Elsevier

Biochemical Pharmacology

Volume 67, Issue 9, 1 May 2004, Pages 1779-1788
Biochemical Pharmacology

Daphnetin induced differentiation of human renal carcinoma cells and its mediation by p38 mitogen-activated protein kinase

https://doi.org/10.1016/j.bcp.2004.01.014Get rights and content

Abstract

Daphnetin has been shown to be a potent in vitro anti-proliferative agent to the human renal cell carcinoma (RCC) cell line, A-498. In the present study, we investigated its effects on mitogen-activated protein kinase (MAPK) signalling along with cell cycle events and cellular differentiation. Daphnetin-activated p38, however, higher concentrations were required to inhibit ERK1/ERK2. In addition, it did not activate SAPK or induce apoptosis, but instead inhibited S phase cell cycle transition of A-498 cells at low concentrations and time of exposure. In addition, a late G1, early S phase inhibition was observed at higher concentrations and time of exposure, indicating that the mechanism of daphnetin-induced differentiation was concentration dependent. Increased expression of the epithelial differentiation markers cytokeratins 8 and 18, correlated with increasing concentrations of daphnetin, while pre-treatment with a specific p38-inhibitor, served to limit this effect. There was no evidence that P-glycoprotein (P-gp) mediated multi-drug resistance (MDR) played a role in the anti-proliferative activity of daphnetin. Consequently, we concluded that p38 MAP kinase is intrinsically involved in mediating the effect of daphnetin in A-498 cells, suggesting that this drug may act by promotion of cellular maturation, and consequently may represent a novel low toxic approach for the treatment of poorly differentiated RCCs.

Introduction

The coumarins comprise a large class of phenolic phytochemical compounds known as benzopyrones, all of which consist of a benzene ring fused to an alpha (α) or gamma (γ) ring containing one oxygen and five carbon atoms. Coumarin (1,2-benzopyrone) belongs to the benzo-α-pyrones, while flavanoids contain the γ-pyrone ring [1], [2]. Coumarins and related compounds have been used clinically in the treatment of thrombotic disorders, chronic infections thermal injuries and immunological disorders and have also been evaluated in the treatment of various malignancies including, renal cell carcinoma (RCC) and malignant melanoma [3], [4], [5], [6], [7], [8], [9]. In the United States alone, the annual diagnosis of RCC in new patients runs to approximately 30,000 and accounts for almost 12,000 deaths [10], [11], [12]. Approximately 85% of RCCs are adenocarcinomas, being predominately proximal tubular in origin. Currently, chemotherapy and cytokine treatments of patients with RCC have produced insufficient response rates with complete remissions being rare [11].

The mitogen-activated protein kinases (MAPKs) are fundamental components of the signalling pathways transducing extracellular stimuli into a variety of cellular responses [13]. Members of the MAPK pathways include the extracellular signal related kinase (ERK), the c-jun kinase (JNK), also referred to as the stress-activated protein kinase (SAPK), and the p38 kinase modules [14], [15]. Although exceptions occur, the majority of research suggests that activation of the ERK pathway is implicated in cellular proliferation, differentiation and survival. Conversely, activation of the SAPK and p38 kinase cascades is generally, although not exclusively, associated with inflammation, apoptosis and cellular differentiation [16], [17]. Activation of the Ras/ERK/MAPK and other signalling cascades, through their translocation to the nucleus, ultimately activate MAPKs, and lead to activation of transcription factors. In turn these transcription factors regulate gene expression of molecules involved in homeostatic responses such as cell growth, proliferation and differentiation and may therefore represent potential novel targets for the prevention and/or treatment of cancers including RCC.

A recent study in our laboratory demonstrated the anti-proliferative effects of the coumarin derivative, daphnetin (7,8-dihydroxycoumarin), using in vitro cytotoxicity assays with two human renal cell lines. The first cell line, A-498 was a human RCC line, while the second was a non-carcinoma proximal tubular cell line termed, HK-2. Results clearly demonstrated that daphnetin was a potent anti-proliferative agent in the RCC cell line, and was significantly less toxic to the HK-2 cells, suggesting that this compound might be capable of selectively inhibiting A-498 rather than HK-2 cells. In addition, mobility shift and BrdU incorporation assays showed that daphnetin did not intercalate DNA but had a concentration-dependent inhibitory effect on its synthesis. Genotoxicity testing using the standard Ames test showed that this compound was not a mutagen either with or without a mammalian metabolic activation system [18]. Taken together, these results suggested that daphnetin warranted further investigation as an agent in the treatment of RCC.

In an attempt to explore the possible therapeutic potential of daphnetin in the treatment of RCC, we have focused our attention on elucidating its effects on a number of key cellular targets. These targets include; MAPK activation, cell cycle and key differentiation parameters along with the possibility that daphnetin may act as a substrate for P-gp mediated MDR. The selection of these molecular targets was based on the knowledge that they are centrally involved in the cause and development of RCC. Results from previous studies have shown that constitutive activation of the Ras/ERK/MAPK pathway has been implicated in the progression of and often the de-differentiated phenotype of RCC [19], [20], [21]. It is thought to be this factor may lead to the almost complete failure of systemic drug therapy regimes for RCC [22]. This may, in the main, be due to the highly drug resistant phenotype and slow growth kinetics observed for RCC. This theory is supported by the observation that RCC, with the exception of adrenal carcinomas, expresses the highest levels of the MDR-1 gene product, P-gp [23], [24], [25]. It has been established that over-expression of P-glycoproteins (P-gp) is responsible for the “classical” type of MDR. The drugs affected by this mode of resistance include: anthracyclines, epipodophyllotoxins (VP-16 and VP-26) and Vinca alkaloids, among others. The multi-drug transporter associated with MDR-1, P-gp-170 prevents the intracellular accumulation and as a consequence inhibits the cytotoxic effects of chemotherapeutic drugs by actively removing them from the cell before they reach their intra-cellular targets. Consequently, as the MDR phenotype of RCC greatly affects clinical prognosis, it should prove valuable to determine the potential of any novel drugs directed against RCC to act as an MDR substrate. Finally, the aim of the current study was to elucidate key aspects of the mechanism of action of daphnetin and provide evidence that this compound may offer a significant advantage in the treatment of RCC.

Section snippets

Test compounds and reagents

Vinblastine, melphalan, dimethyl sulphoxide (DMSO) and SB203580 were purchased from Sigma–Aldrich, Ltd., while 7,8-dihyroxycoumarin (7,8-OHC, Daphnetin) was purchased from Lancaster Synthesis. The chemiluminescence substrate was purchased from Pierce Laboratories Ltd. All phosphorylation state specific antibodies against ERK, p38 and SAPK were purchased from New England Biolabs, Inc. Mouse monoclonal antibodies against cytokeratin 8 and cytokeratin 18 were purchase from Calbiochem. Jsb-1, the

Role of MAPK phosphorylation

Since ERKs, SAPK and p38 modules are the functional convergence points for these three distinct signalling pathways of the MAPK family, the effect of daphnetin on their phosphorylation state was investigated [30]. As shown in Fig. 1A, treatment of A-498 cells with daphnetin over the concentration range 0–500 μM did not inhibit constitutively active ERK1/ERK2 phosphorylation levels at the 48 h time point, when compared to control cells that received drug vehicle alone. However, phosphorylation was

Discussion

Normal cellular growth, proliferation, differentiation and apoptosis is governed by cellular signalling that functions to transduce signals by either allowing or halting progression of a cell through cycle check points ultimately toward their cellular fate [36], [37]. Development of RCC from normal renal epithelium involves alterations in genes whose products control cell division. These include genes that participate directly in controlling cell cycle, such as Rb, the tumour suppressor gene p53

Acknowledgements

This research was supported by the Graduate Training Programme, under the European Social Fund Operational Programme for Industrial Development and the Higher Education Authority, Ireland. This research was carried out in the National Centre for Sensor Research (NCSR), jointly located at Institute of Technology, Tallaght and Dublin City University, Dublin, Ireland.

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