calcimycin and Uterine-Cervical-Neoplasms

Well-run screening programs for cervical cancer in the population at risk have been shown to result in a sharp decrease in the incidence and mortality of cervical cancer in a number of large populations. Expression patterns of a recently identified biomarker family, microRNA, appear to be characteristic of tumor type and developmental origin. Several tumors have been reported to actively release exosomes carrying microRNAs. The present study has determined the association of microRNAs with cervical cancer-derived exosomes. The cervical cancer-derived exosomes were enriched in the cervicovaginal lavages specimens and the abundance of exosomes and exosomal microRNAs was detected by electron microscopy, western blot analysis, RT-qPCR and microRNA target reporter vector. The microRNA-21 and microRNA-146a, which were up-regulated in cervical cancer patients, were associated with the high levels of cervical cancer-derived exosomes. In conclusion, we demonstrated the abundance of exosomes in the cervicovaginal lavage specimens of women with cervical cancer. Furthermore, our results indicated that abnormally high levels of microRNA-21 and microRNA-146a existed in the cervical cancer-derived exosomes and the two microRNAs were functional in 293T cells.

Topics: Calcimycin; Exosomes; Female; HEK293 Cells; HeLa Cells; Humans; MicroRNAs; Neoplasm Staging; Oligonucleotides, Antisense; Papillomavirus Infections; Tetraspanin 29; Tetraspanin 30; Up-Regulation; Uterine Cervical Neoplasms

AMP-activated protein kinase (AMPK) is a critical energy-balancing sensor in the regulation of cellular metabolism in response to external stimuli. Emerging evidence has suggested that AMPK is a potential therapeutic target for human cancers. AICAR, one of the pharmacological AMPK activators, has been widely used to suppress cancer cell growth through activation of LKB1, an upstream kinase of AMPK. However, frequent mutations and deletions of LKB1 found in some cancer cells limit the application of AICAR as an efficient therapeutic drug. Here we show that an alternative pharmacological AMPK activator, A23187, was able to inhibit cervical cancer cell growth through activation of Ca(2+)/calmodulin-dependent protein kinase kinase beta, another upstream kinase of AMPK. Using cervical cancer cell models, we found that HeLa (LKB1-deficient cell) responded less to the anti-proliferative effect exerted by AICAR treatment (p < 0.001) compared with CaSki and C41 (LKB1-expressing cells). Conversely, the anti-proliferative effect was increased significantly in HeLa but not in CaSki and C41 cells under treatment by A23187 (p < 0.001). Moreover, co-treatment of AICAR and A23187 was able to further enhance the inhibitory effect on cell growth of Hela, CaSki and C41 cells. Notably, both AICAR and A23187 exerted the anti-proliferative effect on cervical cancer cells by suppressing AMPK/mTOR signalling activity. These data suggest that A23187 could be an alternative potential therapeutic drug used for anti-proliferation in LKB1-deficient cancer cells.

Topics: Aminoimidazole Carboxamide; AMP-Activated Protein Kinase Kinases; AMP-Activated Protein Kinases; Calcimycin; Calcium-Calmodulin-Dependent Protein Kinase Kinase; Cell Line, Tumor; Cell Proliferation; Enzyme Activation; Enzyme Activators; Female; Growth Inhibitors; HeLa Cells; Humans; Protein Serine-Threonine Kinases; Ribonucleotides; Signal Transduction; Uterine Cervical Neoplasms

Cell adhesion is linked to various regulatory processes of growth as well as apoptotic cell death in normal and transformed epithelial cells. We investigated changes of cellular responses to the deprivation of cell anchorage associated with immortalization or malignant transformation. Normal human ectocervical keratinocytes (NCE cells) deprived of cell anchorage become susceptible to apoptosis, and in parallel they lose their adhesion to the culture substratum. The loss of cell adhesion is not directly due to apoptosis. NCE16 cells, an immortalized but not malignantly transformed subline of NCE, underwent apoptosis and lost cell adhesion in suspension, as the NCE cells did. By contrast, apoptosis was not inducible in human cervical cancer-derived C33A cells in suspension. Of other cell lines derived from human cervical cancer, SiHa cells showed a weak apoptotic response and Caski cells were highly sensitive to apoptosis in the absence of cell anchorage. Unlike NCE or NCE16 cells, all these cancer cells retained cell adhesion as well as growth capability in suspension cultures. These results indicate that retention of cell adhesion and growth capability in the absence of cell anchorage is more closely associated with cancer cell lines than resistance to apoptosis upon the deprivation of cell anchorage.

Topics: Apoptosis; Aspartic Acid; Calcimycin; Calcium; Caspase Inhibitors; Caspases; Cell Adhesion; Cell Culture Techniques; Cell Division; Cell Line; Cervix Uteri; Cycloheximide; DNA Fragmentation; Electrophoresis, Gel, Pulsed-Field; Female; Flow Cytometry; Humans; Infant; Ionophores; Keratinocytes; Protease Inhibitors; Protein Synthesis Inhibitors; Uterine Cervical Neoplasms

The CaSki cell line derived from an epidermoid carcinoma of the uterine cervix produces and releases a tumor associated-antigen, TA-4. The authors have already reported that EGF stimulated the production and secretion of TA-4 by the CaSki cells. EGF receptor is known to be one of the proteins phosphorylated by C-kinase. In order to elucidate a possible role of signal transduction systems (cAMP-A-kinase, diacyglycerol-C-kinase and Ca(2+)-calmodulin) in the regulation of TA-4 production and secretion by human cervical epidermoid carcinoma cells, the effects of cholera toxin (CT), an activator of adenylate cyclase, phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, and Ca2+ ionophore A23187, an activator of Ca2+ modulation on TA-4 production and secretion by CaSki cells were evaluated. TA-4 in the cultured cells and media were measured with a SCC RIA-Kit. The addition of PMA or Ca2+ ionophore to the medium caused increases in the cellular levels of TA-4 and TA-4 levels in the medium in a dose-dependent manner shortly after the addition. Combined treatment with PMA and Ca2+ ionophore did not cause additive increases in TA-4 levels in the cells and medium compared to the treatment with PMA alone or Ca2+ ionophore alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Antigens, Neoplasm; Calcimycin; Carcinoma, Squamous Cell; Cholera Toxin; Female; Humans; Serpins; Signal Transduction; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Uterine Cervical Neoplasms

Epithelial morphogenesis in many organs involves asymmetric microfilament-mediated cellular contractions. Similar contractions, in terms of ultrastructure and cytochalasin B sensitivity, can be induced in the carcinoma cell line C-4II in culture. This line was used to compare total intracellular calcium levels ([Ca]i) in contracted monolayer fragments and in control cultures, and to determine whether epithelial cell contraction depends on influx of extracellular Ca. [Ca]i, defined as Ca not displaceable by lanthanum, was measured by atomic absorption spectrophotometry. Degrees of contraction were determined from shape changes of monolayer fragments. Detachment from the growth surface initiated cellular contractions and caused an immediate increase in [Ca]i, from 1.0 to 4.0-5.0 micrograms Ca/mg protein in early confluent cultures, and from 0.3 to 1.0-2.0 micrograms Ca/mg protein in crowded cultures. This increase was followed by a gradual decline in [Ca]i, though Ca levels remained higher than in controls and contraction progressed for 30 min. Contraction was inhibited completely by cold (7 degrees C) and by Ca-free medium, and in a dose-dependent manner by papaverine (2.5 x 10(-6) M-2.5 x 10(-4) M), lanthanum (1.0 x 10(-6) M-1.0 x 10(-4) M); and D-600 (1.0-2.0 x 10(-4) M). The Ca ionophore A23187 had no effect at 5.0 x 10(-6) M and was inhibitory at higher concentrations. The results provided direct evidence for increased [Ca]i in contracting epithelial cells, and suggest that Ca influx is required for such contraction to take place.

Topics: Calcimycin; Calcium; Cell Line; Cell Movement; Cold Temperature; Cytoskeleton; Epithelium; Female; Gallopamil; Humans; Lanthanum; Magnesium; Papaverine; Uterine Cervical Neoplasms