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HDAC inhibitors (HDACI), a new class of anticancer agents, induce apoptosis in many cancer entities. JNJ-26481585 is a second generation class І HDACI that displays improved efficacy in preclinical studies compared to the established HDACI SAHA (Vorinostat). Therefore, this study aims at evaluating the effects of JNJ-26481585 on human rhabdomyosarcoma (RMS) and at identifying novel synergistic interactions of JNJ-26481585 or the more common HDACI SAHA with different anticancer drugs in RMS cells. Indeed, we show that JNJ-26481585 and SAHA significantly increase chemotherapeutic drug-induced apoptosis in embryonal and alveolar RMS cell lines, when used in combination with chemotherapeutic agents (i.e. doxorubicin, etoposide, vincristine, and cyclophosphamide) which are currently used in the clinic for the treatment of RMS.
We demonstrate that JNJ-26481585 as single agent and in combination with doxorubicin induces apoptosis, which is characterized by activation of the caspase cascade, PARP cleavage, and DNA fragmentation. Induction of caspase-dependent apoptotic cell death is confirmed by the use of the broad-range caspase inhibitor zVAD.fmk, which significantly decreases both JNJ-26481585-triggered and combination treatment-mediated DNA fragmentation, and in addition completely abrogates loss of cell viability. Importantly, JNJ-26481585 significantly inhibits tumor growth in vivo in two preclinical RMS models, i.e. the chicken chorioallantoic membrane (CAM) model and a xenograft mouse model, supporting the notion that JNJ-26481585 hampers tumor maintenance. Also, in combination with doxorubicin JNJ-26481585 significantly reduces tumor growth in in vivo experiments using the CAM model.
Mechanistically, we identify that JNJ-26481585-induced apoptosis is mediated via the intrinsic apoptotic pathway, since we observe increased loss of mitochondrial membrane potential and activation of the proapoptotic Bcl-2 family members Bax and Bak. Interestingly, we find that JNJ-26481585 triggers induction of Bim, Bmf, Puma, and Noxa on mRNA level as well as on protein level, pointing to an altered transcription of BH3-only proteins as important event for the Bax/Bak-mediated loss of mitochondrial membrane potential as well as mitochondrial apoptosis induction upon JNJ-26481585 treatment. JNJ-26481585-initiated activation of Bax and Bak is not prevented with the addition of zVAD.fmk, suggesting that JNJ-26481585 first disrupts the mitochondria and subsequently activates the caspase cascade. When JNJ-26481585 is used in combination with doxorubicin, we observe not only an increase of proapoptotic Bcl-2 proteins, but also a decrease in the level of the antiapoptotic mitochondrial proteins Bcl-2, Mcl-1, and Bcl-xL. This indicates that Bax, Bak, Bim, and Noxa are crucial for JNJ-26481585-induced as well as JNJ/Dox treatment-induced apoptosis, since RNAi mediated silencing of Bax, Bak, Bim, and Noxa significantly impedes DNA fragmentation upon those treatments.
Furthermore, ectopic overexpression of Bcl-2 profoundly impairs both JNJ-26481585 and combination treatment-mediated apoptosis, abrogates caspase cleavage, and reduces activation of Bax and Bak, underlining the hypothesis that JNJ-26481585 initially targets the mitochondria and then activates caspases.
With the more commonly used HDACI SAHA we confirm the results obtained with the HDACI JNJ-26481585, since combination treatment with SAHA and doxorubicin also induces intrinsic apoptosis, which can be significantly diminished by zVAD.fmk or ectopic overexpression of Bcl-2. Treatment with SAHA and doxorubicin also affects expression levels of pro- and antiapoptotic mitochondrial proteins, thus shifting the balance towards the proapoptotic mitochondrial machinery, resulting in Bax/Bak activation, caspase activation, and subsequently apoptosis.
Taken together, we provide evidence that the HDACIs JNJ-26481585 and SAHA are promising therapeutic agents for the treatment of RMS and that combination regimens with HDACIs represent an efficient strategy to prime RMS cells for chemotherapy-induced apoptosis. These findings have important implications for mitochondrial apoptosis-targeted therapies of RMS.
Acute myeloid leukemia is a hematopoietic stem cell disorder and a type of acute leukemia which is characterized by clonal proliferation of myeloid precursors with a reduced capacity to differentiate into more mature cellular elements. Clinically AML is characterized by a high degree of heterogeneity with respect to chromosome abnormalities, gene mutations, and changes in expression of multiple genes and microRNAs. Cytogenetic abnormalities can be detected in approximately 50% to 60% of newly diagnosed AML patients. Majority of AML cases are associated with chromosomal aberrations, more specifically translocations that often result in gene arrangements and expression of aberrant fusion proteins. This study was carried out with two fusion proteins: PML/RARα and DEK/CAN which results from the translocations t(15;17) and t (6,9) respectively. PML/RARα is the most common translocation (97%) and the main driver in Acute Promyelocytic Leukemia (APL), a wellcharacterized and well treatable subtype of AML. In contrast, DEK/CAN occurs in 1-5% of AML, associated with poor prognosis and defines a high risk group in AML. The expression of PML/RARα results in a fusion protein that acts as a transcriptional repressor by interfering with gene expression programs involved in differentiation, apoptosis, and selfrenewal. Current therapy focused on the targeting of PML/RARα fusion protien. Success has been achieved by using either ATRA, anthracyclines and Arsenic trioxide or their combinations. These agents induce differentiation in PML/RARα positive AML and hence called differentiation therapy. In comparison with ATRA, ATO and anthracyclines are poor cellular differentiation agents. Despite early promise, several studies have reported that differentiation therapy is unable to target/eradicate leukemic stem cells or eradicate the disease. Therefore current therapeutic focus is to eliminate leukemic stem cells and achieve complete molecular remission not only in APL but also in acute lymphoblastic leukemia and chronic myeloid leukemia as well. Key enzymes of the eicosanoid pathways in the arachidonic acid metabolism, such as COX1/2 as well as the 5-LO have been shown to be good targets for leukemic stem cell therapy approach in AML by interfering with the Wntsignaling which is known to be indispensable for the pathogenesis of AML. Recently it was reported that the third eicosanoid pathway based on the cytochrome P450 (CYP) enzymes interferes with Wnt-signaling as well as with the proliferation and mobilization of hematopoietic stem cells...