Dual targeting Bcl-2 and Bcl-xL augments osteosarcoma response to doxorubicin.

Chemotherapy resistance is the major cause of treatment failure in osteosarcoma, the most common primary bone malignancy, and sensitizing therapeutic strategy is required to improve the clinical outcome. In this study, we discovered that navitoclax, a selective inhibitor of Bcl-2/Bcl-xL, effectively combats chemoresistance in osteosarcoma. Our research revealed that Bcl-2, but not Bcl-xL, is upregulated in osteosarcoma cells that are resistant to doxorubicin. However, venetoclax, a specific inhibitor of Bcl-2, did not exhibit activity against doxorubicin-resistant cells. Further analysis showed that depleting either Bcl-2 or Bcl-xL alone was insufficient to overcome doxorubicin resistance. Only by depleting both Bcl-2 and Bcl-xL significantly reduce the viability of doxorubicin-resistant cells. Similarly, navitoclax not only decreased the viability of doxorubicin-resistant cells but also acted synergistically with doxorubicin in cells sensitive to the drug. To confirm the ability of navitoclax to overcome doxorubicin resistance, we conducted experiments using multiple mouse models of osteosarcoma, both doxorubicin-sensitive and doxorubicin-resistant. The results provided confirmation that navitoclax is effective in overcoming doxorubicin resistance. Our findings demonstrate that simultaneous inhibition of Bcl-2 and Bcl-xL could serve as a novel strategy to sensitize chemoresistant osteosarcoma cells. Moreover, our study presents preclinical evidence supporting the potential of a navitoclax and doxorubicin combination therapy for the treatment of osteosarcoma, paving the way for future clinical investigations.

BCLXL gene therapy moderates neuropathology in the DBA/2J mouse model of inherited glaucoma.

Axonal degeneration of retinal ganglion cells (RGCs) causes blindness in glaucoma. Currently, there are no therapies that target axons to prevent them from degenerating. Activation of the BAX protein has been shown to be the determining step in the intrinsic apoptotic pathway that causes RGCs to die in glaucoma. A putative role for BAX in axonal degeneration is less well elucidated. BCLXL (BCL2L1) is the primary antagonist of BAX in RGCs. We developed a mCherry-BCLXL fusion protein, which prevented BAX recruitment and activation to the mitochondria in tissue culture cells exposed to staurosporine. This fusion protein was then packaged into adeno-associated virus serotype 2, which was used to transduce RGCs after intravitreal injection and force its overexpression. Transduced RGCs express mCherry-BCLXL throughout their somas and axons along the entire optic tract. In a model of acute optic nerve crush, the transgene prevented the recruitment of a GFP-BAX fusion protein to mitochondria and provided long-term somal protection up to 12 weeks post injury. To test the efficacy in glaucoma, DBA/2J mice were transduced at 5 months of age, just prior to the time they begin to exhibit ocular hypertension. Gene therapy with mCherry-BCLXL did not affect the longitudinal history of intraocular pressure elevation compared to naive mice but did robustly attenuate both RGC soma pathology and axonal degeneration in the optic nerve at both 10.5 and 12 months of age. BCLXL gene therapy is a promising candidate for glaucoma therapy.

Targeting the anti-apoptotic Bcl-2 family members for the treatment of cancer.

Most cells express a variety of both anti-apoptotic and pro-apoptotic Bcl-2 proteins and the interaction within this family dictates whether a cell survives or dies. The dysregulation of the anti-anti-apoptotic Bcl-2 family members is one of the defining features of cancer cells in comparison to normal cells, and significantly contributes to the resistance of cancer cells to current treatment modalities. This anti-apoptotic subfamily of proteins is now a major target in the development of new methods to improve treatment outcomes for cancer patients. Several drugs directed at inhibiting Bcl-2 and related anti-apoptotic proteins have been developed with some showing considerable promise in the clinic. This Review presents the current knowledge of the role of the anti-apoptotic Bcl-2 family in cancer cells, as well as current and future perspectives on targeting this subfamily of proteins for therapeutic intervention in human malignancies. This article is part of a Special Issue entitled "Apoptosis: Four Decades Later".

Mild hypothermia enhanced the protective effect of protein therapy with transductive anti-death FNK protein using a rat focal transient cerebral ischemia model.

We previously reported that the protein transduction domain fused FNK (PTD-FNK) protein, which was derived from anti-apoptotic Bcl-xL protein and thereby gained higher anti-cell death activity, has a strong neuroprotective effect on rat focal brain ischemia models. The aim of this study was to investigate the effect of PTD-FNK protein and hypothermia combined therapy on cerebral infarction. Male SD rats were subjected to 120min middle cerebral artery occlusion (MCAO) with intraluminal thread. Rats were divided into 4 groups: 1) 37°C vehicle administration (37V); 2) 37°C PTD-FNK administration (37F); 3) 35°C vehicle administration (35V); and 4) 35°C PTD-FNK administration (35F). PTD-FNK protein was intravenously administered 60min after the induction of MCAO. Hypothermia (35°C) was applied during 120min MCAO. Rats were sacrificed 24h later; infarct volumes were measured, and Bax, Bcl-2, TUNEL and caspase-12 immunostaining was evaluated. There was significant infarct volume reduction in 37F, 35V, and 35F groups compared to 37V. There was also a significant difference between 37F and 35F. This suggests that hypothermia enhanced the effect of PTD-FNK. Similar results were found in neurological symptoms. Caspase-12 and TUNEL staining showed a significant difference between 37F and 35F; however, Bax and Bcl-2 staining failed to show a difference. In this study we showed an additive protective effect of hypothermia on PTD-FNK treatment, and immunohistological results showed that the protective mechanisms might involve the inhibition of apoptotic pathways through caspase-12, but not through Bcl-2.

Systemic delivery of BH4 anti-apoptotic peptide using CPPs prevents cardiac ischemia-reperfusion injuries in vivo.

There is an obvious need to develop pharmacological strategies to protect the heart in patients suffering from acute myocardial infarction. Apoptosis was evidenced as a main contributor of myocardial ischemia-reperfusion (IR) injury. Our cardioprotective strategy was based on the use of four cell penetrating peptides (CPP: Tat, (RXR)4, Bpep and Pip2b) which were conjugated to the BH4-peptide, derived from the BH4 domain of the Bcl-xL anti-apoptotic protein. These CPP-BH4 conjugates were able to reduce staurosporine-induced apoptosis in primary cardiomyocytes in vitro. Although Pip2b-BH4 was more efficient in terms of cellular uptake, it was as efficient as Tat-BH4 for its anti-apoptotic activity. As required for potential therapeutic application their cardioprotective effects were evaluated in an in vivo mouse model of myocardial IR injury. Our results clearly show that a single low dose (1 mg/kg) injection of Tat-BH4 and Pip2b-BH4 administered intravenously 5 min before reperfusion was able to drastically reduce infarct size (~47%) and to inhibit apoptosis (~60%) in the left ventricle of treated mice. Importantly, these effects are not observed following the injection of CPP alone or scrambled version of BH4. This study evidences that the Pip2b CPP, designed for oligonucleotides translocation, as well as the widely used natural Tat CPP exhibit similar efficacy in vivo to deliver BH4 anti-apoptotic peptide to the reperfused myocardium and may thus become useful therapeutic tools to treat acute myocardial infarction in the clinical setting.

[Adenoviruses mediated BCL-X1 overexpression protects mice from fulminant hepatic failure].

OBJECTIVE: To indentify the relation between hepatic cells apoptosis and the lesion of liver tissue in acute toxic lethal hepatitis. METHODS: 60 Wistar mice were randomly divided into normal control, model group and treatment group. Normal control and model group were pretreated by portal vein injection of normal saline, the treatment group was pretreated by portal vein injection of BCL-X1 adenoviruses. The mice of model group and treatment group were received an injection of D-galn and LPS to establish fulminant hepatic failure models 7 days after pretrement. To observe BCL-X1 expression, serum ALT, AST, hepatocyte apoptosis rate, and mortality rate of the three groups. RESULTS: The BCL-X1 expression was higher in treatment group than in model group; 6 hours after fulminant hepatic failure models were established,the serum ALT, AST level of treatment group was lower than model group;The hepatocyte apoptosis rate of treatment group was lower than model group. The death rate of treatment group was lower than model group. CONCLUSION: In fulminant mice hepatic failure models, the hepatocyte apoptosis rate has a positive correlation with death rate, the overexpression of BCL-X1 can decrease the hepatocyte apoptosis rate and the death rate.

Transplantation of TAT-Bcl-xL-transduced neural precursor cells: long-term neuroprotection after stroke.

Neural precursor cells (NPC) are an interesting tool in experimental stroke research, but their therapeutic potential is limited due to poor long-term survival. We therefore in vitro transduced subventricular zone-(SVZ)-derived NPC with the anti-apoptotic fusion protein TAT-Bcl-x(L) and analyzed NPC survival, differentiation, and post-stroke functional deficits after experimental ischemia in mice. Survival of TAT-Bcl-x(L)-transduced NPC, which were injected at day 7 post-stroke into the ischemic striatum, was significantly increased at 4 weeks after stroke. Increased survival of NPC was associated with reduced infarct injury and decreased post-stroke functional deficits. Animals grafted with TAT-Bcl-x(L)-transduced NPC showed an increased number of immature cells expressing the neuronal marker doublecortin. Since mature neuronal differentiation of NPC was not observed, reduced post-stroke injury cannot be attributed to enhanced neuronal regeneration, but rather to indirect by-stander effects of grafted NPC. In line with this, NPC-mediated neuroprotection of cortical neurons in vitro was associated with increased secretion of growth factors. Thus, in vitro transduction of cultivated NPC with TAT-Bcl-x(L) results in enhanced resistance of transplanted NPC followed by long-term neuroprotection and ameliorated functional deficits after transient focal cerebral ischemia in mice.

Prostate cancer cell death produced by the co-delivery of Bcl-xL shRNA and doxorubicin using an aptamer-conjugated polyplex.

We investigated the synergism between shRNAs against Bcl-xL and doxorubicin (DOX) using aptamer-conjugated polyplexes (APs) in combination cancer therapy. Synergistic and selective cancer cell death was achieved by AP-mediated co-delivery of very small amounts of DOX and Bcl-xL-specific shRNA, which simultaneously activated an intrinsic apoptotic pathway. A branched polyethyleneimine (PEI) was grafted to polyethylene glycol (PEI-PEG) to serve as a vehicle for shRNA delivery, and its surface was further conjugated with an anti-PSMA aptamer (APT) for the selective delivery of APs to prostate cancer cells that express prostate-specific membrane antigens (PSMA) on their cell surface. The APs were finally obtained after intercalation of DOX to form shRNA/PEI-PEG-APT/DOX conjugates. Cell viability assays and FACS analysis of GFP expression against PC3 (PSMA deficient) and LNCaP (PSMA overexpressed) cells demonstrated that the synthesized APs inhibited the growth of PSMA-abundant prostate cancer cells with strong cell selectivity. Consequently, IC(50) values of APs loaded with both DOX and shRNA were approximately 17-fold less than those for the simple mixture of shRNA plus drug (shRNA/Lipofectamine + DOX). These results suggest that AP-mediated co-delivery of an anti-cancer drug and shRNA against Bcl-xL may widen the therapeutic window and allow for the selective destruction of cancer cells.

Enhanced treatment of articular cartilage defect of the knee by intra-articular injection of Bcl-xL-engineered mesenchymal stem cells in rabbit model.

Direct intra-articular injection of mesenchymal stem cells (MSCs) has been proposed as a potential cell therapy for cartilage defects. This cell therapy relies on the survival of the implanted MSCs. However, the arduous local environment may limit cell viability after implantation, which would restrict the cells' regenerative capacity. Thus, it is necessary to reinforce the implanted cells against the unfavourable microenvironment in order to improve the efficacy of cell therapy. We examined whether the transduction of an anti-apoptotic protein, Bcl-xL, into MSCs could prevent cell death and improve the implantation efficiency of MSCs in a rabbit model. Our current findings demonstrate that the group treated with Bcl-xL-engineered MSCs could improve cartilage healing both morphologically and histologically when compared with the controls. These results suggest that intra-articular injection of Bcl-xL-engineered MSCs is a potential non-invasive therapeutic method for effectively treating cartilage defects of the knee.

TAT-Bcl-x(L) improves survival of neuronal precursor cells in the lesioned striatum after focal cerebral ischemia.

Cerebral ischemia activates endogenous neurogenesis in the subventricular zone (SVZ) and the dentate gyrus. Consecutively, SVZ-derived neural precursors migrate towards ischemic lesions. However, functional relevance of activated neurogenesis is limited by poor survival of new-born precursors. We therefore employed the HI-virus-derived fusion protein TAT-Bcl-x(L) to study the effects of acute anti-apoptotic treatment on endogenous neurogenesis and functional outcome after transient cerebral ischemia in mice. TAT-Bcl-x(L) treatment led to significantly reduced acute ischemic cell death (128+/-23 vs. 305+/-65 TUNEL+ cells/mm(2) in controls) and infarct volumes resulting in less motor deficits and improved spatial learning. It significantly increased survival of doublecortin (Dcx)-positive neuronal precursors (389+/-96 vs. 213+/-97 Dcx+ cells in controls) but did not enhance overall post-ischemic cell proliferation or lesion-specific neuronal differentiation 28 days after ischemia. Our data demonstrate that post-stroke TAT-Bcl-x(L)-treatment results in acute neuroprotection, improved functional outcome, and enhanced survival of lesion-specific neuronal precursor cells after cerebral ischemia in mice.

Anti-apoptotic therapy with a Tat fusion protein protects against excitotoxic insults in vitro and in vivo.

A number of gene therapy approaches have been developed for protecting neurons from necrotic neurological insults. Such therapies are limited by the need for transcription and translation of the protective protein, delaying therapeutic impact. As an alternative, we explore the neuroprotective potential of protein therapy, using a fusion protein comprised of the death-suppressing BH4 domain of the Bcl-xL protein and the protein transduction domain of the human immunodeficiency virus Tat protein. This fusion protein decreased neurotoxicity caused by the excitotoxins glutamate and kainic acid in primary hippocampal cultures, and decreased hippocampal damage in vivo in an excitotoxic seizure model.

Corneal graft rejection is accompanied by apoptosis of the endothelium and is prevented by gene therapy with bcl-xL.

Corneal transplants normally enjoy a high percentage of survival, mainly because the eye is an immune-privileged site. When allograft failure occurs, it is most commonly due to rejection, an immune-mediated reaction that targets the corneal endothelium. While the exact mechanism by which the endothelium is targeted is still unknown, we postulate that corneal endothelial cell loss during allograft failure is mediated by apoptosis. Furthermore, because corneal endothelial cells do not normally regenerate, we hypothesize that suppressing apoptosis in the graft endothelium will promote transplant survival. In a murine model of transplantation, TUNEL staining and confocal microscopy showed apoptosis of the graft endothelium occurring in rejecting corneas as early as 2 weeks posttransplantation. We found that bcl-xL protected cultured corneal endothelial cells from apoptosis and that lentiviral delivery of bcl-xL to the corneal endothelium of donor corneas significantly improved the survival of allografts. These studies suggest a novel approach to improve corneal allograft survival by preventing apoptosis of the endothelium.

HIV-Tat-mediated Bcl-XL delivery protects retinal ganglion cells during experimental autoimmune optic neuritis.

In multiple sclerosis (MS), post-mortem studies of human brain tissue as well as data from animal models have shown that apoptosis of neurons occurs to a significant extent during this disease. As neurodegeneration in MS correlates with permanent neurological deficits in patients, understanding the mechanisms would be an important pre-condition for designing appropriate neuroprotective therapies. Myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis often affects the optic nerve and leads to consecutive apoptosis of retinal ganglion cells (RGCs), the neurons that form its axons. In this study, we fused Bcl-XL to the protein transduction domain of the HIV-transactivator of transcription. Thereby, this anti-apoptotic member of the Bcl-2 family was delivered into RGCs of rats with electrophysiologically diagnosed optic neuritis. Transduction of Bcl-XL in our study led to significant rescue of RGCs indicating the relevance of this pathway for neuronal survival under autoimmune inflammatory conditions.