Bcl-xL-mediated ERK2 activation plays a critical role in the inhibition of doxorubicin-induced apoptosis

Bcl-xL is one of the representative anti-apoptotic molecules against various cytotoxic stimuli and it is widely overexpressed in various tumor cells (Kirkin V et al., 2004). Bcl-xL has been demonstrated to exert its anti-apoptotic function through

the prevention of mitochondrial changes such as mitochondrial membrane potential, cytochrome c release, and the production of reactive oxygen species (Kirkin V et al., 2004). However, it is not well characterized whether the anti-apoptotic function of Bcl-xL in response to cytotoxic stimuli is associated with modulation of the activities of signaling molecules. We demonstrate here for the first time that ERK2 activated in Bcl-xL-overexpressing cells treated with doxorubicin significantly contribute to the death blocking effect of Bcl-xL. ERK2 activation in response to doxorubicin was observed not only in overexpressing Huh-7 cells (Fig. 3) but also in Bcl-xL-overexpressing Chang or U87MG cells (Fig. 4), suggesting that this response is generally induced irrespective of cell types and ERK2 activated NF-κB. We tested

whether overexpression of other anti-apoptotic molecules also can inhibit doxorubicin-induced apoptosis through ERK2 activation (Fig. 8). Although overexpression of Bcl-2 but not Akt or survivin, inhibited doxorubicin-induced

apoptosis at lesser extent than Bcl-xL overexpression, ERK2 activation was not observed in Bcl-2 overexpressing cells treated with doxorubicin. These results suggest that ERK2 activation in response to doxorubicin is specifically mediated by Bcl-xL contributing to strong anti-apoptotic activity of Bcl-xL.

Activation of the ERK cascade has been generally known to lead cell proliferation or survival (Chang F et al., 2004), although pro-apoptotic role of ERK has been reported in cisplatin–induced apoptosis in human cervical carcinoma HeLa cells (Wang X et al., 2000) or radiation–induced cell death in HSP25-overexpressed L929 cells (Cho HN et al., 2002). Therefore, ERK may play an anti-apoptotic or pro-apoptotic role depending on the cytotoxic stimuli or cellular contexts. ERK2 has been generally postulated to have proproliferative effects, while ERK1 has antiproliferative effects (Chang F et al., 2004). However, little is known how different in vivo targets of ERK1 and ERK2 are. In our study, mainly ERK2 was activated in Bcl-xL-overexpressing treated with doxorubicin (Fig. 3 and 4).

Functional significance of ERK2 activation in Bcl-xL-mediated blocking of doxorubicin-induced apoptosis was confirmed by the following lines of evidences.

First, overexpression of DN ERK2 sensitized doxorubicin-induced apoptosis in Bcl-xL overexpressing Chang cells (Fig. 5). Second, overexpression of ERK2 in Chang cells alleviated doxorubicin-induced apoptosis (Fig. 6).

MEK is known to be a representative upstream kinase of ERK (Chang F et al., 2004). However, in our study the activity of MEK, a well-known upstream activator of ERK, was not increased in Bcl-xL-overexpressing Huh-7 cells treated with

doxorubicin, whereas p90RSK and CREB, well-known downrstream targets of ERK pathway, were significantly activated (Fig. 3B). Thus, other upstream signaling mechanisms to activate ERK2 may exist in Bcl-xL-overexpressing cells treated with doxorubicin. Possible candidate is MAP kinase phosphatase-1 (MKP), the phosphatase involved in the downregulation of ERK activity (Farooq A and Zhou MM, 2004). Recent report demonstrated that repression of MKP-1 by antharacyclines, doxorubicin and daunorubicin, contributes to their anti-apoptotic activation of ERK in A1N4-myc human mammary epithelial cells (Small GW et al., 2003). Contrastingly with their study, protein levels of MKP-1 or MKP-3 were not significantly decreased by doxorubicin in Bcl-xL overexpressing cells in our study (data not shown), although we cannot exclude the possibility that downregulation of the expression or activities of other phosphatases may be involved in ERK2 activation in xL-overexpressing treated with doxorubicin. In breast cancers, Bcl-xL has been reported to be upregulated (Zapata JM et al., 2003). Moreover, c-Myc overexpression in mammary epithelia cells has been reported to suppress apoptosis and upregulated Bcl-xL at both mRNA and protein levels (Nass SJ et al., 1996).

Therefore, it is very plausible that anti-apoptotic activation of ERK in MDA-MB-231 breast cancer cell lines or in A1N4-myc human mammary epithelial cells in response to doxorubicin may be mediated by high protein levels of Bcl-xL in the study by Small et al.. In addition doxorubicin has been reported to activate ERK in primary rat ventricular myocyte (Adderley SR and Fitzgerald DJ, 1999), SK-N-SH neuroblastoma cells (Guise S et al., 2001) and SiHa human cervical carcinoma cells

(Yeh PY et al., 2002), which are resistant to doxorubicin. In contrast, ERK is not activated in H9 human T-cells (Yu R et al., 1996) and MCF-7 breast cancer cells (Kim J and Freeman MR 2003), which are sensitive to doxorubicin. Although the detailed role of ERK activated by doxorubicin in apoptosis has not been clarified in their respective studies, it’s very possible that the protein levels of endogenous Bcl-xL in the respective cancer cells may affect not only the cellular resistance to doxorubicin but also doxorubicin-induced ERK activation.

Recently, DNA double-strand breaks by doxorubicin have been reported to activate ATM activates MEK/ERK/P90^RSK/IKK signaling pathway, leading to NF-κB activation and cell survival (Panta GR et al., 2004). Although MEK was not activated in Bcl-xL overexpressing cells treated with doxorubicin in our study, ERK, p90^RSK and NF-κB were significantly activated blocking doxorubicin-induced apoptosis (Fig.

3B). Downregulation of ERK2 activity in Bcl-xL-overexpressing Chang cells by the expression of DN ERK2 inhibited doxorubicin-induced activation of p90^RSK and NF-κB, increasing the cellular sensitivity to doxorubicin-induced apoptosis (Fig. 5). In

addition, enhancement of ERK2 activity in control Chang cells by expression of WT ERK2 increased the activities of p90^RSK and NF-κB in response to doxorubicin, alleviating doxorubicin-induced cell death (Fig. 6). Therefore, these results suggest that ERK2/p90^RSK/NF-kB signaling pathway mediated by Bcl-xL is critical for conferring resistance to doxorubicin.

Although overall amino acid sequence of Bcl-xL are very similar to that of Bcl-2, increasing lines of evidences have suggested that pro-survival activities of Bcl-2 and

Bcl-xL are differently regulated. Programmed cell death of WEHI-231 cells upon cross-linking of IgM and upon exposure to immunosuppressants CsA, FK506, and rapamycin could be suppressed by Bcl-xL but no by Bcl-2 (Gottschalk AR et al., 1994). The chemosensitivity of HepG2 cells to taxol and doxorubicin was not affected by Bcl-2 levels, while reduction of Bcl-xL levels rendered the cells more sensitive to the drugs (Luo D et al., 2000). In our study, anti-apoptotic activity of Bcl-xL on doxorubicin-induced apoptosis was more potent that of Bcl-2 (Fig. 8B and 9B), although the protein levels of exogenously expressed Flag-tagged Bcl-xL and Flag-tagged Bcl-2 in the respective stable cell lines were similar in Western blotting data (data not shown). Doxorubicin-induced ERK activation was occurred in Bcl-xL-overexpressing cells but not in Bcl-2-Bcl-xL-overexpressing cells (Fig. 8C and 9C).

Therefore, it remains to be clarified whether this stronger resistance to doxorubicin in Bcl-xL-overexpressing cells is solely caused by ERK2 activation. Moreover, it will be interesting to investigate whether anti-apoptotic action of ERK2 activated in Bcl-xL overexpressing cells treated with doxorubicin is independent of the activity of Bcl-xL controlling mitochondrial function or whether ERK2 directly acts as an upstream signaling pathway of Bcl-xL-mediated control of mitochondria.

Fig. 25. Schematic model for Bcl-xL mediated ERK2 activation in response to doxorubicin.