the β-catenin can’t be tarnished by the destruction complex in this colon cancer cells [
52]. Further we investigated that administration of TAX hindered the mRNA expression of
β-catenin and thus our study strongly illustrated that TAX has compelling anti-proliferation activity in human colon cancer cells, promoting apoptosis and the anti-proliferation effect of TAX may be arbitrated by PI3K/Akt signaling by jamming Wnt/β-catenin signaling transduction, through hampering the β-catenin expression.
Recently studies confirmed that the substantial relations and efficient reticence of NF-κB by β-catenin in colorectal cancer cells requires phosphatidylinositide 3-kinase (PI3K) [
53]. Obstruction of PI3K by compound inhibitors rescinds the configuration of β-catenin and NF-κB protein complexes. In quiescent colorectal cancer cells, β-catenin and NF-κB restricted in the cytoplasm, and administration with PI3K inhibitor give rise to nuclear translocation of NF-κB and membrane retention of β-catenin. Conversely, being not clear whether PI3K directly acts as an association player between β-catenin and NF-κB or otherwise parting a role in β-catenin-mediated suppression of NF-κB triggered by diverse stimuli. Our studies proposed that inhibition of overexpression of β-catenin by administration of TAX in HCT116 and HT29 results in reduced protein expression of NF-κB and PI3K, hence this may be one of the possible mechanism cell death and apoptosis.
Survivin (BIRC5), an associate of the inhibitor of apoptosis (IAP) protein family, is a delegate anti-apoptotic protein which enhance tumor cell expansion [
54]. Survivin is of growing curiosity as a probable therapeutic target to hamper cancer intensification [
55]. Survivin enhances tumor propagation by renovating various significant cell signaling pathways. Our results clearly confirmed a noteworthy inhibition of an anti-apoptotic protein survivin expression in both HCT116 and HT29 cells by administration of TAX.
Acknowledgements
We are grateful to Dr. Javid Dar and Dr. Maria Shabbir who helped in designing the experiment. Furthermore we are grateful to the Deanship of Scientific Research at King Saud University for its funding of this research through Research Group Project number 193.