Role of Bcl-xL in Doxorubicin-Induced Cell Death: Apoptosis and Mitotic Cell Death

Abstract

Bcl-xL overexpressed in various human cancer cells contributes to resistance against various chemotherapeutic agents. In this study, we compared the role of Bcl-xL in high dose (HD) doxorubicin-induced apoptosis and low dose (LD) doxorubicin-induced mitotic cell death. In the first part of this study, we investigated whether anti-apoptotic effect of Bcl-xL on doxorubicin-induced apoptosis was associated with Bcl-xL-mediated modulation of signaling pathways. Bcl-xL overexpression in many cell types effectively blocked HD doxorubicin-induced apoptosis. HD Doxorubicin-induced activation of p38 or JNK was not affected by Bcl-xL overexpression. However, ERK2 was rapidly activated and its high activity was sustained in Bcl-xL-overexpressing cells treated with doxorubicin, whereas its activity was progressively decreased in doxorubicin-treated control cells. CREB, p90RSK, and NF-B, possible target molecules of ERK2, were also activated in Bcl-xL-overexpressing cells treated with doxorubicin. Enhanced expression of wild type ERK2 in control cells alleviated doxorubicin-induced apoptosis. Moreover, forced expression of dominant-negative ERK2 mutant in Bcl-xL overexpressing cells significantly increased the sensitivity to doxorubicin-induced apoptosis, suggesting the critical role of ERK2 in Bcl-xL-mediated blocking of doxorubicin-induced apoptosis. ERK2 activation was not observed in Bcl-2-overexpressing cells treated with doxorubicin, although Bcl-2 overexpression also could inhibit doxorubicin-induced apoptosis. These results suggest that doxorubicin-induced ERK2 activation is specifically mediated by Bcl-xL. Therefore, our results demonstrate that Bcl-xL-mediated ERK2 activation may provide one mechanism by which BcL-xL confers caner cells resistance to doxorubicin-induced apoptosis. In the second part of this study, we investigated whether Bcl-xL overexpression affected senescence and/or mitotic cell death induced by LD of doxorubicin. Bcl-xL overexpression effectively blocked apoptosis induced by high dose HD of doxorubicin, blocking the loss of mitochondrial membrane potential and activation of caspases in Huh-7 cells. In contrast, overexpression of Bcl-xL slightly delayed but not completely blocked doxorubicin-induced senescence-like phenotype (SLP) and the subsequent mitotic cell death. Loss of mitochondrial function and downregulation of mitosis-controlling proteins were also slightly delayed but not abolished by Bcl-xL overexpression. Clonogenicity of hepatoma cells exposed to LD doxorubicin was not enhanced by Bcl-xL overexpression. These results suggest that induction of senescence and/or mitotic cell death by LD doxorubicin may be more effective for Bcl-xL-overexpressing hepatoma cells, which are resistant to the apoptotic effect of chemotherapeutic agents.

Bcl-xL은 많은 종류의 암에 과발현되며 다양한 항암제에 의한 mitochondria의 기능 감소를 막음으로써 항암제 내성을 유발한다. Doxorubicin은 현재 가장 많이 쓰이고 있는 항암제 중 하나이지만, 그 작용 기전이 아직 명확히 규명되지 않고 있다. 본 연구에서는 고농도 doxorubicin에 의해 유도되는 apoptosis와 저농도 doxorubicin에 의해 유도되는 mitotic cell death에서의 Bcl-xL의 기능에 관해 살펴보았다. Bcl-xL이 apoptosis를 막는다는 것은 잘 알려져 있으나, Bcl-xL이 항암제에 반응하여 다른 신호전달체계의 조절을 통해 apoptosis를 막을 수 있음을 보고한 예는 아직까지 없다. 본 연구에서는 Bcl-xL 과발현 세포에 고농도의 doxorubicin을 처리했을 경우 apoptosis가 현저하게 막아졌으며, 이때 MAP kinase 중 ERK2만 활성화되었고 그 downstream 불질인 p90RSK, CREB, NF-B가 활성화되는 것을 확인하였다. Bcl-xL 과발현 세포에서 doxorubicin에 반응하여 활성화되는 ERK2는 doxorubicin에 대한 저항성을 가지게 하는데 중요한 역할을 함을 알 수 있었다. Bcl-2 과발현 또한 doxorubicin에 의한 apoptosis를 막을 수 있으나 ERK2는 활성시키지 못하였다. 다음으로, Bcl-xL의 과발현이 mitotic cell death에 미치는 영향에 대해 조사해보았다. Mitotic cell death 시에는 mitochondria의 기능이 어떻게 변화하는지, 또한 Bcl-xL이 어떤 역할을 하는지 전혀 밝혀져 있지 않은 실정이다. 본 연구를 통해 Bcl-xL 과발현이 저농도 doxorubicin에 의해 유도되는 mitotic cell death를 막지 못한다는 것을 알 수 있었다. Bcl-xL을 과발현시킬 경우 apoptosis 시와는 달리 mitotic cell death 시에 수반되는 mitochondria 기능 감소를 억제하지 못하였다. 따라서, Bcl-xL 과발현으로 인해 apoptosis에 내성을 획득하게 된 암의 치료에 있어서, 저농도 doxorubicin을 이용해 mitotic cell death를 유도하는 것이 효과적인 대안이 될 수 있을 것으로 본다.