[Frontiers in Bioscience 10, 2566-2575, September 1, 2005] 2566 DOCETAXEL INDUCES P53-DEPENDENT APOPTOSIS AND SYNERGIZES WITH FARNESYL TRANSFERASE INHIBITOR R115777 IN HUMAN EPITHELIAL CANCER CELLS M. Caraglia 1 , G. Giuberti 2 , M. Marra 2 , E. Di Gennaro 1 , G. Facchini 3 , F. Caponigro 3 , RV Iaffaioli 3 , A. Budillon 1 , and A. Abbruzzese 2 1 Experimental Pharmacology Unit, Department of Experimental Oncology, National Institute of Tumours Fondazione “G. Pascale”, Naples, Italy; 2 Department of Biochemistry and Biophysics, Second University of Naples, Via Costantinopoli, 16 80138, Naples, Italy; 3 Division of Medical Oncology B, National Institute of Tumours Fondazione “G. Pascale” Naples, Italy TABLE OF CONTENTS 1. Abstract 2. Introduction 3. Materials and methods 3.1. Materials 3.2. Cell culture 3.3. Drug combination studies 3.4. Western blot analysis 3.5. Affinity precipitation of Ras 3.6. Internucleosomal DNA fragmentation (Ladder) 3.7. Flow cytometric analysis of apoptosis 3.8. Fluorescence Microscopy 3.9. Statistical Analysis 4. Results 4.1. Human epithelial cancer cells are differently sensitive to DTX 4.2. DTX causes apoptosis in human epithelial cancer cells 4.3. DTX induces caspase activation and modulates p53 expression and ubiquitination in human cancer cells 4.4. DTX induces ras and erk activation 5. Discussion 6. Acknowledgment 7. References 1. ABSTRACT Docetaxel (Taxotere, DTX) is a promoter of apoptosis in cancer cells. Since cytotoxic mechanisms of DTX are not yet fully understood, we have investigated the effects of DTX on apoptosis and ras->Erk-mediated signal transduction in human epidermoid KB, colon HT-29 and breast HCC1937 cancer cells. We have found that the exposure to 0.78 or 1.56 or 2.5 ng/ml DTX for 48 h induced apoptosis and growth inhibition in about 50 % of KB, HCC1937 and HT-29 cell population, respectively. In these experimental conditions, PARP and caspase 3 cleavage was also showed in all cell lines. KB and HCC1937 cells express a wild type p53 while HT-29 display a mutated form. Interestingly, we have found that DTX reduces the expression of mutated p53 in HT-29 and increases the expression of wild type in KB and HCC1937 cells. Moreover, DTX reduces ubiquitination of the wild type p53 in KB and HCC1937 cells and increases the ubiquitin-conjugated form of mutated p53 in HT-29 cells. Furthermore, exposure of cancer cells to DTX for 48 h increases the expression and activity of Ras and up- regulates Raf-1 and the phosphorylated isoforms of Erk- 1/2. On the bases of these data, we have hypothesized that the increased activity of the ras->erk-dependent pathway induced by DTX could be a protective signalling from the apoptosis caused by the drug. Therefore, we have used R115777, a farnesyl transferase inhibitor that inactivates ras, in combination with DTX. The combined treatment with DTX and R115777 resulted in a strong synergism in growth inhibition in the three cell lines. These data suggest the use of the combination in these therapeutic settings even if further experiments are required for the clinical translation. 2. INTRODUCTION Docetaxel (Taxotere) is a member of the taxane class of anticancer agents to reach clinical use. This semisynthetic analog of paclitaxel (Taxol) is one of the newer potent anti-neoplastic agents now undergoing extensive laboratory and clinical investigations. Several studies indicate that antimicrotubule agents are potent promoters of apoptosis in cancer cells. Cytotoxic mechanisms of antimitotic taxoids are not yet fully understood, but it has been demonstrated that docetaxel