Ovarian cancer stem-like cells show induced translineage-differentiation capacity and are suppressed by alkaline phosphatase inhibitor.

Spheroid formation is one property of stem cells-such as embryo-derived or neural stem cells-that has been used for the enrichment of cancer stem-like cells (CSLCs). However, it is unclear whether CSLC-derived spheroids are heterogeneous or whether they share common embryonic stemness properties. Understanding these features might lead to novel therapeutic approaches. Ovarian carcinoma is a deadly disease of women. We identified two types of spheroids (SR1 and SR2) from ovarian cancer cell lines and patients' specimens according to their morphology. Both types expressed stemness markers and could self-renew and initiate tumors when a low number of cells were used. Only SR1 could differentiate into multiple-lineage cell types under specific induction conditions. SR1 spheroids could differentiate to SR2 spheroids through epithelial-mesenchymal transition. Alkaline phosphatase (ALP) was highly expressed in SR1 spheroids, decreased in SR2 spheroids, and was absent in differentiated progenies in accordance with the loss of stemness properties. We verified that ALP can be a marker for ovarian CSLCs, and patients with greater ALP expression is related to advanced clinical stages and have a higher risk of recurrence and lower survival rate. The ALP inhibitor, levamisole, disrupted the self-renewal of ovarian CSLCs in vitro and tumor growth in vivo. In summary, this research provides a plastic ovarian cancer stem cell model and a new understanding of the cross-link between stem cells and cancers.This results show that ovarian CSLCs can be suppressed by levamisole. Our findings demonstrated that some ovarian CSLCs may restore ALP activity, and this suggests that inhibition of ALP activity may present a new opportunity for treatment of ovarian cancer.

Drug screening identifies niclosamide as an inhibitor of breast cancer stem-like cells.

The primary cause of death from breast cancer is the progressive growth of tumors and resistance to conventional therapies. It is currently believed that recurrent cancer is repopulated according to a recently proposed cancer stem cell hypothesis. New therapeutic strategies that specifically target cancer stem-like cells may represent a new avenue of cancer therapy. We aimed to discover novel compounds that target breast cancer stem-like cells. We used a dye-exclusion method to isolate side population (SP) cancer cells and, subsequently, subjected these SP cells to a sphere formation assay to generate SP spheres (SPS) from breast cancer cell lines. Surface markers, stemness genes, and tumorigenicity were used to test stem properties. We performed a high-throughput drug screening using these SPS. The effects of candidate compounds were assessed in vitro and in vivo. We successfully generated breast cancer SPS with stem-like properties. These SPS were enriched for CD44(high) (2.8-fold) and CD24(low) (4-fold) cells. OCT4 and ABCG2 were overexpressed in SPS. Moreover, SPS grew tumors at a density of 10(3), whereas an equivalent number of parental cells did not initiate tumor formation. A clinically approved drug, niclosamide, was identified from the LOPAC chemical library of 1,258 compounds. Niclosamide downregulated stem pathways, inhibited the formation of spheroids, and induced apoptosis in breast cancer SPS. Animal studies also confirmed this therapeutic effect. The results of this proof-of-principle study may facilitate the development of new breast cancer therapies in the near future. The extension of niclosamide clinical trials is warranted.

LIM-homeobox transcription factor 1, alpha (LMX1A) inhibits tumourigenesis, epithelial-mesenchymal transition and stem-like properties of epithelial ovarian cancer.

OBJECTIVE: We reported recently the hypermethylation of LMX1A, a LIM-homeobox gene, as a prognostic biomarker in ovarian cancer; however, the function of LMX1A in ovarian cancer remains unknown. The present study aimed to evaluate the hypothesized tumour-suppressor functions of LMX1A in ovarian cancer. METHODS: We analysed the function of LMX1A by examining cell lines, animal models and human ovarian cancer tissues. Overexpression of LMX1A in relation to chemotherapy was also analysed. RESULTS: The expression of LMX1A inhibited cell proliferation, migration, invasion and colony formation in vitro, as well as tumourigenicity in a xenotransplantation mouse model. LMX1A also sensitized ovarian cancer cell lines to chemotherapeutics, and affected epithelial-mesenchymal transition (EMT). The restoration of LMX1A down-regulated stem cell markers and inhibited tumour spheroid formation in SKOV3 cells. Univariate analysis of immunohistochemical staining of tissue arrays (n=83) revealed that low LMX1A expression was significantly associated with advanced stages (p=0.001), poor differentiation (p<0.001), early recurrence (p=0.023) and poor overall survival (p=0.042) in ovarian cancer. CONCLUSIONS: The present study demonstrated, for the first time, that LMX1A is a bona fide tumour suppressor of ovarian cancer. The prognostic values of LMX1A may provide a biomarker for personalized treatments of ovarian cancer patients. The mechanisms of LMX1A in EMT and stem-like properties in ovarian cancer warrant further investigation.

Methylomic analysis identifies frequent DNA methylation of zinc finger protein 582 (ZNF582) in cervical neoplasms.

BACKGROUND: Despite of the trend that the application of DNA methylation as a biomarker for cancer detection is promising, clinically applicable genes are few. Therefore, we looked for novel hypermethylated genes for cervical cancer screening. METHODS AND FINDINGS: At the discovery phase, we analyzed the methylation profiles of human cervical carcinomas and normal cervixes by methylated DNA immunoprecipitation coupled to promoter tiling arrays (MeDIP-on-chip). Methylation-specific PCR (MSP), quantitative MSP and bisulfite sequencing were used to verify the methylation status in cancer tissues and cervical scrapings from patients with different severities. Immunohistochemical staining of a cervical tissue microarray was used to confirm protein expression. We narrowed to three candidate genes: DBC1, PDE8B, and ZNF582; their methylation frequencies in tumors were 93%, 29%, and 100%, respectively. At the pre-validation phase, the methylation frequency of DBC1 and ZNF582 in cervical scraping correlated significantly with disease severity in an independent cohort (n = 330, both P<0.001). For the detection of cervical intraepithelial neoplasia 3 (CIN3) and worse, the area under the receiver operating characteristic curve (AUC) of ZNF582 was 0.82 (95% confidence interval= 0.76-0.87). CONCLUSIONS: Our study shows ZNF582 is frequently methylated in CIN3 and worse lesions, and it is demonstrated as a potential biomarker for the molecular screening of cervical cancer.

Growth inhibition of ovarian tumor-initiating cells by niclosamide.

A recent hypothesis for cancer chemoresistance posits that cytotoxic survival of a subpopulation of tumor progenitors drives the propagation of recurrent disease, underscoring the need for new therapeutics that target such primitive cells. To discover such novel compounds active against drug-resistant ovarian cancer, we identified a subset of chemoresistant ovarian tumor cells fulfilling current definitions of cancer-initiating cells from cell lines and patient tumors using multiple stemness phenotypes, including the expression of stem cell markers, membrane dye efflux, sphere formation, potent tumorigenicity, and serial tumor propagation. We then subjected such stem-like ovarian tumor-initiating cells (OTIC) to high-throughput drug screening using more than 1,200 clinically approved drugs. Of 61 potential compounds preliminarily identified, more stringent assessments showed that the antihelmintic niclosamide selectively targets OTICs in vitro and in vivo. Gene expression arrays following OTIC treatment revealed niclosamide to disrupt multiple metabolic pathways affecting biogenetics, biogenesis, and redox regulation. These studies support niclosamide as a promising therapy for ovarian cancer and warrant further preclinical and clinical evaluation of this safe, clinically proven drug for the management of this devastating gynecologic malignancy.

ALDH1-bright epithelial ovarian cancer cells are associated with CD44 expression, drug resistance, and poor clinical outcome.

The role of aldehyde dehydrogenase 1 (ALDH1) as an ovarian cancer stem cell marker and its clinical significance have rarely been explored. We used an Aldefluor assay to isolate ALDH1-bright (ALDH1(br)) cells from epithelial ovarian cancer cell lines and characterized the properties of the stem cells. ALDH1(br) cells were enriched in ES-2 (1.3%), TOV-21G (1.0%), and CP70 (1.2%) cells. Both ALDH1(br) and ALDH1(low) cells repopulated stem cell heterogeneity, formed spheroids, and grew into tumors in immunocompromised mice, although these processes were more efficient in ALDH1(br) cells. In the ES-2 and CP70 cells, ALDH1(br) cells conferred more chemoresistance, and were more enriched in CD44 (by 1.74-fold and 5.18-fold, respectively) than in CD133 (by 1.39-fold and 1.17-fold, respectively), compared with ALDH1(low) cells. Immunohistochemical staining for ALDH1 on a tissue microarray containing 84 epithelial ovarian cancer samples revealed that patients with higher ALDH1 expression (>50%) had poor overall survival, compared with those with lower ALDH1 (P = 0.004) and yielded an odds ratio of death of 2.43 (95% CI = 1.12 to 5.28) by multivariate analysis. The results did not support ALDH1 alone as an ovarian cancer stem cell marker, but demonstrated that ALDH1 is associated with CD44 expression, chemoresistance, and poor clinical outcome. The use of a combination of ALDH1 with other stem cell markers may help define ovarian cancer stem cells more stringently.

Amelioration of rat collagen-induced arthritis through CD4+ T cells apoptosis and synovial interleukin-17 reduction by indoleamine 2,3-dioxygenase gene therapy.

Indoleamine 2,3-dioxygenase (IDO) has been known as an emerging therapeutic target in autoimmunity-related arthritis. The treatment responses of adenoviral vectors encoding IDO (AdIDO) gene therapy in rat collagen-induced arthritis (CIA) were examined in this study. The therapeutic effects on ankle circumference, articular index, and radiographic and histological scores were evaluated in AdIDO-injected ankle joints. We further determined CD4+ T-cell numbers and their apoptotic status, CD68(+) macrophage numbers, kynurenine (a downstream tryptophan metabolite) concentrations, interleukin-17 (IL-17) levels, and retinoic acid-related orphan receptor γt (RORγt) expression in synovial tissues of CIA rats receiving AdIDO treatment. Reduction of ankle circumference, articular index, and radiographic and histological scores were noted in AdIDO-treated ankles, as compared with those receiving injection of control vectors. Furthermore, IDO gene transfer led to decreased infiltrating CD4+ T cells with enhanced apoptosis, reduced CD68+ macrophage numbers, increased kynurenine levels, lower IL-17 concentrations, and decreased RORγt expression within the ankle joints. In addition, such a therapy diminished type II collagen-specific IL-17 production and RORγt expression in CD4+ T cells from draining lymph nodes of CIA rats. Our results demonstrate for the first time that intra-articular delivery of IDO gene ameliorated ankle arthritis of CIA rats by induction of CD4+ T-cell apoptosis and reduction of synovial IL-17 production through the supplement of kynurenine. Taken together, these findings implicate the novel strategy of using IDO gene as a therapeutic approach in treating patients with rheumatoid arthritis.

Licorice and licochalcone-A induce autophagy in LNCaP prostate cancer cells by suppression of Bcl-2 expression and the mTOR pathway.

Licorice is a common Chinese medicinal herb with antitumor activity. Some components in licorice root have been shown to induce cell cycle arrest or apoptosis in cancer cells. This paper demonstrates for the first time that licorice Glycyrrhiza glabra and its component licochalcone-A (LA) can induce autophagy in addition to apoptosis in human LNCaP prostate cancer cells. Exposure of cells to licorice or LA resulted in several confirmed characteristics of autophagy, including the appearance of autophagic vacuoles revealed by monodansylcadaverine (MDC) staining, formation of acidic vesicular organelles (AVOs), and autophagosome membrane association of microtubule-associated protein 1 light chain 3 (LC3) characterized by cleavage of LC3 and its punctuate redistribution, as well as ultrastructural observation of autophagic vacuoles by transmission electron microscopy. Autophagy induction was accompanied by down-regulation of Bcl-2 and inhibition of the mammalian target of rapamycin (mTOR) pathway. In summary, licorice can induce caspase-dependent and autophagy-related cell death in LNCaP cells.

Cathepsin L mediates resveratrol-induced autophagy and apoptotic cell death in cervical cancer cells.

Cathepsins have long been considered as housekeeping molecules. However, specific functions have also been attributed to each of these lysosomal proteases. Squamous cell carcinoma antigen (SCCA) 1, widely expressed in various uterine cervical cells, is an endogenous cathepsin (cat) L inhibitor. In this study, we investigated whether the cat L-SCCA 1 lysosomal pathway and autophagy were involved in resveratrol (RSV)-induced cytotoxicity in cervical cancer cells. RSV induced GFP-LC3 aggregation as well as increased the presence of LC3-II and autophagosomes as was revealed by electron microscopy in cervical cancer cells. Prolonged treatment of RSV induced cytosolic translocation of cytochrome c, caspase 3 activation and apoptotic cell death. This apoptotic effect was abrogated by trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane, an inhibitor of cat B and L, but not by pepstatin A, an inhibitor of cat D. As cervical cancer cells express little cat B, we further studied the role of cat L. RSV induced dissipation of the lysosomal membrane permeability (LMP), leakage and increased cytosolic expression and activity of cat L. Inhibition of cat L by small interference RNA (siRNA) protected cells from RSV-induced cytotoxicity. In contrast, inhibition of SCCA 1 by siRNA promoted RSV-induced cytotoxicity. Inhibition of autophagic response by wortmannin (WT) or asparagine (ASP) resulted in decreased early LC3-II formation, reduced LMP, and abolishment of the increase in RSV-induced cell death. In conclusion, we have identified a new cytotoxic mechanism in which the lysosomal enzyme cat L acts as a death signal integrator in cervical cancer cells. Furthermore, SCCA 1 may play an antiapoptotic role through anti-cat L activity.

Amelioration of collagen-induced arthritis in rats by adenovirus-mediated PTEN gene transfer.

OBJECTIVE: The phosphatidylinositol 3-kinase (PI 3-kinase)/Akt pathway is known to be activated in rheumatoid arthritis (RA) synovial tissue, which impacts cell growth, proliferation, survival, and migration. Phosphatase and tensin homolog deleted from chromosome 10 (PTEN) functions as a negative regulator of PI 3-kinase signaling, thus blocking Akt activation. The aim of this study was to examine the effect of PTEN gene transfer in rats with collagen-induced arthritis (CIA). METHODS: Adenoviral vectors encoding human PTEN (AdPTEN) or beta-galactosidase (AdLacZ) were injected intraarticularly into rats with CIA, and their treatment responses were monitored by measures of clinical, radiographic, and histologic changes. The expression of phosphorylated Akt, total Akt, vascular endothelial growth factor (VEGF), proinflammatory cytokines, and chemokines, as well as the extent of microvessel density in the ankle joints were determined. RESULTS: AdPTEN treatment reduced Akt phosphorylation and decreased VEGF production in human RA synovial fibroblasts. Compared with AdLacZ treatment of the rats with CIA, AdPTEN treatment significantly reduced ankle circumference, articular index scores, radiography scores, and histology scores, and also decreased microvessel density and levels of VEGF and interleukin-1beta. Furthermore, PTEN gene transfer led to down-regulation of Akt activation and increased apoptosis in the ankle joints. CONCLUSION: This study is the first to demonstrate the in vivo effect of intraarticular gene delivery of PTEN on amelioration of arthritis symptoms in rats with CIA, which involved antiangiogenic, antiproliferative, and antiinflammatory effects of PTEN via inhibition of the PI 3-kinase/Akt signaling pathway. Our findings also implicate the PI 3-kinase/Akt pathway as a therapeutic target for the treatment of RA or other inflammatory diseases.

Tumor-selective replication of an oncolytic adenovirus carrying oct-3/4 response elements in murine metastatic bladder cancer models.

PURPOSE: Oncolytic adenoviruses are attractive therapeutics for cancer because they selectively replicate in tumors. However, targeting tumor metastasis remains a major challenge for current virotherapy for cancer. Oct-3/4 is specifically expressed in embryonic stem cells and tumor cells. Oct-3/4 highly expressed in cancer cells may be a potential target for cancer therapy. We developed an E1B-55 kDa-deleted adenovirus, designated Ad.9OC, driven by nine copies of Oct-3/4 response element for treating Oct-3/4-expressing metastatic bladder cancer. EXPERIMENTAL DESIGN: We examined the expression of Oct-3/4 in human bladder tumor tissues and bladder cancer cell lines. We also evaluated the cytolytic and antitumor effects of Ad.9OC on bladder cancer cells in vitro and in vivo. RESULTS: Oct-3/4 expression was detected in bladder cancer cell lines, as well as in human bladder tumor tissues. Notably, Oct-3/4 expression was higher in metastatic compared with nonmetastatic bladder cancer cells. Ad.9OC induced higher cytolytic activity in metastatic bladder cancer cells than in their nonmetastatic counterparts, whereas it did not cause cytotoxicity in normal cells. Pharmacologic and short hairpin RNA-mediated Oct-3/4 inhibition rendered bladder cancer cells more resistant to Ad.9OC-induced cytolysis. Replication of Ad.9OC was detected in murine bladder cancer cells and bladder tumor tissues. We also showed the effectiveness of Ad.9OC for treating bladder cancer in subcutaneous, as well as metastatic, bladder tumor models. CONCLUSIONS: Ad.9OC may have therapeutic potential for treating Oct-3/4-expressing tumors. Especially, metastatic bladder tumors are good target for Ad.9OC treatment. Because Oct-3/4 is expressed in a broad spectrum of cancers, Ad.9OC may be broadly applicable.

Prothymosin alpha lacking the nuclear localization signal as an effective gene therapeutic strategy in collagen-induced arthritis.

Prothymosin alpha (ProT) is regulated by c-Myc, an oncoprotein overexpressed in synovium of rheumatoid arthritis, and is associated with cell proliferation. However, ProT also exerts immunomodulatory activities. The growth-promoting activity of ProT can be abolished by deleting its nuclear localization signal (NLS). In this study, we showed that AdProTDeltaNLS, an adenoviral vector encoding ProT lacking the NLS, did not enhance the proliferation of synovial fibroblasts. AdProTDeltaNLS treatment abolished the up-regulation of the MIP-1alpha promoter activity induced by TNF-alpha in synovial fibroblasts. AdProTDeltaNLS suppressed macrophage chemotaxis and reduced macrophage infiltration into the ankle joints in rats with collagen-induced arthritis (CIA). Neutralization test confirmed the involvement of MIP-1alpha in macrophage chemotaxis. Administration of AdProTDeltaNLS reduced the severity of CIA in the clinical, radiographic, and histological aspects. The levels of TNF-alpha (mean +/- SEM, 1261.9 +/- 107.9 vs 2880.1 +/- 561.4 pg/mg total protein; p < 0.05), IL-1beta (56.8 +/- 8.0 vs 109.2 +/- 4.9 pg/mg total protein; p < 0.01), and MIP-1alpha (41.7 +/- 3.6 vs 55.2 +/- 1.1 pg/mg total protein; p < 0.05) in the ankle joints were lower in the AdProTDeltaNLS-treated rats with CIA than those in their control counterparts. In the AdProTDeltaNLS-treated ankle joints, matrix metalloproteinase-9 expression was decreased by 40% and infiltrating macrophages reduced by 50%. Our results demonstrate that intra-articular delivery of AdProTDeltaNLS significantly ameliorated the clinical course of CIA in rats. This study is the first to suggest that ProT lacking the NLS may have therapeutic potential for the management of rheumatoid arthritis.

Mitochondrial ribosomal protein S36 delays cell cycle progression in association with p53 modification and p21(WAF1/CIP1) expression.

Ribosomal biogenesis is correlated with cell cycle, cell proliferation, cell growth and tumorigenesis. Some oncogenes and tumor suppressors are involved in regulating the formation of mature ribosome and affecting the ribosomal biogenesis. In previous studies, the mitochondrial ribosomal protein L41 was reported to be involved in cell proliferation regulating through p21(WAF1/CIP1) and p53 pathway. In this report, we have identified a mitochondrial ribosomal protein S36 (mMRPS36), which is localized in the mitochondria, and demonstrated that overexpression of mMRPS36 in cells retards the cell proliferation and delays cell cycle progression. In addition, the mMRPS36 overexpression induces p21(WAF1/CIP1) expression, and regulates the expression and phosphorylation of p53. Our result also indicate that overexpression of mMRPS36 affects the mitochondrial function. These results suggest that mMRPS36 plays an important role in mitochondrial ribosomal biogenesis, which may cause nucleolar stress, thereby leading to cell cycle delay.

Low-dose etoposide enhances telomerase-dependent adenovirus-mediated cytosine deaminase gene therapy through augmentation of adenoviral infection and transgene expression in a syngeneic bladder tumor model.

The human telomerase reverse transcriptase (hTERT) promoter can selectively drive transgene expression in many telomerase-positive human cancer cells. Here we evaluated combination therapy of adenoviral vector Ad-hTERT-CD encoding E. coli cytosine deaminase (CD) driven by the hTERT promoter and low-dose etoposide (0.1 microg/mL) for treating bladder cancer. Ad-hTERT-CD conferred sensitivity to 5-fluorocytosine (5-FC) in bladder cancer cells, which could be enhanced by etoposide treatment, but not in normal cells. Such effect was correlated with up-regulation of hypoxia-inducible factor (HIF)-1alpha expression. By contrast, etoposide activated p53 and down-regulated hTERT promoter activity in normal cells. Etoposide also increased adenoviral infection via enhancement of coxsackie-adenovirus receptor expression on bladder cancer and normal cells. Combination index analysis revealed that combined therapy of Ad-hTERT-CD (10(9) plaque-forming units)/5-FC (200 mg/kg) with etoposide (2 mg/kg) synergistically suppressed tumor growth and prolonged survival in mice bearing syngeneic MBT-2 bladder tumors. This combination therapy regimen induced complete tumor regression and generated antitumor immunity in 75% of tumor-bearing mice. Furthermore, increased infiltrating CD4(+) and CD8(+) T cells and necrosis within tumors were found in mice receiving combination therapy of Ad-hTERT-CD and etoposide compared with those treated with either treatment alone. Thus, the potential high therapeutic index of the combination therapy may be an appealing therapeutic intervention for bladder cancer. Furthermore, because a majority of human tumors exhibit high telomerase activity, adenovirus-mediated CD gene therapy driven by the hTERT promoter in combination with low-dose etoposide may be applicable to a broad spectrum of cancers.

Enhancement of humoral and cellular immune responses by an oral Salmonella choleraesuis vaccine expressing porcine prothymosin alpha.

Previously, we showed that murine prothymosin alpha (ProT) enhances the efficacy of a pseudorabies DNA vaccine delivered by bacterial vectors. In this study, we cloned and sequenced the cDNA for porcine ProT. The deduced amino acid sequence of porcine ProT exhibited high homology to ProT from other mammals. Oral Salmonella choleraesuis vaccine carrying the ProT eukaryotic expression plasmid protected mice against virulent S. choleraesuis challenge. The adjuvant effect of ProT on humoral and cellular immune responses enhanced protective efficacy of the vaccine. Furthermore, both humoral and cellular immune responses played roles in the protective immune responses induced by the vaccine. Collectively, our results show that delivery of the ProT gene carried by attenuated S. choleraesuis augmented the immunogenicity of oral S. choleraesuis vaccine.

Transgenic overexpression of prothymosin alpha induces development of polycystic kidney disease.

BACKGROUND: Polycystic kidney disease (PKD) is a genetic disorder characterized by development of renal cysts and progressive renal dysfunction. Renal tissues from both PKD patients and rodent models of PKD show elevated c-myc expression. Prothymosin alpha (ProT) is positively regulated by c-myc through binding to the E box of its promoter. Through creating transgenic mice and clinical studies, we sought to investigate whether ProT overexpression contributes to PKD development. METHODS: ProT heterozygous and homozygous transgenic mice were generated and characterized. Morphologic, histologic, immunohistochemical, and biochemical analyses of the transgenic mice were performed. RESULTS: Two transgenic lines that represented integration at two different loci of the chromosomes were generated. ProT overexpression in the kidneys of homozygous transgenic mice induced a PKD phenotype, which included polycystic kidneys, elevated blood urea nitrogen (BUN), and lethality at about 10 days of age. Similar overexpression pattern of ProT was noted in cystic kidneys of the transgenic mice as well as in human autosomal-recessive PKD (ARPKD) and autosomal-dominant PKD (ADPKD) kidneys. ProT protein levels in the kidneys and urine as well as renal mRNA level of epithelial growth factor receptor (EGFR) of homozygous ProT transgenic mice were significantly higher than heterozygous or nontransgenic littermates. Furthermore, the heterozygous transgenic mice at 17 months of age also developed mild cystic kidneys. CONCLUSION: Transgenic mice overexpressing ProT represent a novel model for PKD and may provide insights into PKD development. ProT, like c-myc and EGFR, may contribute to the development of renal cysts and may be a potential noninvasive diagnostic molecule of PKD.