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Inhibiting CD164 Expression in Colon Cancer Cell Line HCT116 Leads to Reduced Cancer Cell Proliferation, Mobility, and Metastasis in Vitro and in Vivo

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Inhibiting CD164 Expression in Colon Cancer Cell Line HCT116 Leads to Reduced Cancer Cell Proliferation, Mobility, and Metastasis in Vitro and in Vivo Cancer Investigation, 30:380–389, 2012 ISSN: 0735-7907 print / 1532-4192 online Copyright C 2011 Informa Healthcare USA, Inc. DOI: 10.3109/07357907.2012.666692 CELLULAR AND MOLECULAR BIOLOGY Inhibiting CD164 Expression in Colon Cancer Cell Line HCT116 Leads to Reduced Cancer Cell Proliferation, Mobility, and Metastasis in vitro and in vivo Jingqun Tang,1 Liyang Zhang,2 Xiaoling She,2 Guangqian Zhou,3 Fenglei Yu,1 Juanjuan Xiang,2 and Gang Li4,5 1Department of Cardiothoracic Surgery, Xiangya Second Hospital, Central South University, Changsha, Hunan, P.R. China, 2Cancer Research Institute, Key Laboratory of Carcinogenesis and Cancer Invasion of Ministry of Education, Key Laboratory of Carcinogenesis of Ministry of Health, Central South University, Changsha, Hunan, P.R. China, 3School of Medicine, Shenzhen University, Shenzhen, P.R. China, 4Department of Orthopaedics and Traumatology, Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong, 5Stem Cell and Regeneration Program, School of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong served in human and other species (1–4). CD164 was first Background: CD164 (Endolyn) is a sialomucin, which has been identified in CD34+ human hematopoietic progenitor cells found to play roles in regulating proliferation, adhesion, and and bone marrow stromal reticular cells (2, 5, 6). CD164 differentiation of hematopoietic stem cells. Possible association has been implicated in adhesion, proliferation, and differen- of CD164 with solid cancer development remains unknown. tiation of hematopoietic stem and progenitor cells (5, 7), it Methods and Results: We first studied CD164 expression in was suggested to mediate the adhesion of CD34+ hematopoi- biopsies from colorectal cancer, breast, and ovary cancer etic progenitor cells to bone marrow stromal cells and SDF- patients by semi-quantitative immunohistochemistry, and 1 induced binding to bone marrow endothelial cell (2, 8.9). found that CD164 was strongly expressed in all the colorectal CD164 are thought to regulate hematopoiesis by facilitating cancer samples compared to the matching normal colon the adhesion of human CD34+ cells to bone marrow stroma For personal use only. tissues. The possible roles of CD164 in colon cancer (10). Knocking down CD164 expression in Drosophila S2 development were further investigated using a cells increased the cell apoptosis rate (1). Little is known well-established human colon cancer cell line HCT116. We about the relationship between CD164 and solid tumor de- found that knockdown of CD164 expression in HCT116 cells velopment till Havens et al. reported that CD164 may par- significantly inhibited cell proliferation, mobility, and ticipate in mediating prostate cancer bone metastasis in 2006 metastasis in vitro and in vivo. The knockdown of CD164 (11).ContinuousexposuretoanenvironmentofhighZn2+ expression was associated with decreased chemokine receptor canleadtotheupregulationofCD164inprostatecancercells, CXCR4 expression HCT116 cell surface and CD164 is considered to be a cancer promoting gene (12). Re- immunoprecipitation studies showed that CD164 formed cently, CD164 has also been recognized as a potential diag- complexes with CXCR4. nostic marker for acute lymphoblastic leukemia and allergy Conclusions: CD164 is highly expressed in the colon cancer (13, 14). sites, and it promotes HCT116 colon cancer cell proliferation Cancer Invest Downloaded from informahealthcare.com by Chinese University of Hong Kong on 05/29/12 Cancer metastasis is a complex process in which malig- and metastasis both in vitro and in vivo,andtheeffectsmayact nant cells break away from primary tumor, attach to the de- through regulating CXCR4 signaling pathway. Therefore, graded proteins from the surrounding extracellular matrix CD164 may be a new target for diagnosis and treatment for and migrate to other locations via the bloodstream or the colon cancer. lymphatic system. The tumor cell proliferation, adhesion, Keywords CD164; Colon cancer; CXCR4; Metastasis. and migration are involved and tightly regulated during tu- mor metastasis process. The relationship of CD164 and can- INTRODUCTION cer development especially metastasis remains unexplored. In this study, we have investigated the expression of CD164 CD164 (MGC-24v or endolyn) is a member of the sialomucin in human colon cancers and used a well-established human family a mucin containing sialic acid, which is highly con- colon cancer cell line HCT116 to evaluation the roles of Correspondence to: Gang Li, Department of Orthopaedics and Traumatology, Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong. email: [email protected] or Juanjuan Xiang, Cancer Research Institute, Key Laboratory of Carcinogenesis and Cancer Invasion of Ministry of Education, Key Laboratory of Carcinogenesis of Ministry of Health, Central South University, 110 Xiangya Road, Changsha, Hunan, 410078, P.R. China. email: [email protected] CD164 in Colon Cancer CD164 in cancer cell proliferation and migration (metastasis) Cell culture in vitro and in vivo. We also explored the possible underlin- HCT116 cell line was obtained from the European Col- ing mechanisms of CD164 in colon cancer development. lection of Cell Cultures. CD164 knockdown HCT116 (HCT116-CD164-shRNA), knockdown control clone (HCT116-shRNA control) were established by short hairpin MATERIALS AND METHODS RNA (shRNA) transduction method (see below). These cells were cultured in DMEM medium supplemented with Immunohistochemistry on human colon and other cancer penicillin G (100 U/mL Sigam, USA), streptomycin (100 specimens mg/mL),and10%fetalcalfserum.Cellsweregrownat37◦C Twenty (20) biopsies from colorectal cancer patients, 5 sam- in a humidified atmosphere of 5% CO2 and were routinely ples from breast cancer patients and 5 samples from ovary sub-cultured using 0.25% (w/v) trypsin-EDTA solution. cancer patients were obtained with patient’s consent follow- ing excision of the cancers. The specimens were coded and Lentiviral vector and cell transduction fixed in 10% buffered formalin pH 7.0 for 24 hr and then ViraPowerTM lentiviral expression system (Invitrogen, Pais- µ embedded in paraffin wax. The 5- mthicksectionswere ley, UK) was used to introduce Lenti-Luciferase-eGFP- cut using a rotary microtome (Microm HM315, Walldorf, TOPO construct into HCT116 cells. The ViraPowerTM pack- Germany) and the sections were placed on the poly-L-lysine aging mix and Lenti-Luciferase-eGFP-TOPO construct were (Sigma Chemical Co., Poole, UK) coated slides. For immuno- cotransfected using a gene carrier kit (Epoch-Biolabs, USA) histochemistry, sections were dewaxed in xylene and im- into the 293T cell line to produce a lentiviral stock. At 48-hr mersed in graded ethanol and distilled water. Endogenous post transfection, the virus-containing supernatant was har- peroxidase activity was inhibited by immersing the tissue sec- vested by collecting the medium. Viral particles were purified tions in 0.3% hydrogen peroxidase for 20 min at room tem- by ultracentrifugation through a 20% sucrose cushion. For perature. The sections were subsequently rinsed in phosphate infecting HCT116 cells, cells were cultured in 24 well plates buffer solution (PBS). The sections were then incubated with till 80% confluence, the lentivirus was added to the culture purified rabbit anti-human CD164 antibody (1:100 ratio in dishes and incubated for 48 hr, the medium was then replaced PBS) for 45 min at room temperature. Subsequently, the pri- by the selection medium containing 10 µg/mL Blasticidin. mary antibody was blotted and the tissues were rinsed with CD164 knockdown HCT116 (HCT116-CD164-shRNA) PBS. The secondary purified goat anti-rabbit biotinglated an- was established by short hairpin RNA (shRNA) transduction. tibody (DAKO, Dorset, UK, 1:100 in PBS) was applied to A set of MISSION shRNA lentiviral transduction particles thesections,andincubatedfor30minatroomtempera- specific to human CD164 gene was purchased from Sigma ture.SectionswererinsedinPBSandwerethenincubated Aldrich (Poole, UK). The set included five clones targeting For personal use only. with Streptavidin-peroxidase (1:50 in PBS, DAKO, Dorset, different parts of CD164 cDNA sequences. For transduction, UK) for 30 min at room temperature. Diaminobenzidine HCT116 cells were plated in 96-well plate the day to allow chromagen solution (0.5 mg/mL 2,4,2 ,4 –tetrabiphenyl hy- 50% confluence, 100 µLmediumcontaining15µLofthawed drochloride, Sigma Chemical Co., Poole, UK) in 0.1 m im- MISSION shRNA lentiviral particles (MOI 30∼40) as well as idazole in PBS, containing 0.3% hydrogen peroxidase was 8 µg/mL Polybrene were added to the cells for 48 hr. The added to the sections, and incubated at room temperature medium was then changed to media containing 2.5 µg/mL for 5 min. Following a brief rinse in distilled water the tis- Puromysin for stable clone selection. The mock knockdown sue sections were counterstained in Gill’s No. 3 hematoxylin clone was used as control (HCT116-shRNA control). (Sigma Chemical Co., Poole, UK) at a dilution of 1:3 for 5 min, rinsed in distilled water and dehydrated through a RT-PCR series of graded ethanol solutions to xylene, mounted in The RNA samples and cDNA samples were prepared us- DPX and examined by light microscopy. Primary antibody Cancer Invest Downloaded from informahealthcare.com by Chinese University of Hong Kong on 05/29/12
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