Targeting cytosolic phospholipase A2 α in colorectal cancer cells inhibits constitutively activated protein kinase B (AKT) and cell proliferation

A constitutive activation of protein kinase B (AKT) in a hyper-phosphorylated status at Ser473 is one of the hallmarks of anti-EGFR therapy-resistant colorectal cancer (CRC). The aim of this study was to examine the role of cytosolic phospholipase A2α (cPLA2α) on AKT phosphorylation at Ser473 and cell proliferation in CRC cells with mutation in phosphoinositide 3-kinase (PI3K). AKT phosphorylation at Ser473 was resistant to EGF stimulation in CRC cell lines of DLD-1 (PIK3CAE545K mutation) and HT-29 (PIK3CAP499T mutation). Over-expression of cPLA2α by stable transfection increased basal and EGF-stimulated AKT phosphorylation and proliferation in DLD-1 cells. In contrast, silencing of cPLA2α with siRNA or inhibition with Efipladib decreased basal and EGF-stimulated AKT phosphorylation and proliferation in HT-29. Treating animals transplanted with DLD-1 with Efipladib (10 mg/kg, i.p. daily) over 14 days reduced xenograft growth by >90% with a concomitant decrease in AKT phosphorylation. In human CRC tissue, cPLA2α expression and phosphorylation were increased in 63% (77/120) compared with adjacent normal mucosa determined by immunohistochemistry. We conclude that cPLA2α is required for sustaining AKT phosphorylation at Ser473 and cell proliferation in CRC cells with PI3K mutation, and may serve as a potential therapeutic target for treatment of CRC resistant to anti-EGFR therapy.


INTRODUCTION
Colorectal cancer (CRC) is the third most commonly diagnosed cancer in males and the second in females worldwide with over 1.2 million new cases annually [1].Due to the lack of effective treatment for metastatic CRC, there are approximately 600,000 deaths annually [1].Despite the improvement in the clinical outcome following the development of molecular targeted therapy against the epidermal growth factor receptor (EGFR) [2], CRC with mutations of BRAF, RAS, PI3K or PTEN are resistant to anti-EGFR therapy [3,4].RAS and PIK3CA mutation increased protein kinase B (AKT) phosphorylation at Ser 473 [5].Phosphorylation of AKT at Ser 473 is required for tumor progression in colon cancer [6].Therefore, a constitutive activation of AKT in a hyper-phosphorylated status at Ser 473 is one of the hallmarks of anti-EGFR therapy-resistant CRC [7].Hence, identification of pathways that are required for maintaining AKT phosphorylation at Ser 473 in CRC is of clinical importance.
Previous studies have shown the involvement of prostaglandin and its producing enzyme cyclooxygenase (COX) in CRC [8,9].The enthusiasm for the effectiveness of COX-2 inhibitor is hampered by its side effect due to the selective inhibition of COX enzymes.Phospholipase A 2 (PLA 2 ) is a family of enzymes that catalyse the hydrolysis of fatty acid at the sn-2 position of glycerophospholipid on cell membranes [10].Of the family members, cytosolic PLA 2 α (cPLA 2 α) is the only enzyme that catalyses the specific hydrolysis of arachidonic acid (AA) [10].The cleaved free AA is converted to eicosanoids by the COX and lypoxygense (LOX) enzymes [10].As the inhibition of cPLA 2 α reduces the supply of AA to both COX-1 and COX-2 enzymes, it may avoid the side effect of selective COX-2 inhibitors.Moreover, 5-LOX is over-expressed in CRC compared with normal colonic mucosa [11].Blocking 5-LOX reduces CRC cell proliferation in vitro and in vivo [11].Hence, we have evaluated the potential using cPLA 2 α as a therapeutic target for treatment of CRC.This paper describes the effect of ectopic expression, genetic silencing or pharmacological inhibition of cPLA 2 α on AKT phosphorylation at Ser 473 and cell proliferation in vitro and in vivo of CRC cells with constitutive activation of AKT due to gain-of-function mutations in PI3K, as well as cPLA 2 α expression and activation in human CRC tissues.

Silencing of cPLA 2 α decreases EGF-stimulated phospho-AKT at Ser 473 levels and proliferation in CRC cells with mutant PIK3CA P499T
We next determined the effect of genetic silencing of cPLA 2 α with siRNA on p-AKT levels and cell proliferation.Transfection of HT-29 (PIK3CA P499T ) with cPLA 2 α siRNA abolished the t-cPLA 2 α and p-cPLA 2 α protein levels (all P<0.001, Figure 2A and 2B), and significantly decreased both intracellular and extracellular content of arachidonic acid (both P<0.001, Figure 2C).Levels of p-AKT remained unchanged in response to EGF stimulation (final concentration 20 ng/mL, 30 min) in HT-29 (Figure 2A:scramble siRNA with EGF vs. scramble siRNA without EGF).However, Knockdown of cPLA 2 α deceased both basal and EGF stimulated p-AKT levels by 59% (Figure 2A: cPLA 2 αsiRNA without EGF vs. scramble siRNA without EGF) and 30% (cPLA 2 α siRNA with EGF vs. scramble siRNA with EGF), respectively, compared with the scrambled control (all P<0.05, Figure 2B).The levels of t-AKT were unchanged with or without EGF stimulation in the presence or absence of cPLA 2 α siRNA.It indicates that the constitutively-activated AKT as the results of PIK3CA P499T mutation could be inhibited by knockdown cPLA 2 α expression.Again, EGF treatment elicited an increase in p-cPLA 2 α (P<0.05)without affecting t-cPLA 2 α when endogenous cPLA 2 α was unperturbed (Figure 2A and 2B).
Next, we assessed the effect of transient knockdown of cPLA 2 α on cell cycle distribution.There was a clear increase in G 1 /G 0 and corresponding decrease in S phase (all P<0.05, Figure 2D), with no significant change in the proportion of cells in sub-G 1 phase following genetic silencing of cPLA 2 α.We then examined whether Efipladib (a new indole derived cPLA 2 α inhibitor [12,13]) mimics the impact of cPLA 2 α siRNA and exerts the same action on AKT phosphorylation in HT-29 cells.Incubation of HT-29 cells with Efipladib (25 µM, 72 h) indeed decreased basal and EGF-stimulated p-AKT levels without affecting t-AKT (both P<0.05, Figure 2E and 2F).Taken together, targeting cPLA 2 α by genetic silencing or pharmacological inhibition supresses EGF-resistant AKT phosphorylation at Ser 473 and also inhibits cell proliferation in HT-29 cells harbouring mutation in PIK3CA P499T .

Pharmacological inhibition of cPLA 2 α decreases cell proliferation in both DLD-1 and HT-29 cells
Since pharmacological blockade of cPLA 2 α with Efipladib effectively reduced basal and EGF-stimulated AKT phosphorylation, we determined the effect of Efipaldib on cell proliferation in unmodified parental DLD-1 (PIK3CA E545K ) and HT-29 (PIK3CA P499T ) cells.Inhibition of cPLA 2 α with Efipladib reduced cell number (P<0.05, Figure 3A and 3B) and BrdU incorporation (P<0.05, Figure 3C and 3D) in a dose-dependent manner in both DLD-1 and HT-29 cells.Efipladib treatment for 24-48 h blocked DLD-1 cell cycle progression as indicated by an accumulation of cells in the G 0 /G 1 phase with a decrease in the proportion of cells in S phases (all P<0.05, Figure 3E).The decreased G 2 /M phase, however, did not reach statistical significance at 48 h.A similar effect on G 0 /G 1 phase and S phases was noted in HT-29 cells treated with increasing dose of Efipladib after 72 h (all P<0.05, Figure 3F).The fraction of cells in G 2 /M was  also decreased at the highest concentration of Efipladib (25 µM, P<0.05).We found no significant change in cell viability in the presence of Efipladib as assessed by sub-G 1 (Figure 3E and 3F) and Trypan Blue exclusion (data not shown).Hence, consistent with effect of genetic silencing of cPLA 2 α, pharmacological blockade of cPLA 2 α resulted primarily in a cytostatic effect on CRC cells with PIK3CA E545K or PIK3CA P499T mutations.

Pharmacological inhibition of cPLA 2 α reduces p-AKT levels and xenograft growth in mice transplanted with DLD-1 cells
To determine if the marked decrease in p-AKT and cell proliferation in response to Efipladib can be recapitulated in animal, we treated mice carrying unmodified parental DLD-1 xenografts with Efipladib.In vehicle-treated control mice tumour volume increased 4.5-fold at day 14 compared to the day 1 (Figure 4A), but in the Efipladib-treated mice, there was only a 1.4fold increase over 14 days (P<0.001 by two way ANOVA with repeat measurements).Further analysis at each time point revealed a significant difference in tumour volume as early as day 5 of Efipladib treatment (P<0.05, Figure 4A).Mouse body weights did not differ between the two groups.The percentage of Ki-67 positive cells and the levels of p-AKT and p-cPLA 2 α in xenografts were significantly reduced in Efipladib-treated mice compared with the vehicle-treated controls (all P<0.05, Figure 4B-D).The levels of t-AKT and t-cPLA 2 α remained unchanged.Hence, consistent with the in vitro effect of Efipladib on suppressing p-AKT and proliferation, pharmacological inhibition of cPLA 2 α in vivo reduces markedly p-AKT levels and DLD-1 xenograft growth compared with vehicle-treated controls.

The levels of cPLA 2 α and phospho-cPLA 2 α at Ser 505 are increased in colon cancer tissues
To determine the potential of cPLA 2 α as a therapeutic target, we examined cPLA 2 α protein levels in CRC specimens by immunohistochemistry. Compared with adjacent normal epithelial cells, an increase in the extent and/or intensity of immune reactive total cPLA 2 α in malignant epithelial cells was observed in 77/120 cases (64.2%, P<0.001, Figure 5A and 5B).Total cPLA 2 α was mainly located in the cytoplasm in both normal and cancer cells.Although total cPLA 2 α was also present in mesenchymal cells, there was no difference between normal and cancer tissues.Among the clinical parameters analysed, total cPLA 2 α levels were correlated with poor tumour differentiation (p=0.029,Supplemental Table 1).
cPLA 2 α also contains several conserved serine residues as phosphorylation sites.Ser 505 is the most studied and recognised site for phosphorylation of cPLA 2 α.Although phosphorylation is not necessary for basal enzyme activity, phosphorylation at Ser 505 has shown to augment arachidonic acid release [14].Immune reactive phospho-cPLA 2 α at Ser 505 was located in nucleus and cytoplasm in both normal and cancer cells, which is consistent with previous reports in other cell types [15,16].An increase in the extent and/or intensity of phospho-cPLA 2 α at Ser 505 was observed in malignant epithelial cells compared with adjacent normal epithelial cells in 76 out of 120 cases (63.3%, P<0.001, Figure 5C and 5D).Phospho-cPLA 2 α at Ser 505 was also present in mesenchymal cells but not significantly different between normal and cancer.There was no association between phospho-cPLA 2 α and any tumour characteristics (Supplemental Table 1).Taken together, cPLA 2 α expression and activation are increased in nearly two thirds of CRC compared with normal mucosa.

DISCUSSION
We provide three lines of evidence supporting the advantages of targeting cPLA 2 α in colorectal cancer.
Firstly, we have systematically investigated the role of cPLA 2 α in regulation of AKT phosphorylation by ectopic expression, genetic silencing and pharmacological inhibition in CRC cell lines with a constitutive action of AKT at Ser 473 both in vitro and in vivo.Ectopic expression of cPLA 2 α increases basal and EGF-stimulated p-AKT levels.It is interesting to note that without manipulation of cPLA 2 α, AKT phosphorylation does not increase in response to EGF stimulation in both CRC cell lines.This is consistent with the report that constitutively-activated AKT renders cancer cells resistant to manipulation by growth factors [17].However, a marked increase in AKT phosphorylation following EGF stimulation is noted when cPLA 2 α levels are increased by ectopic expression in DLD-1 cells.In contrast, genetic silence or pharmacological blockade of cPLA 2 α decreases basal and EGF-stimulated p-AKT levels in HT-29, which is another CRC cell line harbouring PI3K mutations.Same as DLD-1, p-AKT levels in HT-29 cells are resistant to EGF stimulation.Together with the effect of efipladib on p-AKT in vivo, these findings suggest that cPLA 2 α contributes to basal and EGF-stimulating AKT phosphorylation in CRC cells containing PI3K mutations.It is interesting to mention that we have shown recently that genetic silence or pharmacological blocking of cPLA 2 α decrease phospho-AKT at Ser 473 in prostate cancer cells [18].Hence, the link of cPLA 2 α to AKT appears to be a phenomenon not just limited to colon cancer cell lines.
The mechanism(s) by which cPLA 2 α exerts its action on AKT phosphorylation remains to be elucidated.Based on the significant change in AA concentration in response to cPLA 2 α manipulations, cPLA 2 α may exert its action on AKT via AA and/or its product eicosanoids [19].Eicosanoid receptors can connect to PI3K-AKT pathway via heterotrimeric G proteins [20].PGE 2 may also be able to transactivate EGFR in CRC cells including HT-29 [21,22].As the decrease in proliferation of HT-29 cells by EGFR inhibitor could be abolished in the presence of PGE 2 [23], the possible action site downstream of EGFR cannot be excluded.Furthermore, COX-2 inhibitor has been shown to increase in PTEN expression [24], which could be another mechanism for impinging on AKT.Further study is also needed to determine if cPLA 2 α can affect basal and EGF stimulated other oncogenic pathways such as ERK/MAPK.
Another interesting finding from our study is the phosphorylation of cPLA 2 α at Ser 505 , which is known to increase the AA-releasing activity [14,25].Previous studies have shown an increase in cPLA 2 α phosphorylation in mammalian cells by EGF [26,27].We found in the present study that EGF treatment increases phosphorylation of cPLA 2 α at Ser 505 in both DLD-1 and HT-29 cells.Activation of RAS signalling by mutation or over-expression has been shown to induce PGE 2 secretion in colon cancer [22,28,29].We reported recently that AKT plays a role in stabilising cPLA 2 α protein in prostate cancer cells [30].Hence, it appears that a self-perpetuating loop consisting of AKT and cPLA 2 α is present in CRC and maybe other type of cancer cells.
Secondly, the present study has provided evidence for the first time that pharmacological blockade of cPLA 2 α decreases cell proliferation of CRC cell lines with PI3K mutation both in vitro and in vivo.The presence of somatic PI3K mutations causing constitutive activation of AKT have been regarded as one of the predictive markers of resistance to anti-EGFR therapy [3,4].Therefore inhibition of constitutive activated AKT could be one of the strategies to overcome resistance to anti-EGFR therapy.Our results suggest that in addition to inhibiting AKT phosphorylation at Ser 473 , targeting cPLA 2 α by siRNA or inhibitor can also retard cell-cycle progression and inhibit cell proliferation in CRC cells harbouring PI3K mutations.Similar to Efipladib (an inhibitor of fatty acid cleavage), Cerulenin (a fatty acid synthase inhibitor) decreased AKT phosphorylation at Ser 473 , enhanced antitumor activity of oxaliplatin in human colon cancer cells [31], and suppressed liver metastasis of colon cancer in mice [32].However, it is worth to mention that two published in vivo studies of cPLA 2 α in intestine or colon tumor have yielded inconsistent results.While cross-breeding of Apc min/+ mice with cPLA 2 α knockout suppresses intestine tumorigenesis [33], knockout of cPLA 2 α enhances azoxymethane-induced tumorigenesis in colon [34].Hence, it is likely that azoxymethane-induced CRC may involve signalling pathways that are different from those in Apc min/+ mice and DLD-1 cell xenograft.The prospective of targeting cPLA 2 α is further encouraged by the report that cPLA 2 α knockout mice exhibit a relatively normal phenotype [35].
Thirdly, cPLA 2 α protein is over-expressed and hyper-phosphorylated at Ser 505 in ~60% of colon cancer cases.The mRNA and protein levels of cPLA 2 α have been examined in CRC specimens previously.RT-PCR [36,37], immunoblot [38] or immunohistochemistry [39][40][41] revealed an increased cPLA 2 α in CRC specimens, except two studies conducted by the same group reported a low cPLA 2 α expression in CRC compared to normal mucosa by immunohistochemistry [42,43].In the present study, we examined for the first time the levels of phospho-cPLA 2 α at Ser 505 .Although phosphorylation at Ser 505 is not necessary for basal enzyme activity, phosphorylation at Ser 505 has shown to augment AA release [14,25].In correlation with total cPLA 2 α, phospho-cPLA 2 α at Ser 505 was clearly increased in near two-thirds of the 120 CRC specimens compared with adjacent normal mucosa.As both total and phospho-cPLA 2 α have increased in CRC, it is possible that the increase in phospho-cPLA 2 α results from the increase in total cPLA 2 α expression.Consistent with reports that activated cPLA 2 α translocates to the nucleus following stimulation with calcium ionophore or leukotriene D4 in CRC cells [44], we notice that the phospho-cPLA 2 α is present in nucleus as well, whereas total cPLA 2 α is confined in cytoplasm.
Our study suggests that poorly differentiated tumours, which is associated with unfavourable prognosis [45], are more likely having high cPLA 2 α expression.Two studies have shown that the expression of cPLA 2 α in CRC is correlated with VEGF expression but fail to predict disease-free survival and overall survival [40,41].cPLA 2 α gene polymorphisms has been shown to be associated with patients of familial adenomatous polyposis [46].Since prognostic data of the TMA used in our study are not available, further studies are needed to determine the prognostic value of cPLA 2 α in CRC.
In summary, cPLA 2 α plays a critical role in regulation of AKT phosphorylation and cell proliferation in colon cancer cells in which PIK3CA has a gain-function mutation.We propose that the cPLA 2 α is a potential therapeutic target for treatment of colon cancer that are resistant to anti-EGFR therapy in the results of constitutive activation of AKT.

Ectopic expression and genetic silence of cPLA 2 α
Expression vector containing pCMV-cPLA 2 α or empty vector was stably transfected into DLD-1 cells using Lipofectamine TM 2000 (Invitrogen, Melbourne, VIC, Australia).After 1 day of transfection, media was replenished with fresh medium containing selection antibiotic G418 at 1 mg/mL and cells were allowed to grow for 10 days.Isolated colonies were cultured in the presence of G418 (400 μg/mL).Two clones (Clone 15 and 18) were used for this study.Both show an increase in p-AKT.cPLA 2 α siRNA (TTG AAT TTA GTC CAT ACG AAA) and scramble control (GAA TTT CAA ACT CGA TAT AGT) were transfected into cells (10 nM siRNA duplexes) using HiPerfect Transfection Reagent (QIAGEN, Santa Clarita, CA) as described previously [16].

Arachidonic acid release assay
Fatty acids were extracted from isolated cell pellets or culture media as described by Norris and Dennis [47].A Xevo-Triple quadruple mass spectrometer (Waters, Micromass, UK) coupled to a Phenomenex Kinetex 1.7 µm C18 100A (2.1×150 mm) was used for arachidonic acid analysis.Standard curves were constructed using linear regression of the normalised peak areas of the analyte over internal standard (heptadecanoic acid) against the corresponding nominal concentrations of the arachidonic acid (See Supplemental method).

Cell cycle analysis
CRC cells were plated in triplicate in 6-well plates.After 24 h, cells adherent to plates were exposed to the indicated treatments.Cells were harvested, fixed in 70% v/v ice-cold ethanol, and incubated with propidium iodide (20 μg/mL) and RNase A (100 μg/mL) for 1 h in 37°C incubator.Cells containing propidium iodide-stained DNA were then assessed using FACSCalibur flow cytometer (BD Biosciences, Australia), and the percentage of cells in each phase of the cell cycle was analysed using Flowjo v8.0 (Tree Star, Ashland, OR).

BrdU incorporation
CRC cells were incubated with BrdU at 10 μM in culture medium for 3 h before harvesting.Cells were then trypsinized, fixed in 10% v/v formalin, clotted in agarose gel, and processed for paraffin blocks.Sections of 5 µm thickness were cut and incubated at 60°C for 1 h, deparaffinized in xylene, re-hydrated in graded ethanol and distilled water, and subjected to antigen retrieval in Tris-EDTA solution using a microwave oven.Thereafter, the sections were treated with 2N HCl, blocked with 10% v/v house serum (Sigma-Aldrich) and incubated with anti-BrdU antibody overnight at 4°C.After being rinsed in Tris-buffered saline containing 0.05% Tween-20, the sections were sequentially labelled with a biotinylated secondary antibody and a Vectastain ABC kit from Vector Laboratories.Thereafter, the immunolabelling was visualized with 3,3'-diaminobenzidine tetrahydrochloride from Dako.Sections were scanned and analysed with an automated cellular imaging system (ACIS III, Dako, Denmark).The number of both BrdU-positive and negative cells over 10 randomly selected fields was determined and expressed as a percentage of positive cells in total number of cells.

Xenografts assay
DLD-1 cells (2×10 6 ) were implanted s.c. in the right flanks of 6 week male nude mice.Mice were randomly distributed into two groups once the tumour size reached 50 mm 3 (7 mice/group).One treated with 200 µL of 20% v/v DMSO in PBS i.p. daily (as vehicle control); the other treated with Efipladib (10 mg/kg, i.p. daily) dissolved in DMSO and then diluted in PBS.Tumour growth was assessed every other day by caliper measurement of tumour diameter in the longest dimension (L) and at right angles to that axis (W).Tumour volume was estimated by the formula, L ×W ×W/2.Mice were sacrificed after 14 days of treatment and tumours were excised and the tissue distributed in two halves designated for Ki-67 immunostaining and immunoblotting.The protocol was approved by the Institutional Animal Care and Use Committee (Shanghai Jiao-Tong University).

Immunohistochemistry
Tissue arrays were obtained from Outdo Biotech (Shanghai, China) with 120 individual cases of CRC and adjacent non-cancerous colon tissue from the same individual.Immunohistochemical staining was conducted using a DAKO EnVision+ System HRP as described previously [48].An antibody raised in rabbit against cPLA 2 α (SC-438, 1:400 v/v) was left overnight at 20°C, an antibody in rabbit against phospho-cPLA 2 α (SC-34391-p, 1:150 v/v) was applied at 37°C for 2 h.For Ki-67 immunostaining in xenograft recovered from mice, anti-Ki-67 was applied at 37°C for 2 h and purified rabbit-IgG (Dako, 1:60 v/v) was used as an isotype control.

Statistical analysis
The statistical software SPSS version 14.0 was used for analysis.The scores of total and phospho-cPLA 2 α levels in CRC tissue were analysed by Wilcoxon signed rank test.The nonparametric Mann-Whitney U test was used to test whether the levels of cPLA 2 α and phospho-cPLA 2 α differ in gender, age, or M stage.Gamma regression was used to test the relationship between cPLA 2 α and T, N, TNM stage or differentiation.In vitro data were analysed by one-way ANOVA followed by multiple comparison tests.Xenograft growth was compared between groups by fitting a repeated measures covariate model, where the actual time measurements were viewed as a covariate.Two-tailed P value <0.05 was considered significant.

Figure 3 :
Figure 3: Pharmacological blockade of cPLA 2 α by Efipladib results in decreased cell proliferation.DLD-1 (A) or HT-29 cells (B) were plated in 96-well plates and treated with vehicle control (DMSO) or Efipladib for 72 h.The viable cell number was determined by the MTS assay.DLD-1 (C) or HT-29 (D) cells were plated in 6-well plates and treated with control (DMSO) or Efipladib for 72 h.BrdU was added for 3 h prior to harvesting.BrdU incorporation was determined by immunocytochemistry.Percentage of BrdU positive cells was determined as the average of 10 high-power fields (X40) per sample.* P <0.05 vs. vehicle-treated control, n=3.(E) DLD-1 cells were treated with Efipladib at 25 µM for 1 or 2 days, followed by staining with PI and subsequent analysis with flow cytometry.* P<0.05 vs. vehicle-treated control, n=3.(F) HT-29 cells were treated with Efipladib at indicated doses for 3 days, followed by PI-staining and DNA content analysis.* P<0.05 vs. vehicle-treated control, n=3.All data expressed as Mean ± SD.

Figure 4 :
Figure 4: Pharmacological blockade of cPLA 2 α by Efipladib impedes the growth of DLD-1 xenografts and decreases p-AKT levels in vivo.(A) DLD-1 cells were inoculated into the flanks of nude mice.When xenograft tumours had reached 50 mm 3 in volume, mice were randomised to control (n=7) or Efipladib treatment (7 mice/group) at a dose of 10 mg/kg i.p. daily for 14 days.Inhibition of tumour growth in the Efipladib-treated mice compared with the controls (p<0.001by two way ANOVA with repeat measurement).* p<0.05 vs. control at the same day.(B) The fraction of Ki-67 positive cells was determined from the average number of positive cells in 10 high-power fields (×40).*p < 0.05 vs. control.(C) Xenografts were harvested, fixed and paraffin-embedded, and stained for Ki-67 by immunohistochemistry. Scale bar = 50 µm, magnification 200×.(D) Immunoblot of DLD-1 xenograft tumour and densitometry quantification.*p<0.05 vs. control, n=3.All data expressed as Mean ± SD.