ABSTRACT
Generally, both endoplasmic reticulum (ER) stress and mitochondrial dysregulation are a potential therapeutic target of anticancer agents including bortezomib. The treatment of melanoma cells with bortezomib was found to induce apoptosis together with the upregulation of Noxa, Mcl-1, and HSP70 proteins, and the cleavage of LC3 and autophagic formation. Also, bortezomib induced ER-stress as evidenced by the increase of intracellular Ca(2+) release. In addition, bortezomib enhanced the phosphorylation of inositol-requiring transmembrane kinase and endonuclease 1α (IRE1α), apoptosis signal-regulating kinase 1 (ASK1), c-jun-N-terminal kinase (JNK) and p38, and the activation of the transcription factors AP-1, ATF-2, Ets-1, and HSF1. Bortezomib-induced mitochondrial dysregulation was associated with the accumulation of reactive oxygen species (ROS), the release of both apoptosis inducing factor (AIF) and cytochrome c, the activation of caspase-9 and caspase-3, and cleavage of Poly (ADP-ribose) polymerase (PARP). The pretreatment of melanoma cells with the inhibitor of caspase-3 (Ac-DEVD-CHO) was found to block bortezomib-induced apoptosis that subsequently led to the increase of autophagic formation. In contrast, the inhibition of ASK1 abrogated bortezomib-induced autophagic formation and increased apoptosis induction. Furthermore, the inhibition of JNK, of HSP70 also increased apoptosis induction without influence of bortezomib-induced autophagic formation. Based on the inhibitory experiments, the treatment with bortezomib triggers the activation of both ER-stress-associated pathways, namely IRE1α-ASK1-p38-ATF-2/ets-1-Mcl-1, and IRE1α-ASK1-JNK-AP-1/HSF1-HSP70 as well as mitochondrial dysregulation-associated pathways, namely ROS-ASK1-JNK-AP-1/HSF1-HS70, and AIF-caspase-3-PARP and Cyt.c, and caspase-9-caspase-3-PARP. Taken together, our data demonstrates for the first time the molecular mechanisms, whereby bortezomib triggers both apoptosis and autophagic formation in melanoma cells.
Subject(s)
Antineoplastic Agents/toxicity , Apoptosis/drug effects , Autophagy/drug effects , Boronic Acids/toxicity , Proteasome Inhibitors/toxicity , Pyrazines/toxicity , Bortezomib , Calcium/metabolism , Caspase 3/chemistry , Caspase 3/metabolism , Cell Line, Tumor , Endoplasmic Reticulum Stress/drug effects , HSP70 Heat-Shock Proteins/antagonists & inhibitors , HSP70 Heat-Shock Proteins/genetics , HSP70 Heat-Shock Proteins/metabolism , Humans , MAP Kinase Kinase Kinase 5/metabolism , Melanoma/metabolism , Melanoma/pathology , Membrane Potential, Mitochondrial/drug effects , Microtubule-Associated Proteins/metabolism , Mitochondria/drug effects , Mitochondria/metabolism , Myeloid Cell Leukemia Sequence 1 Protein , Proto-Oncogene Protein c-ets-1/antagonists & inhibitors , Proto-Oncogene Protein c-ets-1/genetics , Proto-Oncogene Protein c-ets-1/metabolism , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , RNA Interference , RNA, Small Interfering/metabolism , Reactive Oxygen Species/metabolism , Signal Transduction/drug effectsABSTRACT
Metastatic cancers including melanoma are frequently associated with increased resistance to apoptosis induced by various therapeutic modalities, and the success of systemic therapy for the treatment of metastatic melanoma is minimal. In the present study, we demonstrated the ability of apoptosis-related protein (APR)-2 to trigger cell death via mechanism mediated by both endoplasmic reticulum (ER) stress [as evidenced by the increase of intracellular Ca(2+) release, the activation of both, inositol-requiring enzyme 1α (IRE1α) and calpain and cleavage of caspase-4] and mitochondrial dysregulation as evidenced by the loss of mitochondrial membrane potential, Cytochrome c release and cleavage of caspases-9 and -3, and poly adenosine diphosphate ribose polymerase (PARP). Also, the activation of apoptosis signal-regulating kinase (ASK) 1, c-jun-N-terminal kinase (JNK) and the transcription factors AP-1 and p53, and the induction of Bax expression were noted in APR-2-expressing cells. Both immune fluorescence staining and western blotting revealed the localization of APR-2 at ER and Bax protein at both mitochondria and ER. However, data of inhibitory experiments demonstrated that APR-2-induced apoptosis of melanoma cells is mediated by three parallel pathways: one of them IRE1/tumour necrosis factor receptor-associated factor 2/ASK1/JNK/Cyt.c/caspase-9/caspase-3/PARP) seems to be mitochondrial dependent, whereas, the other two pathways namely calpain/caspase-4/caspase-9/caspase-3/PARP and protein kinase RNA-like ER kinase/ATF4/C/EBP homologous protein (CHOP)/Bim seem to be mitochondrial independent. In conclusion, our data provide insight into the molecular mechanism of APR-2-induced apoptosis and suggest APR-2 gene transfer as an alternative approach for the treatment of chemoresistance melanoma metastasis.
