ognizant Communication Corporation



Oncology Research, Volume 14, pp. 61-73
0965-0407/03 $20.00 + .00
Copyright © 2003 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Frequent Alterations of Smad Signaling in Human Head and Neck Squamous Cell Carcinomas: A Tissue Microarray Analysis

Wen Xie,1 Savita Bharathy,1 David Kim,3 Bruce G. Haffty,3 David L. Rimm,2 and Michael Reiss1

1Division of Medical Oncology, The Cancer Institute of New Jersey, Robert Wood Johnson Medical School, New Brunswick, NJ
Departments of 2Pathology and 3Therapeutic Radiology, Yale Cancer Center, Yale University School of Medicine, New Haven, CT

Head and neck squamous cell carcinoma (HNSCC) ranks as the sixth most frequent cancer worldwide. HNSCC cell lines are typically refractory to transforming growth factor-b (TGF-b)-mediated cell cycle arrest. A number of these cell lines carry inactivating mutations of the TGF-b type II (TbR-II) receptor, and fail to phosphorylate receptor-associated Smads, Smad2 and Smad3. In addition, we identified several intragenic mutations of the TbR-I gene in a small series of metastatic HNSCC specimens, suggesting that disruptions of TGF-b signaling might contribute to the development and progression of HNSCC. To test this idea, we have now embarked on a larger scale analysis of the patterns of expression and activation of Smads in 170 HNSCC specimens assembled in tissue microarrays. Smad2 protein was expressed by 99% (95% CI: 96-100%) of tumors. The activated form of Smad2, pSmad2, was expressed in 86% (95% CI: 80-91%) of HNSCC, indicating their ability to survive and proliferate in spite of the presence of bioactive TGF-b within the tissue microenvironment. In the 24 remaining cases (14%; 95% CI: 9-20%), pSmad2 was not detected in the tumor cells, although it was expressed by surrounding stromal cells and capillaries. In addition, 38 tumors (22%; 95% CI: 16-29%) failed to express Smad4 protein. Thus, we found evidence of loss of TGF-b/Smad signaling in approximately 15-20% of HNSCC specimens, which is consistent with the phenotype of established human SCC lines. Moreover, we found that these Smad signaling defects were associated with a greater tendency for metastatic spread and regional or distant recurrence of HNSCC. These results indicate that inactivation of TGF-b/Smad signaling occurs frequently in HNSCC and might have an adverse effect on patient outcome.

Key words: Transforming growth factor-b; Smad; Head and neck cancer; Tissue microarray

Address correspondence to Michael Reiss, M.D., Division of Medical Oncology, Department of Internal Medicine, UMDNJ-Robert Wood Johnson Medical School and The Cancer Institute of New Jersey, Room 2007, 195 Little Albany Street, New Brunswick, NJ 08903. Tel: (732) 235-6031; Fax: (815) 333-3972; E-mail: michael.reiss@umdnj.edu

Oncology Research, Volume 14, pp. 75-82
0965-0407/03 $20.00 + .00
Copyright © 2003 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Effect of 3-Fluorothalidomide and 3-Methylthalidomide Enantiomers on Tumor Necrosis Factor Production and Antitumor Responses to the Antivascular Agent 5,6-Dimethylxanthenone-4-Acetic Acid (DMXAA)

Francisco Chung,1 Brian D. Palmer,1 George W. Muller,2 Hon-Wah Man,2 Phillip Kestell,1 Bruce C. Baguley,1 and Lai-Ming Ching1

1Auckland Cancer Society Research Center, Faculty of Medical and Health Sciences, The University of Auckland, Auckland, New Zealand
2Celgene Corporation, Warren, NJ

5,6-Dimethylxanthenone-4-acetic acid (DMXAA) is an antivascular drug that induces tumor necrosis factor (TNF) in mice. Thalidomide inhibits TNF induction by DMXAA and also potentiates its antitumor activity. We investigated whether these effects were enantiomer specific, using the R- or S-enantiomers of two nonracemizable thalidomide analogues. Racemic 3-fluorothalidomide (3FThal) and racemic 3-methylthalidomide (3MeThal) were separated into enantiomers of greater than 98% optical purity using preparative chiral column chromatography. C57Bl/6 mice implanted with subcutaneous Colon 38 tumors were treated with DMXAA (25 mg/kg) alone or together with the pure R- or S-enantiomers by a single IP injection. TNF levels in the serum or tumor tissues 3 h after treatment were measured using ELISAs and tumor growth was also measured. 3FThal and 3MeThal, at their respective single maximum tolerated doses (MTD) of 15 and 50 mg/kg, were more toxic in mice than thalidomide (100 mg/kg). The R- and S-enantiomers of either 3FThal or 3MeThal, at their respective MTD, inhibited DMXAA-induced TNF activity in serum and tumor tissue, but no significant differences were observed between the enantiomers. Coadministration of racemic or enantiomers of 3FThal or 3MeThal at their respective MTD did not potentiate the antitumor responses above that obtained with DMXAA alone, and no enantioselectivity was apparent. We conclude that there is no advantage in using the nonracemizable thalidomide analogues to improve the antitumor activity of DMXAA.

Key words: Thalidomide; DMXAA; Tumor necrosis factor; Antitumor; Colon 38; Enantiomers

Address correspondence to Lai-Ming Ching, Auckland Cancer Society Research Center, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland, New Zealand. Tel: (64-9) 3737 999; Fax: (64-9) 3737-502; E-mail: l.ching@auckland.ac.nz

Oncology Research, Volume 14, pp. 83-91
0965-0407/03 $20.00 + .00
Copyright © 2003 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Epidermal Growth Factor-Responsive Laryngeal Squamous Cancer Cell Line Hep2 Is More Sensitive Than Unresponsive CO-K3 One to Quercetin and Tamoxifen Apoptotic Effects

G. Raspaglio,1 G. Ferrandina,1 C. Ferlini,1 G. Scambia,1 and F. O. Ranelletti2

Departments of 1Gynecology/Obstetrics and 2Histology, Catholic University, L.go A. Gemelli, 8, 00168, Rome, Italy

Epidermal growth factor receptor (EGFR) plays a role in laryngeal squamous cell carcinoma (SCC) development and progression. The flavonoid quercetin (Q) and the antiestrogen tamoxifen (TAM) inhibit proliferation of both primary laryngeal SCC and laryngeal carcinoma cell lines, through still uncharacterized mechanisms. We studied Q and TAM inhibitory effect on epidermal growth factor (EGF)-stimulated Hep2 and CO-K3 laryngeal squamous cell lines. Q and TAM (0.1-1.0 mM) induced more apoptosis in EGF growth-stimulated than in unstimulated Hep2 cells. EGF neither stimulated CO-K3 cell growth nor enhanced Q and TAM-induced apoptosis. Mitogen-activated protein kinase (MAPK) analysis revealed that in Hep2 cells, but not in CO-K3 cells, EGF induced a time-dependent phosphorylation of p42, p44, p38, and p46. In Hep2 cells, but not in CO-K3 cells, Q and TAM produced, upon EGF treatment, a twofold increase of p38 and p46 and an enhancement of p42 and p44 dephosphorylation, suggesting a requirement of EGFR. The enhancing effect was due to a p38 and p46 dephosphorylation delayed kinetics. An antiphosphorylated p38 antibody prevented Q and TAM inhibitory effect on p42 and p44 phosphorylations, suggesting that the EGF-dependent increase in Q and TAM apoptotic effect on Hep2 cells could depend on the p38 inhibition of the survival kinases p42 and p44. In SCC, EGFR overexpression is an early event from dysplasia to neoplasia. We conclude that the capacity of Q and TAM to increase apoptosis in EGFR-activated cells makes these compounds possible chemopreventive drugs in subjects at risk of developing laryngeal cancer.

Key words: Tamoxifen; Quercetin; Epidermal growth factor; Mitogen-activated protein kinases; Apoptosis

Address correspondence to Prof. Franco O. Ranelletti, Department of Histology, Università Cattolica del S. Cuore, L.go F. Vito 1, 00168, Rome, Italy. Fax: +39-06-3051157; E-mail: ranelletti@rm.unicatt.it

Oncology Research, Volume 14, pp. 93-100
0965-0407/03 $20.00 + .00
Copyright © 2003 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Reduction of Bladder Cancer Cell Growth in Response to hCGb CTP37 Vaccinated Mouse Serum

Stephen A. Butler, Edyta M. Staite, and Ray K. Iles

Williamson Laboratory, Department of Obstetrics and Gynecology, Barts and The London, Queen Mary School of Medicine, St. Bartholomew's Hospital, London, UK

The free b-subunit of human chorionic gonadotrophin (hCGb) is well established as an ectopic product of epithelial tumors. Originally explained as an epi-phenomenon, hCGb production by many types of carcinoma is increasingly regarded as a significant tumor event. Studies in bladder cancer have shown that hCGb production, while not diagnostic, is a very good indicator for poor prognosis through correlations with resistance to radiotherapy and rapid metastasis. These clinical findings led to in vitro studies that have shown a direct response to hCGb by bladder carcinoma cell lines. This response is linked by inhibition of apoptosis to an increase in cell population. More recently, studies on hCGb as a marker for poor prognosis in other epithelial cancers now suggest that this phenomenon may not be restricted to bladder carcinoma. Thus, ectopic hCGb represents an ideal target for immunodepletive therapy. Antisera were generated from mice vaccinated with full-length hCGb carboxy terminal peptide (CTP37) and a truncated region comprising 24 of the amino acids of the CTP (CTP24), expressed on the surface of cowpea mosaic virus (CPMV). The effect of the resultant murine antiseras on bladder carcinoma cell growth in vitro was investigated. When CTP37 antisera, at dilutions of 1:50 and 1:100, were incubated with two hCGb-producing cell lines, SCaBER and RT112, significant reductions in cell number, up to 43%, were observed. In the bladder cancer cell line T24, which does not produce hCGb, CTP37 antisera had no growth effects. CTP24 antiserum, like control sera from mice immunized with wild-type CPMV, had no effects on the in vitro growth of any cell lines. This implies that full-length CTP37, but not CTP24, is involved in the oncogenic inhibition of apoptosis by hCGb. hCGb CTP37 vaccines are available as well-tested antifertility vaccines in the Third World. They have now been tested on cancer patients. This study is the only in vitro evidence that such a vaccine would have beneficial antitumor effects via immunodepletion mechanisms. We propose that vaccines such as this could be used as an adjuvant therapy in the treatment of hCGb-producing bladder cancers.

Key words: Free hCGb; Poor prognosis; Cystine knot growth factor; Vaccine development; Anticancer treatment

Address correspondence to R. K. Iles, Williamson Laboratory, East Wing St. Bartholomew's Hospital, West Smithfield, London EC1A 7BE, UK. Tel: 0171 601 8951; Fax: 0171 600 1439; E-mail: r.k.iles@qmul.ac.uk

Oncology Research, Volume 14, pp. 101-112
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Copyright © 2003 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Characterization of Prostate Cancer DU145 Cells Expressing the Recombinant Androgen Receptor

Eugenia Scaccianoce,1 Claudio Festuccia,2 Donatella Dondi,1 Vittoria Guerini,1 Mauro Bologna,2 Marcella Motta,1 and Angelo Poletti1

1Centre for Endocrinological Oncology, Institute of Endocrinology, University of Milan, Italy
2Department of Experimental Medicine, University of L'Aquila, Italy

Prostate cancer (PC) develops as a consequence of abnormal androgenic stimulation. Unfortunately, most of the PC cell lines are androgen independent (like DU145), or express mutated forms of androgen receptor (AR). We have produced and characterized a new stably transfected PC line expressing the AR (DU145-AR). Untreated DU145-AR cells showed a lower proliferation rate than mock transfected cells, but responded to testosterone treatment. PSA mRNA, undetectable in mock DU145 cells, was present and upregulated by testosterone in DU145-AR. About 5% of DU145-AR cells showed modification of morphology and enriched of f-actin after testosterone treatment. Moreover, in DU145-AR plasminogen activator (PA) activity and secreted urokinase type plasminogen activator (uPA) protein were lower than in AR negative cells; again testosterone induced PA activity and uPA protein only in DU145-AR. These results indicate that, in general, the effects of unactivated AR is to suppress function(s) in DU145 cells and the addition of testosterone restores the normal properties associated with the untransfected cells. Some of the effects described may thus be mediated by a ligand-independent activation of AR in DU145 cells.

Key words: Prostate cancer; Androgen receptor; DU145 cells

Address correspondence to Angelo Poletti, Institute of Endocrinology, via Balzaretti 9, 20133 Milano, Italy. Tel: +39-02-5031.8227; Fax: +39-02-5031.8204; E-mail: angelo.poletti@unimi.it