Effects of delta(7)-prostaglandin a(1) methyl-ester on human ovarian-cancer cell-growth in-vitro and in nude-mice

The antitumor activities of Delta(7)-prostaglandin A(1) methyl ester (Delta(7)-PGA(1)) and Delta(7)-PGA(1) emulsified in lecithin oil (lipo Delta(7)-PGA(1)) were studied in nude mice models with ascites or solid tumors formed by i.p. or s.c. inoculation of human ovarian cancer cells (HRA). Inhibitory effects of Delta(7)-PGA(1), on the HRA cell proliferation in vitro were about 3.8-fold higher than those of lipo Delta(7)-PGA(1). In the ascites tumor model, the median survival in a CDDP alone treated group among alone treated groups was longest and followed by a Delta(7)-PGA(1) alone treated group. A combination of CDDP and Delta(7)-PGA(1) resulted in a significant (p<0.05) prolongation of the median survival, compared to that in any alone treated group, while even when CDDP was combined with lipo Delta(7)-PGA(1) the survival was not improved, compared to that in a CDDP alone treated group. In addition, analyses of the survival curve revealed that a combination of CDDP with Delta(7)-PGA(1) resulted in higher survival rate than with lipo Delta(7)-PGA(1). On the other hand, in the s.c. tumor model lipo Delta(7)-PGA(1) (but not Delta(7)-PGA(1)) significantly (p<0.05) inhibited the tumor growth. When combining lipo Delta(7)-PGA(1) with CDDP, its inhibitory effect was further enhanced. Subsequently, the survival time in a lipo Delta(7)-PGA(1)+CDDP treated group was longest and 3 out of 9 mice survived more than 100 days. Taken together, we conclude that Delta(7)-PGA(1) might be suitable for local treatment in i.p, ascites tumors while lipo Delta(7)-PGA(1) is useful for remote treatment in s.c. solid tumors.     

Intracellular calcium and breast-cancer cell-growth and differentiation

The growth of normal human breast epithelial cells in vitro, as well as those of other cell types is strongly influenced by the concentration of calcium in the culture medium [Ca++]e. The aim of this study was to ascertain if calcium also affects breast tumor cell growth in vitro. To address this question, the metastatic breast cancer cells MCF-7 were grown at low (0.04 mM, L-Ca) and high (2.5 mM, H-Ca) [Ca++]e. In each culture condition, we estimated intracellular calcium levels (Ca++]i from Indo-1 fluorescence by the ratio method. We showed that [Ca++]i increased with [Ca++]e, the [Ca++]i values ranging from approximately 50 to 250 nM. Changes of [Ca++]i ware accompanied by changes of cell shape and cell kinetic parameters. In H-Ca, cells were flat and 3 times larger than in L-Ca and the percentage of cells in the S+G2+M phases as well as the percentage of Ki-67 positive cells rapidly dropped on days 3-4 of culture in contrast to cells grown in L-Ca. In H-Ca, the cell growth arrest corresponded to maximal [Ca++]i which was stable during the stationary phase; at that time, a switch from H-Ca to L-Ca resulted in a drop of [Ca++]i and a resumption of cell growth.. In H-Ca, modifications in cell differentiation parameters such as diminution of ER expression and increases of lipid content and EMA expression were observed as compared to cells grown m L-Ca. Our results suggest that MCF-7 cells have retained some calcium dependency and that agents that can increase [Ca++]i in breast tumor cells may limit their proliferation and trigger at least a partial differentiation.     

Quercetin and hyperthermia produce a synergistic inhibitory effect on primary human ovarian-cancer cells

Quercetin (from 0.1 muM to 10 muM) produced a dose dependent inhibition of colony formation of cells from 4 primary ovarian tumors expressing type II estrogen binding sites (type II EBS).The combined effects of quercetin ( 10 muM) and hyperthermia (42-degrees-C) result in a significant synergistic action on three out of four tumors analyzed. Moreover, two other flavonoids tested, rutin and hesperidin, which do not bind to type II EBS, are ineffective in synergizing with hyperthermia. In conclusion our results suggest that hyperthermia could synergize the growth inhibitory activity of quercetin which is probably mediated by the flavonoid interaction with type II EBS.     

Preliminary-study of the effect of selected chinese natural drugs on human ovarian-cancer cells

This study investigated the in vitro anti-cancer effects of four Chinese natural drugs on the human ovarian cancer cell lines A2780 (cisplatin-sensitive) and A2780/CP70 (cisplatin-resistant). Cells were treated with series of concentrations of drug preparations for 24 h. Vincristine prepared by the same pharmaceutical firm in China; was used as an internal control. As assessed by colony formation assays, harringtonine induced similar growth inhibiting effects in A2780 and A2780/CP70 cell lines with a 50% inhibitory dose (IC50) of 0.195 mu g/ml in both. Rabdosia rubescens Hara demonstrated a cytotoxic effect on cisplatin-sensitive A2780 cells with an IC50 of 0.58 mg/ml; but there was no effect on A2780/CP70 cells. The data suggest direct anti-proliferative activity for at least two Chinese natural medicines used in the clinical treatment of cancer in China. Further investigation of Chinese natural medicines may be valuable in the identification of new and effective anti-cancer drugs with minimal side effects.     

Expression and tyrosine phosphorylation of phosphatidyl-inositol-3 kinase in human gastric-cancer cells - its correlation with cell-growth

To reveal the signaling pathway leading to oncogenecity of human cancer cells, we examined the expression and tyrosine-phosphorylation of phosphatidylinositol (PI)-3 kinase in cancer cell lines. Of the 14 cell lines examined, two poorly differentiated human gastric cancer cell lines, NUGC-4 and MKN-45, which were previously found to have aberrant elevation of tyrosine phosphorylation showed elevated levels of PI-3 kinase 85-kDa subunit expression. In these cells, tyrosine-phosphorylation and overall activity of PI-3 kinase were apparently elevated, compared with normal human fibroblasts and another well differentiated gastric cancer cell line, MKN-28. Treatment of these cells with tyrosine kinase inhibitor, genistein, strongly suppressed the PI-3 kinase activity. Furthermore, wortmannin, a potent inhibitor of PI-3 kinase, strongly suppressed the growth of these gastric cancer cells. These results suggest that the growth signaling via tyrosine phosphorylation is required for the activation of PI-3 kinase in NUGC4 and MKN-45, and that this activation plays an important role in oncogenic growth of these cells. However, these two cell lines showed different responses of PI-3 kinase to acid-treatment and tyrosine kinase inhibitors. In MKN-45, activation of PI-3 kinase appeared to be constitutive, and could be relevant to the oncogenic nature of the cell line.     

Stimulation of calcium mobilization but not proliferation by bombesin and tachykinin neuropeptides in human small cell lung cancer cells.

The tachykinin family of neuropeptides, including substance P and neurokinins A and B, induce a transient increase in intracellular free calcium concentration in human small cell lung carcinoma (SCLC) cells, as measured with a calcium indicator fura-2. The effects are dose dependent and even greater than that of bombesin at equimolar concentrations in these cells. The tachykinins, like bombesin, induce calcium mobilization mainly from intracellular store(s). None of the peptides, however, shows a stimulatory effect on DNA synthesis. In addition, exogenously applied bombesin does not stimulate DNA synthesis at any concentration tested. We also examined the effects of a recently reported bombesin antagonist [D-Arg1, D-Phe5, D-Trp7,9, Leu11]substance P in SCLC cells, and compared them to those in Swiss 3T3 fibroblasts in which the mitogenic effect of bombesin is well characterized. The antagonist at 10(-5) M completely abolishes the Ca2+-mobilizing effect of 10(-7) M bombesin in SCLC cells, and that of 10(-9) M but not 10(-7) M bombesin in Swiss 3T3 cells. The antagonist at this concentration effectively inhibits the mitogenic action of bombesin (10(-9) M) in Swiss 3T3 cells; however, much higher doses (approximately 10(-4) M) are needed to inhibit DNA synthesis in SCLC cells. Moreover, the antagonist inhibits DNA synthesis in bombesin/gastrin-releasing peptide-nonproducing cells with a similar dose dependency as in producing cells. These results indicate that bombesin/gastrin-releasing peptide and other calcium mobilizing peptides do not always act as a growth factor in SCLC cells, and that the bombesin antagonist could inhibit growth of SCLC cells through a mechanism other than bombesin antagonism.     

Mechanism of interaction between cisplatin and human recombinant interferon gamma in human ovarian-cancer cell lines

Human ovarian carcinoma cells (2008 and its cisplatin-resistant sub-line 2008/C13*) were sensitized to cisplatin by treatment with human recombinant gamma interferon (IFNγ). IFNγ produced no significant change in the uptake of CDDP. Exposure of 2008 and 2008/C13* cells to IFNγ resulted in a time-dependent decrease of cellular glutathione and total glutathione-S-transferase activity, principally the π isoform. By contrast, the treatment of 2008 and 2008/C13* cell lines with IFNγ induced rather than suppressed metallothionein II_A mRNA levels. IFNγ changed neither the formation of total platinum-DNA adducts, nor DNA repair. A significant decrease in c-erbB-2 expression was observed both in sensitive and in resistant cell lines after treatment with IFNγ, and this decrease was dose-dependent. Our results indicate that the mechanism of IFNγ-induced sensitization in human ovarian-cancer cell lines is multifactorial. © 1995 Wiley-Liss, Inc.     

Tumor-necrosis-factor and DNA topoisomerase-ii inhibitors in human ovarian-cancer - potential role in chemotherapy

Seven ovarian and one cervical human cancer cell lines were examined far their sensitivity or resistance to tumor necrosis factor, to three topoisomerase II inhibitors and to cisplatin. Only one line exhibited the multidrug-resistance phenotype and another one an 'atypical'-MDR phenotype. The combination of TNF and topoisomerase-II inhibitors produced enhanced cytotoxicity and overcame the MDR and the atypical resistance. No potentiation of cisplatin cytotoxicity was observed. These findings suggest that TNF enhances the activity of DNA topoisomerase II both in TNF resistant and sensitive cells.     

Effect of recombinant human tumor-necrosis-factor-alpha and dequalinium chloride on human ovarian-cancer cell-lines in-vitro

Due to preferential uptake and retention, the small molecular weight lipophilic, cationic antimicrobial agent dequalinium chloride (DECA) displays potent in vitro and in vivo antitumor activity against carcinoma cells. The primary mechanism of DECA activity is directed against the mitochondria where it disrupts cellular energy production. One of the direct antitumor effects of tumor necrosis factor (TNF) is also targeted against the mitochondria. The ability of DECA to synergize this effect was examined in vitro against a panel of human ovarian cancer cell lines. The data from single agent and combined drug exposure were analyzed by the isobologram methods of Tsai et al (Cancer Res 49: 2390-2397, 1989). We demonstrate that TNF and DECA strongly synergize in vitro at clinically achievable doses for TNF and potentially clinically achievable doses for DECA. The degree of synergy varied with the cell line tested with UCI-101 being the least responsive and PA-1 cells displaying the greatest synergistic effect. DECA treatment also prolonged animal survival in mice bearing the PA-1 intraperitoneal ovarian carcinoma xenograft. Single agent DECA (5 mg/kg; qod) increased animal survival by 37% (p=0.002) whereas recombinant human TNF (0.5 mug/mouse; qod) increased survival by 12% (p=0.27) in those animals treated 3 days post tumor injection. Sequential DECA/TNF enhanced animal survival by 45% (p=0.0002) in similarly treated animals. DECA, as a mitochondrial poison is an agent capable of potentiating the effects of tumor necrosis factor against ovarian cancer cell lines.     

Expression of urokinase-type plasminogen-activator (upa) and its receptor (upar) in human ovarian-cancer cells and in-vitro invasion capacity

Expression of urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (t-PA), their inhibitor PAI-1 and the uPA-receptor (uPAR) was characterized in six human tumor cell lines (OV-MZ-6, -10, -13, -15, -19 and OVCAR-3) established from patients with cystadenocarcinoma of the ovary. The invasive potential of the ovarian cancer cell lines determined in an in vitro invasion assay did neither correlate with the antigen level of uPA, t-PA, PAI-1 or uPAR nor with the cell surface uPA activity, however, did correlate with the cell surface-bound plasmin activity. The in vitro invasiveness of three cancer cell lines selected displaying a different pattern of uPA and uPAR expression was significantly inhibited by a recombinant soluble truncated form of the uPAR functioning as a scavenger for uPA. Our results suggest that the interference of the uPA/uPAR interaction leads to a reduced in vitro invasiveness of human ovarian cancer cells independent of the level of uPA and uPAR expression.     

High-incidence of allelic loss at the rb gene locus in advanced human ovarian-cancer

We examined 26 ovarian cancers for allelic losses at the loci of five tumor suppressor genes, p53, DCC, RB, APC and WT1. The loss of the p53 gene was most common among these five loci (13/20, 65%). The incidence of allelic loss at the RB locus was significantly higher in advanced stage (III-IV) (6/12, 50%) than in early stage (I-II) tumors (1/14, 7%). There were no cases in which the RB gene was lost but the p53 gene was retained. These results indicate that allelic loss of the RB gene locus occurs later than that of the p53 gene and plays a role in the progression of ovarian cancer.     

Panning and immune electron-microscopic study of mucinous ovarian-cancer antigen-specific cells derived from malignantly transformed bloom-syndrome B-lymphoblastoid cells

We separated characteristic mucinous ovarian cancer (OVC) antigen cells from malignantly transformed Bloom syndrome (BS) cell line (BS-SHI-4M) with the panning procedure using OVC patients sera. We undertook an immune electron-microscopic and scanning electron-microscopic study to acquire information regarding the antigenic determinant of the membrane using pre-embedding method, as well as immunofluorescence (IF) study. The distribution of Protein A colloidal gold (PAG) grains on the cell membrane of mucinous OVC antigen cells paralleled that of fluorescein-conjugated anti-human IgG observed in the IF study. The three patterns of PAG labeling of uniform labeling, uniform partial labeling, and partial labeling of one side of the cell paralleled the three patterns of IF labeling observed under IF. These findings strongly suggest the immunological reaction of BS-SHI-4M OVC-MU antigen cells with the antibody of mucinous OVC patient serum. Western blot analysis demonstrated that the antigen which characterizes mucinous OVC has a band at 84000 MW.     

Vasoactive intestinal polypeptide (VIP) induces calcium mobilization in the human neuroblastoma cell line SK-N-SH

Release of catecholamines, a Ca²⁺-dependent process, is the most useful biochemical marker in the diagnosis of neuroblastoma. Unfortunately, its stimulus is still unknown. We found that vasoactive intestinal polypeptide (VIP), in addition to acetylcholine and muscarine (but not nicotine), causes elevation of the cytoplasmic Ca²⁺-concentration in the highly differentiated human neuroblastoma cell line SK-N-SH, with or without the presence of extracellular Ca²⁺. Additionally, VIP was detected in SK-N-SH cells (0.65 ng/10⁶ cells). Based on these observations and the fact that neuroblastoma is not innervated in vivo, we hypothesize that in this tumor VIP is responsible for Ca²⁺-dependent release of catecholamines in an autocrine or paracrine fashion.     

Cell-mediated-immunity and immunotherapy in ovarian-cancer (review)

The natural containment of ovarian cancer to the peritoneal cavity makes this mileau attractive for examining the immune status within a 'malignant environment'. Such investigations have yielded further insight into host-tumor responses. Immunological strategies have been employed in ovarian cancer either as single agents or combined with chemotherapy and although achieving responses have limited success. The understanding of cytokine inter-relationships, refinement of monoclonal antibody therapy and the potential genetic manipulation will enhance immunological strategies. This review will address aspects of our present knowledge of immune status in ovarian cancer, examine clinical studies incorporating immune agents and potential future therapies.     

Hepatocyte growth-factor and its receptor C-met regulate both cell-growth and invasion of human pancreatic-cancer

Hepatocyte growth factor (HGF) has an important role not only in liver regeneration buy also in the development of cancer metastasis. It has been known that HGF and its receptor/c-MET are overexpressed in human pancreatic cancer in vivo, compared with the normal pancreas. To examine the propensity of pancreatic cancer to metastasize and its association with poor prognosis, we studied the effects of HGF and c-MET on pancreatic cancer cell growth and invasion in vitro. Dose-dependently, HGF promoted both the growth and invasiveness of pancreatic cancer cells that expressed c-MET; as a chemoattractant, the high gradient of HGF determined the direction of the invasiveness of the cells. No stimulant effect, however, was observed in cancer cells that did not express c-MET. These results suggest that HGF and c-MET may play important roles in human pancreatic cancer cell growth and invasion-metastatic potential.     

Nitric oxide mediated photo-induced cell death in human malignant cells

Photodynamic therapy (PDT) is a therapeutic modality used for the treatment of a variety of solid neoplasms. The principle of PDT is based on the selective uptake of a photosensitizing chemical in tumor tissue/cell followed by irradiation of tumors with visible light. The treatment results in a cascade of oxidative events causing cell death both in vitro and in vivo. Nitric oxide (NO) is a gaseous free radical, which is an important modulator of immune, endocrine and neuronal functions and plays an important role in the induction of apoptosis. Hypericin (HY) is a photosensitizing pigment from Hypericum perforatum that displays phototoxic effects in neoplastic cell lines. Our previous studies have shown HY induced apoptotic cell death in nasopharyngeal carcinoma and other tumor cells. To better understand the oxidative mechanism of apoptosis induced by HY, we hypothesized the role of NO in PDT, which is considered to be involved in a variety of physiological and pathological processes. We first demonstrated the presence of nicotinamide adenine dinucleotide hydrogen phosphate-diaphorase (NADPH-d) reactivity, a potential marker of NO synthesizing (NOS) enzyme both at light (LM) and electron microscopic (EM) level. Immunocytochemistry, using specific antibodies for NOS subtypes (constitutive, NOS I and inducible, NOS II), we observed that both NOS I and NOS II was present in all cell lines. The expression of both NOS I and NOS II was further verified using Western blot analysis as early as 15 min post PDT compared to that of drug-treated non-irradiated and light alone treated control cells. Our observation of NO production and distribution using the DAF-2 method is direct evidence of NO production in PDT-treated cells.     

Regulation of cell-growth and apoptosis in synchronized agf cells - involvement of oncogenes and cell-cycle regulatory proteins

Cell synchrony was induced in AGF cells by blocking of the cell cycle at GI-S boundary with high concentrations (2 mM) of thymidine for 11 h. Prolonged arrest of cells in GI-S (15 h-20 h) induced progressive and time dependent apoptosis. Early morphological changes in cellular and nuclear morphology (blebbing) were monitored by Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM) and by staining of nuclei with Hoechst and propidium iodide and stained cells viewed by fluorescence and confocal microscopy. The fluctuations in the levels of the proliferation cell nuclear antigen (PCNA), cyclin A, CDC-2, c-myc and p53 proteins were monitored in synchronized cultures and in cells undergoing apoptosis by immunofluorescence staining and flow cytometry. As expected, the levels of cyclin A and PCNA increased during the S phase and the level of CDC-2 decreased during late S/G2. Similarly, the level of c-myc increased during the S phase, whereas the level of p53 did not change much during S phase. Most importantly, however, the level of staining for c-myc, p53, cyclin A, CDC-2 and PCNA increased markedly during apoptosis. In contrast, the level of actin vimentin and tubulin, although increased during S phase, were markedly decreased during apoptosis. AGF cells stained for c-myc during apoptosis, and viewed by confocal microscopy, revealed increased staining for c-myc in the blebbing nuclei. These results, taken together, suggest a possible active role for c-myc in the process of nuclear blebbing and apoptosis.     

Functional genomics of calcium channels in human melanoma cells

Ca(2+)-signaling of human melanoma is in the focus of intensive research since the identification of the role of WNT-signaling in melanomagenesis. Genomic and functional studies pointed to the important role of various Ca(2+) channels in melanoma, but these data were contradictory. In the present study we clearly demonstrate, in a number of different ways including microarray analysis, DNA sequencing and immunocytochemistry, that various human melanoma cell lines and melanoma tissues overexpress ryanodine receptor type 2 (RyR2) and express P2X(7) channel proteins as compared to melanocytes. These channels, although retain some of their usual characteristics and pharmacological properties, display unique features in melanoma cells, including a functional interaction between the two molecules. Unlike P2X(7), RyR2 does not function as a calcium channel. On the other hand, the P2X(7) receptor has an antiapoptotic function in melanoma cells, since ATP-activation suppresses induced apoptosis, while knock down of the gene expression significantly enhances that.