Selective Killing Effects of Cold Atmospheric Pressure Plasma with NO Induced Dysfunction of Epidermal Growth Factor Receptor in Oral Squamous Cell Carcinoma

The aim of this study is to investigate the effects of cold atmospheric pressure plasma (CAP)-induced radicals on the epidermal growth factor receptor (EGFR), which is overexpressed by oral squamous cell carcinoma, to determine the underlying mechanism of selective killing. CAP-induced highly reactive radicals were observed in both plasma plume and cell culture media. The selective killing effect was observed in oral squamous cell carcinoma compared with normal human gingival fibroblast. Degradation and dysfunction of EGFRs were observed only in the EGFR-overexpressing oral squamous cell carcinoma and not in the normal cell. Nitric oxide scavenger pretreatment in cell culture media before CAP treatment rescued above degradation and dysfunction of the EGFR as well as the killing effect in oral squamous cell carcinoma. CAP may be a promising cancer treatment method by inducing EGFR dysfunction in EGFR-overexpressing oral squamous cell carcinoma via nitric oxide radicals.     

Ubiquitin-specific protease 14 regulates cell proliferation and apoptosis in oral squamous cell carcinoma

Ubiquitin-specific protease 14, a deubiquitinating enzyme, has been implicated in the tumorigenesis and progression of several cancers, but its role in oral squamous cell carcinoma remains to be elucidated. The aim of this study was to explore the expression pattern and roles of Ubiquitin-specific protease 14 in the occurrence and development of oral squamous cell carcinoma. Interestingly, Ubiquitin-specific protease 14 was overexpressed in oral cancer tissues and cell lines at both mRNA and protein levels. b-AP15, a specific inhibitor of Ubiquitin-specific protease 14, significantly inhibited the growth of cancer cells and increased cell apoptosis in a dose-dependent manner. Moreover, knockdown of Ubiquitin-specific protease 14 by shRNA significantly inhibited the proliferation and migration of cancer cells in vitro. Finally, using a xenograft mouse model of oral squamous cell carcinoma, knockdown of Ubiquitin-specific protease 14 markedly inhibited tumor growth and triggered the cancer cell apoptosis in vivo, supporting previous results. In conclusion, for the first time we have demonstrated the expression pattern of Ubiquitin-specific protease 14 in oral squamous cell carcinoma and verified a relationship with tumor growth and metastasis. These results may highlight new therapeutic strategies for tumor treatment, application of Ubiquitin-specific protease 14 selective inhibitor, such as b-AP15, or knockdown by shRNA. Collectively, Ubiquitin-specific protease 14 could be a potential therapeutic target for oral squamous cell carcinoma patients.     

Comparative proteomics approach to screening of potential diagnostic and therapeutic targets for oral squamous cell carcinoma

This work demonstrates that a comprehensive strategy of proteomics identification combined with further validation and detailed functional analysis should be adopted in the field of cancer biomarker discovery. A comparative proteomics approach was utilized to identify differentially expressed proteins in 10 oral squamous carcinoma samples paired with their corresponding normal tissues. A total of 52 significantly and consistently altered proteins were identified with eight of these being reported for the first time in oral squamous carcinoma. Of the eight newly implicated proteins, RACK1 was chosen for detailed analysis. RACK1 was demonstrated to be up-regulated in cancer at both the mRNA and protein levels. Immunohistochemical examination showed that the enhanced expression of RACK1 was correlated with the severity of the epithelial dysplasia as well as clinical stage, lymph node involvement, and recurrence, which are known indicators of a relatively poor prognosis in oral squamous carcinoma patients. RNA interference specifically targeted to silence RACK1 could initiate apoptosis of oral squamous carcinoma cells. Taken together, the results indicate that RACK1 is up-regulated in oral squamous carcinoma, not only being closely related to cell proliferation and apoptosis but also linked to clinical invasiveness and metastasis in carcinogenesis. The observations suggest that RACK1 may be a potential biomarker for early diagnosis, prognosis, and monitoring in the therapy of oral squamous carcinoma. Further this comprehensive strategy could be used for identifying other differentially expressed proteins that have potential to be candidate biomarkers of oral squamous carcinoma.     

Inhibition of lymphokine-activated killer cell generation by blocking factors in sera of patients with head and neck cancer

Summary Cytolytic activation of peripheral blood lymphocytes by recombinant interleukin-2 (rIL-2) in patients with squamous cell carcinoma (SCC) of the head and neck may be inhibited by serum blocking factors, and this could influence therapeutic efficacy. Peripheral blood lymphocytes from 21 patients with this disease and 17 controls were incubated with 10–1000 U rIL-2 for 6 days in supplemented complete medium (containing 10% fetal calf serum) or the same medium plus 10% autologous serum. After washing the effector cells, we determined their cytotoxicity against K562 and MDA1386, a lymphokine-activated-killer(LAK)-sensitive SCC cell line, using a⁵¹Cr-release assay. Patient sera inhibited LAK-generated lysis of both MDA 1386 and K562, while control sera from healthy persons inhibited LAK-generated lysis of MDA1386. The blocking activity of patient sera tended to be greater than that of control sera. The sera of patients with untreated or recurrent disease and those who were free of disease had equivalent inhibitory capacity. The serum blocking factor acted in a dose-dependent manner, and inhibition was overcome by increasing the dose of rIL-2. Levels of circulating immune complexes (measured by the Clq binding method) did not correlate significantly with inhibition. A clinical protocol of repeated plasma exchange in patients with advanced and recurrent squamous cell carcinoma of the head and neck allowed sequential study of one patients's serum before, during, and after treatments. Plasmapheresis removed serum inhibitory factors, albeit temporarily. The activity of serum blocking factors in patients with this disease can be modulated by increasing doses of rIL-2 and by plasma exchange. This modulation may be important to improving clinical response rates for patients undergoing immunotherapy.     

Potential role of P2X7R in esophageal squamous cell carcinoma proliferation

Esophageal cancer is an aggressive tumor and is the sixth leading cause of cancer death worldwide. ATP is well known to regulate cancer progression in a variety of models by different mechanisms, including P2X7R activation. This study aimed to evaluate the role of P2X7R in esophageal squamous cell carcinoma (ESCC) proliferation. Our results show that treatment with high ATP concentrations induced a decrease in cell number, cell viability, number of polyclonal colonies, and reduced migration of ESCC. The treatment with the selective P2X7R antagonist A740003 or siRNA for P2X7 reverted this effect in the KYSE450 cell line. In addition, results showed that P2X7R is highly expressed, at mRNA and protein levels, in KYSE450 lineage. Additionally, KYSE450, KYSE30, and OE21 cells express P2X3R, P2X4R, P2X5R, P2X6R, and P2X7R genes. P2X1R is expressed by KYSE30 and KYSE450, and only KYSE450 expresses the P2X2R gene. Furthermore, esophageal cancer cell line KYSE450 presented higher expression of E-NTPDases 1 and 2 and of Ecto-5′-NT/CD73 when compared to normal cells. This cell line also exhibits ATPase, ADPase, and AMPase activity, although in different levels, and the co-treatment of apyrase was able to revert the antiproliferative effects of ATP. Moreover, results showed high immunostaining for P2X7R in biopsies of patients with esophageal carcinoma, indicating the involvement of this receptor in the growth of this type of cancer. The results suggest that P2X7R may be a potential pharmacological target to treat ESCC and can lead us to further investigate the effect of this receptor in cancer cell progression.     

Growth of normal oral keratinocytes and squamous cell carcinoma cells in a novel protein-free defined medium

Summary A novel protein-free synthetic medium was developed for the culture of normal human oral keratinocytes. This medium, designated PFM-7, supports the serial cultivation of primary or secondary normal oral keratinocytes in protein-free, chemically defined conditions. Normal oral keratinocytes in PFM-7 exhibited nearly equal growth in mass culture without noticeable changes in morphology, response to added growth factors, or gene expression of growth factors and their receptors, compared to cells in Keratinocyte-SFM containing epidermal growth factor and bovine pituitary extract. Furthermore, PFM-7 supported the serial subcultivation of human squamous cell carcinoma cells and enabled both normal and malignant oral squamous cells derived from the same patient to grow under the same protein-free defined conditions. These results indicate that PFM-7 can be used for precise investigations of growth mechanisms, cell products, and gene expression associated with carcinogenesis of human epidermal cells.     

Familial risks of squamous cell carcinoma of the head and neck: retrospective case-control study.

OBJECTIVE: To determine the contribution of inheritance to the incidence of squamous cell carcinoma of the head and neck. DESIGN: Historical cohort study. First degree relatives of cases with squamous cell carcinoma of the head and neck made up the exposed cohort and first degree relatives of spouses of cases made up the comparison unexposed cohort. SETTING: Ear, nose, and throat clinic in a large metropolitan teaching hospital. SUBJECTS: 1429 first degree relatives of 242 index cases of squamous cell carcinoma of the head and neck; as controls, 934 first degree relatives of the spouses of 156 index cases. MAIN OUTCOME MEASURES: Relative risk of developing squamous cell carcinoma in first degree relatives of cases compared with risk in first degree relatives of spouses. RESULTS: The adjusted relative risk for developing head and neck cancer if the index case had squamous cell carcinoma of the head and neck was 3.79 (95% confidence interval 1.11 to 13.0). There were no significantly increased risks associated with a family history of cancer at other sites. The adjusted relative risk for squamous cell carcinoma of the head and neck was 7.89 (1.50 to 41.6) in first degree relatives of patients with multiple primary head and neck tumours. CONCLUSIONS: These data suggest that genetic factors are important in the aetiology of head and neck cancer, in particular for patients with multiple primary cancers. Given the prolonged exposure of these subjects to carcinogens, these genetic factors may have a role in modifying carcinogen activity or in host resistance to carcinogens. Inherited factors may be important in persons with environmentally induced cancers.     

Selection for EGFR gene amplification in a breast epithelial cell line with basal-like phenotype and hereditary background

An epithelial cell line, referred to as A163, was established from breast carcinoma derived from a patient with a strong family history of breast cancer but no known breast cancer susceptibility mutation. A163 was propagated in a serum-free culture medium including the epidermal growth factor. Immunophenotypic characterization demonstrated a mixed luminal and basal-like phenotype. When epidermal growth factor was excluded from the culture medium, A163 entered a quiescent period followed by a period of increased cell proliferation in a subpopulation of the cells. The epidermal growth factor-independent subpopulation retained the basal-like phenotype of the parental cell line. Karyotype and fluorescent in situ hybridization analysis showed an amplification of epidermal growth factor receptor on 7q in A163-S1 only, resulting in high expression of total and phosphorylated epidermal growth factor receptor. The A163-S1 sub-line piles up in culture, indicating a loss of contact inhibition. When grown on transwell filters, A163 shows basal expression of P63 and cytokeratin 14, whereas A163-S1 expresses P63 ubiquitously, and has lost the basal specific expression of cytokeratin 14, indicating a loss of polarity. Furthermore, when cultured in reconstituted basement membrane matrix, A163 form polarized normal like acini. In contrast, A163-S1 form large disorganized structures with lack of polarity. These cell lines may prove useful to understand molecular changes in breast cancer progression, in particular basal-like breast cancer subtype with bad prognosis and no current treatment options.     

The squamous cell carcinoma cell line OM-1 retains both p75-dependent stratified epithelial progenitor potential and cancer stem cell properties

The low-affinity nerve growth factor receptor p75 is a stratified epithelial stem/progenitor marker of human epithelia. We found OM-1, a human squamous cell carcinoma (SCC) cell line, showed distinct cells with p75 cluster, especially located at the center of a growing colony in a monolayer culture. A cell with p75 cluster was surrounded by cytokeratin 14- and cytokeratin 13-expressing cells that settled at the outer margin of the colony. OM-1 cells were also capable of forming tumor spheres in a cell suspension culture, an ability which was attenuated by the inhibition of p75-signaling. Intriguingly, we also found a p75-negative cell population from a growing culture of OM-1 that re-committed to become p75-clustering cells. These results indicated the possibility that SCC with epithelial multi-layering capacity can exploit the p75-dependent stratified epithelial progenitor property for the cancer stemness.     

MicroRNA-181a enhances the chemoresistance of human cervical squamous cell carcinoma to cisplatin by targeting PRKCD

MicroRNAs(miRNAs) are involved in regulating the response of cancer cells to various therapeutic interventions, but their involvement in the chemoresistance of human cervical squamous cell carcinoma is not fully understood. We found miR-181a was significantly up-regulated in specimens from patients with chemoresistant cervical squamous cell carcinoma. In this study, we aimed to clarify the role of miR-181a in regulating the chemoresistance of cervical cancer. Two human cervical squamous cancer cell lines, SiHa and Me180, were used. Enforced expression of miR-181a enhanced chemoresistance to cisplatin in cervical cancer cells through apoptosis reversion. In a nude mouse xenograft model, the overexpression of miR-181a markedly inhibited the therapeutic response to cisplatin. PRKCD, a target gene of miR-181a and a promoter of apoptosis, was negatively regulated by miR-181a. We found that the effect of miR-181a on chemoresistance was mediated by PRKCD. Additionally, silencing of PRKCD yielded an effect similar to that of miR-181a up-regulation and inhibited apoptosis in cervical cancer cells. Our findings suggest that miR-181a may function as an oncogene and induce chemoresistance in cervical squamous cell carcinoma cells at least in part by down-regulating PRKCD, thus may provide a biomarker for predicting chemosensitivity to cisplatin in patients with cervical squamous cancer.     

Procoagulant Activity in Amniotic Fluid Is Associated with Fetal-Derived Extracellular Vesicles

Procoagulant activity in amniotic fluid (AF) is positively correlated with phosphatidylserine (PS) and tissue factor (TF)-expressing(+) extracellular vesicles (EVs). However, it is unknown if pathological fetal conditions may affect the composition, phenotype, and procoagulant potency of EVs in AF. We sought to evaluate EV-dependent procoagulant activity in AF from pregnant people with fetuses with or without diagnosed chromosomal mutations. AF samples were collected by transabdominal amniocentesis and assessed for common karyotype defects (total n = 11, 7 healthy and 4 abnormal karyotypes). The procoagulant activity of AF was tested using a fibrin generation assay with normal pooled plasma and plasmas deficient in factors XII, XI, IX, X, V, and VII. EV number and phenotype were determined by flow cytometry with anti-CD24 and anti-TF antibodies. We report that factor-VII-, X-, or V-deficient plasmas did not form fibrin clots in the presence of AF. Clotting time was significantly attenuated in AF samples with chromosomal mutations. In addition, CD24+, TF+, and CD24+ TF+ EV counts were significantly lower in this group. Finally, we found a significant correlation between EV counts and the clotting time induced by AF. In conclusion, we show that AF samples with chromosomal mutations had fewer fetal-derived CD24-bearing and TF-bearing EVs, which resulted in diminished procoagulant potency. This suggests that fetal-derived EVs are the predominant source of procoagulant activity in AF.     

Prostaglandin E2 concentrations in naturally occurring canine cancer

The purpose of this study was to determine the PGE2 concentration in naturally-occurring cancer in pet dogs and in canine cancer cell lines in order to identify specific types of canine cancer with high PGE2 production which could serve as preclinical models to evaluate anticancer strategies targeting PGE2. PGE2 concentrations were measured by enzyme immunoassay in canine melanoma, soft tissue sarcoma, transitional cell carcinoma, osteosarcoma, and prostatic carcinoma cell lines; in 80 canine tumor tissue samples including oral melanoma (MEL), oral squamous cell carcinoma (SCC), transitional cell carcinoma of the urinary bladder (TCC), lymphoma (LSA), mammary carcinoma (MCA), osteosarcoma (OSA), prostatic carcinoma (PCA); and in corresponding normal organ tissues. High concentrations of PGE(2)(range 400-3300 pg/10(4)cells) were present in cell culture medium from the transitional cell carcinoma, prostatic carcinoma, and osteosarcoma cell lines. PGE2 concentrations in tumor tissues were elevated (tumor PGE2 concentration>mean+2X sd PGE(2)concentration of normal organ tissue) in 21/22 TCC, 5/6 PCA, 7/10 SCC, 5/10 MEL, 3/8 MCA, 4/15 OSA, and 0/9 LSA. Results of this study will help guide future investigations of anticancer therapies that target cyclooxygenase and PGE2.     

Production of soluble matriptase by human cancer cell lines and cell surface activation of its zymogen by trypsin

The membrane-bound serine proteinase matriptase, which is often released from the plasma membrane of epithelial and carcinoma cells, has been implicated to play important roles in both physiological and pathological conditions. However, the regulatory mechanism of its activity is poorly understood. In the present study, we examined expression and activation state of soluble matriptase in 24 human cancer cell lines. Soluble matriptase was detected in the conditioned media from all of 5 colon and 4 breast carcinoma cell lines and 8 of 10 stomach carcinoma cell lines tested. Only two of five lung cancer cell lines released the matriptase protein into the culture media. Out of the five matriptase-negative cell lines, two cell lines expressed the matriptase mRNA. Among 24 cancer cell lines tested, 13 cell lines secreted trypsin in an active or latent form and all of them released matriptase. Most of the 24 cell lines released a latent, single-chain matriptase of 75 kDa as a major form, as well as low levels of complex forms of an activated two-chain enzyme with its specific inhibitor HAI-1. Thus, these soluble matriptases appeared to have little proteolytic activity. Treatment of stomach and colon cancer cell lines with epidermal growth factor stimulated the release of matripatase/HAI-1 complexes. In cancer cell lines secreting active trypsin, however, matriptase was released mostly as an inhibitor-free, two-chain active form. Trypsin seemed to activate the membrane-bound, latent matriptase on the cell surface. These results suggest that matriptase and trypsin cooperatively function for extracellular proteolysis.     

Neoplastic differentiation: characteristics of cell lines derived from a murine teratocarcinoma

Monolayer cultures of a mouse teratocarcinoma were established in vitro. These cultures contained embryonal carcinoma, the malignant stem cell, and its differentiated progeny: parietal yolk sac, neuroepithelial, and mesenchymal cells. Tissues such as squamous epithelium, cartilage, striated muscle, neuroepithelium, and glands were produced from embryonal carcinoma that was maintained under conditions of long term culture. Frequent subcultivation with pancreatin allowed the establishment of cell lines of embryonal carcinoma which have been maintained for more than 18 months in vitro and continue to produce differentiated cells under specific culture conditions. Chromosomally these lines of embryonal carcinoma have a stem line of 39 chromosomes. Two lines of parietal yolk sac cells have been established which produce basement membrane, are not tumorigenic, and chromosomally are hypotetraploid. This system may yield information concerning neoplastic differentiation and its possible use in therapy for cancer.     

Growth of the malignant and nonmalignant human squamous cells in a protein-free defined medium

Summary A novel protein-free synthetic medium has been developed for the culture of human squamous cell carcinoma cells. This medium, designated PF86-1, supports the serial subcultivation of six out of nine human squamous cell carcinoma cell lines in a protein-free, chemically defined condition without the adapting culture from serum-containing conditions. These cell lines growing in PF86-1 exhibited nearly equal potency to grow in massive culture without noticeable changes in morphology but presented a significantly decreased level of colony forming efficiency when compared with the cells cultured in serum-containing media, suggesting the implication of some autocrine mechanism. Interestingly, this medium supported the growth of normal human squamous cells of oral mucosa and skin for more than 2 mo. in the primary explant culture in spite of high levels of calcium ion concentration, where the overgrowth of fibroblasts as contaminant was not observed. These results suggest that PF86-1 supports the growth of cells derived from epidermal tissues selectively and provides the same defined condition for growth of malignant and nonmalignant human squamous cells. It seems, therefore, that PF86-1 allows investigations on the products of squamous cell carcinoma cells or on the differences of growth mechanisms between normal and neoplastic human squamous cells.     

High affinity binding of VIP to human lung cancer cell lines

The binding of ¹²⁵I-VIP to human lung cancer cell lines was investigated. Radiolabeled VIP bound to adenocarcinoma, squamous cell carcinoma, large cell carcinoma and small cell lung cancer (SCLC) cell lines. As SCLC cell line NCI-N592 bound radiolabeled VIP well, its binding was further characterized. ¹²⁵I-VIP bound to membranes in a specific and time dependent manner. ¹²⁵I-VIP bound with high (Kd=0.8 nM) and moderate affinity (Kd=66 nM) to two classes of sites. Pharmacology studies indicated that the order of peptide potency was VIP PHI > secretin > VIP^10–28. Because VIP receptors are present on human lung cancer cells, VIP may function as a regulatory peptide in lung cancer.     

Mitogenic activity elaborated by macrophage-like cell lines acts as competence factor(s) for BALB/c 3T3 cells

The culture medium from several murine macrophage-like cell lines contained a mitogenic activity that functioned synergistically with platelet-poor plasma to induce DNA synthesis in quiescent density-inhibited BALB/c 3T3 fibroblasts. This mitogenic activity was generated by P388D₁ (and other established lines of) macrophage-like cells that were cultured either in medium alone or in medium supplemented with platelet-poor plasma. The amount of mitogenic activity produced was directly related to the length of time the macrophage-like cells were maintained in the medium. Serum-free medium conditioned by macrophage-cells did not stimulate DNA synthesis in density-inhibited 3T3 cells in the absence of plasma; however, a transient (4-hr) exposure to serum-free macrophage-conditioned medium allowed quiescent cells to respond to plasma-derived progression factors. The addition of plasma to 3T3 cells that had been treated with the macrophage-conditioned medium brought about DNA synthesis after a 12-hr lag. The mitogenic activity that was in macrophage-conditioned medium bound to DEAE-Sephadex and eluted in a single peak using a linear NaCl gradient. This macrophage-derived competence factor was not mitogenic for lymphocytes and was clearly separated by DEAE-Sephadex chromatography from the major peak of the previously described mitogenic monokine, Interleukin-I (lymphocyte activating factor).     

Characterization of the state of differentiation of six newly established human non-small-cell lung cancer cell lines

Six new non-small-cell lung cancer (NSCLC) cell lines were established directly from human tissue or indirectly via nude mouse xenografts in serum-supplemented media with success rates of 8% and 13%, respectively. They comprised one adenocarcinoma (ADLC-5M2), two squamous cell carcinomas (EPLC-32M1, EPLC-65H), two large cell carcinomas (LCLC-97TM1, LCLC-103H), and one malignant biphasic mesothelioma (MSTO-211H). All cell lines grew adherent to culture vessels with population doubling times (PDT) of 16-40 h, formed colonies in soft agarose with efficiencies of 0.1%-5.1%, and all grew in athymic nude mice. Xenograft histologies appeared as follows: (a) undifferentiated carcinomas with feeble resemblance to the original tumors in the case of adenocarcinomas and squamous cell carcinomas; (b) large cell carcinoma with high resemblance to the original tumor; (c) an undifferentiated tumor with predominance of large epithelial cells and few fibrous cells in the case of mesothelioma. Human chorionic gonadotropin (HCG) was found by radioimmunoassay and high-affinity binding sites for epidermal growth factor (EGF) by radio-receptor assay in 4/4 cell lines. A very low activity of L-DOPA decarboxylase (DDC) was detectable only in the adenocarcinoma cell line. All cell lines overexpressed the c-myc protooncogene, and no gene rearrangement or amplification was observed. Chromosome analysis revealed modal chromosome numbers of 70-73 in ADLC-5M2, EPLC-32M1, EPLC-65H, and MSTO-211H. Cell lines derived from large cell carcinoma had modal values of 65 and 170 and a wider chromosome distribution than all other cell lines. A NSCLC specific chromosomal aberration has been undetectable until now. These cell lines may aid in elucidating the biology of NSCLC and its interrelationship to other lung tumors.