Elsevier

Lung Cancer

Volume 74, Issue 2, November 2011, Pages 197-205
Lung Cancer

Synergistic cytotoxicity, inhibition of signal transduction pathways and pharmacogenetics of sorafenib and gemcitabine in human NSCLC cell lines

https://doi.org/10.1016/j.lungcan.2011.03.003Get rights and content

Abstract

Introduction

Lung cancer is one of the most lethal tumors and, although standard chemotherapy produces clinical response, there has been little improvement in prognosis. Therefore, research effort has focused on target-specific agents, such as sorafenib, which blocks both the RAF/MEK/ERK signalling pathways and receptors involved in neovascularization and tumor progression, including VEGFR-2 and c-Kit. We investigated whether sorafenib would be synergistic with gemcitabine against NSCLC cell lines.

Materials and methods

Human lung cancer cells A549, CALU-1, CALU-6, H23 and HCC 827 were treated with sorafenib and gemcitabine, alone or in combination, and the cytotoxicity was assessed with CellTiter 96 Non-radioactive cell proliferation kit. Cell cycle and apoptosis were investigated with flow cytometry and fluorescence microscopy, respectively. Moreover, the effects of drugs on Akt (S473), c-Kit (Y823) and ERK (pTpY185/187) phosphorylation were studied with ELISA. Finally, quantitative PCR analysis was performed to assess whether sorafenib and gemcitabine modulated the expression of genes related to drug activity.

Results

Gemcitabine and sorafenib synergistically interacted on the inhibition of cell proliferation, and assessment of apoptosis demonstrated that drug associations increased the apoptotic index. Sorafenib reduced c-Kit and ERK activation and gemcitabine inhibited Akt phosphorylation. Moreover quantitative PCR showed that sorafenib modulated the expression of targets related to gemcitabine activity, while gemcitabine induced the expression of RKIP.

Conclusions

These data demonstrate that sorafenib and gemcitabine synergistically interact against NSCLC cells, through suppression of Akt, c-Kit and ERK phosphorylation, induction of apoptosis and modulation of dCK, RRM1, RRM2 and RKIP gene expression. The association between traditional cytotoxic agents with new target-specific agents, such as sorafenib, is a challenge for both clinical and preclinical future investigations in lung cancer treatments.

Introduction

Lung cancer represents the leading cause of cancer related deaths in many countries. It is estimated that there are 0.2 million diagnosed cases of lung cancer and over 0.16 million patients die in the United States [1]. The most common type of lung cancer is non-small cell lung cancer (NSCLC) which contributes to over 75% of all cases [2]. Despite advances in therapeutic strategies, less than 15% of patients with NSCLC survive beyond 5 years from initial diagnosis.

Chemotherapy is a palliative option for patients with metastatic disease while the administration of concurrent chemotherapy and radiation is indicated for stage III lung cancer and clinical research is investigating the potential of choosing treatment on the basis of mutations or altered expression of critical genes involved in cell growth dysregulation [3].

One of the main reasons for the failure of several clinical trials evaluating targeted therapy in lung cancer is the existence of multilevel cross-stimulation among the targets of the new biological agents along several pathways of signal transduction that lead to neoplastic events; blocking only one of these pathways allows others to act as salvage or escape mechanisms for cancer cells. Preclinical evidence of synergistic antitumor activity achievable by combining targeted agents that block multiple signalling pathways has recently emerged [4], [5]. The complexity of the signalling process in general further supports the need to interfere at different stages to avoid an escape mechanism for the cell. Whether the multitarget approach can be accomplished by using combinations of selective agents or specific agents that intrinsically target various sites is a matter of debate [6].

Such a multitargeted strategy has recently been validated in a number of preclinical and clinical studies using receptor tyrosine kinase (RTK) inhibitors, with broad target selectivity, [7] and classic cytotoxic agents.

Sorafenib (BAY 43-9006) is an oral multikinase inhibitor that decreases the kinase activity of both C-RAF and BRAF and targets the vascular endothelial growth factor receptor family (VEGFR-2 and VEGFR-3) and platelet derived growth factor receptor family (PDGFR and c-Kit) [8]. The in vitro studies are not a suitable model to evaluate the stroma response to treatment but they are useful instruments to study the direct antitumor activity of multikinase inhibitors [9]. Indeed, as reported by Wihlelm et al., sorafenib exerts an antitumor activity by inhibition of MEK and ERK phosphorylation in various cancer cell lines in a broad spectrum of human tumor xenograft models [8]. Together, these data suggest that sorafenib may inhibit tumor growth by a dual mechanism, acting either directly on the tumor (through inhibition of Raf and c-Kit signalling) and on the angiogenesis (through inhibition of VEGFR signalling).

The Raf activation through Ras caused the activation of a mitogenic kinase cascade that ultimately modulates gene expression via the phosphorylation of transcription factors [9], which can have profound effects on cellular proliferation and tumorigenesis. In this regard, KRAS mutations, the most frequent in NSCLC, seem to be promising marker to predict the benefit of MAP Kinase inhibitors [10], [11]. Nonetheless, the introduction of antiangiogenic agents, epidermal growth factor receptor (EGFR) inhibitors and other new anti-cancer agents in the treatment of NSCLC has recently improved the clinical outcome.

Preclinical studies showed that sorafenib displayed a dose-dependent antitumor activity against xenograft models representing human colon, lung, breast, ovarian, pancreatic cancers and melanoma [12]. Results of a phase II trial that investigated the efficacy of sorafenib in patients with stage IIIB/IV NSCLC showed that 12% and 28% obtained partial responses or stable disease, respectively [13]. By contrast, a recent study conducted on sorafenib plus gemcitabine in chemo-naive patients with advanced pancreatic cancer has demonstrated a little effect on OS and on PFS [14]. A cytotoxic chemotherapeutic agent that showed a broad application in multiple solid tumor types including non-small cell lung, breast, pancreatic, bladder and ovarian cancers is gemcitabine (2′,2′-difluoro-2′-deoxycytidine). Gemcitabine is a nucleoside analogue of deoxycytidine, activated by deoxycytidine kinase (dCK), that interferes with DNA synthesis through inhibition of ribonucleotide reductase (RR) and competition with dCTP for incorporation into DNA [15].

An in vivo study demonstrated that the combination of sorafenib with gemcitabine was well tolerated and this association did not abrogate the efficacy of gemcitabine in human pancreatic cancer (MIA-PaCa-2) xenograft model [15]. In particular, gemcitabine and sorafenib resulted in growth delays of 154% and 112%, compared with 221% growth delay when both agents were given in combination [12].

A recent study demonstrated a favorable interaction between sorafenib and gemcitabine in pancreatic cancer cells resulting from a complex modulation of cellular pathways involved in cell proliferation and survival. Sorafenib and gemcitabine synergistically interacted on the inhibition of cell proliferation, and assessment of apoptosis indicated that drug association increased the apoptotic index [16]. Moreover, Hoffman et al. [17] showed that sorafenib decreased the in vitro multi-drug resistance (MDR) by modulating the ABC-protein mRNA expression. In particular, gemcitabine and sorafenib combination produced a significant decrease in ABC_protein expression up to 77% in hepatocellular carcinoma [17].

A phase II trial investigating sorafenib in combination with gemcitabine in patients with advanced ovarian tumor reported few objective responses with 5% of PRs and demonstrated an encouraging incidence of SD (26%). Moreover the median time to progression was 5.4 moths and the median OS time was 13.3 months. [18]. Similar data was obtained from a multicenter study of phase II that investigated the effectiveness of MTD (maximum tolerated dose) of gemcitabine combined with metronomic capecitabine plus sorafenib for the treatment of metastatic RCC [19].

Given the poor prognosis of patients with advanced or metastatic NSCLC, more effective therapies are needed. We have conducted an in vitro study to assess the combination of sorafenib and gemcitabine for the treatment of NSCLC, in A549, CALU-1, CALU-6, H23 and HCC 827 NSCLC cell lines. We evaluated the cytotoxic effects of both drugs as single agents and in combination by analyzing apoptosis, cell cycle, gene expression of drug mechanism enzyme or molecular determinants such as ribonucleotide reductase (RR), deoxycytidine kinase (dCK) and RKIP, a Raf protein inhibitor.

Section snippets

Drugs and chemicals

Sorafenib (BAY 43-9006) was a gift from Bayer Shering Pharma (Leverkusen, Germany). Gemcitabine was generously supplied by Eli Lilly (Indianapolis, IN, USA). Gemcitabine was dissolved in sterile distilled water and sorafenib in DMSO; the compounds were then stored at 4 °C and −20 °C, respectively, and diluted in culture medium immediately before use. RPMI and DMEM media, foetal bovine serum, horse serum, l-glutamine (2 mM), penicillin (50 IU/ml) and streptomycin (50 μg/ml) were from Life

Assay of cytotoxicity

Cells were plated in 6-well sterile plastic plates (Costar, Cambridge, MA, USA) at 104 cells/well and were allowed to attach for 24 h. Cells were treated with (a) gemcitabine (0.001–100 μM) for 24 h followed by a 24-h washout in drug-free medium; (b) sorafenib (0.001–100 μM) for 72 h; (c) gemcitabine for 24 h followed by 24 h of washout in drug-free medium and then sorafenib for 72 h; (d) the reverse sequence of point (c). At the end of drug exposure, 15 μl of MTT solution were added and cells were

Assay of cytotoxicity and interaction between gemcitabine and sorafenib

Gemcitabine was cytotoxic against NSCLC cells with IC50s of 0.10 ± 0.01 (A549), 0.39 ± 0.09 (CALU-1), 0.65 ± 0.08 (CALU-6), 0.10 ± 0.01 (H23) and 0.07 ± 0.01 (HCC827) μM. Cell growth inhibition was also observed in all cell lines after 72 h of exposure to sorafenib, with IC50s of 1.00 ± 0.06, 1.2 ± 0.16, 2.02 ± 0.10, 3.08 ± 0.15 and 0.46 ± 0.07 μM in A549, CALU-1, CALU-6, H23 and HCC827 cells, respectively (Fig. 1).Since the CI method recommends a ratio of IC50s values at which drugs are equipotent, combination

Discussion

Treating lung cancer is a difficult task due to its high frequency for distant metastatization and resistance to both chemo- and radio-therapies. A number of targets have been validated in solid tumors, including the Raf and MAPK transduction pathways [21], [22], and are currently being investigated as possible pharmacological targets. Therefore many new compounds, including sorafenib alone or in combination with traditional agents, are currently undergoing investigation and hold great promise

Conflict of interest statement

All authors have contributed significantly to the work. MDT received an unrestricted research grant from Bayer (Milano, Italy); the other Authors declare no conflict of interest.

Acknowledgement

This study was conducted in the University Hospital Laboratory and it was supported by an unrestricted research grant from Bayer (Milano, Italy).

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