Targeting inhibitors of apoptosis in oral squamous cell carcinoma in vitro

Head and neck cancer, which predominantly arises from the oral mucosa, represents the sixth most common malignancy worldwide. These cancer cells can be resistant to programmed cell death triggered by extrinsic stimuli due to innate overexpression of inhibitor of apoptosis proteins (IAPs). The cellular protein second mitochondria-derived activator of caspases (SMAC) can antagonize IAP-induced caspase inhibition and thus trigger apoptosis. Here, we investigate the cell death-sensitizing effects of the SMAC mimetic LCL161 alone and in combination with Fas ligand (FasL) using a panel of six cell lines. Fas receptor (FasR) expression was analyzed by flow cytometry. Cells were treated with FasL and LCL161 alone or in combination, and cytotoxicity was measured using crystal violet assays. Annexin V and cell viability assays using zVAD-fmk and Necrostatin-1 (Nec-1) were carried out to assess the type of programmed cell death induced by LCL161. To demonstrate the sensitizing effects of LCL161, we employed the t-test to compare the effects of FasL alone and in combination with LCL161. Linear regression analysis was performed to determine initial and half maximal inhibitory concentrations (IC₁₀ and IC₅₀, respectively). Distinct FasR expression was detected in each cell line. Four of six cell lines were significantly sensitized to FasL by LCL161 (p < 0.05), and synergistic effects were observed (y < 1). Moreover, the initially resistant cell line SCC-25 was effectively sensitized to FasL by LCL161. Annexin V FACS analysis demonstrated apoptosis-sensitizing and apoptosis-inducing effects of LCL161 across all cell lines. Using specific cell death inhibitors (zVAD-fmk and Nec-1), we demonstrated that LCL161-initiated apoptosis could not be prevented, highlighting the proapoptotic potential of this mimetic in these cells. Our findings show the effectiveness of apoptotic sensitization of OSCC cells by LCL161 in combination with FasL, thus confirming the importance of an IAP-targeting therapeutic approach for oral squamous cell carcinoma.     

Cationic liposome co-encapsulation of SMAC mimetic and zVAD using a novel lipid bilayer fusion loaded with MLKL-pDNA for tumour inhibition in vivo

The increase in multidrug resistance among colon cancer cells presents a challenge for the development of effective therapies. Small-molecule analogues of second mitochondria-derived activator of caspase (SMAC) mimetic in association with mixed lineage kinase domain-like protein (MLKL)-pDNA and z-VAD-fmk have shown ideal antitumor effects in colon cancer cells in vitro via induction of RIP3-dependent necroptosis. To achieve synergistic antitumor effects in vivo, liposomes loaded with SMAC mimetic, MLKL-pDNA and z-VAD-fmk have been developed using novel lipid fusion methods to co-localise the molecules of interest within the tumour cells. The co-encapsulation liposome (MLKL-zVAD-BV6-LP) had a high entrapment efficiency of approximately 95% for both zVAD and BV6 and was able to condense MLKL-pDNA very well. The vectors showed good biocompatibility, tumour targeting and small-molecule co-localisation. In a CT26 mouse model, the MLKL-zVAD-BV6-LP exhibited a tumour-suppression rate of over 60% in vivo, which was significantly higher than that of both the null-liposome and coadministration groups. Above all, the co-encapsulation system provided a novel approach to combination tumour therapy.     

Cytotoxic lymphocytes; instigators of dramatic target cell death

Most mammalian cells are constantly threatened by viral infection and oncogenic transformation. To maintain healthy function of organs and tissues it is critical that afflicted cells are efficiently detected and removed. Cytotoxic lymphocytes (CL) are chiefly responsible for efficiently seeking out and eliminating damaged or infected cells. It is known that CLs must specifically recognize and bind to their targets, but the molecular events that occur within the target cell that lead to its death are still poorly understood. The two main processes initiated by CLs to induce target cell death are mediated by ligation of surface receptors or release of toxic proteins from secretory granules (granule exocytosis) of the CL. Here we review some of the key findings that have defined our knowledge of the granule exocytosis-mediated pathways to CL-mediated killing and discuss recent insights that challenge conventional views in the important area of CL effector function.     

Initial testing (stage 1) of LCL161, a SMAC mimetic, by the Pediatric Preclinical Testing Program

LCL161, a SMAC mimetic, was tested against the PPTP in vitro panel (1.0 nM to 10.0 µM) and the PPTP in vivo panels (30 or 75 mg/kg [solid tumors] or 100 mg/kg [ALL]) administered orally twice in a week. LCL161 showed a median relative IC₅₀ value of >10 µM, being more potent against several leukemia and lymphoma lines. In vivo LCL161 induced significant differences in EFS distribution in approximately one‐third of solid tumor xenografts (osteosarcoma and glioblastoma), but not in ALL xenografts. No objective tumor responses were observed. In vivo LCL161 demonstrated limited single agent activity against the pediatric preclinical models studied. Pediatr Blood Cancer 2012; 58: 636–639. © 2011 Wiley Periodicals, Inc.     

Therapeutic strategies involving survivin inhibition in cancer

Survivin is a small protein that belongs to the inhibitor of apoptosis protein family. It is abundantly expressed in tumors compared with adult differentiated tissues, being associated with poor prognosis in many human neoplasms. This apoptotic inhibitor has a relevant role in both the promotion of cancer cell survival and in the inhibition of cell death. Consequently, aberrant survivin expression stimulates tumor progression and confers resistance to several therapeutic strategies in a variety of tumors. In fact, efficient survivin downregulation or inhibition results in spontaneous apoptosis or sensitization to chemotherapy and radiotherapy. Therefore, all these features make survivin an attractive therapeutic target to treat cancer. Currently, there are several survivin inhibitors under clinical evaluation, although more specific and efficient survivin inhibitors are being developed. Moreover, novel combination regimens targeting survivin together with other therapeutic approaches are currently being designed and assessed. In this review, recent progress in the therapeutic options targeting survivin for cancer treatment is analyzed. Direct survivin inhibitors and their current development status are explored. Besides, the major signaling pathways implicated in survivin regulation are described and different therapeutic approaches involving survivin indirect inhibition are evaluated. Finally, promising novel inhibitors under preclinical or clinical evaluation as well as challenges of developing survivin inhibitors as a new therapy for cancer treatment are discussed.     

Targeting cell death signalling in cancer: minimising ‘Collateral damage'

Targeting apoptosis for the treatment of cancer has become an increasingly attractive strategy, with agents in development to trigger extrinsic apoptosis via TRAIL signalling, or to prevent the anti-apoptotic activity of BCL-2 proteins or inhibitor of apoptosis (IAP) proteins. Although the evasion of apoptosis is one of the hallmarks of cancer, many cancers have intact apoptotic signalling pathways, which if unblocked could efficiently kill cancerous cells. However, it is becoming increasing clear that without a detailed understanding of both apoptotic and non-apoptotic signalling, and the key proteins that regulate these pathways, there can be dose-limiting toxicity and adverse effects associated with their modulation. Here we review the main apoptotic pathways directly targeted for anti-cancer therapy and the unforeseen consequences of their modulation. Furthermore, we highlight the importance of an in-depth mechanistic understanding of both the apoptotic and non-apoptotic functions of those proteins under investigation as anti-cancer drug targets and outline some novel approaches to sensitise cancer cells to apoptosis, thereby improving the efficacy of existing therapies when used in combination with novel targeted agents.     

小鼠肝细胞SMAC基因特异干扰载体的构建及慢病毒包装

目的构建并鉴定小鼠SMAC特异干扰载体,并进行慢病毒包装。方法针对小鼠SMAC基因设计并合成3对干扰序列siRNA1、siRNA2、siRNA3及一对阴性对照序列siRNAn,脂质体法转染小鼠肝细胞,Real time PCR法筛选出最佳干扰序列。依据该最佳干扰序列合成DNA oligo,连接GV115载体,获得重组干扰质粒,PCR鉴定阳性克隆并测序鉴定构建的正确性。将该重组干扰质粒与辅助质粒pHelper1.0和pHelper2.0共转染293T细胞,进行慢病毒包装,收集细胞上清并浓缩,梯度稀释法测定病毒滴度。多组间比较采用方差分析,进一步两两比较采用SNK-q检验。结果 3条siRNA对肝细胞SMAC mRNA均有不同程度的抑制作用,其中siRNA1的抑制效应最强,抑制效率达到70.3%。依siRNA1片段合成DNA oligo,并与慢病毒载体连接,测序显示其构建正确,梯度稀释法测定慢病毒滴度为6×108TU/ml。结论成功构建了小鼠SMAC基因特异干扰性的慢病毒,为研究SMAC基因在肝衰竭中的作用奠定基础。     

B lymphocyte activation during cognate interactions with CD4+ T lymphocytes: molecular dynamics and immunologic consequences

Productive interaction between T and B lymphocytes is required for humoral immune responses to many foreign protein antigens and production of pathogenic antibodies characteristic of several autoimmune conditions. Thus, much attention has been given in recent years to understand the dynamic molecular interactions and signal transduction required for productive T-B interaction. In this review we highlight current knowledge of signaling and biologic responses that occur in B cells during cognate interactions with helper T cells, focusing on the dynamic function of B cell-surface molecules in T-B synapses.