The Bcl-2 family plays an important role in regulating apoptosis (see review (Thomadaki et al., 2006
)), and targeting anti-apoptotic Bcl-2 family members combats the drug resistance exhibited by many cancer types (see review (Kang and Reynolds, 2009
)). In this study, we investigated the effects and mechanisms of treating melanoma cells with a BH3 mimetic Bcl-2 inhibitor, ABT-737, as a single treatment or combined with a chemo-therapeutic drug already used in clinic, Bortezomib.
We found that knocking-down protein expression of the anti-apoptotic proteins Bcl-2, Bcl-XL, or Mcl-1 individually with siRNAs is not sufficient to induce cell death in melanoma cells. We determined that ABT-737 single treatment induced moderate cytotoxicity at 10 µM, but little or no cytotoxicity at lower doses (). However, knocking-down Mcl-1, but not Bcl-2 or Bcl-XL, dramatically sensitized these melanoma cells to ABT-737 (), demonstrating that Mcl-1 is the critical mediator of melanoma's resistance to ABT-737 treatment. Results here also indicate that melanoma cells have multiple anti-apoptotic defenses, and that neutralizing multiple anti-apoptotic Bcl-2 members at the same time is necessary to induce optimal killing effects for melanoma cells.
The Bcl-2 family can be divided into 3 groups (see review (Thomadaki et al., 2006
)): (1) Anti-apoptotic proteins, including the homologous proteins Bcl-2, Bcl-XL
, Bcl-w, Mcl-1, and A1; (2) Multi-domain pro-apoptotic proteins Bax and Bak (and the less-studied Bok), which are critical downstream mediators of apoptosis; (3) Pro-apoptotic BH3-only proteins, including Noxa, Bad, Bim, Bid, Puma, Bmf, BNIP3, BNIP3L, Hrk, and Bik, which antagonize the function of anti-apoptotic Bcl-2 family members.
Binding affinities between different Bcl-2 family members are not equal and are important for their respective functions. For example, the BH3-only pro-apoptotic protein Bad binds tightly to the anti-apoptotic proteins Bcl-2, Bcl-XL
, and Bcl-w, but not to Mcl-1 or A1, whereas Noxa binds to Mcl-1 and A1 but far less strongly to Bcl-2 and Bcl-XL
, and is not known to bind Bcl-w (Chen et al., 2005
; Willis et al., 2005
; Smith et al., 2011
ABT-737 is a mimetic of Bad, and as such, it binds tightly to Bcl-2, Bcl-XL
, and Bcl-w, antagonizing their anti-apoptotic function (Oltersdorf et al., 2005
). However, ABT-737 does not bind to Mcl-1 or A1, just as Bad does not. Thus, Mcl-1 (or A1) may act as a barrier to ABT-737-induced cell death, and it has been shown that neutralizing Mcl-1 significantly potentiates the cytotoxicity induced by ABT-737 in treating multiple cancer types (Adams et al., 2005
; Certo et al., 2006
; Konopleva et al., 2006
; Letai, 2006
; Lin et al., 2006
; van Delft et al., 2006
; Chen et al., 2007
; Tahir et al., 2007
; Olberding et al., 2010
; Harrison et al., 2011
). Our results for treating melanomas are consistent with these studies, implying that combination treatments that antagonize multiple anti-apoptotic Bcl-2 family members are needed.
We attempted to determine if the anti-apoptotic Bcl-2 family member, A1, regulates melanoma's sensitivity to ABT-737. We found it was difficult to detect A1 protein expression in melanoma cells, probably due to low expression levels (data not shown). In addition, the dramatic increase in melanoma's sensitivity to ABT-737 with Mcl-1 protein knockdown indicates that Mcl-1 is the critical mediator of resistance to ABT-737 in melanoma cells. These results indicate that combination treatments of ABT-737 with drugs that neutralize the anti-apoptotic function of Mcl-1 are good treatment strategies. Moreover, resistance of melanoma cells to Bortezomib has been shown to be mediated by anti-apoptotic Bcl-2 family members (Wolter et al., 2007
), making ABT-737 and Bortezomib a promising combination.
We therefore examined whether combining ABT-737 with the chemotherapeutic drug Bortezomib would synergistically kill melanoma cells. MTS and Annexin V assays showed that combining ABT-737 with Bortezomib synergistically killed melanoma cells (), and immunoblots of cleaved PARP corroborate the fact that the combination treatment induced apoptosis synergistically (). Immunoblot analyses also demonstrated a significant increase in Noxa protein expression and loss of Mcl-1 protein for the combination treatment, significantly increasing the Noxa/Mcl-1 protein ratio (). Bortezomib primarily achieves this through drastic up-regulation of Noxa protein expression, though agents that promote Mcl-1 protein loss in the absence of Noxa up-regulation should have a similar effect.
Interestingly, Bortezomib treatment alone led to a significant increase of Mcl-1 levels at 24 h, most likely due to decreased proteolysis. While this effect has been observed previously in melanoma cells (Qin et al., 2006
), it is in marked contrast to what is observed in multiple myeloma cells, where Mcl-1 undergoes caspase-dependent cleavage (Podar et al., 2008
). Likewise, ABT-737 alone can decrease Mcl-1 in multiple myeloma (Kline et al., 2007
), whereas we did not observe any Mcl-1 decrease with the concentrations used in the present study. These differences are likely due to the fact that apoptosis can induce caspase-dependent degradation of Mcl-1 (Podar et al., 2008
; Miller et al., 2009
), and that ABT-737 alone at 3.3 µM or less did not induce apoptosis in the present study, whereas similar concentrations induce significant cell death in multiple myeloma cell lines (Kline et al., 2007
). Rather, in the present study, Mcl-1 degradation appears to have taken place only upon large-scale apoptosis induced by the combination treatment. These differences underscore the different sensitivities of various cancers to chemotherapeutic drugs, and show why, in the case of melanoma, targeting multiple cellular defenses is likely necessary.
To test whether Noxa up-regulation was responsible for the effects observed in the combination treatment of ABT-737 with Bortezomib, we inhibited Noxa expression via siRNA. This inhibition protected melanoma cells from cytotoxicity induced by the combination treatments (). These results indicate that Bortezomib neutralizes Mcl-1's function specifically through Noxa.
Our in vivo mouse experiments showed that ABT-737 and Bortezomib treatments reduce the rate of tumor growth. Using relatively low doses of the drugs, we found reduced tumor growth for all treatment groups and significant decreases in the tumor doubling rate (), suggesting that each drug is promising as a candidate for treating melanoma. The combination treatment increased this time further still, and was statistically significant compared to both Bortezomib and ABT-737 individual treatments.
One critical limiting factor with the use of Bortezomib and ABT-737 in our mouse model was acute toxicity, particularly of Bortezomib. We therefore used relatively low doses that did not cause weight loss and found that the effect of the drug combination was still significant. However, without this dosing limitation, it is likely that we would observe a much more pronounced effect on tumor growth in the mice treated with the drug combination. This implies that newer proteasome inhibitors that are less toxic than Bortezomib, and can be used at higher doses, may work even better with ABT-737 and hold more promise for clinical trials.
The results from our in vitro
Bortezomib treatments are consistent with our previous study on the combination treatment of ABT-737 with another proteasome inhibitor, MG-132 (Miller et al., 2009
). In that study, we found that the combination treatment of MG-132 and ABT-737 antagonizes Mcl-1 through increased Noxa expression and caspase-dependent Mcl-1 cleavage (Miller et al., 2009
). Noxa induction appears to be a consistent mechanism for cell death in melanoma cells treated with various proteasome inhibitors (Qin et al., 2005
). These data indicate that various proteasome inhibitors function through the same mechanisms when combined with ABT-737 in treating melanoma cells. Currently, more potent and less toxic, next-generation proteasome inhibitors are in development for clinical use (Orlowski and Kuhn, 2008
; Kuhn et al., 2011
). Our data suggest that combining these newer proteasome inhibitors with ABT-737 would be an effective means of treating melanomas, if they are less toxic, and that this is worthy of further investigation.
In conclusion, the data reported here indicate that drugs that neutralize Mcl-1's function are good candidates for combination therapy with ABT-737 for treating melanomas. This validates rational molecular approaches for targeting multiple anti-apoptotic Bcl-2 members in developing cancer treatments.