ognizant Communication Corporation



Oncology Research, Vol. 16, pp. 497-506
0965-0407/07 $90.00 + .00
E-ISSN 1555-3906
Copyright © 2007 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

The Proteomic Analysis of Cisplatin Resistance in Breast Cancer Cells

Laura Smith,1 Kevin J. Welham,2 Mark B. Watson,1 Philip J. Drew,1 Michael J. Lind,1 and Lynn Cawkwell1

1The Cancer Biology Proteomics Group, Postgraduate Medical Institute of the University of Hull in Association with the Hull York Medical School, Hull, UK
2Department of Chemistry, University of Hull, Hull, UK

Resistance to cisplatin represents a major obstacle in the effective management of many cancers, including metastatic breast cancer. We aimed to gain further understanding of the mechanisms underlying development of cisplatin resistance using an in vitro cell line model. The MCF-7 breast cancer cell line and a novel derivative displaying significant resistance to cisplatin were analyzed using two-dimensional gel electrophoresis. The protein profiles were compared and 15 differentially expressed proteins were identified by matrixassisted laser desorption/ionization time-of-flight mass spectrometry. The downregulation of beta-tubulin type 3, cytokeratin 17, tropomyosin 1-alpha, peroxiredoxin 4, heat shock 27-kDa protein 1, glutathione-S-transferase mu 3, ribosomal protein P0, isocitrate dehydrogenase 3, and peptidyl-prolyl isomerase A isoform 1 was associated with cisplatin-resistant cells. In contrast, the expression of hydroxyprostaglandin dehydrogenase 15-(NAD), matrix metalloproteinase 9, heterogeneous nuclear ribonucleoprotein A3, proteasome beta 1 subunit, electron transfer flavoprotein beta-polypeptide isoform 1, and peptidyl-propyl isomerase B precursor was upregulated in cisplatin-resistant cells. The downregulation (at least twofold) of glutathione-S-transferase mu 3, cytokeratin 17, and peroxiredoxin 4 was confirmed by Western blotting. We have identified alterations in the expression levels of several proteins that may be associated with cisplatin resistance and are candidates for further validation in clinical samples.

Key words: Apoptosis; Biomarkers; Breast cancer; Cisplatin; Chemotherapy; Proteomics

Address correspondence to Dr. Lynn Cawkwell, Ph.D., R&D Building, Castle Hill Hospital, Hull, HU16 5JQ, UK. Tel: (+44) 1482 875875, ext. 3617; Fax: (+44) 1482 622398; E-mail: L.Cawkwell@hull.ac.uk

Oncology Research, Vol. 16, pp. 507-516
0965-0407/07 $90.00 + .00
E-ISSN 1555-3906
Copyright © 2007 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Actin-Sequestering Protein, Thymosin-Beta-4 (TB4), Inhibits Caspase-3 Activation in Paclitaxel-Induced Tumor Cell Death

Eun-Yi Moon,1,2 Ji-Hee Song,2 and Kyu-Hwan Yang3

1Department of Bioscience and Biotechnology, Sejong University, Seoul 143-747, Korea
2Department of Functional Genomics, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Taejeon 305-806, Korea
3Gachon Bionano Research Institute, Kyungwon University, Kyunggi 461-701, Republic of Korea

Thymosin-beta-4 (TB4) as an actin-sequestering peptide has been detected outside of cells in blood plasma or in wound fluid. TB4 induces tumor metastasis and paclitaxel resistance, which is the most significant obstacle to successful therapy in tumors. Here we investigated the inhibitory effect of TB4 peptides on tumor cell death by paclitaxel. The effect of TB4 peptides was assayed by the measurement of caspase-3 activity, G2/M arrest, and Bcl-2 phosphorylation. Cell survival rate was increased and caspase-3 activity was decreased by the treatment with TB4 peptides. In contrast, small interfering RNA (siRNA) of TB4 inhibited cell viability and augmented caspase-3 activity. Significant changes in Bcl-2 phosphorylation were detected by TB4 peptide treatment or by the overexpression of TB4 gene in Hela cells. The reduced population in G2/M phase by TB4 peptide treatment was correlated with the decreased expression of cyclin B1. The data were confirmed in gastric tumor cell lines, SNU 638 (low TB4 level) and SNU 668 (high TB4 level), which were established from clinically isolated gastric tumors. In conclusion, soluble TB4 peptides produced in cancer cells could be an obstacle to treat tumors with paclitaxel. Therefore, TB4 could be a novel target to control paclitaxel resistance.

Key words: Thymosin-beta-4 (TB4); Paclitaxel; Hela cell; siRNA; Caspase-3; Bcl-2

Address correspondence to Eun-Yi Moon, Department of Bioscience and Biotechnology, Sejong University, Seoul 143-747, Korea. Fax: +82-2-466-8768; E-mail: eunyimoon@sejong.ac.kr or eunyimoon@yahoo.com

Oncology Research, Vol. 16, pp. 517-526
0965-0407/07 $90.00 + .00
E-ISSN 1555-3906
Copyright © 2007 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

The Effect of Combined Treatment on Head and Neck Human Cancer Cell Lines With Novel Analogs of Calcitriol and Cytostatics

Joanna Wietrzyk,1 Magdalena Milczarek,1 and Andrzej Kutner2

1Institute of Immunology and Experimental Therapy, 53-114 Wroclaw, Poland
2Pharmaceutical Research Institute, 01-793 Warsaw, Poland

New vitamin D analogs are interesting candidates for anticancer treatment, including squamous cell carcinomas (SCCs), especially in combination with cytostatics. In order to evaluate the effect of combined application of cisplatin, imatinib, or docetaxel and new vitamin D analogs [PRI-1906: (24E)-24a-homo-(1S)-1,25-dihydroxyergocalciferol, and PRI-2191: (24R)-1,24-dihydroxyvitamin D3], against the cells of two human SCCs lines (SCC-25 and FaDu), cytotoxic activity and the effect on cell cycle and apoptosis were determined. The synergistic or additive antiproliferative effect was observed for all cytostatics used after treatment of FaDu cell line with calcitriol or its analogs. Antagonism caused by combination of calcitriol and docetaxel was shown only in the lowest dose. FaDu cells treated with cytostatics and vitamin D analogs cumulated in G0/G1 stage. A statistically significant decrease (2×) in the percentage of apoptotic cells was observed only in combination of imatinib and calcitriol or PRI-1906. On the other hand, when the SCC-25 cell line incubated with cisplatin and imatinib in combination with calcitriol or PRI-2191 (100 nM) was used, the quantitative method of Chou and Talalay indicated antagonism. In the lower doses of calcitriol or PRI-2191 combined with imatinib, the synergistic effect was observed, but in the case of combination with cisplatin or docetaxel only weak additivity was detected. Moreover, a significant decrease (2×) of the percentage of SCC-25 cells undergoing apoptosis induced by docetaxel, cisplatin, and imatinib was observed. The combination of all cytostatic drugs applied with PRI-1906 in all doses caused synergism or additivity. These results might indicate that PRI-1906 is more effective than calcitriol or PRI-2191 as a potential anticancer agent, when used in combination therapy with cytostatic agents. To our knowledge, this is the first observation of interaction with calcitriol or its analogs and imatinib.

Key words: Calcitriol and its analogs; Imatinib mesylate; Cisplatin; Docetaxel; Combined antitumor therapy

Address correspondence to Joanna Wietrzyk, Institute of Immunology and Experimental Therapy, R. Weigla St. 12, 53-114 Wroclaw, Poland. Tel: 4871/3371172; Fax: 4871/3709942; E-mail: wietrzyk@iitd.pan.wroc.pl

Oncology Research, Vol. 16, pp. 527-533
0965-0407/07 $90.00 + .00
E-ISSN 1555-3906
Copyright © 2007 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Alterations Found in p16/Rb/Cyclin D1 Pathway in the Dysplastic and Malignant Cervical Epithelium

N. Vijayalakshmi,1 G. Selvaluxmi,2 U. Majhi,3 and T. Rajkumar1

1Department of Molecular Oncology, Cancer Institute (WIA), Tamilnadu, India
2Department of Radiation Oncology, Cancer Institute (WIA), Tamilnadu, India
3Department of Pathology, Cancer Institute (WIA), Tamilnadu, India

Cervical cancer is the one of the most common cancers affecting the south Indian women. The objective of the present study was to analyze the p16/Rb/cyclin D1 pathway in the normal (n = 30), dysplastic (n = 56), and malignant cervical epithelium (n = 142) using immunohistochemistry. The positive immunoreactivity for p16 was as follows: CIN I-1/12 (8.3%), CIN II-2/8 (25%), CIN III-31/36 (86.1%), and in invasive tumors-121/142 (85.1%); for cyclin D1 it was CIN I-4/12 (66.6%), CIN II-5/8 (62.5%), CIN III-0%, and invasive tumors-5/142 (3.5%); and for pRb it was CIN I-9/12 (75%), CIN II-5/8(62.5%), CIN III-1/36 (97.2%), and in invasive tumors-41/142 (28.8%). Expression of cyclin D1 and p16 in the CINs were mutually exclusive and the correlation between the two biomarkers was found to be statistically significant (p = 0.0009). There was downregulation of pRb in invasive cancers, with only 32% (39/121) of the p16-positive tumors being positive for pRb (p = 0.032). Analysis of the pattern of expression of these biomolecules showed increased p16-positive phenotypes and decreased cyclin D1- and pRB-positive phenotype among the invasive tumors compared to low-grade CIN lesions.

Key words: Retinoblastoma protein (pRb); p16; Cyclin D1; Cyclin D1/p16/Rb pathway; Cervical cancer; HPV

Address correspondence to Prof. T. Rajkumar, Director and Scientific Director, Cancer Institute (WIA), Adyar,Chennai-600 020, Tamilnadu, India. Tel: 91-44-22350340; Fax: 91-44-22201121; E-mail: cancer_institute_wia@vsnl.com

Oncology Research, Vol. 16, pp. 535-548
0965-0407/07 $90.00 + .00
E-ISSN 1555-3906
Copyright © 2007 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Analysis of Gene Expression Profiles Reveals Novel Correlations With the Clinical Course of Colorectal Cancer

Duccio Cavalieri,1 Piero Dolara,1 Enrico Mini,1 Cristina Luceri,1 Cinzia Castagnini,1 Simona Toti,1,2 Karolina Maciag,1 Carlotta De Filippo,1 Stefania Nobili,1 Maria Morganti,1 Cristina Napoli,1 Giulia Tonini,2 Michela Baccini,2 Annibale Biggeri,2 Francesco Tonelli,3 Rosa Valanzano,3 Claudio Orlando,3 Stefania Gelmini,3 Fabio Cianchi,4 Luca Messerini,5 and Lucio Luzzatto6

1Department of Pharmacology, University of Florence, Florence, Italy
2Department of Statistics, University of Florence, Florence, Italy
3Department of Physiopathology, University of Florence, Florence, Italy
4Dipartimento di Area Critica Medico Chirurgica, University of Florence, Florence, Italy
5Department of Human Pathology and Oncology, University of Florence, Florence, Italy
6Istituto Toscano Tumori, Florence, Italy

In order to discover potential markers of prognosis in colorectal cancer (CRC) we have determined gene expression profiles, using cDNA microarrays in CRC samples obtained from 19 patients in Dukes stages C and D, with favorable clinical course (Dukes C patients, survival >5 years after surgery, group A, n = 7) or unfavorable clinical course (Dukes stage C and D patients, survival <5 years after surgery, group B, n = 12). Gene expression was measured in RNA from each tumor, using a pool of equal amounts of RNA from all tumors as a reference. To identify and rank differentially expressed genes we used three different analytical methods: (i) Significance Analysis of Microarrays (SAM), (ii) Cox's Proportional Hazard Model, and (iii) Trend Filter (a mathematical method for the assessment of numerical trends). The level of expression of a gene in an individual tumor was regarded as of interest when that gene was identified as differentially expressed by at least two of these three methods. By these stringent criteria we identified eight genes (ITGB2, MRPS11, NPR1, TXNL2, PHF10, PRSS8, KCNK3, JAK3) that were correlated with prolonged survival after surgery. Pathway analysis showed that patients with favorable prognosis had several activated metabolic pathways (carbon metabolism, transcription, amino acid and nitrogen metabolism, signaling and fibroblast growth factor receptor pathways). To further validate individual gene expression findings, the RNA level of each gene identified as a marker with microarrays was measured by real-time RT-PCR in CRC samples from an independent group of 55 patients. In this set of patients the Cox Proportional Hazard Model analysis demonstrated a significant association between increased patient survival and low expression of ITGB2 (p = 0.011) and NPR1 (p = 0.023) genes.

Key words: Colorectal cancer prognosis; Gene expression; Microarrays; Real-time RT-PCR

Address correspondence to Prof. Enrico Mini, Department of Pharmacology, Viale Pieraccini 6, 50139, Firenze, Italy. Fax: +39 055 4271280; E-mail: enrico.mini@unifi.it