non-SP Research Articles

Foot-and-mouth disease seroprevalence is higher in young female crossbred sheep and goats compared to their counterparts in the border area between Pakistan and Afghanistan.

Foot-and-mouth disease (FMD) is a highly contagious disease in ruminants that causes significant economic losses worldwide. However, the prevalence of FMD virus (FMDV) in small ruminants has been overlooked in Pakistan. This study aimed to determine the prevalence of FMD in sheep and goats in the border area between Pakistan and Afghanistan. 800 sheep and goats belongs to age groups of 6 month to > 2 years. A total of 800 serum samples were collected from sheep (n = 424) and goats (n = 376) and subjected to structural protein (SP) and 3ABC non-SP (NSP) ELISAs for the detection of antibodies against SP and NSP of the FMDV. For NSP, 340/800 (42.5%) of samples were positive, while SP analysis revealed that serotype O (44.5%) was the most common in sheep and goats, followed by Asia-1 (42%) and A (32%) serotypes. Sheep (39%; 95% CI, 34 to 44) had a higher (P < .05) prevalence of FMD than goats (46%; 95% CI, 41 to 51). Statistically significant (P < .05) differences in the seroprevalence of FMD-SP and FMD-NSPs were observed between various agencies (areas) of the study area. Risk factors such as age, sex, breed, season, flock size, body condition, animal movement, and production system were significantly (P < .05) associated with FMDV prevalence. This study showed that FMD is highly prevalent in sheep and goats in the border areas of Pakistan and Afghanistan. Therefore, outbreak investigation teams should be arranged at the border level to develop FMD risk-based surveillance and control plans for small ruminants in order to mitigate infection risks.

Genetics of the rabbit haemorrhagic disease virus strains responsible for rabbit haemorrhagic disease outbreaks in Japan between 2000 and 2020.

Rabbit haemorrhagic disease (RHD) is a highly contagious and fatal disease in rabbits caused by the rabbit haemorrhagic disease virus (RHDV), which includes two genotypes, RHDV-GI.1 and RHDV2-GI.2. RHDVs tend to recombine among different strains, resulting in significant genetic evolution. This study evaluated the genetics of Japanese RHDV strains associated with six outbreaks between 2000 and 2020 using whole-genome sequencing, genomic recombination and phylogenetic analyses. Genomic recombination analysis using near-complete genomic sequences revealed that two Japanese strains detected in 2000 and 2002 were non-recombinant GI.1 (variant RHDVa-GI.1a) strains of different origins, most closely related to strains identified in PR China in 1997 and the USA in 2001, respectively. In contrast, four recent Japanese GI.2 strains detected between 2019 and 2020 were recombinant viruses harbouring structural protein (SP) genes from GI.2 strains and non-SP (NSP) genes from a benign rabbit calicivirus (RCV) strain of genotype RCV-E1-GI.3 (GI.3P-GI.2) or an RHDV G1-GI.1b variant (GI.1bP-GI.2). Phylogenetic analysis based on SP and NSP regions revealed that the GI.1bP-GI.2 recombinant virus detected in Ehime prefecture and the GI.3P-GI.2 recombinant viruses detected in Ibaraki, Tochigi and Chiba prefectures were most closely related to recombinant viruses identified in Australia in 2017 and Germany in 2017, respectively. These results suggested that past RHD outbreaks in Japan did not result from the evolution of domestic RHDVs but rather represented incursions of foreign RHDV strains, implying that Japan is constantly at risk of RHDV incursion from other countries.

An active RUNX1-ID1/ID3 axis governs differentiation and chemoresistance of cancer stem cell population in epithelial ovarian cancer cells

Progression, relapse, and therapy resistance are the most challenging features of cancer therapy that have been postulated to be driven by Cancer Stem Cell (CSC) population. This enigmatic subpopulation of cancer cells has therefore emerged as promising therapeutic candidate. We earlier reported enrichment of CSC-like side population (SP) with increasing resistance towards Cisplatin and Paclitaxel either alone or in combination in epithelial ovarian cancer (EOC) cells. This SP population is a small proportion of the total population of cancer cells characterised with high expression of drug transporters, a unique feature of stem cells and thereby can be isolated through their efflux properties of DNA binding dyes. While the bulk non-SP (NSP) population of the cancer cells lack overexpression of the drug transporters and thus can be identified as the dye containing population. In this study, we show that increased expression of Runt related transcription factor 1 (RUNX1) maintains undifferentiated state of CSC-like SP cells through upregulation of inhibitors of DNA binding/differentiation genes (ID1 and ID3) in late cisplatin-paclitaxel resistant cells. Higher RUNX1 expression was found to correlate with decreased median overall survival and disease-free survival in The Cancer Genome Atlas (TCGA) data set of high grade serous ovarian cancer (HGSOC) patients. The protein-protein interaction network analysis of 397 upregulated genes in RUNX1-high samples of TCGA data show significant enrichment of pathways known to negatively regulate CSC differentiation. Intriguingly RUNX1 inhibition not only induces CSC differentiation but also downregulates anti-apoptotic protein BCL2 in both SP and NSP cells and potentiates cytotoxic effects of Cisplatin-Paclitaxel in chemoresistant EOC cells. Inhibition of BCL2 through Venetoclax treatment, a small molecule BH3 mimic, sensitized these cells to platinum taxol treatment. Altogether, our data reveal new regulatory roles by RUNX1 to modulate CSC differentiation via ID1 and ID3 and to promote chemoresistance through BCL2 upregulation.

Overexpression of DUSP6 enhances chemotherapy-resistance of ovarian epithelial cancer by regulating the ERK signaling pathway.

Objective: DUSP6 is a negative regulator of the ERK signaling pathway and plays an important role in chemotherapy-resistance. Previously we showed that DUSP6 is overexpressed in ovarian cancer side population (SP) cells that possess cancer stem cell-like properties and are quiescent and chemotherapy-resistant. Here, we explore the effects of DUSP6 on chemotherapy-resistance by examining its regulation of the ERK signaling pathway and G0/G1 cell cycle arrest.Methods: mRNA and protein expression of DUSP6 and G0/G1 cell cycle checkpoint regulating proteins (CyclinD1, CyclinD3 and CyclinE2) was evaluated among ovarian cancer cell lines and tissue samples. Ovarian cancer cells were transiently transfected to overexpress DUSP6. After treatment with cisplatin, cell viability was measured by the MTS assay at 48 hours and the half maximal inhibitory concentration (IC50) for each cell line was calculated. Subcellular localization and cell cycle analysis were determined by using immunofluorescence and FACS, respectively.Results: SKOV3 and OVCAR8 SP cells were shown to express higher levels of DUSP6 and lower levels of CyclinD3 compared with non-SP (NSP) cells (P<0.001). Among 39 ovarian cancer tissue samples, expression of DUSP6 in the chemotherapy-resistant group (12 samples) was higher than in the chemotherapy-sensitive group (27 samples) (P<0.05). While a lower level of expression of CyclinD3 was seen in the chemotherapy-resistant group, it was not statistically different from the chemotherapy-sensitive group. HO8910 cells where shown to have higher IC50 to cisplatin than SKOV3 or OVCAR8 cells, and this correlated with higher levels of DUSP6 expression. Overexpression of DUSP6 in SKOV3 cells led to an increase in cisplatin IC50 values (P<0.05), and also markedly reduced the expression levels of phospho-ERK1/2 and CyclinD3 and to the predominance of cells in the G0/G1 phase.Conclusion: Our findings reveal an enhancement of chemotherapy-resistance and a predominance of cells in G1 cell cycle arrest in DUSP6-overexpressing ovarian cancer cells. This suggests that overexpression of DUSP6 promotes chemotherapy-resistance through the negative regulation of the ERK signaling pathway, increasing the G0/G1 phase ratio among ovarian cancer cells, and leading to cellular quiescence.

OCT4 accelerates tumorigenesis through activating JAK/STAT signaling in ovarian cancer side population cells.

BackgroundAlthough surgery, chemotherapy, and radiotherapy eliminate clinically apparent ovarian tumor, the 5-year survival rate is no more than 45%. Cancer stem cells (CSCs) have been identified for precaution of tumor metastasis and recurrence in many kinds of cancers including ovarian cancer.AimThis study aims to explore the function of OCT4, a CSC marker, in ovarian cancer progression and to investigate its underlying mechanism.Materials and methodsBy Hoechst side population (SP) technique, CSC-like SP cells from human ovarian cancer SKOV3 and A2780 cells were isolated and used for this study. shRNA and lentivirus targeting human OCT4 gene were used to knock down OCT4 in SP cells and upregulate OCT4 in non-SP (NSP) cells stably. Peficitinib was used to inhibit JAK/STAT signaling. Cell counting kit-8, flow cytometry, and in vivo xenograft model were used to evaluate the effects of OCT4/JAK/STAT on the viability, drug resistance, apoptosis, cycle, and tumorigenesis of the SP cells. Immunofluorescence staining was used to detect the location of STAT6.ResultsResults showed that OCT4 was upregulated in the SP of SKOV3 and A2780 cells when compared with the NSP cells. Downregulation of OCT4 inhibited SP cell viability, tumorigenesis, and reduced cell drug resistance and induced a G2/M phase arrest, while upregulation of OCT4 conferred NSP cell malignant features. Besides, OCT4 upregulation in NSP cells increased the phosphorylated levels of proteins in JAK and STAT families, especially in JAK1 and STAT6. Furthermore, the roles of apoptosis inhibition and viability, invasion, and tumorigenesis promotions induced by OCT4 in NSP cells were all abolished when adding peficitinib.ConclusionOur study demonstrated that OCT4 accelerated ovarian cancer progression through activating JAK/STAT signaling pathway.

Anticancer effects of curcumin on nude mice bearing lung cancer A549 cell subsets SP and NSP cells.

Curcumin is a key polyphenolic curcuminoid extracted from the root of turmeric rhizome Curcuma longa Linn, which is a frequently used Chinese herb for the treatment of cancer. The aim of the present study was to investigate the mechanism of the inhibitory effects of curcumin on nude mice with lung cancer A549 cell subsets side population (SP) and non-SP (NSP) cells. BALB/c mice were subcutaneously injected with the tumor cells of A549 SP or NSP subsets consisting of 1×109 cells/l (0.2 ml in total). After 16 days of inoculation with A549, the mice were intraperitoneally injected with curcumin (100 mg/kg, 0.2 ml) once every other day, eight times in total. A series of assays were performed to detect the effects of curcumin on: i) Tumor weight and size; ii) Notch and hypoxia inducible factor 1 (HIF-1) mRNA expression by quantitative polymerase chain reaction; and iii) vascular endothelial growth factor (VEGF) and nuclear factor-κB (NF-κB) by immunohistochemistry. It was determined that curcumin decreased the tumor weight and size, downregulated the expression of Notch and HIF-1 mRNA and suppressed the VEGF and NF-κB expression. These results indicated that curcumin inhibited lung cancer growth through the regulation of angiogenesis mediated by VEGF signaling.

Identification of side population cells in human lung adenocarcinoma A549 cell line and elucidation of the underlying roles in lung cancer.

The present study aimed to isolate and characterize side population (SP) cells in the human lung cancer A549 cell line, and elucidate the molecular mechanism of SP cells underlying lung cancer. The SP and non-SP (NSP) cells in A549 cells were isolated and their differentiation was analyzed by fluorescence-activated cell sorting. An in vitro plate clone assay, Matrigel® Transwell assay and chemoresistance analysis of the sorted SP and NSP cells were performed. In addition, the sorted SP and NSP cells were injected into BALB/c nude mice to detect their tumorigenic potential in vivo. The expression of ATP-binding cassette sub-family G member 2 (ABCG2) in transplanted tumors was detected by immunohistochemistry. The SP and NSP cells were successfully isolated. The results demonstrated that SP cells accounted for 1.09% of live A549 cells. SP cells produced SP and NSP cells, while NSP cells only produced NSP cells. In addition, SP cells formed more colonies, exhibited improved invasive ability and increased levels of chemoresistance compared with NSP cells in vitro. SP cells demonstrated a higher tumorigenic potential in BALB/c nude mice, and the number of ABCG2-positive cells in the SP xenograft tumors were significantly increased compared with that in the NSP xenograft tumors. The present study indicated that SP cells isolated from the human lung cancer A549 cell line demonstrated increased tumorigenicity, and improved invasive ability and chemoresistance compared with NSP cells. In addition, detection of ABCG2 expression may assist in predicting the chemotherapeutic outcome of patients, and serve as a target for treating lung cancer.

Characteristics of primary side population cervical cancer cells.

The aim of the present study was to identify and characterize side population (SP) cells in primary cervical cancer. A primary culture was successfully established, and the SP cells were isolated via fluorescence-activated cell sorting. Subsequently, in vitro analysis of clonogenic capacity by soft agar assay and in vivo analysis of tumorigenicity were performed. The isolated SP cells accounted for ~4.73% of the total primary culture cells. The SP cells had a decreased proliferation rate and an increased distribution in G0/G1 compared with non-SP (NSP) cells. Following isolation, SP cells exhibited increased proliferative and self-renewal potency compared with NSP cells. Furthermore, significant ATP binding cassette subfamily G member 2 (ABCG2) expression was detected in SP cells but not in NSP cells. The tumor formation rate of SP cells was longer, and the tumor size and tumor formation rate of SP cells were increased in non-obese diabetic/severe combined immunodeficiency mice. In conclusion, the present study demonstrated that SP cells can be isolated from primary cervical cancer cell culture, and SP cells are enriched with stem cell-like cells that have a high capacity for colony formation and tumorigenesis.

Targeting BCRP/ABCG2 by RNA interference enhances the chemotherapy sensitivity of human colon cancer side population cells.

Relapse and metastasis are frequent in colon cancer and may be linked to stem cell characteristics. This study isolated side population (SP) cells from a colon cancer cell line (Colo-320) and examined their self-renewal and differentiation abilities. Compared to non-SP (NSP) cells, SP colon cancer cells were more tumorigenic in vivo and exhibited more invasive characteristics and a greater ability to form colonies. Additionally, more cells were in G0/G1 phase and more highly expressed the multidrug resistance protein BCRP/ABCG2. We achieved enhanced chemotherapy sensitivity by transfecting SP cells with a hairpin-like, small interfering RNA (siRNA) eukaryotic expression plasmid targeting BCRP/ABCG2.

Abstract 2495: hPaf1/PD2 interacts with OCT3/4 in maintenance of the self-renewal process of ovarian cancer stem cells

Abstract Background: Ovarian cancer (OC) is the most lethal gynecological malignancy among women with more than 85% of the patients manifesting tumor recurrence. Emerging evidence suggests that a small population of cells within the tumor - the ‘cancer stem cells (CSCs)’ is capable to giving rise to the entire histopathology of the tumor and is responsible for mediating drug resistance, recurrence, and disease aggressiveness. Previously, hPaf1(human RNA polymerase II associated factor1)/PD2 (Pancreatic Differentiation2) - a core component of RNA polymerase II associated factor (PAF) complex, was shown to be overexpressed in pancreatic CSCs and involved in the maintenance of mouse ESCs. Hence, we hypothesized that hPaf1 is involved in the maintenance of self-renewal property of ovarian CSCs (OCSCs). In this study, we investigated the functional role of hPaf1 in OCSCs which has not been explored before. Methods: Expression of hPaf1, cancer stem cell marker ESA, and self-renewal protein OCT3/4 was analyzed using confocal microscopy on OC tissue array. Side population (SP) was isolated from two OC cell lines OVCAR3 and A2780 by Hoechst staining using flow-cytometer, and characterized using tumor sphere assay. Immunoblotting was performed on characterized SP and NSP (non-SP) for OCSC and self-renewal markers. Transient knockdown of hPaf1 in SP was performed to understand how hPaf1 affects the CSC phenotype through immunoblotting, confocal microscopy, colony formation assay, and tumor sphere formation assay. To determine the interaction between hPaf1 and OCT 3/4, reciprocal co-immunoprecipitation was performed in SP/OCSCs. Results: There was a significant overexpression and considerable co-localization of hPaf1/PD2 with ESA and OCT3/4 in OC tissues compared to normal ovary tissues. SP from OVCAR3 formed larger and greater number of tumor spheres compared to NSP cells. Moreover, SP isolated from OVCAR3 and A2780 showed higher expression of hPaf1 along with CSC markers (CD44, CD133, ESA, CD24), and self- renewal proteins (β-CATENIN, SOX2, OCT3/4, SHH, and HER2). Transient knockdown of hPaf1 resulted in a significant decrease in expression of CSC markers and self-renewal proteins. In addition, functional characteristics of CSCs such as in vitro tumor formation capacity in non-adherent media and colony formation ability were impaired upon knockdown of hPaf1 suggesting that hPaf1 is involved in maintenance of the CSC phenotype. We also observed that hPaf1 physically interacts with OCT3/4 in OVCAR3 SP cells which provides a mechanism for the maintenance of OCSCs by hPaf1. Conclusion: Altogether, hPaf1/PD2 is overexpressed in OCSCs and its knockdown resulted in loss of OCSC phenotype. Moreover, hPaf1 is responsible for the maintenance of OCSCs through its interaction with OCT3/4. Hence, therapies that are able to abrogate hPaf1 mediated self-renewal of OCSCs represent potential therapeutic avenues to overcome tumor relapse in OC. Citation Format: Saswati Karmakar, Parthasarathy Seshacharyulu, Arokia Priyanka Vaz, Imayavaramban Lakshmanan, Moorthy Palanimuthu Ponnusamy, Surinder Kumar Batra. hPaf1/PD2 interacts with OCT3/4 in maintenance of the self-renewal process of ovarian cancer stem cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2495.

Biological characteristics of side population cells in a self-established human ovarian cancer cell line.

The aim of the present study was to establish an ovarian cancer (OC) cell line from ascites of an ovarian serous cystadenocarcinoma patient and investigate the biological characteristics of its side population (SP) cells. The OC cell line was established by isolating, purifying and subculturing primary cells from ascites of an ovarian serous cystadenocarcinoma patient (stage IIIc; grade 3). SP and non-SP (NSP) cells were isolated by fluorescence-activated cell sorting and cultured in serum-free medium and soft agar to compare the tumorsphere and colony formation capacities. Furthermore, SP and NSP cell tumorigenesis was examined by subcutaneous and intraperitoneal injection of the cells to non-obese diabetic/severe combined immune deficiency (NOD/SCID) mice. Drug resistance to cisplatin was examined by cell counting kit-8. The OC cell line was successfully established from ascites of an ovarian serous cystadenocarcinoma patient, which exhibited properties similar to primary tumors subsequent to >50 passages and >2 years of culture. The SP cell ratio was 0.38% in the OC cell line, and a similar SP cell ratio (0.39%) was observed when sorted SP cells were cultured for 3 weeks. Compared with NSP cells, SP cells exhibited increased abilities in differentiation and tumorsphere and colony formation, in addition to the formation of xenografted tumors and ascites and metastasis of the tumors in NOD/SCID mice, even at low cell numbers (3.0×103 cells). The xenografted tumors demonstrated histological features similar to primary tumors and expressed the ovarian serous cystadenocarcinoma marker CA125. In addition, SP cells demonstrated a significantly stronger drug resistance to cisplatin compared with NSP and unsorted cells, while treatment with verapamil, an inhibitor of ATP-binding cassette transporters, potently abrogated SP cell drug resistance. In conclusion, the present study verified SP cells from an established OC cell line and characterized the cells with self-renewal, differentiation, proliferation, tumorigenesis and stronger drug resistance capacities.

The Metastatic Potential and Chemoresistance of Human Pancreatic Cancer Stem Cells.

Cancer stem cells (CSCs) typically have the capacity to evade chemotherapy and may be the principal source of metastases. CSCs for human pancreatic ductal carcinoma (PDAC) have been identified, but neither the metastatic potential nor the chemoresistance of these cells has been adequately evaluated. We have addressed these issues by examining side-population (SP) cells isolated from the Panc-1 and BxPC3 lines of human PDAC cells, the oncogenotypes of which differ. SP cells could be isolated from monolayers of Panc-1, but only from spheroids of BxPC3. Using orthotopic xenografts into the severely immunocompromised NSG mouse, we found that SP cells isolated from both cell lines produced tumors that were highly metastatic, in contrast to previous experience with PDAC cell lines. SP cells derived from both cell lines expressed the ABCG2 transporter, which was demonstrably responsible for the SP phenotype. SP cells gave rise to non-SP (NSP) cells in vitro and in vivo, a transition that was apparently due to posttranslational inhibition of the ABCG2 transporter. Twenty-two other lines of PDAC cells also expressed ABCG2. The sensitivity of PDAC SP cells to the vinca alkaloid vincristine could be greatly increased by verapamil, a general inhibitor of transporters. In contrast, verapamil had no effect on the killing of PDAC cells by gemcitabine, the current first-line therapeutic for PDAC. We conclude that the isolation of SP cells can be a convenient and effective tool for the study of PDAC CSCs; that CSCs may be the principal progenitors of metastasis by human PDAC; that the ABCG2 transporter is responsible for the SP phenotype in human PDAC cells, and may be a ubiquitous source of drug-resistance in PDAC, but does not confer resistance to gemcitabine; and that inhibition of ABCG2 might offer a useful adjunct in a therapeutic attack on the CSCs of PDAC.

Abstract A127: Butyrylcholinesterase (BChE) expression as a marker of cellular proliferation across neuroblastoma cells lines: new prognostic marker and therapeutic target

Abstract Butrylcholinesterase (BChE) is a glycoprotein found in the peripheral and central nervous system including in neuroblastoma, a tumor of the peripheral sympathetic nervous system. The use of BChE as a tumor biomarker and potential therapeutic target has been proposed. The purpose of this study was to assess the association between BChE and tumorigenicity in two NB cell lines and their respective stem cell populations. Two MYCN amplified neuroblastoma cell lines, BE-2 (highly MYCN amplified) and IMR-32 (intermediately MYCN amplified) were studied along with neuroblastoma stem cells identified by side population (SP) in flow cytometry. MYCN protein, which is a poor prognostic proliferative marker in neuroblastoma, was measured by western blot in each of the two cell lines to characterize and confirm the amplification phenotype. Cell cycle characteristics of each cell line was evaluated using a propidium iodide based analysis by flow cytometry. BChE protein was measured using western blot in the two cell lines as well as in their SP and non-SP (NSP) populations after sorting. An MYCN non-amplified neuroblastoma cell line (SH-SY5Y) was tested for both MYCN and BChE protein as a potential low MYCN and BChE expressing tumor. The metabolic profile (glycolysis and OXPHOS) was determined for both unsorted, SP and NSP cells using Seahorse® metabolic analysis. BE-2 and IMR-32 tumorigenicity was characterized by growing cells subcutaneously in the flanks of NSG immunodeficient mice. BE-2c tumors developed earlier and grew more rapidly than IMR-32 tumors after injection of a similar number of cells. MYCN protein was similarly expressed in BE-2c and IMR-32 despite higher in-vivo tumorigenicity of BE-2c. In contrast, BChE was higher in BE-2 compared to IMR-32 corresponding to the order of tumorigenicity in-vivo. BE-2 and IMR-32 SP stem cells were relatively quiescent as measured by lower BChE protein compared to NSP cells and lower metabolic rates as measured by extracellular acidification (glycolysis) and oxygen consumption (OXPHOS metabolism). Cell cycle analysis and cell growth curves support BE-2 being a more proliferative cell line than IMR-32. MYCN has been the traditional marker to identify aggressive poor prognostic NB but data presented suggests BChE may also identify high risk disease generally correlating with growth of tumor in NSG mice and cellular growth characteristics. The use of small molecule inhibitors of BChE are currently under pre-clinical evaluation and given the relationship between BChE expression and tumorigenicity, BChE may be a promising new target. Ongoing work in our laboratories is investigating mechanisms of association between BChE and high-risk features of NB. Citation Format: Katie Greenwood, Timothy R. McGuire, Donald Coulter, Erin McIntyre, Nagendra K. Chaturvedi, Paul Kortylewicz, Shantaram S. Joshi, Xiaoyu Chen, John G. Sharp, Janina Baranowska-Kortylewicz. Butyrylcholinesterase (BChE) expression as a marker of cellular proliferation across neuroblastoma cells lines: new prognostic marker and therapeutic target. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A127.

Up-regulation of lymphocyte antigen 6 complex expression in side-population cells derived from a human trophoblast cell line HTR-8/SVneo.

The continual proliferation and differentiation of trophoblasts are critical for the maintenance of pregnancy. It is well known that the tissue stem cells are associated with the development of tissues and pathologies. It has been demonstrated that side-population (SP) cells identified by fluorescence-activated cell sorting (FACS) are enriched with stem cells. The SP cells in HTR-8/SVneo cells derived from human primary trophoblast cells were isolated by FACS. HTR-8/SVneo-SP cell cultures generated both SP and non-SP (NSP) subpopulations. In contrast, NSP cell cultures produced NSP cells and failed to produce SP cells. These SP cells showed self-renewal capability by serial colony-forming assay. Microarray expression analysis using a set of HTR-8/SVneo-SP and -NSP cells revealed that SP cells overexpressed several stemness genes including caudal type homeobox2 (CDX2) and bone morphogenic proteins (BMPs), and lymphocyte antigen 6 complex locus D (LY6D) gene was the most highly up-regulated in HTR-8/SVneo-SP cells. LY6D gene reduced its expression in the course of a 7-day cultivation in differentiation medium. SP cells tended to reduce its fraction by treatment of LY6D siRNA indicating that LY6D had potential to maintain cell proliferation of HTR-8/SVneo-SP cells. On ontology analysis, epithelial-mesenchymal transition (EMT) pathway was involved in the up-regulated genes on microarray analysis. HTR-SVneo-SP cells showed enhanced migration. This is the first report that LY6D was important for the maintenance of HTR-8/SVneo-SP cells. EMT was associated with the phenotype of these SP cells.

Dysregulation of the PI3K/Akt signaling pathway affects cell cycle and apoptosis of side population cells in nasopharyngeal carcinoma.

Increasing evidence has suggested that certain types of cancer possess their own stem-like cells, and that one subset of these cells, termed the side population (SP), may have an important role in tumorigenesis and cancer therapy. However, the molecular mechanisms underlying the modulation of SP cells in nasopharyngeal carcinoma (NPC) have remained elusive. In the present study, it was hypothesized that dysregulation of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt signaling pathway may influence SP and non-SP (NSP) phenotype. SP cells from the HK-1 NPC cell line were identified, and cancer stem cell markers were found to be highly expressed in SP cells compared with that of NSP cells. Freshly sorted SP cells demonstrated a significant increase in the proportion of cells in G0/G1 phase, while the majority of NSP cells were in the proliferative phase. Following 48 h of culture subsequent to cell sorting, the differences in cell cycle distribution between the SP and NSP cells converged. In addition, the apoptotic ratio of NSP cells was higher than that of SP cells at 24 h following sorting, but had no significant differences 48 h following sorting. To elucidate the potential mechanism mediating the cell cycle and apoptosis in SP cells, the expression levels of key molecules in the PI3K/Akt signaling pathway were evaluated. PI3K and Akt were upregulated, while 14-3-3σ protein was downregulated in SP cells when freshly sorted (0 h). However, there was no significant difference in the expression of these molecules between SP and NSP cells following 48 h of culture. These results suggested that dysregulation of the PI3K/Akt signaling pathway may be associated with the cell cycle and apoptosis of SP cells in NPC. However, further investigation is required to elucidate the detailed mechanisms underlying these effects.

Evaluation of stem-like side population cells in a recurrent nasopharyngeal carcinoma cell line.

BackgroundSide population (SP) assay identifies cells with dye/drug extrusion ability, a characteristic of stem cells. Here, we determined if SP cells exist in a verified cell line originating from recurrent nasopharyngeal carcinoma (NPC) and a xenograft established from recurrent metastatic NPC. These cells were evaluated for stem-like properties via functional assays as well as for tumourigenicity.MethodsWe used Hoechst 33342 to identify the SP from non-SP (NSP) cells in HK1 NPC cell line and xeno-284 NPC xenograft. The cells were assayed for in vitro characteristics of cancer stem cells (CSC), gene expression and tumourigenicity ability. Student’s t test was used to test for significance.ResultsFive to ten percent and less than 0.5% of HK1 and xeno-284 NPC cells, respectively, were SP cells. Fumitremorgin C (FTC), as opposed to verapamil, was effective in causing the cells to retain Hoechst 33342 dye. HK1 SP cells formed more holoclones, had more aldehyde dehydrogenase (ALDH) activity, divided asymmetrically and contained slow-proliferating cells. ABCG2, SOX2, TERT, MYC, Hedgehog, Notch, TGFβ and Wnt signalling pathway genes were significantly upregulated in the SP cells. However, despite these differences in vitro, both HK1 SP and NSP cells had an overall similar tumourigenic potential in vivo.ConclusionsHK1 SP cells were ABCG2-specific as confirmed by FTC inhibition and gene expression data. Despite data from in vitro and gene expression experiments suggesting stem-like features, there was no significant difference in tumourigenic potential between SP and NSP cells. We conclude that SP assay alone is not sufficient to identify CSCs in HK1 cells. Our work also suggests the presence of a stem-cell like population among NPC cells which do not display increased tumourigenicity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12935-014-0101-0) contains supplementary material, which is available to authorized users.

Characterization of side population cells isolated from the colon cancer cell line SW480.

Side population (SP) cells may play a crucial role in tumorigenesis and the recurrence of cancer. Many types of cell lines and tissues have demonstrated the presence of SP cells, including colon cancer cell lines. This study aimed to identify cancer stem cells (CSCs) in the SP of the colon cancer cell line SW480. SP cells were isolated by fluorescence-activated cell sorting (FACS), followed by serum-free medium (SFM) culture. The self-renewal, differentiated progeny, clone formation, proliferation, invasion ability, cell cycle, chemosensitivity and tumorigenic properties in SP and non-SP (NSP) cells were investigated through in vitro culture and in vivo serial transplantation. The expression profiles of ATP-binding cassette (ABC) protein transporters and stem cell-related genes were examined by RT-PCR and western blot analysis. The human colon cancer cell lines SW480, Lovo and HCT116 contain 1.1 ± 0.10, 0.93 ± 0.11 and 1.33 ± 0.05% SP cells, respectively. Flow cytometry analysis revealed that SP cells could differentiate into SP and NSP cells. SP cells had a higher proliferation potency and CFE than NSP cells. Compared to NSP cells, SP cells were also more resistant to CDDP and 5-FU, and were more invasive and displayed increased tumorigenic ability. Moreover, SP cells showed higher mRNA and protein expression of ABCG2, MDR1, OCT-4, NANOG, SOX-2, CD44 and CD133. SP cells isolated from human colon cancer cell lines harbor CSC properties that may be related to the invasive potential and therapeutic resistance of colon cancer.

Enhanced Bone-Forming Activity of Side Population Cells in the Periodontal Ligament

Regeneration of alveolar bone is critical for the successful treatment of periodontal diseases. The periodontal ligament (PDL) has been widely investigated as a source of cells for the regeneration of periodontal tissues. In the present study where we attempted to develop an effective strategy for alveolar bone regeneration, we examined the osteogenic potential of side population (SP) cells, a stem cell-containing population that has been shown to be highly abundant in several kinds of tissues, in PDL cells. Isolated SP cells from the rat PDL exhibited a superior ability to differentiate into osteoblastic cells compared with non-SP (NSP) and unsorted PDL cells in vitro. The mRNA expressions of osteoblast markers and bone morphogenetic protein (BMP) 2 were significantly upregulated in SP cells and were further increased by osteogenic induction. To examine the bone-forming activity of SP cells in vivo, PDL SP cells isolated from green fluorescent protein (GFP)-transgenic rats were transplanted with hydroxyapatite (HA) disks into wild-type animals. SP cells exhibited a high ability to induce the mineralized matrix compared with NSP and unsorted PDL cells. At 12 weeks after the implantation, some of the pores in the HA disks with SP cells were filled with mineralized matrices, which were positive for bone matrix proteins, such as osteopontin, bone sialoprotein, and osteocalcin. Furthermore, osteoblast- and osteocyte-like cells on and in the bone-like mineralized matrices were GFP positive, suggesting that the matrices were directly formed by the transplanted cells. These results suggest that PDL SP cells possess enhanced osteogenic potential and could be a potential source for cell-based regenerative therapy for alveolar bone.

SPARC was overexpressed in human endometrial cancer stem-like cells and promoted migration activity

ObjectivesWe previously demonstrated that side-population (SP) cells found in human endometrial cancer tissue have features of cancer stem cells (CSCs). Endometrial cancer SP cells show enhanced migration, the potential to differentiate into the mesenchymal cell lineage, and they are associated with the epithelial–mesenchymal transition (EMT). In this study, we analyzed the expression and function of a specific protein, SPARC (secreted protein acidic and rich in cysteine) which we found to be up-regulated in endometrial cancer. MethodsWe performed microarray expression analysis to screen for up-regulated genes in CSCs using a set of RK12V-SP cells and -non-SP (NSP) cells. We used the MetaCore package to identify the Gene GO pathway MAPs associated with the up-regulated genes. Here, we investigated the expression and functions of SPARC, one of the genes up-regulated in endometrial CSCs.We established SPARC-overexpressing cells by transfecting endometrial cancer cells (Ishikawa cells [IK-SPARC cells]). We characterized these cells' growth rate, tumorigenicity, migration and invasion activity. The levels and locations of SPARC protein expression in Hec1SP cells-derived tumors and endometrial cancer tissues were examined by immunohistochemistry. ResultsSPARC was detected by microarray expression analysis during screens for up-regulated genes in SP and NSP CSC. The level of SPARC expression was enhanced in Hec1 SP cells compared with that in Hec1 non-SP cells. SPARC enhanced fibronectin expression and promoted migration activity in IK cells. SPARC expression suppressed tumor growth but promoted formation of tumor stroma.SPARC was expressed in endometrial cancer tissues, in particular, poorly differentiated endometrioid adenocarcinoma, clear and serous adenocarcinoma,but not in normal endometrial tissue. ConclusionThis is the first report of overexpression of SPARC in endometrial cancer stem-like cells. SPARC expression is associated with cell migration and stroma formation.

Possible association between stem-like hallmark and radioresistance in human cervical carcinoma cells.

We aimed to investigate the possibility of an association between a stem-like hallmark and radiotherapeutic sensitivity in human cervical carcinoma cells. Side-population (SP) cells and non-SP (NSP) cells in HeLa cells were isolated using flow cytometry and Hoechst 33342 efflux. We performed Western blot analysis to evaluate the expression of stem cell markers (CXCR4, Oct3/4, CD133, and SOX2) and apoptosis markers after irradiation. In addition, SP and NSP cells were injected into nude mice and we assessed subcutaneous tumor formation. To examine tolerance of irradiation, colony formation and apoptosis change were confirmed in the SP and NSP cells. SP cells showed a higher expression of CXCR4, Oct3/4, CD133, and SOX2 than NSP cells. The colony size of SP cells cultured on non-coated dishes was larger than that of NSP cells, and NSP cells were easily induced to undergo apoptosis. SP cells tended to form spheroids and showed a higher level of tumorigenicity compared with NSP cells. In addition, nude mice inoculated with SP cells showed greater tumor growth compared with NSP cells. SP cells showed a higher tumorigenicity and lower apoptotic potential, leading to enhanced radiotolerance. Tumor SP cells showed higher-level stem-cell-like characters and radioresistance than NSP cells. SP cells may be useful for new therapeutic approaches for radiation-resistant cervical cancer.