British Journal of Cancer

Rauth, S; Kichina, J; Green, A

doi: 10.1038/bjc.1997.268pmid: 9184169

We have investigated the effect of the soybean isoflavone genistein on the growth and differentiation of human melanoma cells. Four human melanoma cell lines, either completely lacking or containing different levels of wild-type p53, were treated with genistein in vitro in culture. It has been found that genistein significantly inhibited cell growth and that the chemosensitivity might depend on cellular p53 content. Specifically, the data suggest that high levels of wild-type p53 expression make cells resistant to genistein's growth-inhibitory action. Further support for this observation came from the stable transfection studies in which p53 transfectants expressing high levels of wild-type p53 became resistant to genistein. With respect to cell differentiation, our study showed that genistein increased melanin content and tyrosinase activity and caused the cells to form dendrite-like structures. Cells lacking p53 responded more than cells with p53 to dendrite-like structure formation. We also observed that genistein-induced differentiation involved an increase in tyrosinase mRNA level; the mechanisms by which genistein increases tyrosinase transcripts remain to be elucidated. Genistein treatment of the melanoma cell lines resulted in cell cycle arrest at G2/M check point and no significant apoptosis was observed.

Bansal, GS; Cox, HC; Marsh, S; Gomm, JJ; Yiangou, C; Luqmani, Y; Coombes, RC; Johnston, CL

doi: 10.1038/bjc.1997.269pmid: 9184170

The level of expression of keratinocyte growth factor (KGF) mRNA has been measured in human breast cell lines, purified populations of epithelial cells, myoepithelial cells and fibroblasts from reduction mammoplasty tissue and a panel of 42 breast cancers and 30 non-malignant human breast tissues using a semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) procedure. We found similar levels of KGF mRNA in malignant and non-malignant breast tissues. The study of the amount of KGF mRNA in breast cell lines and purified populations of cells revealed that fibroblasts are the predominant source of KGF with malignant and non-malignant epithelial cells containing very low levels of KGF mRNA. We have examined the distribution of fibroblast growth factor receptor (FGFR)-2-IIIb, which is a high-affinity receptor for KGF and find that it is present on malignant and non-malignant epithelial cells. The level of FGFR-2-IIIb present on breast cancer cell lines was sufficient for KGF stimulation of breast cancer cell proliferation. Other members of the fibroblast growth factor family have been either not expressed in the human breast (FGF3, FGF4) or have been found at much reduced levels in breast cancer (FGF1, FGF2) and this is the first member of the family to potentially influence the progression of breast cancer through stimulation of cell division.

Schmidt, M; Vakalopoulou, E; Schneider, DW; Wels, W

doi: 10.1038/bjc.1997.270pmid: 9184171

Epidermal growth factor (EGF) receptor-overexpression is characteristic of many human tumours of epithelial origin and has been correlated with unfavourable patient prognosis. Its involvement in the malignant process, its elevated expression in tumours and its accessibility on the tumour cell surface make the EGF receptor a potential target for directed tumour therapy. We have previously characterized a recombinant antibody - Pseudomonas exotoxin A fusion protein, scFv(225)-ETA, which displayes antitumoral activity towards EGF receptor-overexpressing tumour cells but is less potent in tumour cell killing than TGF-alpha-ETA, a recombinant toxin using the natural EGF receptor ligand transforming growth factor alpha (TGF-alpha) as a targeting domain. Here, we describe the construction and functional characterization in vitro of a novel single-chain antibody-toxin, scFv(14E1)-ETA, based on the independently isolated EGF receptor-specific monoclonal antibody 14E1. ScFv(14E1)-ETA binds to an EGF receptor epitope that is very similar or identical to that of scFv(225)-ETA with nine times higher affinity than the latter and displays more than tenfold higher cytotoxic activity on EGF receptor-overexpressing tumour cells. ScFv(14E1)-ETA cell killing activity was very similar to that of TGF-alpha-ETA on receptor-overexpressing cells but, in contrast to the latter, scFv(14E1)-ETA was much more selective and did not display significant cytotoxic activity on cells expressing moderate EGF receptor levels.

Jungwirth, A; Schally, AV; Pinski, J; Halmos, G; Groot, K; Armatis, P; Vadillo-Buenfil, M

doi: 10.1038/bjc.1997.271pmid: 9184172

Tumour-inhibitory effects of a new antagonist of growth hormone-releasing hormone (GH-RH), MZ-4-71, were evaluated in nude mice bearing androgen-independent human prostate cancer cell lines DU-145 and PC-3 and in Copenhagen rats implanted with Dunning R-3327 AT-1 prostatic adenocarcinoma. After 6 weeks of therapy, the tumour volume in nude mice with DU-145 prostate cancers treated with 40 microg day(-1) MZ-4-71 was significantly decreased to 37 +/- 13 mm3 (P < 0.01) compared with controls that measured 194 +/- 35 mm3. A similar inhibition of tumour growth was obtained in nude mice bearing PC-3 cancers, in which the treatment with MZ-4-71 for 4 weeks diminished the tumour volume to 119 +/- 35 mm3 compared with 397 +/- 115 mm3 for control animals. Therapy with MZ-4-71 also significantly decreased weights of PC-3 and DU-145 tumours and increased tumour doubling time. Serum levels of GH and IGF-I were significantly decreased in animals treated with GH-RH antagonist. In PC-3 tumour tissue, the levels of IGF-I and IGF-II were reduced to non-detectable values after therapy with MZ-4-71. The growth of Dunning R-3327 AT-1 tumours in rats was also significantly inhibited after 3 weeks of treatment with 100 microg of MZ-4-71 day(-1) i.p. as shown by a reduction in tumour volume and weight (both P-values < 0.05). Specific high-affinity binding sites for IGF-I were found on the membranes of DU-145, PC-3 and Dunning R-3327 AT-1 tumours. Our results indicate that GH-RH antagonist MZ-4-71 suppresses growth of PC-3, DU-145 and Dunning AT-1 androgen-independent prostate cancers, through diminution of GH release and the resulting decrease in the secretion of hepatic IGF-I, or through mechanisms involving a lowering of tumour IGF-I levels and possibly an inhibition of tumour IGF-I and IGF-II production. GH-RH antagonists could be considered for further development for the therapy of prostate cancer, especially after the relapse.

de Souza, PL; Castillo, M; Myers, CE

doi: 10.1038/bjc.1997.272pmid: 9184173

Cytoplasmic phospholipase A2 (PLA2) is known to be phosphorylated and activated by MAP kinase (Lin et al 1993, Cell 72: 269-278), an important downstream component of signal transduction, whereas paclitaxel has been shown to inhibit isoprenylation of ras proteins (Danesi et al 1995, Mol Pharmacol 47: 1106-1111). Given that quinacrine (Q), a PLA2 inhibitor, and paclitaxel (P) might act at different sites in the cell signalling pathway, our aim was to test whether they were synergistic in combination against prostate cancer cells. Cell viability of PC-3, PC-3M and DU145 cells in 96 - well plates was assessed 96 h after drugs were added concurrently. Using Chou analysis, we demonstrated synergy for the combination against all three cell lines. Further, synergy was present under both conservative (mutually non-exclusive) and non-conservative (mutually exclusive) models. Studies in the nude mouse xenograft model support the finding of synergy in vitro. In DU145-bearing mice, Q (50 mg kg(-1)) and P (0.5 mg kg(-1)) given daily for 12 consecutive days, either concurrently or sequentially, was more effective than either drug alone, at twice the dose intensity. In an enzyme-linked immunosorbent (ELISA) apoptosis assay, arachidonic acid was able to partially reverse Q- and P-induced apoptosis, suggesting PLA2 pathway involvement. Finally, the combination of lovastatin, another inhibitor of ras isoprenylation, and quinacrine had synergistic inhibitory effects on the growth of PC-3 cells in vitro, suggesting that the combination of these two classes of compounds might serve as an attractive therapeutic approach for prostate cancer.

Koizumi, M; Maeda, H; Yoshimura, K; Yamauchi, T; Kawai, T; Ogata, E

doi: 10.1038/bjc.1997.273pmid: 9184174

To clarify the meaning and clinical value of bone formation markers in bone metastasis from prostate cancer, we investigated the bone formation markers carboxy-terminal propeptide of type I procollagen (PICP), bone-specific alkaline phosphatase (BA1-p) and osteocalcin, so-called bone gla protein (BGP) in 43 prostate cancer patients with and 46 patients without overt bone metastasis. Patients with bone metastasis were evaluated repeatedly by bone scan at intervals of 3-6 months. The expression patterns of bone formation markers in patients with progression of bone metastasis became dissociated; BA1-p and PICP were elevated in patients with progression of bone metastasis but BGP was not. Instead, BGP showed slight elevation in patients with improvement and complete remission of bone metastasis. PICP, BA1-p and BGP are all bone formation markers, but each marker appears in a different phase of bone formation: PICP appears in proliferation phase, BA1-p appears in matrix maturation phase and BGP appears in late bone formation phase. Our findings that BGP was not elevated in progression of bone metastasis and that it increased slightly with improvement and complete remission of bone metastasis may imply that the bone formation that occurs in blastic bone metastasis is different from normal bone formation.

Poirson-Bichat, F; Gonfalone, G; Bras-Gonçalves, RA; Dutrillaux, B; Poupon, MF

doi: 10.1038/bjc.1997.274pmid: 9184175

Methionine (MET) is required for cell metabolism. MET endogenously synthesized from homocysteine (HCY) supports the proliferation of normal cells, but not that of numerous malignant cells, as shown previously. MET starvation should have an anti-tumour effect, and its deleterious effects on the hosts might be prevented by HCY. Anti-tumour effects of MET starvation must be reinforced by ethionine (ETH), a MET analogue. MET dependency of PC-3, a human prostate cancer cell line, was studied in vitro. Proliferation of PC-3 cells, cultivated in MET-free medium, was 29% compared with growth in MET+HCY- medium. Addition of HCY to MET-free medium increased the proliferation rate to 56%. The concentration of ETH required to decrease the PC-3 cell proliferation rate to 50% (IC50) was 0.5 mg ml(-1) in MET-HCY- medium. ETH-induced inhibition was abolished by MET addition and was reinforced by HCY. PC-3 cell cycle was blocked in the S-G2-phase after 30 h culture in the absence of MET; this blockage was not reversed by addition of HCY. ETH at the IC50 in MET-HCY+ medium blocked DNA replication. Apoptotic cells appeared after 30 h incubation in MET-HCY+ medium only when ETH was added. ATP pools were decreased after 15 h of culture in MET-free medium. In vivo, MET starvation was obtained by feeding tumour-bearing mice a diet containing a synthetic amino acid mixture as the protein supply, in which HCY replaced MET. Given to nude mice bearing xenografted PC-3, from day 1 after grafting and for 3 weeks, this diet inhibited tumour growth (34% on day 20, P < 0.007); this effect was potentiated by ETH (200 mg kg(-1) day(-1) i.p.) (56% on day 20, P < 5 x 10(-5)). The differences between the effects of these two treatments were significant (P < 0.017) and optimal on day 20. These data showed that combination of ETH and HCY slowed the proliferation of prostate cancer cells in vitro and in vivo, decreased ATP synthesis and caused cell cycle arrest and apoptosis. Experimental therapy based on cancer cell MET metabolism deficiency could be efficient for treating advanced prostate cancers refractory to current therapies.

Okutomi, T; Ubukata, T; Yamaoka, K; Abe, S; Yamaguchi, H

doi: 10.1038/bjc.1997.275pmid: 9184176

Effects of amphotericin B on production of endogenous tumour necrosis factor alpha (TNF-alpha) and anti-tumour activity in mice was examined. Intravenous administration of Fungizone, an amphotericin B preparation complexed with deoxycholate, augmented the induction of endogenous TNF in response to a second stimulus with intravenous doses of FK23 (heat-killed Enterococcus faecalis). This augmentation was observed when more than 1.8 microg of Fungizone was injected intravenously before intravenous dosing of FK23. The time interval between priming injection of Fungizone and secondary injection of FK23 for the maximal effect was 3 h. Similar augmentation of TNF production was also observed in amphotericin B-primed and FK23-injected mice. Correspondingly, anti-tumour activity of the combined, intravenous injection of Fungizone and FK23 with a 3-h interval was examined. Growth of Meth A fibrosarcoma was clearly inhibited by this combination but not by administration of either one alone. These results suggest that amphotericin B is able to elicit anti-tumour activity, perhaps through activation of the immune system, and in particular augmentation of the induction of endogenous TNF.

Ogawa, M; Maeda, K; Onoda, N; Chung, Y-S; Sowa, M

doi: 10.1038/bjc.1997.276pmid: 9184177

Previous studies have shown that tumour-suppressor genes play an important role in the progression of solid tumours. Recently, the p21WAF1/CIP1 tumour-suppressor protein has been reported to work as a critical downstream effector of p53 and a potent inhibitor of cyclin-dependent kinases. Thus, the p21WAF1/CIP1 gene is thought to play a central role in tumour suppression. In this study we investigated p21 protein expression in gastric carcinomas. A total of 172 primary gastric carcinoma specimens were immunohistochemically stained for p21 protein expression. Correlations between p21 expression and clinicopathological features were examined. Loss of p21 expression was observed in 104 of 172 tumour tissues (60.4%), and the frequency of p21 loss increased as the stage progressed. Expression of p21 in the primary tumour was frequently lost in patients with either lymph node, liver or peritoneal metastases as compared with patients without metastases. In patients with p21-negative tumours, the risk of recurrence following curative surgery was significantly higher, and the prognosis was significantly poorer than in patients with p21-positive tumours. Loss of p21 expression in primary gastric carcinoma correlates with disease progression. The status of p21 gene expression may have prognostic value in this disease.

Coope, RC; Browne, PJ; Yiangou, C; Bansal, GS; Walters, J; Groome, N; Shousha, S; Johnston, CL; Coombes, RC; Gomm, JJ

doi: 10.1038/bjc.1997.277pmid: 9184178

Acidic fibroblast growth factor (FGF1) and two of its receptors, FGFR1 and FGFR4, were localized in cryostat sections of normal, benign and malignant human breast tissue by immunohistochemistry. Without pretreatment, FGF1 staining was mainly seen in normal epithelial cells. However, polymerase chain reaction (PCR) analysis and immunoblotting of isolated normal epithelial and myoepithelial cells showed FGF1 mRNA and protein to be present in both cell types. Following incubation of frozen sections at 37 degrees C in phosphate-buffered saline, FGF1 staining was also revealed in myoepithelial cells and basement membrane adjacent to carcinoma cells. Treatment with protease inhibitors demonstrated that this effect was due to the activity of an endogenous protease. In contrast, FGF1 staining was found to be associated with the stroma adjacent to malignant cells only in the presence of protease inhibitors. FGFR1 and FGFR4 immunostaining was localized to both normal and malignant epithelial cells and to a lesser extent to myoepithelial cells. There was no difference in the staining intensity for the FGF receptors between normal and cancer samples. The change in location of FGF1 between normal and malignant tissues and the sensitivity of stored FGF1 to the action of endogenous proteases raises the possibility of both autocrine and paracrine roles for FGF1 in the normal and malignant human breast.