Abstract #1075: PP2A induction by FTY720 inhibits survival and self-renewal of CD34+/CD38- CML stem cell through the simultaneous suppression of BCR/ABL and BCR/ABL-independent signals. | Cancer Research
Abstract #1075: PP2A induction by FTY720 inhibits survival and self-renewal of CD34+/CD38- CML stem cell through the simultaneous suppression of BCR/ABL and BCR/ABL-independent signals.
Paolo Neviani, Ramasamy Santhanam, Yihui Ma, Bin Zhang, Hsiaoyin Mao, Stefano Volinia, Guido Marcucci, Ching-Shih Chen, Jorge Cortes, Claudia Huettner, Steffen Koschmieder, Peter Hokland, Ravi Bhatia, Denis Roy, Michael Caligiuri and Danilo Perrotti
Paolo NevianiThe Ohio State Univ. Medical Ctr., Columbus, OH; City of Hope National Medical Center, Duarte, CA; University of Ferrara, Ferrara, I M.D. Anderson Cancer Center, Houston, TX; Dana Farber Cancer Institute, Boston, MA; University of Muenster, Muenster, G %C5rhus University Hospital, %C5rhus-C, D Hopital Maisonneuve-Rosemont, Montreal, CanadaRamasamy SanthanamThe Ohio State Univ. Medical Ctr., Columbus, OH; City of Hope National Medical Center, Duarte, CA; University of Ferrara, Ferrara, I M.D. Anderson Cancer Center, Houston, TX; Dana Farber Cancer Institute, Boston, MA; University of Muenster, Muenster, G %C5rhus University Hospital, %C5rhus-C, D Hopital Maisonneuve-Rosemont, Montreal, CanadaYihui MaThe Ohio State Univ. Medical Ctr., Columbus, OH; City of Hope National Medical Center, Duarte, CA; University of Ferrara, Ferrara, I M.D. Anderson Cancer Center, Houston, TX; Dana Farber Cancer Institute, Boston, MA; University of Muenster, Muenster, G %C5rhus University Hospital, %C5rhus-C, D Hopital Maisonneuve-Rosemont, Montreal, CanadaBin ZhangThe Ohio State Univ. Medical Ctr., Columbus, OH; City of Hope National Medical Center, Duarte, CA; University of Ferrara, Ferrara, I M.D. Anderson Cancer Center, Houston, TX; Dana Farber Cancer Institute, Boston, MA; University of Muenster, Muenster, G %C5rhus University Hospital, %C5rhus-C, D Hopital Maisonneuve-Rosemont, Montreal, CanadaHsiaoyin MaoThe Ohio State Univ. Medical Ctr., Columbus, OH; City of Hope National Medical Center, Duarte, CA; University of Ferrara, Ferrara, I M.D. Anderson Cancer Center, Houston, TX; Dana Farber Cancer Institute, Boston, MA; University of Muenster, Muenster, G %C5rhus University Hospital, %C5rhus-C, D Hopital Maisonneuve-Rosemont, Montreal, CanadaStefano VoliniaThe Ohio State Univ. Medical Ctr., Columbus, OH; City of Hope National Medical Center, Duarte, CA; University of Ferrara, Ferrara, I M.D. Anderson Cancer Center, Houston, TX; Dana Farber Cancer Institute, Boston, MA; University of Muenster, Muenster, G %C5rhus University Hospital, %C5rhus-C, D Hopital Maisonneuve-Rosemont, Montreal, CanadaGuido MarcucciThe Ohio State Univ. Medical Ctr., Columbus, OH; City of Hope National Medical Center, Duarte, CA; University of Ferrara, Ferrara, I M.D. Anderson Cancer Center, Houston, TX; Dana Farber Cancer Institute, Boston, MA; University of Muenster, Muenster, G %C5rhus University Hospital, %C5rhus-C, D Hopital Maisonneuve-Rosemont, Montreal, CanadaChing-Shih ChenThe Ohio State Univ. Medical Ctr., Columbus, OH; City of Hope National Medical Center, Duarte, CA; University of Ferrara, Ferrara, I M.D. Anderson Cancer Center, Houston, TX; Dana Farber Cancer Institute, Boston, MA; University of Muenster, Muenster, G %C5rhus University Hospital, %C5rhus-C, D Hopital Maisonneuve-Rosemont, Montreal, CanadaJorge CortesThe Ohio State Univ. Medical Ctr., Columbus, OH; City of Hope National Medical Center, Duarte, CA; University of Ferrara, Ferrara, I M.D. Anderson Cancer Center, Houston, TX; Dana Farber Cancer Institute, Boston, MA; University of Muenster, Muenster, G %C5rhus University Hospital, %C5rhus-C, D Hopital Maisonneuve-Rosemont, Montreal, CanadaClaudia HuettnerThe Ohio State Univ. Medical Ctr., Columbus, OH; City of Hope National Medical Center, Duarte, CA; University of Ferrara, Ferrara, I M.D. Anderson Cancer Center, Houston, TX; Dana Farber Cancer Institute, Boston, MA; University of Muenster, Muenster, G %C5rhus University Hospital, %C5rhus-C, D Hopital Maisonneuve-Rosemont, Montreal, CanadaSteffen KoschmiederThe Ohio State Univ. Medical Ctr., Columbus, OH; City of Hope National Medical Center, Duarte, CA; University of Ferrara, Ferrara, I M.D. Anderson Cancer Center, Houston, TX; Dana Farber Cancer Institute, Boston, MA; University of Muenster, Muenster, G %C5rhus University Hospital, %C5rhus-C, D Hopital Maisonneuve-Rosemont, Montreal, CanadaPeter HoklandThe Ohio State Univ. Medical Ctr., Columbus, OH; City of Hope National Medical Center, Duarte, CA; University of Ferrara, Ferrara, I M.D. Anderson Cancer Center, Houston, TX; Dana Farber Cancer Institute, Boston, MA; University of Muenster, Muenster, G %C5rhus University Hospital, %C5rhus-C, D Hopital Maisonneuve-Rosemont, Montreal, CanadaRavi BhatiaThe Ohio State Univ. Medical Ctr., Columbus, OH; City of Hope National Medical Center, Duarte, CA; University of Ferrara, Ferrara, I M.D. Anderson Cancer Center, Houston, TX; Dana Farber Cancer Institute, Boston, MA; University of Muenster, Muenster, G %C5rhus University Hospital, %C5rhus-C, D Hopital Maisonneuve-Rosemont, Montreal, CanadaDenis RoyThe Ohio State Univ. Medical Ctr., Columbus, OH; City of Hope National Medical Center, Duarte, CA; University of Ferrara, Ferrara, I M.D. Anderson Cancer Center, Houston, TX; Dana Farber Cancer Institute, Boston, MA; University of Muenster, Muenster, G %C5rhus University Hospital, %C5rhus-C, D Hopital Maisonneuve-Rosemont, Montreal, CanadaMichael CaligiuriThe Ohio State Univ. Medical Ctr., Columbus, OH; City of Hope National Medical Center, Duarte, CA; University of Ferrara, Ferrara, I M.D. Anderson Cancer Center, Houston, TX; Dana Farber Cancer Institute, Boston, MA; University of Muenster, Muenster, G %C5rhus University Hospital, %C5rhus-C, D Hopital Maisonneuve-Rosemont, Montreal, CanadaDanilo PerrottiThe Ohio State Univ. Medical Ctr., Columbus, OH; City of Hope National Medical Center, Duarte, CA; University of Ferrara, Ferrara, I M.D. Anderson Cancer Center, Houston, TX; Dana Farber Cancer Institute, Boston, MA; University of Muenster, Muenster, G %C5rhus University Hospital, %C5rhus-C, D Hopital Maisonneuve-Rosemont, Montreal, Canada
AACR Annual Meeting-- Apr 18-22, 2009; Denver, COAbstractWe recently reported that BCR/ABL-SET-dependent inhibition of protein phosphatase PP2A tumor suppressor activity is essential for the leukemogenic potential of imatinib/dasatinib-sensitive and -resistant CD34+ CML progenitors, Here we show that SET-dependent suppression of PP2A is also a feature of imatinib/dasatinib-insensitive CD34+/CD38- BCR/ABL+ stem cells (HSC) but not of normal HSC. To determine the biological importance and therapeutic implications of impaired PP2A activity in Ph(+) HSC, we evaluated the effects of FTY720 (2.5 mM), a PP2A activator currently in phase III trials for MS patients, and lentiviral-mediated ectopic PP2Ac expression on survival/self-renewal of BCR/ABL+ HSC isolated from bone marrow of CML blast crisis patients (n=8; Ph1\#8805;90%) and/or SCL-tTA-BCR/ABL transgenic animals (n=10). FTY720 treatment (2.5 mM; 72h) severely suppressed the clonogenicity, self-renewal and long-term repopulating potential of CD34+/CD38- CML HSC. In fact, the CFC output of LTC-IC cultures deriving from FTY720-treated Ph(+) CD34+ cells was more than 95% inhibited if compared to that of cultures from untreated CML cells. By contrast, imatinib (5 uM) and dasatinib (200 nM) led to a 3.5 and 5-fold increase in CFC output, respectively. Accordingly, a 50-90% reduction of the CFSEMAX/quiescent cells was observed in FTY720-treated CD34+ CML cells. Notably, FTY720 did not exert any significant effect on CFSE+ normal CD34+ cells whereas imatinib and dasatinib treatment led to a 22 and 27% increase in CFSEMAX CML cells, respectively. Interestingly, only FTY720 triggered apoptosis of CFSEMAX CML cells (41% Annexin V+ ) although BCR/ABL activity (phospho-ABL staining) in CFSEMAX cells was inhibited by FTY720, Imatinib and dasatinib, suggesting that BCR/ABL-independent PP2A-regulated signals control the survival/self-renewal of CML HSC. Indeed, lentiviral-driven PP2Ac-overexpression and FTY720, but not imatinib, significantly decreased (40-90% reduction) CFC/serial replating efficiency, colony size and CFSEMAX fraction (66-96% reduction) of Lin-/Sca+/Kit+ (LSK) cells isolated from BM of leukemic SCL-tTA-BCR/ABL mice. Mechanistically, the PP2A detrimental effect on survival/self-renewal of CML HSC might depend on the ability of PP2A to inactivate b-catenin that, reportedly, is a PP2A target essential for self-renewal of CML-BC progenitors. In fact, immunoblotting, immunofluorescence and LET/TCF luciferase assays showed that ectopic PP2Ac expression and FTY720, but not imatinib, induce b-catenin degradation in BCR/ABL+ mouse LSK and CML HSC, suggesting that BCR/ABL-independent, PP2A-sensitive and b-catenin-mediated signals may account for resistance of CML quiescent HSC to TKI. Thus, FTY720 has the potential to eradicate CML by targeting both stem and progenitor Ph(+) cells.Citation Information: In: Proc Am Assoc Cancer R 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 1075.Footnotes100th AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO
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Paolo Neviani, Ramasamy Santhanam, Yihui Ma, Bin Zhang, Hsiaoyin Mao, Stefano Volinia, Guido Marcucci, Ching-Shih Chen, Jorge Cortes, Claudia Huettner, Steffen Koschmieder, Peter Hokland, Ravi Bhatia, Denis Roy, Michael Caligiuri and Danilo Perrotti
Cancer Res (69) (9 Supplement) 1075;
Citation Manager Formats
Abstract #1075: PP2A induction by FTY720 inhibits survival and self-renewal of CD34+/CD38- CML stem cell through the simultaneous suppression of BCR/ABL and BCR/ABL-independent signals.
Paolo Neviani, Ramasamy Santhanam, Yihui Ma, Bin Zhang, Hsiaoyin Mao, Stefano Volinia, Guido Marcucci, Ching-Shih Chen, Jorge Cortes, Claudia Huettner, Steffen Koschmieder, Peter Hokland, Ravi Bhatia, Denis Roy, Michael Caligiuri and Danilo Perrotti
Cancer Res (69) (9 Supplement) 1075;
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Related Articles Cited By... More in this TOC Section吲哚美辛对慢性粒细胞白血病急变期CD34~+细胞的影响研究--《解放军医学杂志》2016年06期
吲哚美辛对慢性粒细胞白血病急变期CD34~+细胞的影响研究
【摘要】：目的探讨吲哚美辛(IN)对慢性粒细胞白血病(简称慢粒)急变期CD34+细胞凋亡和周期的影响,并从Wnt/β-catenin信号通路初步探讨其可能的分子机制。方法采用免疫磁珠分选技术分选慢粒慢性期、急变期患者骨髓标本和正常脐带血标本中的CD34+细胞,流式细胞术鉴定其分选纯度,瑞氏染色观察其细胞形态,采用免疫荧光技术检测CD34+细胞中β-catenin和BCR/ABL的表达及定位。使用IN联合伊马替尼(IM)处理CD34+细胞,免疫荧光技术检测β-catenin蛋白变化,瑞氏染色和流式细胞术观察细胞凋亡及细胞周期,定量PCR检测靶基因c-myc和cyclin D1的m RNA水平,流式细胞术和免疫荧光技术检测BCR/ABL蛋白变化。结果成功分选出CD34+细胞,纯度达90%以上;β-catenin和BCR/ABL均在慢粒急变期CD34+细胞中高表达,主要定位于胞质。IN与IM联用能够显著抑制慢粒急变期CD34+细胞中β-catenin的表达,使慢粒急变期CD34+细胞的细胞周期被阻滞在G0/G1期,明显增加细胞的凋亡,明显降低c-myc和cyclin D1的m RNA水平,并使BCR/ABL的蛋白水平显著下降,但对正常CD34+细胞没有影响。结论 IN通过影响细胞周期和细胞凋亡,增强IM对慢粒急变期CD34+细胞的杀伤力,其机制可能是与降低β-catenin的表达,抑制c-myc和cyclin D1的转录及BCR/ABL的蛋白水平有关。
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400-819-9993Mesalazine inhibits the &-catenin signalling pathway acting through the upregulation of &-protocadherin gene in colo-rectal cancer cells - PARENTI - 2009 - Alimentary Pharmacology & Therapeutics - Wiley Online Library
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Background& Several reports indicate that mesalazine (5-aminosalicylic acid, 5-ASA) is a promising candidate for the chemoprevention of colo-rectal cancer because of its ability to reach the purpose avoiding the unwanted side effects usually associated with prolonged administration of nonsteroidal anti-inflammatory drugs. This activity of 5-ASA is probably the consequence of a number of effects determined on colo-rectal cancer cells, consisting of reduced proliferation, increased apoptosis and activation of cell cycle checkpoints and DNA repair processes. A recent observation has suggested that inhibition of &-catenin signalling could induce these cellular effects.Aim& To characterize better the capacity of 5-ASA to inhibit the &-catenin signalling pathway.Methods& Genes belonging to the &-catenin signalling pathway were analysed in colo-rectal cancer cell lines treated with 5-ASA using a combination of laboratory assays that are able to detect their phenotypic expression and functional activity.Results& The results obtained indicated that 5-ASA induces the expression of a protein called &-protocadherin that belongs to the cadherin superfamily and is able to sequester &-catenin on the plasmatic membrane of treated cells hampering its function.Conclusion& These findings suggest that &-protocadherin might be employed as a biological marker to monitor the chemopreventive efficacy of 5-ASA.IntroductionNonsteroidal anti-inflammatory drugs (NSAIDs) are characterized by a well-recognized chemopreventive activity against colo-rectal cancer (CRC). This activity has been observed in general population as well as in patients exhibiting an increased risk to develop the considered disease. Unfortunately, the systemic and gastrointestinal toxicity of NSAIDs drastically limits their administration in the context of clinical protocols requiring a long-term treatment of patients. Both therapeutic and toxic effects elicited by these compounds are largely dependent on the inhibition of cyclooxygenase (COX)-1 and COX-2 enzymes that, in turn, is responsible for a reduced synthesis of prostaglandins normally sustaining a number of physiological functions. Several reports indicate that mesalazine (5-aminosalicylic acid or 5-ASA) can be a promising alternative to achieve a comparable anti-CRC chemopreventive activity. The main advantage of this drug is represented by the observation that, with the only exception of a modest and occasional nefrotoxicity, it is substantially devoid of the severe side effects usually provoked by NSAIDs. In fact, despite the chemical similarity with a typical NDAID such as aspirin, 5-ASA is characterized by a weak COX inhibitory activity and a poor systemic availability, i.e. pharmacological properties that convincingly account for its clinical safety. Not surprisingly, distinct mechanisms appear to mediate the anti-inflammatory effect of 5-ASA and, among them, an important role is probably played by the inhibition of transcription factors regulating the immune response such as peroxisome proliferator-activated receptors and nuclear factor (NF)kB. It has to be pointed out that the chemopreventive efficacy of 5-ASA has been, to date, exclusively demonstrated in patients affected by inflammatory bowel diseases, while it remains to be confirmed in other individual categories such as healthy people or patients carrying genetic tumour syndromes. Although this issue can be addressed only through specifically designed clinical trials, an extensive characterization of the anti-tumour effects that 5-ASA exerts at the cellular and molecular level would greatly contribute to such studies. In more detail, information achieved by this investigation could help to: (i) corroborate the biological rationale supporting 5-ASA chemoprevention of CRC; (ii)
(iii) identify biological markers that are able to monitor the entity of pharmacological response. In this regard, a growing body of evidence indicates that stimulation with 5-ASA determines a number of biological effects on CRC cells such as inhibition of proliferation, induction of apoptosis and enhancement of cell cycle checkpoints and DNA repair processes. Interestingly, 5-ASA has been recently demonstrated to interfere with the &-catenin signalling pathway by inhibiting the nuclear translocation of &-catenin, necessary to permit its transcription activity. This finding could, in principle, explain virtually all the effects that 5-ASA induces on CRC cells, as &-catenin has been implicated in the molecular control of G1/S and G2/M cell cycle transitions and indirectly also of apoptosis. An even more relevant aspect of the issue is represented by the consolidated demonstration that &-catenin signalling is constitutively activated in CRC therefore implying a specific action of 5-ASA in the chemoprevention of this neoplastic condition. In our report, we present the results of a set of experiments performed on the CaCo2 CRC cell line to characterize better the molecular mechanisms by which 5-ASA inhibits the &-catenin signalling pathway. The data obtained demonstrated that, at least partly, this effect is mediated by the upregulation of a protein called &-protocadherin that belongs to the cadherin superfamily and is able to sequester &-catenin on the plasmatic membrane of 5-ASA-treated cells, thus hampering its transcription activity.Materials and methodsColon cancer cell linesCaCo2 and HT29 cell lines were obtained from ATCC (Rockville, MD, USA) and cultured in DMEM medium (Euroclone, Devon, UK), supplemented with 10% heat inactivated foetal bovine serum (Lonza, Walkersville, MD, USA) and 1&mm l-glutamine (Euroclone). 5-Aminosalicylic acid (SOFAR S.p.A., Milano, Italy) was dissolved in cell culture medium at concentrations that ranged from 10 to 20&mm.Flow cytometry analysisDistribution of cells in the different phases of cell cycle was analysed by bi-parametric flow cytometry analysis performed as described. Briefly, cells were preincubated with 10&&m BrdU (Sigma Aldrich, St Louise, MO, USA) and stained with a purified mouse primary monoclonal antibody (MoAb) directed against BrdU (BD Biosciences, Erembodegem, Belgium) followed by a rabbit anti-mouse immunoglobulin (Ig)G secondary antibody conjugated with fluorescein isothiocyanate (FITC) (Dako A/S, Glostrup, Denmark). Samples were then resuspended in a 50&&g/mL PI water solution. Apoptotic cells were stained with the Annexin V&FITC Apoptosis Detection Kit I (BD Biosciences) following the manufacturer&s guidelines. Analysis of cells labelled for the assessment of cell cycle and apoptosis was then accomplished using a Coulter Epics XL flow cytometer (Coulter Electronics Inc., Hialeah, FL, USA).Protein extract preparation and western blot analysisPreparation of total, cytoplasmic and nuclear extracts and Western blot analysis of protein expression were carried out as previously described. Electrophoresis was performed on 7.5&10% sodium dodecyl sulphate (SDS)&polyacrylamide gel electrophoresis (PAGE) followed by electroblotting to nitrocellulose sheets. Blotted membranes were preblocked with a solution containing 5% nonfat milk (Regilait, Saint-Martin-Belle-Roche, France) and incubated with the primary antibody that was specific for each analysed protein, and with a common secondary antibody conjugated to horse-radish peroxidase. The following primary antibodies were used for Western blot analysis: mouse anti-human &-catenin MoAb (BD Biosciences), mouse anti-human &-protocadherin polyclonal antibody (Abnova Corporation, Walnut, CA, USA), mouse anti-human p21 MoAb (Cell Signaling Technology, Denver, MA, USA) and mouse anti-human c-myc MoAb (Sigma Aldrich). As secondary antibody, we used a goat anti-mouse Ig (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Expression of actin was also analysed with a mouse anti-human pan actin MoAb (Sigma Aldrich) to normalize analysed protein samples. Detection of Western blot signals was carried out using the BM Chemiluminescence Blotting Substrate (Roche Diagnostics, Mannheim, Germany). Densitometric analysis of Western blot results was carried out using the Quantity One software (Bio-Rad Laboratories Ltd., Hemel Hempstead, UK).Coimmunoprecipitation analysisTo perform this assay, cells were lysed in PBSTDS buffer consisting of phosphate-buffered saline (PBS) pH 7.4, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 1&&&complete (Roche Diagnostics), containing a mix of protease inhibitors, 1&mm sodium orthovanadate and 1&mm sodium molybdate, added as phosphatase inhibitors. A total of 250&500&&g of cell lysate was then incubated overnight at 4&&C with 1 volume of HNTG Buffer (20&mm hepes pH 7.5, 150&mm sodium chloride, 0.1% Triton X-100, 10% glycerol, 1&mm sodium orthovanadate and 1&mm sodium molybdate), 30&&L of protein G agarose (KPL, Gaithersburg, MD, USA) and 2&&g of mouse anti-human &-catenin MoAb (BD Biosciences). Immunoprecipitate was recovered by centrifugation, washed extensively with HNTG buffer and loaded on 7.5% SDS&PAGE. Western blot analysis was performed with a mouse anti-& protocadherin polyclonal antibody (Abnova Corporation) as described above. Samples undergoing glycosylation analysis were resuspended in RIPA buffer, boiled for 5&min and then incubated o/n at 37&&C with 2&U of Peptide N-Glycosidase F (PNGase F; Sigma) or, as a control, processed with the same modalities, but in the absence of the enzyme.RNA extraction and Microarray analysisTotal ribonucleic acid (RNA) was extracted from the various analysed cell populations using the Qiagen total RNA purification kit (Qiagen, Valencia, CA, USA). RNA integrity and concentration were verified using the Bio-Analyzer technique (Applied Biosystem, Foster City, CA). Three micrograms of total RNA were then converted into labelled complementary RNA (c)RNA using the one-cycle target labelling assay and hybridized to Affymetrix HGU133plus2 GeneChip arrays as already described. Images obtained by scanning chips were processed using the Affymetrix GeneChip Operating Software (GCOS, Affymetrix, Santa Clara, CA, USA). This software allows the operator to: (i) determine the presence of a messenger (m)RNA in the analysed sample by assigning an &Absolute Call&&Absent& or &Present&; (ii) quantify mRNA levels in terms of a &Signal& (iii) attribute a &Change&&Increased& (I), &Decreased& (D) or &Not Changed& (NC), indicating mRNA variations between two compared cell populations. Changes in mRNA levels, between the compared expression profiles, were quantified in terms of &Fold Change&.Quantitative real-time PCROne hundred nanograms of total RNA was reverse transcribed using High Capacity cDNA Archive Kit (Applied Biosystems). Quantitative real-time PCR (QRT&PCR) was then performed using an ABI PRISM 7900 sequence detection system (Applied Biosystems). Primers and probes for mRNA amplification of E-cadherin, &-protocadherin, &-catenin, Axin1, inhibitor of &-catenin (ICAT) and TCF-4, p21waf-1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were designed by Applied Biosystems. Each cDNA sample was run in triplicate using the Taqman Universal PCR Master Mix (Applied Biosystems). Quantification of QRT&PCR signals was performed using the &D&DCt relative quantification method. This procedure calculates the relative changes in gene expression of the target gene normalized to the endogenous control and compared with a calibrator sample. The values obtained were represented in terms of relative quantity of mRNA level variation.Immunofluorescence and colocalization assayAnalysed cells were seeded in 35&mm Petri plates (Euroclone), fixed in 4% paraformaldehyde for 10&min and permeabilized in freshly prepared 0.5% Triton X-100/PBS for 10&min. After an initial blocking treatment performed with 1% bovine serum albumin (BSA)/PBS for 15&min, they were incubated with purified primary antibodies, initially, and with the proper secondary antibodies conjugated to different fluorochromes, subsequently. Both incubations were carried out for 1&h in 1% BSA/PBS. Immunofluorescence analysis of &-catenin expression was performed using a mouse anti-human &-catenin MoAb (BD Biosciences) and a FITC conjugated rabbit anti-mouse IgG (Dako). Colocalization assay of &-protocadherin and &-catenin proteins was conducted with a goat anti-human &-protocadherin polyclonal antibody (R&D System, Minneapolis, MN, USA) and a mouse anti-human &-catenin MoAb (BD Biosciences), both used as primary antibodies. Secondary antibodies were then added in the following order: Alexa Fluor 488 conjugated donkey anti-goat IgG (1& Invitrogen, Carlsbad, CA, USA), goat serum (15& Euroclone), Alexa Fluor& 568 conjugated goat anti-mouse IgG (1& Invitrogen). Nuclear counter-staining was achieved with DAPI. Labelled samples were analysed using a Carl Zeiss Axioskop 40 fluorescent microscope (Carl Zeiss, Milano, Italy) and Leica SP2-AOBS confocal microscope (Leica Microsystems GmbH, Wetzlar, Germany). Densitometric analysis of confocal microscopy images was performed using the LCS Lite software (Leica Microsystem GmbH).Statistical analysisAll experiments were repeated at least three times and results presented in terms of mean&&&S.E.M. values. The number of replicates (n) is indicated in the figure legends or in the . Multiple comparisons were performed by one way analysis of variance (anova). Pairwise comparisons were carried out using the Student&s t-test procedure. Results of statistical analysis were considered significant when exhibiting P-values &0.05 corrected, where indicated, according to the Bonferroni&s method.ResultsProliferation effects determined by 5-ASA on CaCo2 cellsTo characterize the biological effects exerted by 5-ASA on CRC cells, we used the colon adenocarcinoma CaCo2 cell line. A set of preliminary dose&response experiments were performed to verify the capacity of 5-ASA to inhibit the proliferation activity of these cells. For this purpose, they were exposed for 96&h to 10 and 20&mm concentrations of the tested compound, corresponding to the levels achieved inside the colon lumen of patients undergoing administration of 1 and 2&g/day of 5-ASA respectively. Proliferation activity was then estimated performing a daily cell count and a flow cytometry analysis of PI/BrdU positivity. The former revealed a remarkable inhibition of cell growth exhibiting the best efficiency (&4-fold) at 96&h treatment with 20&mm 5-ASA () (see
for statistical analysis). Under these treatment conditions, the latter disclosed an evident increase of cells in the G2/M phase of cell cycle (mean&&&S.E.M. values: 27.3&&&8.2 vs. 8.2&&&0.9 of untreated cells, P&= 0.0084), indicating a block of proliferation occurring at this level (). Flow cytometry analysis of Annexin V positivity evidenced that the inhibitory effect, elicited by 5-ASA on proliferation activity of CaCo2 cells, was accompanied by a significant induction of the apoptotic process (data not shown).Figure&1. &Analysis of effects exerted by 5-ASA treatment on proliferation activity of CaCo2 cells. (A) Line histogram presenting the results of daily cell counts performed, in four independent experiments, on untreated and 5-ASA-treated CaCo2 cells. Treatment times and cell numbers are reported on x- and y-axes respectively. Results are represented as mean&&&S.E.M. values (n&=&4). (B) Bi-parametric flow cytometry analysis of cell cycle performed on untreated (CONT.) and 5-ASA-treated CaCo2 cells. Results are presented as dot plot reporting PI values on x-axis and BrdU positivity on y-axis. Yellow, green and red dots indicate cells in the G0/G1, S and G2/M phases of cell cycle respectively. Percentage of cells in each cell cycle phase is reported inside the corresponding gate. Asterisks indicate statistically significant results (P&&&0.05). The data presented are representative of three experiments performed.Table&1.&
&Statistical analysis of cell growth data in untreated and 5-ASA-treated CaCo2 cells
anova analysis0.018140.012510.000010.00005Student&s t-test 10&mm 5-ASA vs. UT0.06340.16720.00140.0159Student&s t-test 20&mm 5-ASA vs. UT0.02460.01270.00030.0033These experiments confirmed the expected antiproliferative/pro-apoptotic effects of 5-ASA and therefore validated the CaCo2 cell-based model for further analysis on the investigated issue.Effect of 5-ASA on endocellular &-catenin levels and distribution in CaCo2 cellsDue to the peculiar regulatory mechanisms controlling the &-catenin signalling pathway, levels of &-catenin protein contained in the various subcellular compartments represent reliable indicators of its activation. On this ground, the expression of &-catenin protein was assessed by Western blot in different cell fractions of CaCo2 cells exposed to 20&mm 5-ASA for 96&h. Densitometric analysis of this set of experiments showed a &2.5-fold decrease in &-catenin in nuclear extract of 5-ASA-treated cells as compared to control untreated cells (P&=&0.0042; ). The amount of analysed protein in total and cytoplasmic extracts, containing both plasmatic membrane and cytosolic content under the adopted experimental conditions, remained, on the contrary, basically unaffected (P&=&0.0005 and 0.2982 ). These data were consistent with a re-distribution of endocellular &-catenin, rather than a downregulation of its expression. To dissect this point better, we also analysed subcellular &-catenin localization using an immunofluorescence assay approach. The results obtained, presented in , provided a clear demonstration that at the end of stimulation, &-catenin signal was localized in the nucleus and, to a lesser extent, in the cytoplasm of untreated cells, whereas it was almost exclusively localized in the plasmatic membrane of 5-ASA-treated cells. Densitometric analysis of confocal microscopy images confirmed this finding indicating that the nuclear/membrane ratio of fluorescence signal, expressed as mean&&&S.E.M. values, was 1.90&&&0.17 for untreated cells and 0.40&&&0.05 for 5-ASA-treated cells (P&=&0.0005).Figure&2. &Analysis of &-catenin protein expression in subcellular compartments of CaCo2 cells treated with 5-ASA. (A, upper): Western blot analysis of &-catenin protein in total, cytoplasmic and nuclear extracts of CaCo2 cells untreated and treated with 20&mm 5-ASA for 96&h. Analysed cell samples are indicated on the top. Investigated proteins and their molecular weights are indicated on the left and on the right respectively. Normalization of the protein amount loaded in each lane was achieved using a pan-actin antibody that is able to detect either cytoplasmic or nuclear actin. Data presented are representative of five experiments performed. kDa, kiloDalton. (A, lower): Densitometric analysis of Western blot results (see upper panel). This analysis was performed on five independent experiments normalizing &-catenin protein expression on the levels of the actin housekeeping protein. The data obtained were presented as bar histogram reporting normalized &-catenin fold change on y-axis and the analysed conditions on x-axis. The entity of fold change was represented using arbitrary units and expressed as mean&&&S.E.M. values (n&=&5). (B) Immunofluorescence analysis of &-catenin protein on CaCo2 cells exposed to a 20&mm 5-ASA treatment for 96&h. Cells treated with 5-ASA were analysed upon staining with an anti-&-catenin primary antibody and a FITC conjugated secondary antibody (green fluorescence). Untreated control cells (CONT.) were also analysed. Nuclei were counterstained with DAPI (blue fluorescence). Merge images of the two analysed fluorescence signals are also shown. The data presented are representative of three experiments performed. At least 10 high power fields for each specimen were examined.These results suggested that treatment of CaCo2 cells with 5-ASA interferes with nuclear translocation of &-catenin by sequestering this protein on the plasmatic membrane of treated cells.Expression profiling of the cadherin gene superfamily in 5-ASA-treated CaCo2 cellsIt is recognized that localization of &-catenin on plasmatic membrane is mediated by the association with cadherin superfamily proteins, the most important being represented by E-cadherin. Several reports have also demonstrated that these proteins are able to sequester &-catenin on plasmatic membrane, inhibiting its nuclear translocation and transcription activity. The upregulation of a cadherin gene could consequently provide a satisfactory explanation to our data. To address this issue, we evaluated by means of the Affymetrix microarray methodology, the transcriptome changes determined on these cells by a 96-h treatment with 20&mm 5-ASA. Bioinformatic analysis of the obtained expression profiles allowed the detection of 10 genes coding for cadherin and cadherin-like proteins (). With the only exception of &-protocadherin, undergoing a threefold upregulation, all other genes (including E-cadherin) exhibited only a moderate variation of mRNA expression upon treatment with 5-ASA ().Figure&3. &Expression profiling of 5-ASA-treated CaCo2 cells performed to estimate the mRNA expression variations of genes belonging to the cadherin superfamily. The mRNA expression of cadherin superfamily genes was assessed by DNA microarray in untreated and 5-ASA-treated CaCo2 cells under the experimental conditions indicated in . The results obtained are represented as bar histogram indicating the fold change values (x-axis) of detected cadherin genes (y-axis). The data presented are representative of three experiments performed. Cadherin genes have been all indicated using gene symbols: CDH1, cadherin 1 or E- CDH10, cadherin 10; CDH17, cadherin 17; CELSR1, cadherin EGF LAG seven-pass G-type receptor 1; CELSR2, cadherin EGF LAG seven-pass G-type receptor 2; MUCDHL, mucin and cadherin like or &- PCDH1, protocadherin 1; PCDH18, protocadherin 18; PCDHA1, protocadherin alpha 1; PCDHGA1 protocadherin gamma subfamily A 1.This finding clearly indicated &-protocadherin as the candidate interactor of &-catenin on the plasmatic membrane of 5-ASA-treated CaCo2 cells.QRT&PCR analysis of genes regulating the &-catenin signalling pathway in 5-ASA-treated CaCo2 cellsTo corroborate this hypothesis, we analysed, by QRT&PCR, the mRNA expression of E-cadherin, &-protocadherin, and other genes involved in the regulation of the &-catenin signalling pathway, in CaCo2 cells untreated and treated with 20&mm 5-ASA for 48 and 96&h. Among the latter, we included genes coding for: &-catenin, i.e. the main comp Axin1, because of its capacity to promote the activity of &-catenin ICAT, responsible for the inhibition of &-catenin-dep p21waf-1, i.e. a negatively regulated target of &-catenin, mediating growth arrest at cell cycle checkpoints. The results obtained revealed that as expected, expression of E-cadherin and &-catenin genes were not modified by treatment with 5-ASA. On the contrary, &-protocadherin and p21waf-1 gene expression underwent a 12-fold and a 16-fold upregulation respectively (P&=&0.03619 and 0.0257 upon a 96-h treatment), whereas Axin1 and ICAT genes exhibited only a two- to threefold induction of their expression (P&=&0.0266 and 0.0028 upon a 48-h treatment) ().Figure&4. &QRT&PCR analysis of genes belonging to the &-catenin signalling pathway in CaCo2 cells treated with 5-ASA. CaCo2 cells stimulated with 20&mm 5-ASA for 48 and 96&h were analysed by QRT&PCR in three independent experiments to estimate the mRNA expression of genes belonging to the &-catenin signalling pathway, all indicated on y-axis. Transcript levels were measured in terms of relative quantity (RQ) and reported on x-axis as mean&&&S.E.M. values (n&=&3). Asterisks indicate statistically significant results (P&&&0.05).These results confirmed microarray data and demonstrated that the increase in &-protocadherin expression, observed in 5-ASA-treated CaCo2 cells, is accompanied by the expected inhibition of &-catenin pathway.Western blot analysis of &-protocadherin expression in 5-ASA-treated CaCo2 cellsTo confirm QRT&PCR data at the protein level, we performed a time course Western blot analysis on CaCo2 cells undergoing exposure to 20&mm 5-ASA for up to 96&h. Under these experimental conditions, we assessed the expression of &-protocadherin, &-catenin, c-myc and p21waf-1 proteins. This study included the c-myc protooncogene as it was previously demonstrated to mediate the transcriptional repression that &-catenin exerts on the p21waf-1 gene. The results of these experiments evidenced that &-protocadherin and p21waf-1 proteins were gradually but remarkably induced in the cytoplasmic extract of analysed cells (). Upregulation of these proteins was coupled with a consistent and parallel decrease of &-catenin and c-myc proteins in the nuclear compartment of the same cells (). The increase in &-protocadherin protein expression, assessed by densitometric analysis of Western blot films, averaged &7- to &12-fold depending on the considered treatment times ().Figure&5. &Results of a time course Western blot analysis evaluating the expression of &-protocadherin and other &-catenin pathway related proteins in CaCo2 cells treated with 5-ASA. Depending on the considered protein, this analysis was performed on cytoplasmic (upper panel) and/or nuclear extracts (lower panel) of studied cells, at 24&h intervals following treatment with 5-ASA. Analysed cell samples are indicated on the top. Assayed proteins and their molecular weights are indicated on the left and on the right respectively. Treatment modalities and normalization of protein amounts have been already described in . The data presented are representative of three experiments performed.Table&2.&
&Densitometric analysis of &-protocadherin protein expression in untreated and 5-ASA-treated CaCo2 cells
Densitometric analysis (arbitrary units, mean&&&S.E.M.)1&&&0.47.1&&&0.110.8&&&0.79.5&&&0.612.4&&&0.2anova (P-value)Student&s t-test, 5-ASA vs. UT (P-value)0.&0.00210.00070.00040.0001These results further supported the possibility that induction of &-protocadherin expression, determined by 5-ASA treatment of CaCo2 cells, could be responsible for a cascade of molecular events ultimately leading to increased transcription of p21waf-1 gene, followed by growth arrest of analysed cells.Coimmunoprecipitation analysis of &-protocadherin/&-catenin protein interactionThe hypothesis arising from our data would directly imply that &-protocadherin, as other proteins belonging to the cadherin superfamily, is able to bind &-catenin on the plasmatic membrane of studied cells. To verify this interaction, we designed a co-immunoprecipitation assay in which immunoprecipitates obtained from lysates of CaCo2 cells using an anti-&-catenin antibody were analysed by Western blot performed with an anti-&-protocadherin antibody. This procedure allowed us to demonstrate the presence of two immunoreactive &-protocadherin bands, of 93 and 110&kDa, in 5-ASA-treated CaCo2 cells (). Treatment of the same immunoprecipitate with the PNGase F glycosidase determined a partial conversion of the 110&kDa to the 93&kDa protein band indicating, as already suggested by other authors, that the former was a glycosylated version of the latter (). Interestingly, the 110&kDa form was exclusively observed in anti-&-catenin immunoprecipitate, while it was undetectable in the input control ().Figure&6. &Analysis of &-protocadherin/&-catenin protein interaction. This analysis was performed by coimmunoprecipitation and colocalization assay performed in CaCo2 cells under the treatment modalities described in . The data presented are representative of three experiments performed. (A) Coimmunoprecipitation analysis demonstrating the existence of a &-protocadherin/&-catenin protein complex in 5-ASA-treated CaCo2 cells. Western blot analysis of &-protocadherin was performed on non-immunoprecipitated cell extracts used as control (input), on immunoprecipitates obtained with a control antibody (normal mouse IgG) and on anti-&-catenin immunoprecipitates. The latter samples also underwent glycosylation analysis carried out by treatment with the PNGase F glycosidase (Glyase). Control of digestion was achieved under the same experimental conditions, but in absence of the enzyme (Mock). IP, WB, W A kDa, kiloDalton. (B) Colocalization analysis of &-protocadherin and &-catenin on plasmatic membrane of 5-ASA-treated CaCo2 cells. Cells were labelled with combinations of antibodies staining &-protocadherin with alexa fluor 488 (green fluorescence, a) and &-catenin with alexa fluor 568 (red fluorescence, b). Nuclei were counterstained with Dapi (blue fluorescence, c). Results of immunofluorescence labelling were analysed using a confocal microscope. Colocalization of the studied proteins was assessed evaluating the presence of a fluorescence merge (yellow fluorescence, d) resulting by the overlap of green and red fluorescence signals. At least 10 high power fields for each specimen were examined.This observation suggested that the 110&kDa form of &-protocadherin might be a post-translational modified version of the wild-type protein, characterized by a preferential binding activity to &-catenin.Confocal microscopy analysis of &-catenin/&-protocadherin colocalization in 5-ASA-treated CaCo2 cellsTo confirm &-catenin/&-protocadherin interaction and to determine its localization at the subcellular level, we performed a confocal microscopy analysis of 5-ASA-treated CaCo2 cells in which the investigated proteins were stained using antibodies conjugated to distinct fluorochromes and colocalization was assessed by merging their immunofluorescence images (green and red respectively). As shown in , this assay demonstrated that both signals were almost exclusively localized on the plasmatic membrane of the analysed cells (panels a and b) on which the overlay image (panel d) disclosed the presence of the expected yellow/orange fluorescence, indicating a colocalization of &-catenin and &-protocadherin proteins at this cellular site.These results provided, in our opinion, a definitive demonstration that upregulated &-protocadherin expression, occurring in response to treatment of CaCo2 cells with 5-ASA, is responsible for sequestration of &-catenin on plasmatic membrane.Analysis of biological effects promoted by 5-ASA treatment on HT29 cellsTo verify the results described so far in a distinct cell population, we stimulated with 5-ASA the HT29 colon adenocarcinoma cell line under the same experimental conditions used for CaCo2 cells. As shown in , a 96-h treatment of HT29 cells with 20&mm 5-ASA resulted in about a twofold decrease of proliferation activity, estimated with a daily cell count ( for statistical analysis), and a three- and fivefold upregulations of &-protocadherin and p21waf-1 mRNA expression respectively (P&=&0.0469 and 0.0200), assessed by QRT&PCR.Figure&7. &Biological effects exerted on HT29 cells by 5-ASA treatment. (A) The effect of 5-ASA treatment on HT29 cells was assessed performing a dose-response experiment carried out as described in
(n&=&6). (B) The mRNA expression variations of gene belonging to the &-catenin signalling pathway were analysed in HT29 cells using the QRT&PCR reaction under the experimental conditions described in
(n&=&3).Table&3.&
&Statistical analysis of cell growth data in untreated and 5-ASA-treated HT29 cells anova analysis (P-values)0.585850.100810.037680.00679Student&s t-test 10&mm 5-ASA vs. UT (P-values)0.73800.29840.19170.0970Student&s t-test 20&mm 5-ASA vs. UT (P-values)0.31020.04900.01160.0111These experiments indicated that the effects exerted by 5-ASA on HT29 cells were substantially comparable to those observed in CaCo2 cells. The lower response of HT29 cells to 5-ASA treatment could be related to the more transformed phenotype of these cells.DiscussionA better comprehension of the molecular mechanisms underlying the anti-CRC chemopreventive activity of 5-ASA could provide crucial information to improve and/or to monitor the clinical efficacy of the drug. To clarify this issue, we studied the biological effects determined by 5-ASA on the CaCo2 CRC cell line. Preliminary experiments performed in our laboratory using this cell model evidenced that 5-ASA treatment leads to a reduced proliferation activity coupled with a block in the G2/M phase of cell cycle. These results confirmed some previous observations, contradicting, at the same time, other reports indicating an intra-S phase proliferation block. This discrepancy is, in our opinion, only apparent as the studies under discussion globally suggest a capacity of 5-ASA to potentiate cell cycle checkpoints and DNA repair activities. The different action levels by which 5-ASA affects these processes could simply reflect the biological heterogeneity of the investigated CRC cell lines. Subsequent experimental work developed in our laboratory clearly demonstrated that treatment of CaCo2 cells with 5-ASA is accompanied by sequestration of &-catenin on plasmatic membrane leading to a reduced nuclear translocation of this transcription factor, in turn associated with a decreased expression of the c-myc protooncogene and a remarkable upregulation of the p21waf-1 growth arrest gene. This cascade of events, disclosing an interference with the &-catenin signalling pathway, is perfectly consistent with the accumulation of 5-ASA-treated cells in the G2/M phase of cell cycle. In fact, several reports indicate that, depending on the functional state of the cell, &-catenin pathway is able to control either the G1/S or G2/M checkpoint of cell cycle and both effects are virtually mediated by p21waf-1 protein. Sequestration of &-catenin on plasmatic membrane was not mediated by E-cadherin that, at least in epithelial cells, is commonly considered as the preferential interactor of &-catenin in the mentioned cellular site. Conversely, further investigation allowed us to identify &-protocadherin as the protein that was primarily responsible for this effect. This protein had been previously isolated by two distinct research groups and, based on its structural properties, assigned to the cadherin protein superfamily. Experimental work developed by these authors allowed them to demonstrate that &-protocadherin is prevalently expressed in a number of epithelial tissues, including intestinal mucosa, where it promotes cell adhesion processes such as those responsible for homophilic cell-cell interactions. Taken together, these observations suggested an involvement of &-protocadherin in the biological functions that are normally regulated by other cadherins such as proliferation, apoptosis and development. Our results indicated that &-protocadherin is remarkably induced by stimulation with 5-ASA, colocalized with &-catenin on plasmatic membrane and coimmunoprecipitated together with this protein from lysates of treated cells. These data provide, in our opinion, a convincing indication that upregulation of &-protocadherin could underlie the capacity of 5-ASA to inhibit &-catenin signalling in CaCo2 cells. Several reports ascribe the same activity to both classical (E-cadherin) and variant (fat1 proto-cadherin) cadherin proteins. The most intriguing among such studies evidenced that treatment of the SW480 CRC cell line with vitamin D3 upregulates the expression of E-cadherin leading to sequestration of &-catenin on plasmatic membrane. This finding was accompanied by biological effects that are very similar to those observed in 5-ASA-treated CaCo2 cells. A side consideration arising from this report is that vitamin D3 is endowed with a recognized anti-CRC chemopreventive activity, implying that these compounds could determine their effects through similar molecular mechanisms but acting on distinct targets. Taken together, these data allow one to hypothesize that 5-ASA might synergize with vitamin D3, resulting in a potentiation of the final chemopreventive effect. It is worth to underline that induction of &-protocadherin expression in response to 5-ASA treatment is not restricted to a single CRC cell context as, although initially detected in CaCo2 cells, it was subsequently confirmed in the HT29 cell line. The relevance of our results also resides in the possibility that, similar to other cadherins, &-protocadherin might be the product of a tumour suppressor gene destined to a downregulated expression during the process of CRC tumour transformation and playing a specific role in the metastatic phase of the disease. Future studies will help verify this hypothesis. A general conclusion arising from the data presented here is that, if confirmed through appropriate clinical studies, &-protocadherin and other downstream components of the &-catenin signalling pathway might be employed as biological markers to monitor the chemopreventive efficacy of 5-ASA.AcknowledgementsDeclaration of personal interests: Dr Brenno Canovi is an employee of Sofar S.p.A. Declaration of funding interests: This study was funded in full by a Grant from Sofar S.p.A.
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