Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

Senescent mesenchymal stem cells promote colorectal cancer cells growth via galectin-3 expression

Senescent mesenchymal stem cells promote colorectal cancer cells growth via galectin-3 expression Background: Cellular senescence is linked to aging and tumorigenesis. The senescence of mesenchymal stem cells (MSCs) may influence the tumor growth, metastasis, and angiogenesis by secreting a variety of cytokines and growth factors. Results: The conditioned media of adipose derived MSCs (AD-MSCs) stimulated the proliferation of human LoVo colorectal-cancer cells, and the replicative senescent MSCs had the more obvious effects in comparison to that of premature AD-MSCs. Analysis of the factors secreted in the MSCs culture media determined that senescent MSCs expressed and secreted high levels of galectin-3. Galectin-3 expression correlated with the stimulatory effect of senescent AD-MSCs on LoVo cells proliferation, as knockdown of galectin-3 in senescent AD-MSCs significantly reversed the effect of MSCs–mediated growth stimulation of LoVo cells. Furthermore, the simultaneous addition of recombinant galectin-3 to the co-culture systems partially restored the tumor-promoting effect of the senescent AD-MSCs. Analysis of the mechanisms of senescent MSCs and galectin-3 on LoVo cells signal transduction determined that senescent MSCs and exogenous galectin-3 promoted cell growth by activating the mitogen-activated protein kinase (MAPK) (extracellular signal-regulated kinase [ERK]1/2) pathway. Conclusions: Senescent MSCs may alter the tissue microenvironment and affect nearby malignant cells via cytokine secretion, and galectin-3 is an important mediator of senescent AD-MSC–mediated stimulation of colon cancer cell growth. Therefore, thorough assessment of AD-MSCs prior to their implementation in clinical practice is warranted. Keywords: Cellular senescence, Mesenchymal stem cells, Galectin-3, LoVo cells Background cells during the aging process in vivo may contribute to Cellular senescence, a state of irreversible growth arrest, the age-related increase in cancer incidence [4]. can be triggered by many mechanisms, including telo- Mesenchymal stem cells (MSC), which possess broad mere shortening, epigenetic derepression of the cyclin- and potent multilineage differentiation potential and dependent kinase inhibitor 2A/ADP ribosylation factor potent immunoregulatory properties, are the optimal (INK4a/ARF) locus, and DNA damage [1]. Cellular sen- source for stem cell transplant in clinical applications [5, 6]. escence is linked to aging and tumorigenesis, and has Similar to other adult somatic cells, long-term culture been proposed as a suppressive mechanism against the in vitro leads to MSC senescence, which results in development of cancer [2, 3]. However, recent studies growth arrest and reduced differentiation [7, 8]. The have revealed that cellular senescence also has tumor- replicative senescence of MSCs is evinced by telomere promoting effects, and the accumulation of senescent shortening, enlargement and flattening of the charac- teristic stem cell morphology, reduced proliferation * Correspondence: [email protected] rate, and altered secretory profile [8, 9]. The function Department of Hematology, Affiliated Hospital of Guiyang Medical College, of adult tissue-specific stem cells declines with age, and No. 28, Guiyi Street, Yunyan District, Guiyang, Guizhou Province 550004, aged MSCs could be more deleterious as they can greatly China Full list of author information is available at the end of the article © 2015 Li et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Li et al. Cell & Bioscience (2015) 5:21 Page 2 of 9 impair tissue homeostasis and repair [9, 10]. MSCs from colon cancer cell groups achieved similar proliferation aged patients with coronary artery disease have impaired levels. angiogenic potential and reduced proangiogenic factor se- cretion [11]. Senescent MSCs may contribute to the Galectin-3 contributed to P30-MSC stimulation of colon physiological decline in tissue homeostasis and to the in- cancer cell growth creased risk of neoplasm during aging [9]. Accumulated evidence indicates that galectin-3 is closely Recently report showed that MSCs stimulated inva- involved in tumor cell proliferation, transformation, migra- sion, survival and tumorigenesis of colorectal cancer tion, invasion, and metastasis [13]. We analyzed galectin-3 cells through the release of soluble NRG1, activating the expression in CM-P3 or CM-P30 with quantitative PCR HER2/HER3-dependent PI3K/AKT signalling cascade in (Q-PCR) and ELISA, and found that both mRNA and pro- colorectal cancer cells [12]. In this study, we showed that tein levels of galectin-3 were significantly upregulated in replicative senescent AD-MSCs significantly promoted the AD-MSCs during senescence (Fig. 3a-b), suggesting the proliferation of LoVo colorectal-cancer cells in com- that galectin-3 may have been involved in the P30-MSC– parison to premature AD-MSCs, and the expression of mediated growth stimulation of LoVo cells. galectin-3, a powerful modulator of cell migration and To examine whether galectin-3 secretion in P30-MSCs spread in carcinoma cells, correlated with the stimula- stimulates LoVo cell proliferation, we blocked galectin-3 tory effect of senescent AD-MSCs on colorectal cancer expression in P30-MSCs with a galectin-3–specific cells proliferation. Therefore, thorough assessment of siRNA. Q-PCR and ELISA data showed that LGALS3 AD-MSCs prior to their implementation in clinical prac- mRNA expression was decreased and that galectin-3 se- tice is warranted. cretion in CM-P30 was significantly reduced following siRNA treatment (Fig. 3c, d). Results LoVo cells were incubated with 50 or 100 ng/ml recom- Characterization of P30-MSCs binant galectin-3 for 24 hours, and the proliferation of In the first few generations, the AD-MSCs exhibited LoVo cells were evaluated by CCK-8. As Fig. 4a showed fibroblast-like morphology. After 30 passages, the MSCs that the rgalectin-3 enhanced the growth of LoVo cells. appeared longer and larger, with accumulation of granu- LoVo cells were then incubated with CM-P30 pre-treated lar cytoplasmic inclusions. The percentage of positive with the galectin-3 siRNA or NC, and the knockdown of SA-β-Gal staining was increased significantly in the rep- galectin-3 in senescent AD-MSCs significantly reversed licative P30-MSCs (Fig. 1a). The AD-MSCs were also the effect of MSCs–mediated growth stimulation of LoVo capable of osteogenic and adipogenic differentiation cells (Fig. 4b). Furthermore, the simultaneous addition of when cultured in the appropriate inducing media. As- 100 ng/ml recombinant galectin-3 to the co-culture sys- sessment of the degree of osteogenic and adipogenic dif- tems partially restored the tumor-promoting effect of the ferentiation via Alizarin Red S and Oil Red O staining, senescent AD-MSCs. respectively, revealed a sharp decline in the adipogenic and osteogenic potential of P30-MSCs compared to P3- MSCs (Fig. 1b). CCK-8 analysis showed that the prolifer- P30-MSCs promoted ERK1/2 activation in colon cancer ation potential of AD-MSCs declined significantly with cells cell replicative passaging (Fig. 1c). Western blot analysis As reported previously [14], exogenous galectin-3 induces of p53 and p21 expression in the AD-MSCs showed that the extracellular signal-regulated kinases (ERK1/2) phos- p53 and p21 expression increased gradually with passage phorylation in cancer cells, and the activation of ERK1/2 (Fig. 1d). Flow cytometric analysis demonstrated that are associated with cancer cell proliferation and survival both P3-MSCs and P30-MSCs were positive for CD73, [15, 16]. Our western blot data were showed in Fig. 5a, the CD90, and CD105, and negative for CD34, CD45, and CM of MSCs promoted ERK1/2 phosphorylation in the HLA-DR (data not shown). LoVo cells and that CM-P30 had a greater stimulative ef- fect on ERK1/2 activation. Moreover, the phosphorylation CM-P30 promoted colon cancer cell proliferation of ERK1/2 induced by CM-P30 of MSCs were aborted by To determine the effect of P30-MSCs on colon cancer U0126, the specific inhibitor of MEK1/2, suggesting that cell growth, LoVo cells were cultured with concentrated the signal was transferred through a specific Raf-MEK1/2- CM-P3 or CM-P30 for 48 h. As shown in Fig. 2a-c, the ERK1/2 pathway to activate ERK1/2. We then knocked conditioned medium of MSCs can promoted LoVo cell down galectin-3 expression in the P30-MSCs and com- proliferation, and the LoVo cells cultured with CM-P30 pared the promoter effect of the CM-P30 on ERK1/2 spread and grew faster than cells cultured with culture phosphorylation to that of the CM of MSCs treated with medium alone or with CM-P3. However if the cells were MSC . Galectin-3 knockdown diminished the CM-P30– NC incubated for 48 h, both of CM-P3 and CM-P30 treated induced ERK1/2 phosphorylation; however, the addition of Li et al. Cell & Bioscience (2015) 5:21 Page 3 of 9 Fig. 1 Characterization of senescent AD-MSCs. (a) SA-β-Gal staining of senescent MSCs (P30 AD-MSCs) or pre-senescent MSCs (P3 AD-MSCs). (b) Oil Red O and Alizarin red staining of differentiated AD-MSCs. (c) The proliferation potential of AD-MSCs by CCK-8 analysis. (d) Western blot analysis of AD-MSC P21 and P53 expression. GAPDH, glyceraldehyde-3-phosphate dehydrogenase internal control exogenous galectin-3 to the CM restored ERK1/2 activa- associated morphological features, decreased proliferation, tion in the LoVo cells (Fig. 5b). SA-β-Gal positivity, induced p53 and p21 expression, and increased galectin-3 expression. We then showed that Discussion CM-P30 promoted colon cancer cell proliferation. In the Recent studies have shown that a pool of molecules se- co-culture experiments, we demonstrated that galectin-3 creted by senescent cells, referred to as having the mediated the promoter effects of AD-MSCs on colon can- senescence-associated secretory phenotype (SASP), is cer cell proliferation to some extent, as specific knock- associated with arrest of cell proliferation and may down of galectin-3 with siRNA significantly reversed the contribute to it via the autocrine/paracrine pathways MSC-mediated stimulation of colon cancer cell growth. [10, 17]. Our data revealed that the MSCs had the typical The tumor microenvironment is increasingly regarded senescence-associated characteristics and SASP after re- as an important regulator of malignant progression of can- peated passage, marked by the appearance of senescence- cer cells [18]. MSCs may secrete a variety of cytokines and Li et al. Cell & Bioscience (2015) 5:21 Page 4 of 9 Fig. 2 The CM of AD-MSCs promotes LoVo cell proliferation in vitro. (a) Morphology of LoVo cells incubated with the CM of AD-MSCs for 24 h. (b) The proliferation assays of LoVo cell with CM of AD-MSCs for 24 h. (c) The proliferation assays of LoVo cell with CM of AD-MSCs for 12 h, 24 h and 48 h. All experiments were repeated at least three times, normal DMEM without cell medium (DMEM) was used as the control. ***P < 0.001 versus control; P < 0.05, P3-MSC versus P30-MSC growth factors that influence tumor growth, metastasis, immunosuppressive function of UC-MSCs [25]. In and angiogenesis [19, 20]. Karnoub et al. [19] showed that addition, galectin-3 expression in most cancer cells is in- MSCs play a pivotal role in colon cancer progression and creased, and is associated with growth and metastases in metastasis; when recruited into breast cancer stroma, pancreatic and breast cancer systems [26]. A recent report bone marrow MSCs tended to facilitate breast cancer cell [27] showed that galectin-3 is involved in the nicotine- metastasis and regulate cancer stem cell behavior via the induced promotion of apoptosis resistance of breast cancer secretion of the chemokine CCL5. Recent studies have cells and that it promotes cancer cell growth and protects shown that the CM of MSCs enhances tumor growth, in- cells from apoptosis induced by chemotherapeutic drugs. dicating that the factors secreted by MSCs have profound Baptiste et al. [28] showed that galectin-3 was a powerful effects on “reprogramming” tumor growth [18, 21], and modulator of cell adhesion and spread in breast carcinoma the senescent umbilical cord MSCs promoted the prolifer- cells and that the exogenous addition of recombinant ation and migration of breast cancer cells [22]. galectin-3 promoted the growth of galectin-3–null cells. Of the MSC SASP factors, we found that galectin-3 was However, our data showed that recombinant galectin-3 has an important mediator of cancer-promoting activity. A a weaker effect on cancer cell growth than senescent unique chimera-type member of the β-galactoside–binding MSCs, suggesting that other cytokines secreted from sen- soluble lectin family with a molecular mass of 29–35 kD, escent MSCs may be associated with the stimulation of galectin-3 is implicated in a variety of biological functions colon cancer cell growth. [23, 24]. Previously, Liu et al. showed that galectin-3 is se- Lastly, we analyzed the mechanisms of senescent MSCs creted into the CM of UC-MSCs and is involved in the and galectin-3 on colon cancer cell signal transduction, Li et al. Cell & Bioscience (2015) 5:21 Page 5 of 9 Fig. 3 Galectin-3 expression in P3-MSC or P30-MSC. (a) LGALS3 mRNA expression in P3-MSCs or P30-MSCs. (b) Galectin-3 protein levels in the CM of AD-MSCs. (c) Decreased expression of LGALS3 mRNA in P30-MSCs following treatment with galectin-3 siRNA. (d) Decreased expression of galectin-3 protein in CM-P30 following treatment with galectin-3 siRNA. MSC , MSCs treated with galectin-3 siRNA; MSC , MSCs treated gal-3▼ NC ### with negative control of siRNA. All experiments were repeated at least three times. **P < 0.01, P3-MSC versus P30-MSC; P < 0.001, MSC NC versus MSC gal-3▼ finding that senescent MSCs and exogenous galectin-3 Materials and methods promoted colon cancer cell growth by activating the Isolation and preparation of senescent AD-MSCs MAPK (ERK1/2) pathway. The extracellular signal- The AD-MSCs were isolated and obtained by density gradi- regulated kinases (ERK1/2) which are serine/threonine ent centrifugation, approved by the Ethics Committee of protein kinases, as one of the most important regulator of the Affiliated Hospital of Guiyang Medical College. Briefly, key cellular processes [15], involved in carcinogenesis AD-MSCs were isolated and cultured according to a colla- due to their ability to stimulate cell proliferation and genase digestion protocol. Mononuclear cells were plated survival [16]. The MAPK pathways are activated by di- in α-minimum essential medium (Invitrogen, Carlsbad, verse extracellular and intracellular stimuli including CA, USA) containing 10 % fetal bovine serum (FBS) peptide growth factors, cytokines, hormones, and vari- (HyClone, Utah, USA), and cultured in a humidified incu- ous cellular stressors [29]. Gao et al. [14] showed that bator containing 5 % CO2 at 37 °C. exogenous galectin-3 induces ERK1/2 phosphorylation AD-MSCs were further cultivated to confluence and were in cancer cells, and our results are consistent with this amplified, and replicative senescent cells were passaged reports. serially over 30 passages when the typical senescence- In summary, our study suggests that senescent MSCs associated morphological features appeared. Passage 3 (P3) may alter the tissue microenvironment and affect nearby and P30 cells were referred to as “young” (P3-MSCs) and malignant cells via cytokine secretion, and that galectin-3 “senescent” MSCs (P30-MSCs), respectively. is an important mediator of senescent AD-MSC–mediated stimulation of colon cancer cell growth. Therefore, thor- Characterization of senescent AD-MSCs ough assessment of AD-MSCs prior to their implementa- Senescence-associated β-galactosidase (SA-β-Gal) stain- tion in clinical practice is warranted. ing was performed using an SA-β-Gal staining kit (Cell Li et al. Cell & Bioscience (2015) 5:21 Page 6 of 9 Fig. 4 Galectin-3 is an important mediator of P30-MSC–mediated stimulation of LoVo cell growth. (a) Proliferation of LoVo cells incubated with 50 or 100 ng/ml recombinant galectin-3. (b) Proliferation of LoVo cells incubated with CM of P30-MSCs, which treated with galectin-3 siRNA or NC. Control, normal DMEM without cell medium; siGal-3, galectin-3 siRNA; rgalectin-3, recombinant galectin-3. All experiments were repeated at ## least three times. *P < 0.05,**P < 0.01 versus control; P < 0.01, versus siGAL-3 Signaling Technology, Beverly, MA, USA) according to Biosciences, San Jose, CA, USA); cells were stained with the manufacturer’s instructions. For the proliferation phycoerythrin-conjugated antibodies against CD105, CD73, analysis, MSCs were counted every 24 hours for 5 days CD90, CD45 and HLA-DR (Becton Dickinson Biosciences). using a cell counting kit-8 (CCK-8) (DoJinDo, ShangHai, For osteogenic and adipogenic differentiation, MSCs were China). All experiments were performed three times. incubated with MesenCult Osteogenic or Adipogenic AD-MSCs surface marker analysis by flow cytometry was Stimulatory Medium (STEMCELL Technologies, Vancouver, performed on a FACSCalibur unit (Becton Dickinson Canada) for 2–3 weeks. Osteogenic and adipogenic Fig. 5 Western blot analysis of P30-MSC and exogenous galectin-3 promotion of ERK1/2 activation in LoVo cells. (a) the CM of MSCs promoted ERK1/2 phosphorylation in the LoVo cells, which were aborted by U0126 for 60 min treatment. (b) LoVo cells incubated with CM of P30-MSCs, which treated with or without galectin-3 siRNA.siGal-3, galectin-3 siRNA; rgalectin-3, recombinant galectin-3; GAPDH was used as the internal ## control. All experiments were repeated at least three times. P < 0.01 versus control; **P < 0.01 versus siGAL-3 Li et al. Cell & Bioscience (2015) 5:21 Page 7 of 9 differentiation was evaluated using Alizarin Red S and Oil CA, USA) using Fast SYBR Green PCR Master Mix (Ap- Red O (Sigma-Aldrich, St. Louis, MO, USA) staining, plied Biosystems). We used the following primer pairs as respectively. reported previously [31]: β-actin (forward: 5′-GAAGGTG p21 and p53 protein levels were analyzed by western AAGGTCGGAGTCA-3′,reverse: 5′-GAAGATGGTGA blotting. Proteins were extracted from 1 × 10 P3-MSCs or TGGGATTTC-3′)and LGALS3 (forward: 5′-CCAAAGA P30-MSCs with 1 ml radioimmunoprecipitation assay GGGAATGATGTTGCC-3′, reverse: 5′-TGATTGTACT (RIPA) buffer (Sigma-Aldrich), separated by 10 % sodium GCAACAAGTGAGC-3′). dodecyl sulfate–polyacrylamide gel electrophoresis (SDS- Galectin-3 levels in CM-P3 or CM-P30 were measured PAGE), and transferred to Immobilon polyvinyl difluoride as described previously [25]. Briefly, the concentrated CM (PVDF) membranes. The membranes were incubated with was analyzed using a Human Galectin-3 Platinum ELISA antibody against p21 and p53 (Abcam, Cambridge, MA, (enzyme-linked immunosorbent assay) Kit (eBioscience, USA), followed by goat anti-rabbit horseradish peroxid- San Diego, CA, USA) according to the manufacturer’s ase (HRP)-conjugated antibody (Santa Cruz Biotech- instructions. nology,Santa Cruz, CA, USA),and visualized with SuperSignal West Pico Chemiluminescent Substrate Galectin-3 knockdown (Millipore, Billerica, MA, USA). To knockdowngalectin-3expression,P30-MSCswere transfected with predesigned small interfering RNA (siRNA) Preparation of conditioned medium (Sigma-Aldrich) using Lipofectamine RNAiMAX reagent To harvest the MSC conditioned medium (CM), 80 % (Invitrogen). The respective siRNA sequences of MSCs confluent P3-MSC and P30-MSC cultures were exten- are as follows: galectin-3 siRNA target site (MSC ): gal-3▼ sively washed with phosphate-buffered saline (PBS) and 5′-CAC UUU AAC CCA CGC UUC AdTdT-3′ and 5′- incubated in serum-free culture medium for 24 h. Then, UGA AGC GUG GGU UAA AGU GdTdT-3′; negative the CM from the P3-MSCs (CM-P3) and P30-MSCs control sequences (MSC ): 5′-UUC UCC GAA CGU NC (CM-P30) was collected and concentrated to 1/2× volume GUC ACG UTT-3′ and 5′-ACG UGA CAC GUU CGG with an ultra-filtration membrane with a molecular weight AGA ATT-3′. cut-off of 3 kD by centrifuging at 5000 rpm for 30 min at P30-MSCs were seeded in 6-well plates, and then trans- 4 °C. The concentrated CM was then filtered through a fected with a final concentration of 40pmol/μlMSC gal-3▼ 0.22-μm membrane and stored at −80 °C until used. or MSC and incubated for 24 h. Following the incuba- NC tion and supernatant collection, total RNA or proteins Cell proliferation assay of colon cancer cells were prepared and quantified as described above. The human LoVo colon cancer cell line was obtained Galectin-3 was quantified using the Human Galectin-3 from the American Type Culture Collection (ATCC). Platinum ELISA Kit. LoVo cells were established from a metastatic nodule Moreover, the addition of recombinant galectin-3 resected from a 56-year-old Caucasian male colorectal (R&D Systems, Minneapolis, MN, USA) with a final con- adenocarcinoma patient [30]. centration of 50-100nmol/ml to the CM of the galectin- The LoVo cells were cultured in phenol red-free 3 knockdown MSCs, and the proliferation of LoVo cells Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, were analyzed. Carlsbad, CA, USA) supplemented with 10 % FBS; 2.5 × 10 cells/well were plated in 200μlgrowthmedium in 96- Western blotting for mitogen-activated protein kinase well plates. After 24 h, the medium was replaced with con- (MAPK) centrated conditioned medium of CM-P3 and CM-P30 as LoVo cells were incubated with CM of MSCs treated for 12h, 24 h or 48 h. Tumor cell proliferation was evalu- with siRNA in the presence or absence of U0126, the ated using Cell Counting Kit-8 according to the manufac- specific inhibitor of MEK1/MEK2. Proteins were ex- turer’sinstructions. tracted from LoVo cells using RIPA buffer, separated by 10 % SDS-PAGE, and transferred to Immobilon PVDF Analysis of galectin-3 expression in MSCs membranes (Millipore). The membranes were blocked We analyzed galectin-3 (LGALS3) mRNA expression with with PBS containing 5 % non-fat dry milk and 0.1 % real-time PCR: total RNA was isolated from 1 × 10 P3- Tween 20 overnight. After washing, the membranes MSCs or P30-MSCs using TRIzol (Invitrogen) according were incubated with antibodies against phosphorylated to the manufacturer’s instructions. First-strand comple- extracellular signal–regulated kinase 1/2 (pERK1/2) and mentary DNA was reverse-transcribed using a Reverse total ERK1/2 (tERK1/2) (Cell Signaling Technology, Inc., Transcriptase MIX Kit (Toyobo, Osaka, Japan). Quantita- Danvers, MA, USA), incubated with HRP-conjugated tive real-time PCR was performed in an ABI 7500 Fast secondary antibody and visualized with SuperSignal Real-Time PCR System (Applied Biosystems, Foster City, West Pico Chemiluminescent Substrate (Millipore). Li et al. Cell & Bioscience (2015) 5:21 Page 8 of 9 Statistical analysis cells and their differentiation to adipocytes and osteoblasts. Mol Biol Rep. 2011;38(8):5161–8. The data are presented as the mean ± standard deviation 8. Bonab MM, Alimoghaddam K, Talebian F, Ghaffari SH, Ghavamzadeh A, from ≥3 experiments analyzed using SPSS 17.0 statistical Nikbin B. Aging of mesenchymal stem cell in vitro. BMC Cell Biol. 2006;7:14. software (IBM, New York, NY, USA). Analysis of signifi- 9. Baxter MA, Wynn RF, Jowitt SN, Wraith JE, Fairbairn LJ, Bellantuono I. Study of telomere length reveals rapid aging of human marrow stromal cells cance between two groups was performed with an un- following in vitro expansion. Stem Cells. 2004;22(5):675–82. paired 2-tailed Student t-test; 1-way analysis of variance 10. Severino V, Alessio N, Farina A, Sandomenico A, Cipollaro M, Peluso G, et al. was used for multiple comparisons. Differences were Insulin-like growth factor binding proteins 4 and 7 released by senescent cells promote premature senescence in mesenchymal stem cells. Cell Death considered significant when P < 0.05. Dis. 2013;4:e911. 11. Efimenko A, Dzhoyashvili N, Kalinina N, Kochegura T, Akchurin R, Tkachuk V, Abbreviations et al. Adipose-derived mesenchymal stromal cells from aged patients with ARF: ADP ribosylation factor; CM: Conditioned media; DMEM: Dulbecco’s coronary artery disease keep mesenchymal stromal cell properties but modified Eagle’s medium; ELISA: Enzyme-linked immunosorbent assay; exhibit characteristics of aging and have impaired angiogenic potential. ERK: Extracellular signal-regulated kinase; FBS: Fetal bovine serum; Stem Cells Transl Med. 2014;3(1):32–41. HLA-DR: Human leukocyte antigen-DR; HRP: Horseradish peroxidase; 12. De Boeck A, Pauwels P, Hensen K, Rummens JL, Westbroek W, Hendrix A, INK4a: Cyclin-dependent kinase inhibitor 2A; MAPK: Mitogen-activated protein et al. Bone marrow-derived mesenchymal stem cells promote colorectal kinase; MSCs: Mesenchymal stem cells; P3: Passage 3; P30: Passage 30; cancer progression through paracrine neuregulin 1/HER3 signalling. Gut. PBS: Phosphate-buffered saline; PVDF: Polyvinyl difluoride; Q-PCR: Quantitative 2013;62(4):550–60. polymerase chain reaction; RIPA: Radioimmunoprecipitation assay; SA-β- 13. Song L, Tang JW, Owusu L, Sun MZ, Wu J, Zhang J. Galectin-3 in cancer. Gal: Senescence-associated β-galactosidase; SASP: Senescence-associated Clin Chim Acta. 2014;431:185–91. secretory phenotype; SDS-PAGE: Sodium dodecyl sulfate–polyacrylamide gel 14. Gao X, Balan V, Tai G, Raz A. Galectin-3 induces cell migration via a electrophoresis; siRNA: Small interfering RNA; AD-MSCs: Adipose derived calcium-sensitive MAPK/ERK1/2 pathway. Oncotarget. 2014;5(8):2077–84. mesenchymal stem cells. 15. Datta A, Loo SY, Huang B, Wong L, Tan SS, Tan TZ, et al. SPHK1 regulates proliferation and survival responses in triple-negative breast cancer. Competing interests Oncotarget. 2014;5(15):5920–33. The authors declare that they have no competing interests. 16. Santen RJ, Song RX, McPherson R, Kumar R, Adam L, Jeng MH, et al. The role of mitogen-activated protein (MAP) kinase in breast cancer. J Steroid Authors’ contributions Biochem Mol Biol. 2002;80(2):239–56. Yanju L designed the overall study, supervised the project, analyzed data, 17. Kuilman T, Peeper DS. Senescence-messaging secretome: SMS-ing cellular and wrote the manuscript. XX provided suggestions for the project, stress. Nat Rev Cancer. 2009;9(2):81–94. prepared the manuscript and edited the manuscript. LW and Yanqi L 18. Zhang C, Zhai W, Xie Y, Chen Q, Zhu W, Sun X. Mesenchymal stem cells performed siRNA assay and ELISA analysis. GL and HL performed Q-PCR and derived from breast cancer tissue promote the proliferation and migration western blot analysis, XW and YJ. All authors read and approved the final of the MCF-7 cell line. Oncol Lett. 2013;6(6):1577–82. manuscript. 19. Karnoub AE, Dash AB, Vo AP, Sullivan A, Brooks MW, Bell GW, et al. Mesenchymal stem cells within tumour stroma promote breast cancer Acknowledgements metastasis. Nature. 2007;449(7162):557–63. This work was supported by the State High-tech Research and Development 20. Walter M, Liang S, Ghosh S, Hornsby PJ, Li R. Interleukin 6 secreted from Plans (Grants: 2011AA020109). adipose stromal cells promotes migration and invasion of breast cancer cells. Oncogene. 2009;28(30):2745–55. Author details 21. Zhu W, Huang L, Li Y, Qian H, Shan X, Yan Y, et al. Mesenchymal stem Department of Hematology, Affiliated Hospital of Guiyang Medical College, cell-secreted soluble signaling molecules potentiate tumor growth. No. 28, Guiyi Street, Yunyan District, Guiyang, Guizhou Province 550004, Cell Cycle. 2011;10(18):3198–207. China. Skin Regeneration Department of General Hospital of Chinese Armed 22. Di GH, Liu Y, Lu Y, Liu J, Wu C, Duan HF. IL-6 secreted from senescent Police, No. 69 Yongding Road, Haidian District, Beijing 100039, China. mesenchymal stem cells promotes proliferation and migration of breast Department of Hematology, The Second Hospital of Hebei Medical cancer cells. PLoS One. 2014;9(11):e113572. University, Hebei Key Laboratory of Hematology, Hebei Institution of 23. Radosavljevic G, Volarevic V, Jovanovic I, Milovanovic M, Pejnovic N, Hematology, 215 West Heping Road, Shijiazhuang City, Hebei Province Arsenijevic N, et al. The roles of Galectin-3 in autoimmunity and tumor 050000, China. Yanzhou Hospital of Traditional Chinese Medicine, No. 43 progression. Immunol Res. 2012;52(1-2):100–10. Dongqiao North Road, Yanzhou District, Jining, Shandong Province 272100, 24. Wan SY, Zhang TF, Ding Y. Galectin-3 enhances proliferation and angiogenesis China. of endothelial cells differentiated from bone marrow mesenchymal stem cells. Transplant Proc. 2011;43(10):3933–8. Received: 20 January 2015 Accepted: 23 April 2015 25. Liu GY, Xu Y, Li Y, Wang LH, Liu YJ, Zhu D. Secreted galectin-3 as a possible biomarker for the immunomodulatory potential of human umbilical cord mesenchymal stromal cells. Cytotherapy. 2013;15(10):1208–17. References 26. Song S, Ji B, Ramachandran V, Wang H, Hafley M, Logsdon C, et al. 1. Collado M, Blasco MA, Serrano M. Cellular senescence in cancer and aging. Overexpressed galectin-3 in pancreatic cancer induces cell proliferation and Cell. 2007;130(2):223–33. invasion by binding Ras and activating Ras signaling. PLoS One. 2. Campisi J, d’Adda di Fagagna F. Cellular senescence: when bad things 2012;7(8):e42699. happen to good cells. Nat Rev Mol Cell Biol. 2007;8(9):729–40. 27. Guha P, Bandyopadhyaya G, Polumuri SK, Chumsri S, Gade P, Kalvakolanu 3. Feldser DM, Greider CW. Short telomeres limit tumor progression in vivo by DV, et al. Nicotine promotes apoptosis resistance of breast cancer cells inducing senescence. Cancer Cell. 2007;11(5):461–9. and enrichment of side population cells with cancer stem cell-like 4. Ohtani N, Takahashi A, Mann DJ, Hara E. Cellular senescence: a double-edged properties via a signaling cascade involving galectin-3, alpha9 nicotinic sword in the fight against cancer. Exp Dermatol. 2012;21 Suppl 1:1–4. acetylcholine receptor and STAT3. Breast Cancer Res Treat. 5. Bianco P, Robey PG, Simmons PJ. Mesenchymal stem cells: revisiting history, 2014;145(1):5–22. concepts, and assays. Cell Stem Cell. 2008;2(4):313–9. 28. Baptiste TA, James A, Saria M, Ochieng J. Mechano-transduction mediated 6. Akiyama K, Chen C, Wang D, Xu X, Qu C, Yamaza T, et al. secretion and uptake of galectin-3 in breast carcinoma cells: implications in Mesenchymal-stem-cell-induced immunoregulation involves FAS-ligand-/ the extracellular functions of the lectin. Exp Cell Res. 2007;313(4):652–64. FAS- mediated T cell apoptosis. Cell Stem Cell. 2012;10(5):544–55. 7. Cheng H, Qiu L, Ma J, Zhang H, Cheng M, Li W, et al. Replicative senescence 29. Kim EK, Choi EJ. Pathological roles of MAPK signaling pathways in human of human bone marrow and umbilical cord derived mesenchymal stem diseases. Biochim Biophys Acta. 2010;1802(4):396–405. Li et al. Cell & Bioscience (2015) 5:21 Page 9 of 9 30. Hsu HH, Kuo WW, Ju DT, Yeh YL, Tu CC, Tsai YL, et al. Estradiol agonists inhibit human LoVo colorectal-cancer cell proliferation and migration through p53. World J Gastroenterol. 2014;20(44):16665–73. 31. Gieseke F, Bohringer J, Bussolari R, Dominici M, Handgretinger R, Muller I. Human multipotent mesenchymal stromal cells use galectin-1 to inhibit immune effector cells. Blood. 2010;116(19):3770–9. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Cell & Bioscience Springer Journals

Senescent mesenchymal stem cells promote colorectal cancer cells growth via galectin-3 expression

Download PDF
9 pages

Loading next page...
 
/lp/springer-journals/senescent-mesenchymal-stem-cells-promote-colorectal-cancer-cells-tntzdsWRcE

References (31)

Publisher
Springer Journals
Copyright
Copyright © 2015 by Li et al.
Subject
Life Sciences; Cell Biology; Microbiology
eISSN
2045-3701
DOI
10.1186/s13578-015-0012-3
pmid
26273429
Publisher site
See Article on Publisher Site

Abstract

Background: Cellular senescence is linked to aging and tumorigenesis. The senescence of mesenchymal stem cells (MSCs) may influence the tumor growth, metastasis, and angiogenesis by secreting a variety of cytokines and growth factors. Results: The conditioned media of adipose derived MSCs (AD-MSCs) stimulated the proliferation of human LoVo colorectal-cancer cells, and the replicative senescent MSCs had the more obvious effects in comparison to that of premature AD-MSCs. Analysis of the factors secreted in the MSCs culture media determined that senescent MSCs expressed and secreted high levels of galectin-3. Galectin-3 expression correlated with the stimulatory effect of senescent AD-MSCs on LoVo cells proliferation, as knockdown of galectin-3 in senescent AD-MSCs significantly reversed the effect of MSCs–mediated growth stimulation of LoVo cells. Furthermore, the simultaneous addition of recombinant galectin-3 to the co-culture systems partially restored the tumor-promoting effect of the senescent AD-MSCs. Analysis of the mechanisms of senescent MSCs and galectin-3 on LoVo cells signal transduction determined that senescent MSCs and exogenous galectin-3 promoted cell growth by activating the mitogen-activated protein kinase (MAPK) (extracellular signal-regulated kinase [ERK]1/2) pathway. Conclusions: Senescent MSCs may alter the tissue microenvironment and affect nearby malignant cells via cytokine secretion, and galectin-3 is an important mediator of senescent AD-MSC–mediated stimulation of colon cancer cell growth. Therefore, thorough assessment of AD-MSCs prior to their implementation in clinical practice is warranted. Keywords: Cellular senescence, Mesenchymal stem cells, Galectin-3, LoVo cells Background cells during the aging process in vivo may contribute to Cellular senescence, a state of irreversible growth arrest, the age-related increase in cancer incidence [4]. can be triggered by many mechanisms, including telo- Mesenchymal stem cells (MSC), which possess broad mere shortening, epigenetic derepression of the cyclin- and potent multilineage differentiation potential and dependent kinase inhibitor 2A/ADP ribosylation factor potent immunoregulatory properties, are the optimal (INK4a/ARF) locus, and DNA damage [1]. Cellular sen- source for stem cell transplant in clinical applications [5, 6]. escence is linked to aging and tumorigenesis, and has Similar to other adult somatic cells, long-term culture been proposed as a suppressive mechanism against the in vitro leads to MSC senescence, which results in development of cancer [2, 3]. However, recent studies growth arrest and reduced differentiation [7, 8]. The have revealed that cellular senescence also has tumor- replicative senescence of MSCs is evinced by telomere promoting effects, and the accumulation of senescent shortening, enlargement and flattening of the charac- teristic stem cell morphology, reduced proliferation * Correspondence: [email protected] rate, and altered secretory profile [8, 9]. The function Department of Hematology, Affiliated Hospital of Guiyang Medical College, of adult tissue-specific stem cells declines with age, and No. 28, Guiyi Street, Yunyan District, Guiyang, Guizhou Province 550004, aged MSCs could be more deleterious as they can greatly China Full list of author information is available at the end of the article © 2015 Li et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Li et al. Cell & Bioscience (2015) 5:21 Page 2 of 9 impair tissue homeostasis and repair [9, 10]. MSCs from colon cancer cell groups achieved similar proliferation aged patients with coronary artery disease have impaired levels. angiogenic potential and reduced proangiogenic factor se- cretion [11]. Senescent MSCs may contribute to the Galectin-3 contributed to P30-MSC stimulation of colon physiological decline in tissue homeostasis and to the in- cancer cell growth creased risk of neoplasm during aging [9]. Accumulated evidence indicates that galectin-3 is closely Recently report showed that MSCs stimulated inva- involved in tumor cell proliferation, transformation, migra- sion, survival and tumorigenesis of colorectal cancer tion, invasion, and metastasis [13]. We analyzed galectin-3 cells through the release of soluble NRG1, activating the expression in CM-P3 or CM-P30 with quantitative PCR HER2/HER3-dependent PI3K/AKT signalling cascade in (Q-PCR) and ELISA, and found that both mRNA and pro- colorectal cancer cells [12]. In this study, we showed that tein levels of galectin-3 were significantly upregulated in replicative senescent AD-MSCs significantly promoted the AD-MSCs during senescence (Fig. 3a-b), suggesting the proliferation of LoVo colorectal-cancer cells in com- that galectin-3 may have been involved in the P30-MSC– parison to premature AD-MSCs, and the expression of mediated growth stimulation of LoVo cells. galectin-3, a powerful modulator of cell migration and To examine whether galectin-3 secretion in P30-MSCs spread in carcinoma cells, correlated with the stimula- stimulates LoVo cell proliferation, we blocked galectin-3 tory effect of senescent AD-MSCs on colorectal cancer expression in P30-MSCs with a galectin-3–specific cells proliferation. Therefore, thorough assessment of siRNA. Q-PCR and ELISA data showed that LGALS3 AD-MSCs prior to their implementation in clinical prac- mRNA expression was decreased and that galectin-3 se- tice is warranted. cretion in CM-P30 was significantly reduced following siRNA treatment (Fig. 3c, d). Results LoVo cells were incubated with 50 or 100 ng/ml recom- Characterization of P30-MSCs binant galectin-3 for 24 hours, and the proliferation of In the first few generations, the AD-MSCs exhibited LoVo cells were evaluated by CCK-8. As Fig. 4a showed fibroblast-like morphology. After 30 passages, the MSCs that the rgalectin-3 enhanced the growth of LoVo cells. appeared longer and larger, with accumulation of granu- LoVo cells were then incubated with CM-P30 pre-treated lar cytoplasmic inclusions. The percentage of positive with the galectin-3 siRNA or NC, and the knockdown of SA-β-Gal staining was increased significantly in the rep- galectin-3 in senescent AD-MSCs significantly reversed licative P30-MSCs (Fig. 1a). The AD-MSCs were also the effect of MSCs–mediated growth stimulation of LoVo capable of osteogenic and adipogenic differentiation cells (Fig. 4b). Furthermore, the simultaneous addition of when cultured in the appropriate inducing media. As- 100 ng/ml recombinant galectin-3 to the co-culture sys- sessment of the degree of osteogenic and adipogenic dif- tems partially restored the tumor-promoting effect of the ferentiation via Alizarin Red S and Oil Red O staining, senescent AD-MSCs. respectively, revealed a sharp decline in the adipogenic and osteogenic potential of P30-MSCs compared to P3- MSCs (Fig. 1b). CCK-8 analysis showed that the prolifer- P30-MSCs promoted ERK1/2 activation in colon cancer ation potential of AD-MSCs declined significantly with cells cell replicative passaging (Fig. 1c). Western blot analysis As reported previously [14], exogenous galectin-3 induces of p53 and p21 expression in the AD-MSCs showed that the extracellular signal-regulated kinases (ERK1/2) phos- p53 and p21 expression increased gradually with passage phorylation in cancer cells, and the activation of ERK1/2 (Fig. 1d). Flow cytometric analysis demonstrated that are associated with cancer cell proliferation and survival both P3-MSCs and P30-MSCs were positive for CD73, [15, 16]. Our western blot data were showed in Fig. 5a, the CD90, and CD105, and negative for CD34, CD45, and CM of MSCs promoted ERK1/2 phosphorylation in the HLA-DR (data not shown). LoVo cells and that CM-P30 had a greater stimulative ef- fect on ERK1/2 activation. Moreover, the phosphorylation CM-P30 promoted colon cancer cell proliferation of ERK1/2 induced by CM-P30 of MSCs were aborted by To determine the effect of P30-MSCs on colon cancer U0126, the specific inhibitor of MEK1/2, suggesting that cell growth, LoVo cells were cultured with concentrated the signal was transferred through a specific Raf-MEK1/2- CM-P3 or CM-P30 for 48 h. As shown in Fig. 2a-c, the ERK1/2 pathway to activate ERK1/2. We then knocked conditioned medium of MSCs can promoted LoVo cell down galectin-3 expression in the P30-MSCs and com- proliferation, and the LoVo cells cultured with CM-P30 pared the promoter effect of the CM-P30 on ERK1/2 spread and grew faster than cells cultured with culture phosphorylation to that of the CM of MSCs treated with medium alone or with CM-P3. However if the cells were MSC . Galectin-3 knockdown diminished the CM-P30– NC incubated for 48 h, both of CM-P3 and CM-P30 treated induced ERK1/2 phosphorylation; however, the addition of Li et al. Cell & Bioscience (2015) 5:21 Page 3 of 9 Fig. 1 Characterization of senescent AD-MSCs. (a) SA-β-Gal staining of senescent MSCs (P30 AD-MSCs) or pre-senescent MSCs (P3 AD-MSCs). (b) Oil Red O and Alizarin red staining of differentiated AD-MSCs. (c) The proliferation potential of AD-MSCs by CCK-8 analysis. (d) Western blot analysis of AD-MSC P21 and P53 expression. GAPDH, glyceraldehyde-3-phosphate dehydrogenase internal control exogenous galectin-3 to the CM restored ERK1/2 activa- associated morphological features, decreased proliferation, tion in the LoVo cells (Fig. 5b). SA-β-Gal positivity, induced p53 and p21 expression, and increased galectin-3 expression. We then showed that Discussion CM-P30 promoted colon cancer cell proliferation. In the Recent studies have shown that a pool of molecules se- co-culture experiments, we demonstrated that galectin-3 creted by senescent cells, referred to as having the mediated the promoter effects of AD-MSCs on colon can- senescence-associated secretory phenotype (SASP), is cer cell proliferation to some extent, as specific knock- associated with arrest of cell proliferation and may down of galectin-3 with siRNA significantly reversed the contribute to it via the autocrine/paracrine pathways MSC-mediated stimulation of colon cancer cell growth. [10, 17]. Our data revealed that the MSCs had the typical The tumor microenvironment is increasingly regarded senescence-associated characteristics and SASP after re- as an important regulator of malignant progression of can- peated passage, marked by the appearance of senescence- cer cells [18]. MSCs may secrete a variety of cytokines and Li et al. Cell & Bioscience (2015) 5:21 Page 4 of 9 Fig. 2 The CM of AD-MSCs promotes LoVo cell proliferation in vitro. (a) Morphology of LoVo cells incubated with the CM of AD-MSCs for 24 h. (b) The proliferation assays of LoVo cell with CM of AD-MSCs for 24 h. (c) The proliferation assays of LoVo cell with CM of AD-MSCs for 12 h, 24 h and 48 h. All experiments were repeated at least three times, normal DMEM without cell medium (DMEM) was used as the control. ***P < 0.001 versus control; P < 0.05, P3-MSC versus P30-MSC growth factors that influence tumor growth, metastasis, immunosuppressive function of UC-MSCs [25]. In and angiogenesis [19, 20]. Karnoub et al. [19] showed that addition, galectin-3 expression in most cancer cells is in- MSCs play a pivotal role in colon cancer progression and creased, and is associated with growth and metastases in metastasis; when recruited into breast cancer stroma, pancreatic and breast cancer systems [26]. A recent report bone marrow MSCs tended to facilitate breast cancer cell [27] showed that galectin-3 is involved in the nicotine- metastasis and regulate cancer stem cell behavior via the induced promotion of apoptosis resistance of breast cancer secretion of the chemokine CCL5. Recent studies have cells and that it promotes cancer cell growth and protects shown that the CM of MSCs enhances tumor growth, in- cells from apoptosis induced by chemotherapeutic drugs. dicating that the factors secreted by MSCs have profound Baptiste et al. [28] showed that galectin-3 was a powerful effects on “reprogramming” tumor growth [18, 21], and modulator of cell adhesion and spread in breast carcinoma the senescent umbilical cord MSCs promoted the prolifer- cells and that the exogenous addition of recombinant ation and migration of breast cancer cells [22]. galectin-3 promoted the growth of galectin-3–null cells. Of the MSC SASP factors, we found that galectin-3 was However, our data showed that recombinant galectin-3 has an important mediator of cancer-promoting activity. A a weaker effect on cancer cell growth than senescent unique chimera-type member of the β-galactoside–binding MSCs, suggesting that other cytokines secreted from sen- soluble lectin family with a molecular mass of 29–35 kD, escent MSCs may be associated with the stimulation of galectin-3 is implicated in a variety of biological functions colon cancer cell growth. [23, 24]. Previously, Liu et al. showed that galectin-3 is se- Lastly, we analyzed the mechanisms of senescent MSCs creted into the CM of UC-MSCs and is involved in the and galectin-3 on colon cancer cell signal transduction, Li et al. Cell & Bioscience (2015) 5:21 Page 5 of 9 Fig. 3 Galectin-3 expression in P3-MSC or P30-MSC. (a) LGALS3 mRNA expression in P3-MSCs or P30-MSCs. (b) Galectin-3 protein levels in the CM of AD-MSCs. (c) Decreased expression of LGALS3 mRNA in P30-MSCs following treatment with galectin-3 siRNA. (d) Decreased expression of galectin-3 protein in CM-P30 following treatment with galectin-3 siRNA. MSC , MSCs treated with galectin-3 siRNA; MSC , MSCs treated gal-3▼ NC ### with negative control of siRNA. All experiments were repeated at least three times. **P < 0.01, P3-MSC versus P30-MSC; P < 0.001, MSC NC versus MSC gal-3▼ finding that senescent MSCs and exogenous galectin-3 Materials and methods promoted colon cancer cell growth by activating the Isolation and preparation of senescent AD-MSCs MAPK (ERK1/2) pathway. The extracellular signal- The AD-MSCs were isolated and obtained by density gradi- regulated kinases (ERK1/2) which are serine/threonine ent centrifugation, approved by the Ethics Committee of protein kinases, as one of the most important regulator of the Affiliated Hospital of Guiyang Medical College. Briefly, key cellular processes [15], involved in carcinogenesis AD-MSCs were isolated and cultured according to a colla- due to their ability to stimulate cell proliferation and genase digestion protocol. Mononuclear cells were plated survival [16]. The MAPK pathways are activated by di- in α-minimum essential medium (Invitrogen, Carlsbad, verse extracellular and intracellular stimuli including CA, USA) containing 10 % fetal bovine serum (FBS) peptide growth factors, cytokines, hormones, and vari- (HyClone, Utah, USA), and cultured in a humidified incu- ous cellular stressors [29]. Gao et al. [14] showed that bator containing 5 % CO2 at 37 °C. exogenous galectin-3 induces ERK1/2 phosphorylation AD-MSCs were further cultivated to confluence and were in cancer cells, and our results are consistent with this amplified, and replicative senescent cells were passaged reports. serially over 30 passages when the typical senescence- In summary, our study suggests that senescent MSCs associated morphological features appeared. Passage 3 (P3) may alter the tissue microenvironment and affect nearby and P30 cells were referred to as “young” (P3-MSCs) and malignant cells via cytokine secretion, and that galectin-3 “senescent” MSCs (P30-MSCs), respectively. is an important mediator of senescent AD-MSC–mediated stimulation of colon cancer cell growth. Therefore, thor- Characterization of senescent AD-MSCs ough assessment of AD-MSCs prior to their implementa- Senescence-associated β-galactosidase (SA-β-Gal) stain- tion in clinical practice is warranted. ing was performed using an SA-β-Gal staining kit (Cell Li et al. Cell & Bioscience (2015) 5:21 Page 6 of 9 Fig. 4 Galectin-3 is an important mediator of P30-MSC–mediated stimulation of LoVo cell growth. (a) Proliferation of LoVo cells incubated with 50 or 100 ng/ml recombinant galectin-3. (b) Proliferation of LoVo cells incubated with CM of P30-MSCs, which treated with galectin-3 siRNA or NC. Control, normal DMEM without cell medium; siGal-3, galectin-3 siRNA; rgalectin-3, recombinant galectin-3. All experiments were repeated at ## least three times. *P < 0.05,**P < 0.01 versus control; P < 0.01, versus siGAL-3 Signaling Technology, Beverly, MA, USA) according to Biosciences, San Jose, CA, USA); cells were stained with the manufacturer’s instructions. For the proliferation phycoerythrin-conjugated antibodies against CD105, CD73, analysis, MSCs were counted every 24 hours for 5 days CD90, CD45 and HLA-DR (Becton Dickinson Biosciences). using a cell counting kit-8 (CCK-8) (DoJinDo, ShangHai, For osteogenic and adipogenic differentiation, MSCs were China). All experiments were performed three times. incubated with MesenCult Osteogenic or Adipogenic AD-MSCs surface marker analysis by flow cytometry was Stimulatory Medium (STEMCELL Technologies, Vancouver, performed on a FACSCalibur unit (Becton Dickinson Canada) for 2–3 weeks. Osteogenic and adipogenic Fig. 5 Western blot analysis of P30-MSC and exogenous galectin-3 promotion of ERK1/2 activation in LoVo cells. (a) the CM of MSCs promoted ERK1/2 phosphorylation in the LoVo cells, which were aborted by U0126 for 60 min treatment. (b) LoVo cells incubated with CM of P30-MSCs, which treated with or without galectin-3 siRNA.siGal-3, galectin-3 siRNA; rgalectin-3, recombinant galectin-3; GAPDH was used as the internal ## control. All experiments were repeated at least three times. P < 0.01 versus control; **P < 0.01 versus siGAL-3 Li et al. Cell & Bioscience (2015) 5:21 Page 7 of 9 differentiation was evaluated using Alizarin Red S and Oil CA, USA) using Fast SYBR Green PCR Master Mix (Ap- Red O (Sigma-Aldrich, St. Louis, MO, USA) staining, plied Biosystems). We used the following primer pairs as respectively. reported previously [31]: β-actin (forward: 5′-GAAGGTG p21 and p53 protein levels were analyzed by western AAGGTCGGAGTCA-3′,reverse: 5′-GAAGATGGTGA blotting. Proteins were extracted from 1 × 10 P3-MSCs or TGGGATTTC-3′)and LGALS3 (forward: 5′-CCAAAGA P30-MSCs with 1 ml radioimmunoprecipitation assay GGGAATGATGTTGCC-3′, reverse: 5′-TGATTGTACT (RIPA) buffer (Sigma-Aldrich), separated by 10 % sodium GCAACAAGTGAGC-3′). dodecyl sulfate–polyacrylamide gel electrophoresis (SDS- Galectin-3 levels in CM-P3 or CM-P30 were measured PAGE), and transferred to Immobilon polyvinyl difluoride as described previously [25]. Briefly, the concentrated CM (PVDF) membranes. The membranes were incubated with was analyzed using a Human Galectin-3 Platinum ELISA antibody against p21 and p53 (Abcam, Cambridge, MA, (enzyme-linked immunosorbent assay) Kit (eBioscience, USA), followed by goat anti-rabbit horseradish peroxid- San Diego, CA, USA) according to the manufacturer’s ase (HRP)-conjugated antibody (Santa Cruz Biotech- instructions. nology,Santa Cruz, CA, USA),and visualized with SuperSignal West Pico Chemiluminescent Substrate Galectin-3 knockdown (Millipore, Billerica, MA, USA). To knockdowngalectin-3expression,P30-MSCswere transfected with predesigned small interfering RNA (siRNA) Preparation of conditioned medium (Sigma-Aldrich) using Lipofectamine RNAiMAX reagent To harvest the MSC conditioned medium (CM), 80 % (Invitrogen). The respective siRNA sequences of MSCs confluent P3-MSC and P30-MSC cultures were exten- are as follows: galectin-3 siRNA target site (MSC ): gal-3▼ sively washed with phosphate-buffered saline (PBS) and 5′-CAC UUU AAC CCA CGC UUC AdTdT-3′ and 5′- incubated in serum-free culture medium for 24 h. Then, UGA AGC GUG GGU UAA AGU GdTdT-3′; negative the CM from the P3-MSCs (CM-P3) and P30-MSCs control sequences (MSC ): 5′-UUC UCC GAA CGU NC (CM-P30) was collected and concentrated to 1/2× volume GUC ACG UTT-3′ and 5′-ACG UGA CAC GUU CGG with an ultra-filtration membrane with a molecular weight AGA ATT-3′. cut-off of 3 kD by centrifuging at 5000 rpm for 30 min at P30-MSCs were seeded in 6-well plates, and then trans- 4 °C. The concentrated CM was then filtered through a fected with a final concentration of 40pmol/μlMSC gal-3▼ 0.22-μm membrane and stored at −80 °C until used. or MSC and incubated for 24 h. Following the incuba- NC tion and supernatant collection, total RNA or proteins Cell proliferation assay of colon cancer cells were prepared and quantified as described above. The human LoVo colon cancer cell line was obtained Galectin-3 was quantified using the Human Galectin-3 from the American Type Culture Collection (ATCC). Platinum ELISA Kit. LoVo cells were established from a metastatic nodule Moreover, the addition of recombinant galectin-3 resected from a 56-year-old Caucasian male colorectal (R&D Systems, Minneapolis, MN, USA) with a final con- adenocarcinoma patient [30]. centration of 50-100nmol/ml to the CM of the galectin- The LoVo cells were cultured in phenol red-free 3 knockdown MSCs, and the proliferation of LoVo cells Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, were analyzed. Carlsbad, CA, USA) supplemented with 10 % FBS; 2.5 × 10 cells/well were plated in 200μlgrowthmedium in 96- Western blotting for mitogen-activated protein kinase well plates. After 24 h, the medium was replaced with con- (MAPK) centrated conditioned medium of CM-P3 and CM-P30 as LoVo cells were incubated with CM of MSCs treated for 12h, 24 h or 48 h. Tumor cell proliferation was evalu- with siRNA in the presence or absence of U0126, the ated using Cell Counting Kit-8 according to the manufac- specific inhibitor of MEK1/MEK2. Proteins were ex- turer’sinstructions. tracted from LoVo cells using RIPA buffer, separated by 10 % SDS-PAGE, and transferred to Immobilon PVDF Analysis of galectin-3 expression in MSCs membranes (Millipore). The membranes were blocked We analyzed galectin-3 (LGALS3) mRNA expression with with PBS containing 5 % non-fat dry milk and 0.1 % real-time PCR: total RNA was isolated from 1 × 10 P3- Tween 20 overnight. After washing, the membranes MSCs or P30-MSCs using TRIzol (Invitrogen) according were incubated with antibodies against phosphorylated to the manufacturer’s instructions. First-strand comple- extracellular signal–regulated kinase 1/2 (pERK1/2) and mentary DNA was reverse-transcribed using a Reverse total ERK1/2 (tERK1/2) (Cell Signaling Technology, Inc., Transcriptase MIX Kit (Toyobo, Osaka, Japan). Quantita- Danvers, MA, USA), incubated with HRP-conjugated tive real-time PCR was performed in an ABI 7500 Fast secondary antibody and visualized with SuperSignal Real-Time PCR System (Applied Biosystems, Foster City, West Pico Chemiluminescent Substrate (Millipore). Li et al. Cell & Bioscience (2015) 5:21 Page 8 of 9 Statistical analysis cells and their differentiation to adipocytes and osteoblasts. Mol Biol Rep. 2011;38(8):5161–8. The data are presented as the mean ± standard deviation 8. Bonab MM, Alimoghaddam K, Talebian F, Ghaffari SH, Ghavamzadeh A, from ≥3 experiments analyzed using SPSS 17.0 statistical Nikbin B. Aging of mesenchymal stem cell in vitro. BMC Cell Biol. 2006;7:14. software (IBM, New York, NY, USA). Analysis of signifi- 9. Baxter MA, Wynn RF, Jowitt SN, Wraith JE, Fairbairn LJ, Bellantuono I. Study of telomere length reveals rapid aging of human marrow stromal cells cance between two groups was performed with an un- following in vitro expansion. Stem Cells. 2004;22(5):675–82. paired 2-tailed Student t-test; 1-way analysis of variance 10. Severino V, Alessio N, Farina A, Sandomenico A, Cipollaro M, Peluso G, et al. was used for multiple comparisons. Differences were Insulin-like growth factor binding proteins 4 and 7 released by senescent cells promote premature senescence in mesenchymal stem cells. Cell Death considered significant when P < 0.05. Dis. 2013;4:e911. 11. Efimenko A, Dzhoyashvili N, Kalinina N, Kochegura T, Akchurin R, Tkachuk V, Abbreviations et al. Adipose-derived mesenchymal stromal cells from aged patients with ARF: ADP ribosylation factor; CM: Conditioned media; DMEM: Dulbecco’s coronary artery disease keep mesenchymal stromal cell properties but modified Eagle’s medium; ELISA: Enzyme-linked immunosorbent assay; exhibit characteristics of aging and have impaired angiogenic potential. ERK: Extracellular signal-regulated kinase; FBS: Fetal bovine serum; Stem Cells Transl Med. 2014;3(1):32–41. HLA-DR: Human leukocyte antigen-DR; HRP: Horseradish peroxidase; 12. De Boeck A, Pauwels P, Hensen K, Rummens JL, Westbroek W, Hendrix A, INK4a: Cyclin-dependent kinase inhibitor 2A; MAPK: Mitogen-activated protein et al. Bone marrow-derived mesenchymal stem cells promote colorectal kinase; MSCs: Mesenchymal stem cells; P3: Passage 3; P30: Passage 30; cancer progression through paracrine neuregulin 1/HER3 signalling. Gut. PBS: Phosphate-buffered saline; PVDF: Polyvinyl difluoride; Q-PCR: Quantitative 2013;62(4):550–60. polymerase chain reaction; RIPA: Radioimmunoprecipitation assay; SA-β- 13. Song L, Tang JW, Owusu L, Sun MZ, Wu J, Zhang J. Galectin-3 in cancer. Gal: Senescence-associated β-galactosidase; SASP: Senescence-associated Clin Chim Acta. 2014;431:185–91. secretory phenotype; SDS-PAGE: Sodium dodecyl sulfate–polyacrylamide gel 14. Gao X, Balan V, Tai G, Raz A. Galectin-3 induces cell migration via a electrophoresis; siRNA: Small interfering RNA; AD-MSCs: Adipose derived calcium-sensitive MAPK/ERK1/2 pathway. Oncotarget. 2014;5(8):2077–84. mesenchymal stem cells. 15. Datta A, Loo SY, Huang B, Wong L, Tan SS, Tan TZ, et al. SPHK1 regulates proliferation and survival responses in triple-negative breast cancer. Competing interests Oncotarget. 2014;5(15):5920–33. The authors declare that they have no competing interests. 16. Santen RJ, Song RX, McPherson R, Kumar R, Adam L, Jeng MH, et al. The role of mitogen-activated protein (MAP) kinase in breast cancer. J Steroid Authors’ contributions Biochem Mol Biol. 2002;80(2):239–56. Yanju L designed the overall study, supervised the project, analyzed data, 17. Kuilman T, Peeper DS. Senescence-messaging secretome: SMS-ing cellular and wrote the manuscript. XX provided suggestions for the project, stress. Nat Rev Cancer. 2009;9(2):81–94. prepared the manuscript and edited the manuscript. LW and Yanqi L 18. Zhang C, Zhai W, Xie Y, Chen Q, Zhu W, Sun X. Mesenchymal stem cells performed siRNA assay and ELISA analysis. GL and HL performed Q-PCR and derived from breast cancer tissue promote the proliferation and migration western blot analysis, XW and YJ. All authors read and approved the final of the MCF-7 cell line. Oncol Lett. 2013;6(6):1577–82. manuscript. 19. Karnoub AE, Dash AB, Vo AP, Sullivan A, Brooks MW, Bell GW, et al. Mesenchymal stem cells within tumour stroma promote breast cancer Acknowledgements metastasis. Nature. 2007;449(7162):557–63. This work was supported by the State High-tech Research and Development 20. Walter M, Liang S, Ghosh S, Hornsby PJ, Li R. Interleukin 6 secreted from Plans (Grants: 2011AA020109). adipose stromal cells promotes migration and invasion of breast cancer cells. Oncogene. 2009;28(30):2745–55. Author details 21. Zhu W, Huang L, Li Y, Qian H, Shan X, Yan Y, et al. Mesenchymal stem Department of Hematology, Affiliated Hospital of Guiyang Medical College, cell-secreted soluble signaling molecules potentiate tumor growth. No. 28, Guiyi Street, Yunyan District, Guiyang, Guizhou Province 550004, Cell Cycle. 2011;10(18):3198–207. China. Skin Regeneration Department of General Hospital of Chinese Armed 22. Di GH, Liu Y, Lu Y, Liu J, Wu C, Duan HF. IL-6 secreted from senescent Police, No. 69 Yongding Road, Haidian District, Beijing 100039, China. mesenchymal stem cells promotes proliferation and migration of breast Department of Hematology, The Second Hospital of Hebei Medical cancer cells. PLoS One. 2014;9(11):e113572. University, Hebei Key Laboratory of Hematology, Hebei Institution of 23. Radosavljevic G, Volarevic V, Jovanovic I, Milovanovic M, Pejnovic N, Hematology, 215 West Heping Road, Shijiazhuang City, Hebei Province Arsenijevic N, et al. The roles of Galectin-3 in autoimmunity and tumor 050000, China. Yanzhou Hospital of Traditional Chinese Medicine, No. 43 progression. Immunol Res. 2012;52(1-2):100–10. Dongqiao North Road, Yanzhou District, Jining, Shandong Province 272100, 24. Wan SY, Zhang TF, Ding Y. Galectin-3 enhances proliferation and angiogenesis China. of endothelial cells differentiated from bone marrow mesenchymal stem cells. Transplant Proc. 2011;43(10):3933–8. Received: 20 January 2015 Accepted: 23 April 2015 25. Liu GY, Xu Y, Li Y, Wang LH, Liu YJ, Zhu D. Secreted galectin-3 as a possible biomarker for the immunomodulatory potential of human umbilical cord mesenchymal stromal cells. Cytotherapy. 2013;15(10):1208–17. References 26. Song S, Ji B, Ramachandran V, Wang H, Hafley M, Logsdon C, et al. 1. Collado M, Blasco MA, Serrano M. Cellular senescence in cancer and aging. Overexpressed galectin-3 in pancreatic cancer induces cell proliferation and Cell. 2007;130(2):223–33. invasion by binding Ras and activating Ras signaling. PLoS One. 2. Campisi J, d’Adda di Fagagna F. Cellular senescence: when bad things 2012;7(8):e42699. happen to good cells. Nat Rev Mol Cell Biol. 2007;8(9):729–40. 27. Guha P, Bandyopadhyaya G, Polumuri SK, Chumsri S, Gade P, Kalvakolanu 3. Feldser DM, Greider CW. Short telomeres limit tumor progression in vivo by DV, et al. Nicotine promotes apoptosis resistance of breast cancer cells inducing senescence. Cancer Cell. 2007;11(5):461–9. and enrichment of side population cells with cancer stem cell-like 4. Ohtani N, Takahashi A, Mann DJ, Hara E. Cellular senescence: a double-edged properties via a signaling cascade involving galectin-3, alpha9 nicotinic sword in the fight against cancer. Exp Dermatol. 2012;21 Suppl 1:1–4. acetylcholine receptor and STAT3. Breast Cancer Res Treat. 5. Bianco P, Robey PG, Simmons PJ. Mesenchymal stem cells: revisiting history, 2014;145(1):5–22. concepts, and assays. Cell Stem Cell. 2008;2(4):313–9. 28. Baptiste TA, James A, Saria M, Ochieng J. Mechano-transduction mediated 6. Akiyama K, Chen C, Wang D, Xu X, Qu C, Yamaza T, et al. secretion and uptake of galectin-3 in breast carcinoma cells: implications in Mesenchymal-stem-cell-induced immunoregulation involves FAS-ligand-/ the extracellular functions of the lectin. Exp Cell Res. 2007;313(4):652–64. FAS- mediated T cell apoptosis. Cell Stem Cell. 2012;10(5):544–55. 7. Cheng H, Qiu L, Ma J, Zhang H, Cheng M, Li W, et al. Replicative senescence 29. Kim EK, Choi EJ. Pathological roles of MAPK signaling pathways in human of human bone marrow and umbilical cord derived mesenchymal stem diseases. Biochim Biophys Acta. 2010;1802(4):396–405. Li et al. Cell & Bioscience (2015) 5:21 Page 9 of 9 30. Hsu HH, Kuo WW, Ju DT, Yeh YL, Tu CC, Tsai YL, et al. Estradiol agonists inhibit human LoVo colorectal-cancer cell proliferation and migration through p53. World J Gastroenterol. 2014;20(44):16665–73. 31. Gieseke F, Bohringer J, Bussolari R, Dominici M, Handgretinger R, Muller I. Human multipotent mesenchymal stromal cells use galectin-1 to inhibit immune effector cells. Blood. 2010;116(19):3770–9. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit

Journal

Cell & Bioscience – Springer Journals

Published: May 6, 2015

There are no references for this article.