title: Sequence-dependent antitumor effects of differentiation agents in combination with cell cycle-dependent cytotoxic drugs.
abstract: OBJECTIVE
  Combination of two differentiation agents such as phenylbutyrate (PB) and 13-cis-retinoic acid (CRA) has been shown to have an additive inhibitory effect on tumor growth in preclinical studies. In this report we explored the hypotheses that these "cytostatic" agents may have a greater antitumor activity in combination with "cytotoxic" compounds and their biological effect may be sequence-dependent.
  
  
  METHODS
  The antitumor activity of combination of PB and CRA with paclitaxel (TX ) and doxorubicin (DOXO) on human prostate and colon carcinoma cell lines was assessed both in vitro and in vivo. The effect on cell cycle, apoptotic rate, cyclin expression and induction of p21 expression was also determined.
  
  
  RESULTS
  Following treatment of tumor cells with PB + CRA + TX or DOXO, inhibition of tumor cell growth was greatly enhanced as compared to PB + CRA, TX or DOXO alone, with >90% growth inhibition. However, when the cells were pretreated with PB + CRA followed by TX or DOXO, the enhanced inhibition was abolished suggesting a protective effect to this sequence. Interestingly treatment with PB + CRA restored sensitivity to DOXO in PC-3 human prostate cancer cell line. PB + CRA induced p21 expression and cell-cycle arrest in G1 phase, while TX and DOXO induced G2/M arrest. p21 and p53-deficient colon carcinoma cell lines were more sensitive to the effect of PB + CRA and TX as single agents and in combination, as compared to the wild type cells. When p21-deficient cells were pretreated with PB + CRA followed by TX the protective effect was still observed. Treatment of tumor cells with combination of these drugs induced cell cycle delay at multiple mitotic checkpoints before undergoing apoptosis. Tumor growth was significantly inhibited and delayed in animals treated with either TX or concomitantly with TX and PB + CRA as compared to control. Animals treated with all three agents demonstrated further growth inhibition or delay than the TX alone or PB + CRA arm.
  
  
  CONCLUSIONS
  These results suggest a rational therapeutic approach for combination of differentiation-inducing agents with cytotoxic drugs given concomitantly, but not sequentially.
authors.affiliation: The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, 1650 Orleans Street, Cancer Research Building, 1M52, Baltimore, MD 21231, USA.
authors.forename: Henk M W
authors.initials: HM
authors.lastname: Verheul
authors.forename: David Z
authors.initials: DZ
authors.lastname: Qian
authors.forename: Michael A
authors.initials: MA
authors.lastname: Carducci
authors.forename: Roberto
authors.initials: R
authors.lastname: Pili
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology* / Q000633:toxicity; D017209:Apoptosis / Q000187:drug effects*; D002277:Carcinoma / Q000473:pathology; D002453:Cell Cycle / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects*; D002455:Cell Division / Q000187:drug effects*; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000473:pathology; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics; D016213:Cyclins / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D015474:Isotretinoin / Q000633:toxicity; D008297:Male; D010654:Phenylbutyrates / Q000633:toxicity*; D016133:Polymerase Chain Reaction; D011471:Prostatic Neoplasms / Q000473:pathology
chemical_list: D000970:Antineoplastic Agents; C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D010654:Phenylbutyrates; D015474:Isotretinoin