Inhibition of AKT induces p53/SIRT6/PARP1-dependent parthanatos to suppress tumor growth.

BACKGROUND: Targeting AKT suppresses tumor growth through inducing apoptosis, however, during which whether other forms of cell death occurring is poorly understood. METHODS: The effects of increasing PARP1 dependent cell death (parthanatos) induced by inhibiting AKT on cell proliferation were determined by CCK-8 assay, colony formation assay, Hoechst 33,258 staining and analysis of apoptotic cells by flow cytometry. For the detailed mechanisms during this process, Western blot analysis, qRT-PCR analysis, immunofluorescence and co-immunoprecipitation were performed. Moreover, the inhibition of tumor growth by inducing p53/SIRT6/PARP1-dependent parthanatos was further verified in the xenograft mouse model. RESULTS: For the first time, we identified that inhibiting AKT triggered parthanatos, a new form of regulated cell death, leading to colon cancer growth suppression. For the mechanism investigation, we found that after pharmacological or genetic AKT inhibition, p53 interacted with SIRT6 and PARP1 directly to activate it, and promoted the formation of PAR polymer. Subsequently, PAR polymer transported to outer membrane of mitochondria and resulted in AIF releasing and translocating to nucleus thus promoting cell death. While, blocking PARP1 activity significantly rescued colon cancer from death. Furthermore, p53 deletion or mutation eliminated PAR polymer formation, AIF translocation, and PARP1 dependent cell death, which was promoted by overexpression of SIRT6. Meanwhile, reactive oxygen species production was elevated after inhibition of AKT, which might also play a role in the occurrence of parthanatos. In addition, inhibiting AKT initiated protective autophagy simultaneously, which advanced tumor survival and growth. CONCLUSION: Our findings demonstrated that AKT inhibition induced p53-SIRT6-PARP1 complex formation and the activation of parthanatos, which can be recognized as a novel potential therapeutic strategy for cancer. Video Abstract.

Anti-Neoplastic Cytotoxicity of SN-38-Loaded PCL/Gelatin Electrospun Composite Nanofiber Scaffolds against Human Glioblastoma Cells In Vitro.

Electrospun poly(ε-caprolactone) (PCL)/gelatin (GT) scaffolds were developed to provide controlled release of 7-ethyl-10-hydroxy camptothecin (SN-38). Acetic acid was introduced to improve the miscibility of PCL and GT to produce a homogeneous nanofiber membrane mixture. The effect of SN-38 content in binary mixtures on processability, fiber morphology, water sorption, swelling, and drug release was investigated. Electrospun PCL/GT blend nonwoven fibers showed fiber surface roughness, decreased PCL crystallinity, and increased swelling with increasing drug content of 1, 2, and 4 wt %. Additionally, increasing the SN-38 concentration reduced the degradation rate of the GT. Furthermore, we hypothesize the existence of a drug content saturation point in the monoaxial fiber to explain the different drug release patterns of PG2 compared with those of PG1 and PG4. The matrix also showed good biodegradation and anti-tumor function. Our results demonstrate that SN-38-loaded PCL/GT fibers can be obtained by electrospinning. The SN-38-loaded fibers merit further evaluation as a means to potentially prevent locoregional recurrence following surgical tumor resection.

Patient-specific cardiovascular progenitor cells derived from integration-free induced pluripotent stem cells for vascular tissue regeneration.

Tissue-engineered blood vessels (TEBVs) are promising in regenerating a live vascular replacement. However, the vascular cell source is limited, and it is crucial to develop a scaffold that accommodates new type of vascular progenitor cells and facilitates in vivo lineage specification of the cells into functional vascular smooth muscle cells (VSMCs) to regenerate vascular tissue. In the present study, integration-free human induced pluripotent stem cells (hiPSCs) were established from patient peripheral blood mononuclear cells through episomal vector nucleofection of reprogramming factors. The established hiPSCs were then induced into mesoderm-originated cardiovascular progenitor cells (CVPCs) with a highly efficient directed lineage specification method. The derived CVPCs were demonstrated to be able to differentiate into functional VSMCs. Subcutaneous implantation of CVPCs seeded on macroporous nanofibrous poly(l-lactide) scaffolds led to in vivo VSMC lineage specification and matrix deposition inside the scaffolds. In summary, we established integration-free patient-specific hiPSCs from peripheral blood mononuclear cells, derived CVPCs through directed lineage specification, and developed an advanced scaffold for these progenitor cells to further differentiate in vivo into VSMCs and regenerate vascular tissue in a subcutaneous implantation model. This study has established an efficient patient-specific approach towards in vivo regeneration of vascular tissue.

Sustained delivery of dbcAMP by poly(propylene carbonate) micron fibers promotes axonal regenerative sprouting and functional recovery after spinal cord hemisection.

This study describes the use of poly(propylene carbonate) (PPC) electrospun fibers as vehicle for the sustained delivery of dibutyryl cyclic adenosine monophosphate (dbcAMP) to the hemisected spinal cord. The dbcAMP and PPC were uniformly mixed with acetonitrile; then, electrospinning was used to generate micron fibers. The release of dbcAMP was assessed by ELISA in vitro. Our results showed that the encapsulation of dbcAMP in the fibers led to stable and prolonged release in vitro. The PPC micron fibers containing dbcAMP and the PPC micron fibers without dbcAMP were then implanted into the hemisected thoracic spinal cord, followed by testing of the functional recovery and immunohistochemistry. Compared with the control group, sustained delivery of dbcAMP promoted axonal regenerative sprouting and functional recovery and reduced glial scar formation, and the PPC micron fibers without dbcAMP did not have these effects. Our findings demonstrated the feasibility of using PPC electrospun fibers containing dbcAMP for spinal cord injury. The approach described here also will provide a platform for the potential delivery of other axon-growth-promoting or scar-inhibiting agents.

A preferable approach to clone hLIF cDNA from the genomic DNA

Complementary DNA (cDNA) is valuable for investigating protein structure and function in the research of life science, but it is difficult to obtain by traditional reverse transcription. In this study, we employed a novel strategy to clone the human leukemia inhibitory factor (hLIF) gene cDNA from genomic DNA directly isolated from the mucous membrane of mouth. The hLIF sequence can be acquired within a few hours by means of amplification of each exon and splicing using overlap-PCR. Thus, the new approach developed in this study is simple, time- and cost-effective, and it is not limited to particular gene expression levels of each tissue.