Vasoactive intestinal polypeptide binds with high affinity to non-small cell lung cancer cells and elevates cyclic AMP levels

Vasoactive intestinal polypeptide (VIP) receptors were characterized on non-small cell lung cancer (NSCLC) cells. ¹²⁵I-VIP bound specifically to membranes derived from 6 NSCLC cell lines. Specific ¹²⁵I-VIP was time dependent and a linear function of EPLC-65H membrane concentration. ¹²⁵I-VIP bound with high (K_d=0.2 nM) and moderate (K_d=39 nM) affinity to two classes of sites. Pharmacology studies indicated that the order of peptide potency was VIP > rGHRH > PHI = helodermin > secretin > glucagon. Also VIP elevated the cAMP levels 10-fold using cell line ADLC-5M2. These data indicate that functional VIP receptors are present on NSCLC cells.     

Alterations in manganese, copper, and zinc contents, and intracellular status of the metal-containing superoxide dismutase in human mesothelioma cells

BACKGROUND AND AIM: Molecular diagnostics and therapeutics of human mesothelioma using disease-related markers present major challenges in clinical practice. To identify biochemical alternations that would be markers of human mesothelioma, we measured the intracellular steady-state levels of biologically important trace metals such as manganese (Mn), copper (Cu), and zinc (Zn) in a human mesothelial cell line, MeT-5A, and in five human mesothelioma cell lines (MSTO-211H, NCI-H226, NCI-H2052, NCI-H2452, ACC-MESO-1) by inductively coupled plasma-mass spectrometry (ICP-MS). We also aimed to investigate whether the alterations were related to the intracellular status of metal-containing superoxide dismutase (SOD). RESULTS: There were no significant differences in the contents of the trace metals among MeT-5A, MSTO-211H, and ACC-MESO-1 cells. However, each of the other three mesothelioma cell lines had a unique characteristic in terms of the intracellular amounts of the metals; NCI-H226 contained an extremely high level of Mn, an amount 7.3-fold higher than that in MeT-5A. NCI-H2052 had significantly higher amounts of Cu (3.4-fold) and Zn (1.3-fold) compared with MeT-5A. NCI-H2452 contained about 5.8-fold the amount of Cu and 2.5-fold that of Mn compared with MeT-5A. As for the intracellular levels of copper/zinc-SOD (Cu/Zn-SOD) and manganese-SOD (Mn-SOD), those of Cu/Zn-SOD were relatively unchanged among the cells tested, and no notable correlation with Cu or Zn contents was observed. On the other hand, all mesothelioma cells highly expressed Mn-SOD compared with MeT-5A, and a very high expression of the enzyme with a robust activity was observed in the two mesothelioma cells (NCI-H226, NCI-H2452) containing a large amount of Mn. CONCLUSIONS: In comparison with MeT-5A human mesothelial cells, some human mesothelioma cells had significantly higher amounts of Mn or Cu and one mesothelioma cell had a significantly higher amount of Zn. Interestingly, all mesothelioma cells overexpressed Mn-SOD compared with MeT-5A, and the cells whose Mn-SOD activity was increased contained higher amounts of Mn. It seemed that intracellular Mn content was positively correlated with Mn-SOD, suggesting that the intracellular Mn level is associated with Mn-SOD activity. These biochemical signatures could be potential disease-related markers of mesothelioma.     

Resveratrol and clofarabine induces a preferential apoptosis-activating effect on malignant mesothelioma cells by Mcl-1 down-regulation and caspase-3 activation

We previously demonstrated that resveratrol and clofarabine elicited a marked cytotoxicity on malignant mesothelioma (MM) MSTO-211H cells but not on the corresponding normal mesothelial MeT-5A cells. Little is known of the possible molecules that could be used to predict preferential chemosensitivity on MSTO-211H cells. Resveratrol and clofarabine induced down-regulation of Mcl-1 protein level in MSTO-211H cells. Treatment of cells with cycloheximide in the presence of proteasome inhibitor MG132 suggested that Mcl-1 protein levels were regulated at the post-translational step. The siRNA-based knockdown of Mcl-1 in MSTO-211H cells triggered more growth-inhibiting and apoptosis-inducing effects with the resultant cleavages of procaspase-3 and its substrate PARP, increased caspase-3/7 activity, and increased percentage of apoptotic propensities. However, the majority of the observed changes were not shown in MeT-5A cells. Collectively, these studies indicate that the preferential activation of caspase cascade in malignant cells might have important applications as a therapeutic target for MM. [BMB Reports 2015; 48(3): 166-171]     

Natural diterpenes from coffee,cafestol and kahweol induce apoptosis through regulation of specificity protein 1 expression in human malignant pleural mesothelioma

Background

Malignant pleural mesothelioma (MPM) is a highly aggressive cancer with a very poor prognosis. Several clinical studies such as immunotherapy, gene therapy and molecular targeting agents have been tried for treatment of malignant mesothelioma, however, there is no application for effective clinical treatment. Coffee has various biological functions such as anti-oxidant, anti-inflammatory, anti-mutagenic and anti-carcinogenic activities. The therapeutic activities of the bioactive compounds in coffee was sugested to influence intracellular signaling of MPM. Regarding to the cancer-related functions, In this study, suppression of Sp1 protein level followed by induction of MSTO-211H cell apoptosis by cafestol and kahweol were investigated in oreder to determine Sp1''s potential as a significant target for human MPM therapy as well.

Methods

Cells were treated separately with final concentration of cafestol and kahweol and the results were analyzed by MTS assay, DAPI staining, PI staining, luciferase assay, RT-PCR, and immunoblotting.

Results

Viability of MSTO-211H and H28 cells were decreased, and apoptotic cell death was increased in MSTO-211H as a result of cafestol and kahweol treatment. Cafestol and kahweol increased Sub-G₁ population and nuclear condensation in MSTO-211H cells. Roles of Sp1 in cell proliferation and apoptosis of the MSTO-211H cells by the Sp1 inhibitor of Mithramycin A were previously confirmed. Cafestol and kahweol significantly suppressed Sp1 protein levels. Kahweol slightly attenuated Sp1 mRNA, while Cafestol did not affect in MSTO-211H cells. Cafestol and kahweol modulated the promoter activity and protein expression level of the Sp1 regulatory genes including Cyclin D1, Mcl-1, and Survivin in mesothelioma cells. Apoptosis signaling cascade was activated by cleavages of Bid, Caspase-3, and PARP with cafestol and by upregulation of Bax, and downregulation of Bcl-_xl by kahweol.

Conclusions

Sp1 can be a novel molecular target of cafestol and kahweol in human MPM.     

Synthesis and biological evaluation of novel tamoxifen analogues

A collection of compounds, structurally related to the anticancer drug tamoxifen, used in breast cancer therapy, were designed and synthesized as potential anticancer agents. McMurry coupling reaction was used as the key synthetic step in the preparation of these analogues and the structural assignment of E, Z isomers was determined on the basis of 2D-NOESY experiments. The compounds were evaluated for their antiproliferative activity on breast cancer (MCF-7), cervix adenocarcinoma (HeLa) and biphasic mesothelioma (MSTO-211H) human tumor cell lines. The estrogen like properties of the novel compounds were compared with those of the untreated controls using an estrogen responsive element-based (ERE) luciferase reporter assay and compared to 17β-estradiol (E2). Finally, with the aim to correlate the antiproliferative activity with an intracellular target(s), the effect on relaxation activity of DNA topoisomerases I and II was assayed.     

Simultaneous immunoenzyme staining of vimentin and cytokeratins with monoclonal antibodies as an aid in the differential diagnosis of malignant mesothelioma from pulmonary adenocarcinoma

The occurrence and coexpression of the cytoskeletal proteins vimentin and cytokeratins were studied in malignant mesotheliomas and pulmonary carcinomas. For this purpose a double immunoenzyme staining with monoclonal antibodies was developed which made it possible to visualize vimentin and cytokeratins simultaneously within the same cell. A clear distinction between stromal cells (vimentin only) and tumour cells was also obtained. A total of 12 mesotheliomas (six mixed type and six epithelioid type) and 13 carcinomas (eight adenocarcinomas and five large cell undifferentiated carcinomas) were studied. The results revealed a clear difference between mesotheliomas and adenocarcinomas: 11 of 12 mesotheliomas showed coexpression of vimentin and cytokeratins in at least 50% of the tumour cells, while in seven of the eight adenocarcinomas none or only a few cells could be seen with this coexpression. In the undifferentiated large cell carcinomas three of five expressed both components, but in less than 25% of the cells. It is concluded that a reliable double immunoenzyme staining of vimentin and cytokeratins can be used as an additional means to distinguish malignant mesothelioma from pulmonary adenocarcinoma.     

Pharmacological targeting of p53 through RITA is an effective antitumoral strategy for malignant pleural mesothelioma

Malignant mesothelioma, a very aggressive tumor associated to asbestos exposure, is expected to increase in incidence, and unfortunately, no curative modality exists. Reactivation of p53 is a new attractive antitumoral strategy. p53 is rarely mutated in mesothelioma, but it is inactivated in most tumors by the lack of p14^ARF. Here, we evaluated the feasibility of this approach in pleural mesothelioma by testing RITA and nutlin-3, two molecules able to restore p53 function through a different mechanism, on a panel of mesothelioma cell lines representing the epithelioid (NCI-H28, NCI-H2452, IST-MES 2), biphasic (MSTO-211H), and sarcomatoid (NCI-H2052) histotypes compared with the normal mesothelial HMC-hTERT. RITA triggered robust caspase-dependent apoptosis specifically in epithelioid and biphasic mesothelioma cell lines, both through wild-type and mutant p53, concomitant to p21 downregulation. Conversely, nutlin-3 induced a p21-dependent growth arrest, rather than apoptosis, and was slightly toxic on HMC-hTERT.

Interestingly, we identified a previously undetected point mutation of p53 (p.Arg249Ser) in IST-MES 2, and showed that RITA is also able to reactivate this p53 mutant protein and its apoptotic function. RITA reduced tumor growth in a MSTO-211H-derived xenograft model of mesothelioma and synergized with cisplatin, which is the mainstay of treatment for this tumor.

Our data indicate that reactivation of p53 and concomitant p21 downregulation effectively induce cell death in mesothelioma, a tumor characterized by a high intrinsic resistance to apoptosis. Altogether, our findings provide the preclinical framework supporting the use of p53-reactivating agents alone, or in combination regimens, to improve the outcome of patients with mesothelioma.     

Can cytology reliably subtype non-small cell lung carcinomas?

Cytology specimens play an important role in the diagnosis and predictive testing of lung cancer. While morphological characterisation of small cell and non-small cell lung carcinomas (NSCLC) on cytology is possible, further subtyping of NSCLC into adenocarcinoma and squamous cell carcinoma morphology is also mandatory in the current era of personalised medicine. Notably, cytology specimens in different forms (fine needle aspiration, exfoliative, and cell block) with or without immunocytochemistry are reliable sources for accurate diagnosis of adenocarcinoma and squamous cell carcinoma as evidenced by numerous studies present in the literature. However, there are instances where subtyping of NSCLC based on morphology alone is challenging on cytology samples, especially non-cell block preparations. In this paper, we will discuss current concepts, advances, and challenges of subtyping NSCLC in cytology specimens.     

Establishment of a human malignant fibrous mesothelioma cell line and the biological characteristics compared with malignant epithelial mesothelioma cell line

Two human malignant mesothelioma cell lines, which we designated "epithelial mesothelioma cells" and "fibrous mesothelioma cells", were established from the pleural fluid containing malignant mesothelial cells of a 72-year-old Japanese man. These cell lines were separated by the colonial techniques from the initiation of the primary cultures and grew well without interruption for 12 years. They were characterized as producing hyaluronic acid. These cell lines displayed different biological characteristics, including morphology, heterotransplantability and genetics using with BAC array CGH. The epithelial mesothelioma cells were epithelial in shape and transplantable into the subcutis of nude mice, while the cells of the fibrous mesothelioma line were fibroblast-like and transplantable into the submucosa of Hamster's cheek pouches but not into the subcutis of nude mice. The mesotheliomas are classified into three types: epithelial mesothelioma, fibrous mesothelioma and mixed type. The gene copy number losses observed on 9p21.3, 9p21.2, 9p21.1, among others may be a major mechanism of malignant mesothelioma carcinogenesis. We considered and supported the combination theory for the histogenesis of malignant mesothelioma.     

CD9 Negatively Regulates CD26 Expression and Inhibits CD26-Mediated Enhancement of Invasive Potential of Malignant Mesothelioma Cells

CD26/dipeptidyl peptidase IV is a cell surface glycoprotein which consists of multiple functional domains beside its ectopeptidase site. A growing body of evidence indicates that elevated expression of CD26 correlates with disease aggressiveness and invasive potential of selected malignancies. To further explore the molecular mechanisms involved in this clinical behavior, our current work focused on the interaction between CD26 and CD9, which were recently identified as novel markers for cancer stem cells in malignant mesothelioma. We found that CD26 and CD9 co-modulated and co-precipitated with each other in the malignant mesothelioma cell lines ACC-MESO1 and MSTO-211H. SiRNA study revealed that depletion of CD26 led to increased CD9 expression, while depletion of CD9 resulted in increased CD26 expression. Consistent with these findings was the fact that gene transfer of CD26 into CD26-negative MSTO-211H cells reduced CD9 expression. Cell invasion assay showed that overexpression of CD26 or gene depletion of CD9 led to enhanced invasiveness, while CD26 gene depletion resulted in reduced invasive potential. Furthermore, our work suggested that this enhanced invasiveness may be partly mediated by α5β1 integrin, since co-precipitation studies demonstrated an association between CD26 and α5β1 integrin. Finally, gene depletion of CD9 resulted in elevated protein levels and tyrosine phosphorylation of FAK and Cas-L, which are downstream of β1 integrin, while depletion of CD26 led to a reduction in the levels of these molecules. Collectively, our findings suggest that CD26 potentiates tumor cell invasion through its interaction with α5β1 integrin, and CD9 negatively regulates tumor cell invasion by reducing the level of CD26-α5β1 integrin complex through an inverse correlation between CD9 and CD26 expression. Our results also suggest that CD26 and CD9 serve as potential biomarkers as well as promising molecular targets for novel therapeutic approaches in malignant mesothelioma and other malignancies.     

Centrifugal separation of cells in sputum specimens from patients with undifferentiated carcinoma

Ethanol-fixed cells in sputum from patients with undifferentiated carcinoma of the lung were separated in aqueous Ficoll using a discontinuous density gradient centrifugation technique. The selective enrichment of small cell undifferentiated (e.g., oat cell) or large cell undifferentiated carcinoma cells was achieved while removing most of the leukocytes (80-90%) and macrophages (65-75%) from specimen fractions containing the greatest relative frequencies of cancer cells. The maximum purity of small cell carcinoma cells (0.04%) occurs in moderate density (rho = 1.121 g/ml) gradient fractions and results in a 2.4-fold enrichment relative to unprocessed specimens. In contrast, the maximum purity of large cell carcinoma cells (0.22%) is obtained in very high density (rho = 1.172 g/ml) gradient fractions and results in a 1.2-fold enrichment in comparison with unprocessed specimens. Microscopic examination of Papanicolaou-stained specimen fractions reveals that these enrichments were achieved while retaining diagnostically significant cytomorphologic and tinctorial features necessary for cancer screening and diagnosis. Peak purity ranges of undifferentiated cancer cells significantly overlap comparable ranges for material from bronchogenic adenocarcinoma and squamous cell carcinoma.     

Cell biologic properties in explant culture and heterotransplantation of malignant uterine cervical cells

The cell of origin of uterine cervical cancer was studied by using culture, enzyme histochemistry and heterotransplantation. Twenty-seven epidermoid carcinomas (8 large cell keratinizing squamous, 12 large cell nonkeratinizing squamous and 7 small cell nonkeratinizing squamous) and 2 adenocarcinomas of the uterine cervix were placed in culture. An outgrowth of carcinoma cells in vitro was observed in 22 of 29 cases: 6 keratinizing, 8 large cell nonkeratinizing and 6 small cell nonkeratinizing carcinomas and 2 adenocarcinomas. The squamous carcinomas showed a squamous-cell outgrowth pattern, except for one large cell nonkeratinizing and three small cell nonkeratinizing carcinomas that showed a glandular-cell outgrowth pattern. One of three keratinizing carcinomas was transplantable into the subcutis of BALB/c nude mice, producing keratinizing tumors; three of six large cell and one of three small cell nonkeratinizing carcinomas reproduced themselves, while the other two small cell carcinomas produced poorly differentiated adenocarcinomas in mice. The transplanted adenocarcinoma produced a well-differentiated adenocarcinoma resembling the original tumor. Small cell carcinomas and adenocarcinomas contained a heat-stable, L-phenylalanine-sensitive alkaline phosphatase. These results suggest that many uterine cervical cancers originate from the reserve cell.     

Studies on the biology,histology and cytogenetics of two sublines of a murine ovarian reticular cell sarcoma

Two sublines of the ovarian reticular cell sarcoma M5 in C57BL mice respond differently to cyclophosphamide and other alkylating agents. The subline R16, which is resistant to cyclophosphamide, was obtained by treating M5 mice repeatedly with this compound and subsequently transplanting the regrowing tumor for 16 passages. The R16 subline shows biological characteristics perfectly superimposable to those of the parent line and histologically resembles an undifferentiated mesenchymal neoplasia with numerous atypical nuclei and karyokinetic figures with large necrotic areas. The cytogenetic examination of the distribution in the chromosomal number of R16 indicates that this subline may be considered a clone of the parent line with a modal class of 35 chromosomes (34–37) versus a class of 34 (31–37) in the M5 tumor line. The presence of metacentric chromosomes characterizes the modal class of the two lines, 23 in the R16, and 25 in the M5 tumor lines.Abbreviations M5 murine ovarian reticular cell sarcoma - M5-CTX-16R(R16) subline of M5 made resistant to cyclophosphamide - CTX cyclophosphamide     

Neurotensin expression and outcome of malignant pleural mesothelioma

Malignant pleural mesothelioma is a frequently fatal disease and the impact of available treatments is globally poor. Identification of new prognostic factors would help in the understanding of disease progression and, possibly, patient management. Here, we evaluate the prognostic impact of the neurotensin (NTS) and its cognate receptor (NTSR1) known for mediating cellular proliferation, survival, invasiveness, and mobility. We studied a series of 52 consecutive patients with epithelioid malignant mesothelioma undergoing management with curative intent, by immunohistochemistry for the expression of NTS and NTSR1. Specimens were scored as 0, 1, or 2 for less than 10%, between 10 and 50%, or more than 50% of NTS positive staining in tumor cells, respectively. Immunohistochemistry revealed that NTS and NTSR1 expression was found in 71.1% and 90.4% of malignant mesotheliomas, respectively. Using univariate analysis, expression of NTS was significantly (p = 0.015) related with a poor prognosis, with median survivals of 11.0 months, 18.4 months, and 29.8 months in patients showing expression scored as 2, 1, and 0, respectively. Multivariate analysis showed that expression of NTS (p = 0.007) and non-surgical therapy (p = 0.004) were independent predictors of poor prognosis. In order to evaluate the role of NTS/NTSR1 complex in mesothelioma progression, in vitro cell invasion assays and wound healing were performed on the mesothelioma cell line, MSTO-211H, and showed that inhibition of the NTS system resulted in a significant reduction of both migration and collagen invasion of mesothelioma cells. The expression of NTS is identified as a prognostic marker in patients with malignant pleural mesothelioma (Patent EP 08305971.7).     

Establishment and characterization of human uterine poorly differentiated adenocarcinoma cell line (HHUABM)

The cell line designated HHUABM was established from the metastatic region (left Bartholin gland) of human endometrial adenocarcinoma. The cell line grew well, multilayering rapidly without contact inhibition, and 72 serial passages were successively done within 25 months. The cultured cells of HHUABM line were round and spindle in shape, and showed a pavement-like arrangement. The distribution of chromosome number varied narrowly at the diploid range, and the modal chromosome number was 46. The 90% of metaphase cells showed normal karyotype. The HHUABM cells were transplanted easily into the subcutis of BALB/c nude mice and produced poorly differentiated adenocarcinoma resembling the original tumor. The conditioned medium promoted the proliferation of CPAE (endothelial cells). The estradiol-17 beta and progesterone receptors were not detected.     

Characteristics of two cell lines derived from a histologically undifferentiated bronchial carcinoma expressing neuroendocrine markers

Summary This study investigates the characteristics of two human cell lines—1PT and 1PT VARIANT A—both derived from the same histologically undifferentiated, neuroendocrine positive, non-small cell lung carcinoma (NSCLC) and capable of growth in unsupplemented serum-free minimum essential medium. In stationary culture, the cells of both lines grew both attached to a plastic substratum and in suspension; the 1PT VARIANT A line formed three-dimensional clusters of loosely adherent cells. The cell lines differed in their DNA content, the 1PT having 1.44 times and the 1PT VARIANT A having 2.39 times the normal human diploid DNA content. Chromosome counts supported this observation, the ploidy of the 1PT and VARIANT A lines being 1.11 and 1.64, respectively. On transmission electron microscopy the cells of both lines had dense core granules and immature desmosomes, whereas only the 1PT VARIANT A line had mucin granules. Both lines formed, in nude mice, tumors that, like the original tumor from which they were derived, were histologically undifferentiated and showed local invasion. The original tumor and both lines had demonstrable neuroendocrine markers. Cytokeratins were apparent in the tumor but not the cell lines, and neurofilaments were present in the cell lines only. Staining for epithelial membrane antigen, neural cell adhesion molecule, and desmoplakin differentiated between the two lines. These lines provide a useful model for the investigation of the biology of the neuroendocrine positive subgroup of NSCLC, which is clinically important because of the possible responsiveness of these tumors to chemotherapy.     

Ten years of respiratory cytopathology at Duke University Medical Center. II. The cytopathologic diagnosis of lung cancer during the years 1970 to 1974, with a comparison between cytopathology and histopathology in the typing of lung cancer

In 1975 Duke University Medical Center, a retrospective and prospective survey of respiratory cytopathologic specimens was undertaken for the ten-year period 1970 to 1979. The purpose of this study was to document the role of cytopathology in the diagnosis of lung cancer at this institution. This paper presents the results of the cytopathologic and histopathologic typing of cases of lung cancer seen at Duke University Medical Center from 1970 to 1974. During this period, 9,892 cytologic specimens from the lower respiratory tract were processed. Cytopathologic diagnoses of cancer with tissue confirmation were made on 483 specimens from 232 patients. Because original cytologic diagnoses, but not histopathologic diagnoses, had been made in conformity with a modified WHO classification of lung neoplasms, all histopathologic material was reviewed and reclassified when necessary. This was carried out by one of the authors (E.H.B.) as a blind review without benefit of knowledge of either preexisting cytopathologic or histopathologic diagnoses. Twenty-six patients were excluded from the current study because of lack of satisfactory histologic material. In 94 patients classified by histopathology as having squamous cell carcinoma, 76.4% of the positive cytologic specimens were also called squamous cell carcinoma; 18.6% were interpreted as large cell undifferentiated carcinoma. In 39 patients classified by tissue as having large cell undifferentiated carcinoma, the cytology agreed in 42.4% of the positive specimens. For the 29 patients thought histologically to have small cell undifferentiated carcinoma, the same diagnosis was rendered in 95.5% of the cytologically positive specimens from these patients. For the adenocarcinoma group of 43 patients, a cytopathologic diagnosis of adenocarcinoma was made in 67.8% of the positive specimens.