Stromal interaction molecule (STIM) proteins are parts of elaborate eukaryotic Ca2+ signaling systems and are considered to be important players in regulating neuronal Ca2+ homeostasis under normal ageing and pathological conditions. Here, we investigated the potential role of STIM1 in 6-hydroxydopamine (6-OHDA)-induced toxicity in undiff erentiated PC12 cell lines. Cells exposed to 6-OHDA demonstrated alterations in the generation of reactive oxygen species (ROS) in a Ca2+-dependent manner. Downregulation of STIM1 expression by specifi c small interfering RNA (siRNA) attenuated apoptotic cell death, reduced intracellular ROS production, and partially prevented the impaired endogenous antioxidant enzyme activities after 6-OHDA treatment. Furthermore, STIM1 knockdown signifi cantly attenuated 6-OHDA-induced intracellular Ca2+ overload by inhibiting endogenous store-operated calcium entry (SOCE). The eff ect of STIM1 siNRA on SOCE was related to orai1 and L-type Ca2+ channels, but not to transient receptor potential canonical type 1 (TRPC1) channel. In addition, silencing of STIM1 increased the Ca2+ buffering capacity of the endoplasmic reticulum (ER) in 6-OHDA-injured cells. ER vacuoles formed from the destruction of ER structural integrity and activation of ER-related apoptotic factors (CHOP and Caspase-12) were partially prevented by STIM1 knockdown. Moreover, STIM1 knockdown attenuated 6-OHDA-induced mitochondrial Ca2+ uptake and mitochondrial dysfunction, including the collapse of mitochondrial membrane potential (MMP) and the decrease of ATP generation. Taken together, our data provide the fi rst evidence that inhibition of STIM1-meditated intracellular Ca2+ dyshomeostasis protects undiff erentiated PC12 cells against 6-OHDA toxicity and indicate that STIM1 may be responsible for neuronal oxidative stress induced by ER stress and mitochondrial dysfunction in PD.

Objective: Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) has recently been reported to be a marker of cancer stem cells (CSCs) in colorectal cancer (CRC), and the prognostic value of LGR5 in CRC has been evaluated in several studies. However, the conclusions remain controversial. In this study, we aimed to evaluate the association between the expression of LGR5 and the outcome of CRC patients by performing a meta-analysis.Methods: We systematically searched for relevant studies published up to February 2014 using the PubMed, Web of Science, EMBASE and Wangfang databases. Only articles in which LGR5 expression was detected by immunohistochemistry were included. A meta-analysis was performed using STATA 12.0, and pooled hazard ratios (HRs) with 95% confidence intervals (CIs) were used to estimate the strength of the association between LGR5 expression and the prognosis of CRC patients.Results: A total of 7 studies comprising 1833 CRC patients met the inclusion criteria, including 6 studies comprising 1781 patients for overall survival (OS) and 3 studies comprising 528 patients for disease-free survival (DFS). Our results showed that high LGR5 expression was significantly associated with poor prognosis in terms of OS (HR: 1.87, 95% CI: 1.23-2.84; P = 0.003) and DFS (HR: 2.44, 95% CI: 1.49-3.98; P<0.001). Further subgroup analysis revealed that many factors, including the study region, number of patients, follow-up duration and cutoff value, affected the significance of the association between LGR5 expression and a worse prognosis in patients with CRC. In addition, there was no evidence of publication bias, as suggested by Begg's and Egger's tests.Conclusions: The present meta-analysis indicated that high LGR5 expression was associated with poor prognosis in patients with CRC and that LGR5 is an efficient prognostic factor in CRC.

Grape seed proanthocyanidins (GSPs), a biologically active component of grape seeds, have been reported to possess a wide array of pharmacological and biochemical properties. Recently, the inhibitory effects of GSPs on various cancers have been reported, but their effects on cervical cancer remain unclear. Here, we explored the effect of GSPs on cervical cancer using in vitro and in vivo models. In vitro, the treatment of HeLa and SiHa cells with GSPs resulted in a significant inhibition of cell viability. Further investigation indicated that GSPs led to the dose-dependent induction of apoptosis in cancer cells. The underlying mechanism was associated with increased expression of the pro-apoptotic protein Bak-1, decreased expression of the anti-apoptotic protein Bcl-2, the loss of mitochondrial membrane potential, and the activation of caspase-3, suggesting that GSPs induced cervical cancer cell apoptosis through the mitochondrial pathway. In addition, the administration of GSPs (0.1%, 0.2%, and 0.4%, w/v) as a supplement in drinking water significantly inhibited the tumor growth of HeLa and SiHa cells in athymic nude mice, and the number of apoptotic cells in those tumors was also increased significantly. Taken together, our studies demonstrated that GSPs could inhibit the growth of cervical cancer by inducing apoptosis through the mitochondrial pathway, which provides evidence indicating that GSPs may be a potential chemopreventive and/or chemotherapeutic agent for cervical cancer.

High aldehyde dehydrogenase (ALDH) activity characterizes a subpopulation of cells with cancer stem cell (CSC) properties in several malignancies. To clarify whether ALDH can be used as a marker of cervical cancer stem cells (CCSCs), ALDH(high) and ALDH(low) cells were sorted from 4 cervical cancer cell lines and 5 primary tumor xenografts and examined for CSC characteristics. Here, we demonstrate that cervical cancer cells with high ALDH activity fulfill the functional criteria for CSCs: (1) ALDH(high) cells, unlike ALDH(low) cells, are highly tumorigenic in vivo; (2) ALDH(high) cells can give rise to both ALDH(high) and ALDH(low) cells in vitro and in vivo, thereby establishing a cellular hierarchy; and (3) ALDH(high) cells have enhanced self-renewal and differentiation potentials. Additionally, ALDH(high) cervical cancer cells are more resistant to cisplatin treatment than ALDH(low) cells. Finally, expression of the stem cell self-renewal-associated transcription factors OCT4, NANOG, KLF4 and BMI1 is elevated in ALDH(high) cervical cancer cells. Taken together, our data indicated that high ALDH activity may represent both a functional marker for CCSCs and a target for novel cervical cancer therapies.

Octamer-binding transcription factor 4 (OCT4) is a key regulatory gene that maintains the pluripotency and self-renewal properties of embryonic stem cells. Although there is emerging evidence that it can function as oncogene in several cancers, the role in mediating cervical cancer remains unexplored. Here we found that OCT4 protein expression showed a pattern of gradual increase from normal cervix to cervical carcinoma in situ and then to invasive cervical cancer. Overexpression of OCT4 in two types of cervical cancer cells promotes the carcinogenesis, and inhibits cancer cell apoptosis. OCT4 induces upregulation of miR-125b through directly binding to the promoter of miR-125b-1 confirmed by chromatin immunoprecipitation analysis. MiRNA-125b overexpression suppressed apoptosis and expression of BAK1 protein. In contrast, miR-125b sponge impaired the anti-apoptotic effect of OCT4, along with the upregulated expression of BAK1. Significantly, Luciferase assay showed that the activity of the wild-type BAK1 30-untranslated region reporter was suppressed and this suppression was diminished when the miR-125b response element was mutated or deleted. In addition, we observed negative correlation between levels of BAK1 and OCT4, and positive between OCT4 and miR-125b in primary cervical cancers. These findings suggest an undescribed regulatory pathway in cervical cancer, by which OCT4 directly induces expression of miR-125b, which inhibits its direct target BAK1, leading to suppression of cervical cancer cell apoptosis.

Undifferentiated embryonic cell transcription factor-1 (UTF1) is an important transcription factor during development, which plays critical roles in cell fate determination. However, its expression and function in somatic tissues remain unclear. Here, we investigated the expression pattern of the UTF1 in the human normal and cancerous lesions of cervix and found that UTF1 was downregulated in cervical carcinogenesis, which was related to the hypermethylation of UTF1 promoter. Exogenous expression of UTF1 resulted in the significant inhibition of cell proliferation in vitro and tumorigenesis in vivo through attenuating cell cycle arrest via increasing the level of p27(Kip1). Luciferase reporter assay indicated that the region containing an intact activating transcription factor site between nucleotides 517 and 388 of the p27(Kip1) promoter was indispensable for its activation by UTF1. Chromatin immunoprecipitation analysis confirmed the physical interaction between UTF1 and the p27(Kip1) promoter. Taken together, our findings reveal that UTF1 attenuates cell proliferation and is inactivated in cervical carcinogenesis through epigenetic modification, which strongly supports that UTF1 is a potential tumor suppressor.

B-lymphoma Moloney murine leukemia virus insertion region-1 is an oncogene in various human tumors, and overexpression correlates with a poor clinical outcome. Sex-determining region of Y chromosome related high mobility group box-2, coding for a critical transcription factor determining the fate of stem cells, was recently identified as an oncogene in human cervical carcinoma and other tumors. However, the roles of B-lymphoma Moloney murine leukemia virus insertion region-1 and sex-determining region of Y chromosome related high mobility group box-2 in the pathogenesis of cervical carcinoma are poorly understood. We initially observed a more pronounced increase in B-lymphoma Moloney murine leukemia virus insertion region-1 protein expression in primary cervical carcinoma than in normal cervical tissues, and B-lymphoma Moloney murine leukemia virus insertion region-1 protein expression correlated significantly with sex-determining region of Y chromosome related high mobility group box-2 protein expression, as seen by Western blotting (r = 0.75; P < .01 ). Furthermore, B-lymphoma Moloney murine leukemia virus insertion region-1 and sex-determining region of Y chromosome related high mobility group box-2 both bad higher expression in cervical carcinoma than in normal cervical tissue, and the amounts correlated with pathologic grade. Immunofluorescence analysis showed that B-lymphoma Moloney murine leukemia virus insertion region-1 colocalized in the nucleus with sex-determining region of Y chromosome related high mobility group box-2 in both normal cervical tissue and cervical carcinoma. From the cervical carcinoma cell line SiHa,we isolated 2 clones, B-lymphoma Moloney murine leukemia virus insertion region-1(+)/sex-determining region of Y chromosome-related high mobility group box-2(+) (SiHa-3) and B-lymphoma Moloney murine leukemia virus insertion region-1(-)/sex-determining region of Y chromosome-related high mobility group box-2(-) (SiHa-2). The SiHa-3 cells grew better in vitro and formed tumors more readily in vivo than did SiHa-2. Knockout of sex-determining region of Y chromosome-related high mobility group box-2 inhibited cell growth in vitro with a block at G1/S. In contrast, knockout of B-lymphoma Moloney murine leukemia virus insertion region-1 did not affect either cell growth in vitro or the cell cycle. Interference with either B-lymphoma Moloney murine leukemia virus insertion region-1 or sex-determining region of Y chromosome-related high mobility group box-2 in SiHa-3 significantly inhibited tumorigenesis (P < .05). Coexpression of B-lymphoma Moloney murine leukemia virus insertion region-1 and sex-determining region of Y chromosome-related high mobility group box-2 may promote cervical carcinogenesis. (c) 2013 Elsevier Inc. All rights reserved.

The miR-302-367 cluster is specifically expressed in human embryonic stem cells and has been shown to convert human somatic cells into induced pluripotent stem cells. Here, we investigated the role of the miR-302-367 cluster in cervical carcinoma. The cluster was not endogenously expressed in cervical cancer cells, and its ectopic expression did not reprogram the cervical cancer cells to an embryonic stem cell-like state. However, ectopic expression of the miR-302-367 cluster in He La and Si Ha cervical cancer cells inhibited cell proliferation and tumor formation by blocking the G1/S cell cycle transition. We identified a new cell cycle regulatory pathway in which the miR-302-367 cluster directly down-regulated both cyclin D1 and AKT1 and indirectly up-regulated p271(Kip1) and p21(Cip1), leading to the suppression of cervical cancer cell proliferation. Our findings suggest that the miR-302-367 cluster may be used as a therapeutic reagent for the treatment of cervical carcinoma.

BACKGROUND: The Kruppel-like factor 4 (KLF4) protein, a zinc finger transcription factor that is highly expressed in epithelial tissues such as the gut and skin, has been implicated in both tumor suppression and progression. However, the role of KLF4 in human cervical carcinoma is still unclear. METHODS: The expression of KLF4 in cervical carcinoma tissues and cervical cancer cell lines was examined with immunohistochemistry and Western blot assay. The effects of KLF4 silencing and overexpression on the cell proliferation, cell viability, and tumor formation of cervical cancer cells were investigated. RESULT: KLF4 protein expression showed a pattern of gradual decrease from normal cervix to cervical carcinoma in situ and then to invasive cervical carcinomas (P < .05). Overexpression of KLF4 in SiHa and C33A cells resulted in significantly inhibited cell growth and significantly attenuated tumor formation. Consistently, KLF4 silencing in HeLa cells significantly promoted cell growth and tumor formation. Furthermore, KLF4 overexpression caused cell cycle arrest at the G1/S transition, along with the up-regulated expression of p27Kip1 protein. Promoter analysis revealed that KLF4 transactivated the expression of p27Kip1 through the specific motif that is between the nucleotides of -435 and -60 in its promoter. The results from chromatin immunoprecipitation assays demonstrated the physical interaction between KLF4 protein and this specific motif in p27Kip1 promoter. CONCLUSIONS: KLF4 may function as a tumor suppressor in cervical carcinoma by inhibiting cell growth and tumor formation. Cancer 2012. (C) 2011 American Cancer Society.

Tumor development has long been known to resemble abnormal embryogenesis. The embryonic stem cell gene NANOG, a divergent homeodomain transcription factor that is independent of leukemia inhibitory factor, has been reported to be expressed in germ cells and in several tumor types. However, the short-term expression and role of NANOG in cervical cancer remain unclear. In the present study, we demonstrate that NANOG exhibits cellular shuttling behavior and increasing stromal distribution during the progression of cervical cancer. Our molecular data using RT-PCR and restriction enzyme digestion show that NANOG is mainly transcribed from the NANOG gene in cervical cancer. In addition, MC using confocal microscopy suggests that mesenchymal stem cells (MSCs) are one type of cytoplasmic NANOG-positive cells in cervical cancer stroma. Co-culture of cervical cancer derived MSCs with SiHa cells showed increased proliferation characteristics in vitro and enhanced tumor growth in vivo. Our results show, for the first time to our knowledge, that MSCs are a source of cytoplasmic NANOG expression in the cervical cancer stroma and that they participate in the progression of cervical cancer both in vitro and in vivo. Our study provides evidence that NANOG is a cervical cancer progression marker and also serves as a starting point for a more extensive exploration of the cellular translocation of NANOG and the multifunctionality of the stromal fnicroenvironment (Am J Path, 2012, 181:652-661; bttp://dx.doi.org/10.1016/j.ajpath.2012.04.008)