Low-dose taxotere enhances the ability of sorafenib to induce apoptosis in gastric cancer models

Abstract

Despite the low efficacy of conventional antitumour drugs, chemotherapy remains an essential tool in controlling advanced gastric and oesophageal cancers. We aimed to provide a biological rationale based on the sorafenib-taxotere interaction for the clinical treatment of gastric cancer. In vitro experiments were performed on four human gastric cancer cell lines (GK2, AKG, KKP and NCI-N87). Cytotoxicity was evaluated by sulforhodamine B (SRB) assay, cell cycle perturbations, apoptosis and mitotic catastrophe were assessed by flow cytometric and microscopic analyses, and protein expression was studied by Western blot. In the in vivo experiments, nude mice xenografted with the most resistant line were treated with sorafenib and docetaxel singly or in association. Sorafenib inhibited cell growth (IG(50) values ranged from 3.4 to 8.1 μM) and caused down-regulation of MAP-K/ERK phosphorylation and of mcl-1 and p-bad expression after a 48-hr exposure. Apoptosis induction was associated with caspase-3 and -9 activation and mitochondrial membrane depolarization. The drug combination enhanced apoptosis (up to 80%) and produced a synergistic interaction when low doses of the taxane preceded administration of the antityrosine kinase. This synergism was probably due to the induction of an anomalous multidiploid G0-G1 peak and to consequent mitotic catastrophe, which increased sensitivity to sorafenib. Consistent with in vitro results, the docetaxel-sorafenib sequence exhibited high therapeutic efficacy in NCI-N87 mouse xenografts producing tumour weight inhibition (> 65%), tumour growth delay (up to 25 days) and increased mouse survival (30%). Our findings suggest the potential clinical usefulness of treatment with sorafenib and docetaxel for advanced gastric cancer.

Fig 1

(A) Western blot analysis of baseline expression of sorafenib targets (c-raf, b-raf, EGFR, PDGFR-β, VEGFR) in gastric cancer lines, and κ-ras mutation analysis (mut., mutated; w.t., wild-type). (B) Cytotoxic activity of sorafenib after 24, 48 and 72-hr exposure in gastric cancer cell lines. (C) Effect of 10 μM sorafenib concentration on MAP kinase pathway after 48-hr exposure.

Fig 2

(A) Representative images of cytofluorimetric analysis of apoptosis in GK2, AKG, KKP and NCI N87 cells after 48-hr exposure to 10 μM sorafenib concentration. (B) Changes in apoptotic-related markers after 48-hr exposure to 10 μM sorafenib concentration. (C) Percentage of mitochondrial membrane potential depolarization (ΔΨ) after different exposure times to 10 μM sorafenib concentration.

Fig 3

(A) In vitro analysis of interaction between sorafenib and docetaxel after different treatment schedules. In all combination experiments, sorafenib was tested at concentrations of 0.1, 1 and 10 μM, while docetaxel was used at concentrations of 0.001, 0.01, 0.1 μM for KKP and AKG cells, and 0.0001, 0.001 and 0.01 μM for GK2 line. (B) Apoptosis induction and influence on caspase-3 activation caused by different sorafenib (Sor) – docetaxel (Doc) treatment schedules.

Fig 4

(A) Cell cycle perturbation induced by 0.01 μM concentration of docetaxel (Doc) after 1-hr exposure followed by 24-hr washout. (B) Representative images of cytofluorimetric analysis of apoptosis in untreated GK2 and after exposure to 0.01 μM of docetaxel. (C) Biparametric analysis of GK2 cells based on cyclin B1 detection and propidium iodide staining to highlight potential docetaxel-induced abnormalities in mitotic exit. (D) Representative images of morphologic microscope analysis of GK2 cells exposed to docetaxel showing cell populations with DNA condensation, abnormal mitotic figures, multinucleation and large viable cell formation.

Fig 5

Antitumour efficacy of combination of docetaxel and sorafenib in NCI-N87 tumour xenografts. Mice were injected i.m. with NCI-N87 cells and starting from sixth day after implant were treated with docetaxel and sorafenib alone or in combination. (♦), untreated; (□), doc-etaxel; (▴), sorafenib; (○), docetaxel followed by sorafenib, the latter given for 14 days; (*), docetaxel followed by sorafenib, the latter given for 21 days; (•), docetaxel followed by sorafenib, the latter given for 28 days. Arrows indicate the start of treatments. Data obtained in combination treatment groups were significantly different (P < 0.001) from those obtained in groups treated with a single agent and from untreated groups. Similarly, results obtained in groups treated with single agent were significantly different (P < 0.001) from those observed in untreated groups.