Elsevier

Biochemical Pharmacology

Volume 71, Issue 11, 28 May 2006, Pages 1540-1550
Biochemical Pharmacology

The BH3-only protein, PUMA, is involved in oxaliplatin-induced apoptosis in colon cancer cells

https://doi.org/10.1016/j.bcp.2006.02.011Get rights and content

Abstract

Oxaliplatin, the first line chemotherapeutic of colon cancer, induces damage to tumors via induction of apoptosis. PUMA (p53 up-regulate modulator of apoptosis) is an important pro-apoptotic member of Bcl-2 family and regulated mainly by p53. Here we investigated the role of PUMA in oxalipaltin-induced apoptosis and the potential mechanism. We showed that oxaliplatin-induced PUMA expression in a time- and dose-dependent manner and suppression of PUMA expression by stable transfecting anti-sense PUMA plasmid decreased oxaliplatin-induced apoptosis in colon cancer cells. By abrogating the function of p53, we further demonstrated that the induction was p53-independent. We also found that oxaliplatin could inactivate ERK and suppression of ERK activity by its specific inhibitor (PD98059), and dominant negative plasmid (DN-MEK1) enhanced the oxaliplatin-induced PUMA expression and apoptosis in a p53-independent manner. Taken together, our data suggest that PUMA plays an important role in oxaliplatin-induced apoptosis and the induction could be both p53-dependent and p53-independent. Moreover, PUMA expression and apoptosis in oxaliplatin-treated colon cancer cells could be regulated partly by ERK inactivation. Identification of the molecular components involved in regulating the cellular sensitivity to oxaliplatin may provide potential targets for development of novel compounds that may be useful in enhancement of oxaliplatin cytotoxicity in p53 deficient colon cancer.

Introduction

Oxaliplatin (1R, 2R-diaminocyclohexane) is the first of several analogs from the diaminocyclohexane platinum family to be successfully developed in the clinic and has a non-hydrolyzable diaminocyclohexane (DACH) carrier ligand which is maintained in the final cytotoxic metabolites of the drug [1], [2]. It produces the same type of inter- and 1, 2-GG intrastrand cross-links like cisplatin but has a spectrum of activity and mechanisms of action and resistance different from those of cisplatin and carboplatin [3]. Oxaliplatin has shown a wide spectrum of antitumor effects both in vitro and in vivo and has a better safety profile than cisplatin. A lack of cross-resistance with cisplatin and carboplatin makes it a promising chemotherapy agent [4], [5].

Inducing cancer cells to apoptosis is an important action of oxaliplatin [6], but the precise molecular mechanism is not very clear. As Bcl-2 family members play a pivotal role in the intrinsic apoptotic cascade [7], [8], [9], therefore, we investigated the roles of PUMA (p53 up-regulate modulator of apoptosis) in oxiliplatin-induced apoptosis. PUMA was identified by Yu and Nakano et al. in 2001 [10]. It belongs to BH3-only protein family and is found to locate at mitochondrial membrane and interacts with Bcl-2 and Bcl-XL through a BH3 domain [11], [12]. When apoptotic stimuli induces PUMA expression, it will make Bax translocates to the mitochondria membrane and multimerizes [13], cytochrome c releases and apoptosis appears. Previous studies have shown that PUMA is a direct mediator of p53 through the cytochrome c/Apaf-1-dependent pathway [14] and plays an important role in stress-induced apoptosis [15], [16]. The balance between p21WAF1/CIP1 and PUMA is pivotal in determining the cells to cycle arrest or apoptosis in human cancer.

Mitogen-activated protein kinases (MAPKs) are serine/threonine kinases that play an important role in signal transduction from the cell surface to the nucleus. The mammalian MAPKs can be divided into extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs), and p38 MAPKs. In response to a wide range of extracellular stimuli, the MAPK cascades determine cell fate, including cell growth, differentiation, and apoptosis [17]. Generally, the ERK cascade is mainly involved in the regulation of cell proliferation in a variety of cells [18], while the JNK and p38 MAPK pathways are responsible for induction of differentiation and apoptosis [19]. In our present study, we determined the role of PUMA in oxaliplatin-induced colon cancer cell apoptosis and attempted to find the potential role of MAPK signaling pathway in this process. Our data showed that oxaliplatin induced PUMA expression in a p53-independent manner. Suppression of PUMA expression decreased oxaliplatin-induced apoptosis in colon cancer cells. Blockade of ERK activity increased the PUMA expression and apoptosis in oxaliplatin-treated colon cancer cells in a p53-independent manner. These results suggest that oxaliplatin-induced ERK inactivation is involved in the regulation of oxaliplatin-induced PUMA expression and apoptosis.

Section snippets

Materials

RPMI 1640 medium, FBS (fetal bovine serum) were purchased from GIBCO. Oxaliplatin (1R, 2R-diaminocyclohexane) and pifithrin-alpha (p53 inhibitor) were obtained from Sigma (Poole, Dorset, UK). SP600125 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one) and PD98059 (2V-amino-3V-methoxyflavone) were purchased from Calbiochem (La Jolla, CA). p53 (DO-1) antibody, p73 antibody, p21/Waf-1 antibody, p38 MAPK (Tyr183/Tyr185) antibody, phospho-specific JNK (Tyr183/Tyr185) antibody, phospho-specific

Oxaliplatin induced PUMA expression in colon cancer cells in a time- and dose-dependent manner

PUMA plays an important role in stress-induced apoptosis, but its role in oxaliplatin-induced apoptosis is unknown. To assess the effects of oxliplatin on PUMA expression, we exposed lovo cells to a dose range of oxaliplatin at different time points as described in Section 2 and measured its expression by Western blotting. As shown in Fig. 1, oxaliplatin induced PUMA expression in colon cancer cells and the induction was in a time- and dose-dependent manner. The PUMA expression reached a

Discussion

In the present study, we determined the role of PUMA in oxaliplatin-induced colon cancer cell apoptosis and the potential mechanism in this process. Our data showed for the first time that oxaliplatin could induce PUMA expression and the induction is p53 independent. ERK signaling pathway also appears to be essential in the regulation of oxaliplatin-induced PUMA expression and apoptosis.

Induction of apoptosis in cancer cells is a critical feature of chemotherapeutics [23]. The major mechanism

Acknowledgments

The authors thank Dr. Thijn R. Brummelkamp for providing the pSUPER-p53 plasmid. This work was supported by the Science and Technology Program of Guangdong province, China (2002C30307). This work was also supported in part by an AoE scheme of UGC and grants from the Research Grants Council of the Hong Kong Special Administrative Region, China (HKU 7243/02M to MCL) and grants from Li Ka Shing Institute of Health Sciences, Hong Kong, China (to HFK).

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