Cancer-specific toxicity of apoptin is independent of death receptors but involves the loss of mitochondrial membrane potential and the release of mitochondrial cell-death mediators by a Nur77-dependent pathway

Abstract

Apoptin, a small proline-rich protein derived from the chicken anaemia virus, induces cell death selectively in cancer cells. The signalling pathways of apoptin-induced, cancer cell-selective apoptosis are not well understood. Here, we demonstrate that apoptin triggers apoptosis by activating the mitochondrial/intrinsic pathway, and that it acts independently of the death receptor/extrinsic pathway. Jurkat cells deficient in either FADD or caspase-8 (which are both necessary for the extrinsic pathway) were equally as sensitive to apoptin as their parental clones. This demonstrates that apoptin is likely to act through the mitochondrial death pathway. Apoptin treatment causes a loss of mitochondrial membrane potential, and release of the mitochondrial proteins cytochrome c and apoptosis-inducing factor. Apoptin-induced cell death is counteracted by the anti-apoptotic Bcl-2 family members, Bcl-2 itself and Bcl-XL, as shown in Jurkat leukaemia cells. In addition, we describe the processing and activation of caspase-3. By contrast, cleavage of caspase-8, which is predominantly triggered by the death receptor pathway, is not observed. Furthermore, apoptin triggers the cytoplasmic translocation of Nur77, and the inhibition of Nur77 expression by siRNA significantly protects MCF7 cells from apoptin-triggered cell death. Thus, our data indicate that the apoptin death signal(s) ultimately converges at the mitochondria, and that it acts independently of the death receptor pathway.

6-Mercaptopurine, an activator of Nur77, enhances transcriptional activity of HIF-1α resulting in new vessel formation

“…1, 5 0 -CAGUCCA GCCAUGCUCCUTT-3 0 , 5 0 -AGGAGCAUGGCU GGACU GTT-3 0 (Maddika et al, 2005); si-Nur77 no.2, 5 0 -GAAG GAAGUUGUCCGAACATT-3 0 , 5 0 -UGUUCGGACAACU UCCUUCTT-3 0 ), and the nonspecific control si-RNA (si-GL3, 5 0 -CUUACGCUGAGUACUUCGATT-3 0 , 5 0 -UCGAAGUA CUCAGCGUAAGTT-3 0 ) were synthesized and purified by Shamchully Pharm Co. (Korea). The transfection of si-RNAs was performed twice at 24 h intervals with Oligofectamine reagent (Invitrogen) as described previously (Yoo et al, 2006).…”

Inhibition of hepatocarcinoma by systemic delivery of Apoptin gene via the hepatic asialoglycoprotein receptor

“…33 Previously, intratumoral treatment of hepatocarcinoma 9,11 or breast tumor 34 with recombinant Apoptin-expressing adenovirus vectors resulting in clear signs of tumor regression were reported. Recently, Guelen et al 6 and Maddika et al 35 have provided evidence that at least under in vitro conditions TAT-Apoptin protein can be transduced into human normal and tumor cells, resulting in induction of cell death in tumor cells only. In addition to therapy resistance, toxicity to normal tissues often hampers cancer therapies such as chemotherapy and radiation.…”

Towards novel paradigms for cancer therapy

“…Such activities have been demonstrated for the adenovirusderived E4orf4, the parvovirus H1 protein NS1 and chicken anemia virus-derived apoptin (Guelen et al, 2004;Maddika et al, 2005;Maddika et al, 2006;Rohn and Noteborn, 2004) (Table 1). During natural infection, these proteins orchestrate the cytolytic events that are requisite for successful spreading of the virus.…”

“…Finally, apoptin induces apoptosis in a wide variety of human cancer cell lines via classical apoptotic pathways (Rohn and Noteborn, 2004;Maddika et al, 2005Maddika et al, , 2006Backendorf et al, 2008). Several independent in vivo analyses using human xenografted tumors have proven that administration of apoptin, as well as its recombinant version protein transduction domain 4 (PTD4)-apoptin, results in a significant reduction of tumor growth without major side effects (Peng et al, 2007;Sun et al, 2009).…”