MK5 is degraded in response to doxorubicin and negatively regulates doxorubicin-induced apoptosis in hepatocellular carcinoma cells

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Abstract

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide. The mechanisms by which hepatoma cells resist apoptosis induced by doxorubicin are largely unknown. MAPKAPK5 (MK5), also named as p38-regulated/activated protein kinase (PRAK), has been identified as a crucial mediator of skin tumorigenesis in mouse and colon cancerogenesis in human. Here, we describe a novel role of MK5 in doxorubicin-induced apoptosis in human hepatoma cells. Expression of MK5 was highly upregulated in hepatoma cell lines. Doxorubicin rather than other chemotherapeutic drugs reduced MK5 protein level in a time- and concentration-dependent manner in hepatoma cells (HepG2 and Hep3B). We further showed that MK5 degradation induced by doxorubicin was via the 26S proteasome. Remarkably, stable overexpression of MK5 led to decreased cleavage of caspase-3 and PARP and attenuated doxorubicin-induced apoptosis, while stable knockdown of endogenous MK5 sensitized hepatoma cells to doxorubicin, which was coupled with increased cleavage of caspase-3 and PARP. Taken together, our results firstly demonstrate that MK5 is degraded in response to doxorubicin and negatively regulates doxorubicin-induced apoptosis, providing novel insights into the molecular mechanism of doxorubicin resistance in hepatoma cells.

Highlights

► MK5 expression is upregulated in hepatoma cells. ► Doxorubicin promotes MK5 degradation via the 26S proteasome. ► MK5 regulates doxorubicin-induced apoptosis in hepatoma cells. ► MK5 regulates the cleavage of caspase-3 and PARP in doxorubicin-induced apoptosis.

Introduction

Hepatocellular carcinoma (HCC) ranks as the third leading cause of cancer mortality worldwide, with more than 600,000 new cases diagnosed annually [1]. Over the past decade, curative surgery has become the mainstay of treatment for HCC and the first option for patients with early-stage tumors [2], [3]. However, many patients have been diagnosed with unresectable HCC at the time of initial diagnosis due to lack of early detection methods [2].

Chemotherapy has proven to be an effective treatment modality especially for patients with inoperable HCC. Doxorubicin (DOX), an anthracycline-based anticancer drug, is widely used for HCC among various chemotherapeutic drugs [4]. DOX acts as a DNA topoisomerase II inhibitor and blocks DNA synthesis through intercalation into the DNA strands, triggering intracellular apoptotic pathways in cancer cells [4]. Apoptosis is a tightly regulated cell suicide process and plays a vital role in tumor regression. DOX-induced apoptosis has been characterized by activation of caspase-3 which subsequently leads to cleavage of PARP [4]. Recent studies have demonstrated that the response rate to DOX becomes lower and lower [5], [6]. Therefore, it is of great significance to search for novel molecular targets for developing more effective treatment methods.

Mitogen-activated protein kinase (MAPK)-activated protein kinase 5 (MK5), also described as p38-regulated/activated protein kinase (PRAK), has been identified as a serine/threonine kinase involved in p38 MAPK signaling pathway [7], [8]. Previous researches on MK5-deficiency mice indicated that MK5 was an essential factor in tumor suppression [9], [10]. The MK5-deficient mice were susceptible to dimethylbenzanthracene (DMBA)-induced skin carcinoma [9]. Intriguingly, a recent study on the same model revealed that once the tumor was formed, MK5 functioned as a critical factor to promote the progression of skin cancer [11]. Activation of MK5 was induced by tumor-secreted proangiogenic factors, resulting in endothelial cell migration and tumor angiogenesis [11]. All these evidence suggest that MK5 plays a dual role in the development of cancer, depending on the tissue type and the stage of carcinogensis. Moreover, MK5 was reported to be down-regulated in colon carcinomas and the negative feedback loop formed by MK5 and Myc was disrupted during colorectal tumorigenesis [10]. Despite the clear roles of MK5 in skin carcinogenesis and colorectal tumorigenesis, there is no evidence indicating that MK5 is involved in hepatocarcinogenesis.

MK5 was originally identified as a downstream target of p38 MAPK [7], [12], [13]. The members of p38 MAPK group have been shown to regulate apoptosis in response to various stimuli [14], [15]. Previous findings indicated the involvement of p38 MAPK in both cisplatin-provoked apoptosis in epithelial renal tubule cells and TNF-α-induced apoptosis in differentiated PC12 cells [16], [17]. On the basis of these results, we hypothesize that MK5 is likely to be associated with the stress-induced apoptosis.

We describe here the involvement of MK5 in DOX-triggered apoptosis. High expression of MK5 was detected in hepatoma cells. Proteasomal degradation of MK5 was specifically induced by DOX. Stable overexpression or knockdown of MK5 significantly attenuated or promoted, respectively, DOX-induced apoptosis in hepatoma cells.

Section snippets

Cell lines and cell culture

The human hepatoma cell lines HepG2, Hep3B and SK-Hep-1 were purchased from the American Type Culture Collection. Other cell lines L02, SMMC-7721, BEL-7402, QGY-7703 and Huh7 were obtained from State Key Laboratory of Genetic Engineering, Fudan University (Shanghai, China). All these cell lines have been previously published [18], [19]. Cells were maintained in DMEM with 10% FBS at 37 °C in an atmosphere of 5% CO2.

Plasmid construction

The full-length MK5 cDNA was cloned into the pcDNA 3.1/Myc-His vector for

MK5 expression is upregulated in hepatoma cells

Previous studies have suggested the involvement of MK5 in skin carcinogenesis and colorectal tumorigenesis. To test whether MK5 plays a role in HCC, we firstly determined the expression of endogenous MK5 in hepatoma cell lines and an immortalized normal human liver cell line L02 by Western blot. As shown in Fig. 1A and B, MK5 protein levels were significantly higher in seven hepatoma cell lines than in L02 cells. These data indicate that expression of MK5 protein is frequently upregulated in

Discussion

In the present study, we have identified MK5 expression in hepatoma cells and the functional role of MK5 in DOX-induced hepatoma cell apoptosis. We have showed that MK5 protein is frequently highly expressed in hepatoma cells (Fig. 1). Previous studies have demonstrated that MK5 functions as an important regulator of skin carcinogenesis and colorectal tumorigenesis [10], [11]. Our study firstly implies a potential novel function of MK5 in hepatocarcinogenesis. We further show that hepatoma

Acknowledgments

This work was supported by the National Key Sci-Tech Special Project of China (2008ZX10002-020 and 2013ZX10002-010) and the National Natural Science Foundation for Young Scholar of China (Grant No. 81101810).

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