Squamous cell carcinoma (SCC) and melanoma are malignant human cancers of the skin with an annual mortality that exceed 10,000 cases every year in the USA alone. In this study, the lysosomal protein saposin C (SapC) and the phospholipid dioloylphosphatidylserine (DOPS) were assembled into cancer-selective nanovesicles (SapC-DOPS) and successfully tested using several in vitro and in vivo skin cancer models. Using MTT assay that measures the percentage of cell death, SapC-DOPS cytotoxic effect on three skin tumor cell lines (squamous cell carcinoma, SK-MEL-28, and MeWo) was compared to two normal nontumorigenic skin cells lines, normal immortalized keratinocyte (NIK) and human fibroblast cell (HFC). We observed that the nanovesicles selectively killed the skin cancer cells by inducing apoptotic cell death whereas untransformed skin cancer cells remained unaffected. Using subcutaneous skin tumor xenografts, animals treated with SapC-DOPS by subcutaneous injection showed a 79.4 % tumor reduced compared to the control after 4 days of treatment. We observed that the nanovesicles killed skin cancer cells by inducing apoptotic cell death compared to the control as revealed by TUNEL staining of xenograft tumor sections.

γ-Aminobutyric acid type A (GABAA) receptor plasticity participates in mediating adaptation to environmental change. Previous studies in rats demonstrated that extrasynaptic GABAA receptor subunits and receptors in the pons, a brainstem region involved in respiratory control, are upregulated by exposure to sustained hypobaric hypoxia. In these animals, expression of the mRNA encoding the extrasynaptic α4 subunit rose after 3 days in sustained hypoxia, while those encoding the α6 and δ subunits increased dramatically by 2 weeks. However, the participation of extrasynaptic subunits in maintaining respiration in normoxic conditions remains unknown. To examine the importance of α4 in a normal environment, respiratory function, motor and anxiety-like behaviors, and expression of other GABAA receptor subunit mRNAs were compared in wild-type (WT) and α4 subunit-deficient mice. Loss of the α4 subunit did not impact frequency, but did lead to reduced ventilatory pattern variability. In addition, mice lacking the subunit exhibited increased anxiety-like behavior. Finally, α4 subunit loss resulted in reduced expression of other extrasynaptic (α6 and δ) subunit mRNAs in the pons without altering those encoding the most prominent synaptic subunits. These findings on subunit-deficient mice maintained in normoxia, in conjunction with earlier findings on animals maintained in chronic hypoxia, suggest that the expression and regulation of extrasynaptic GABAA receptor subunits in the pons is interdependent and that their levels influence respiratory control as well as adaptation to stress.

Introduction: Acidic phosphatidylserine-enriched membrane microdomains (PS-EMMs) are exposed on the membrane surface of cancer cells and tumor blood vessels. We have explored a novel PS-EMMs targeting mechanism by saposin C (SapC)-coupled dioleoylphosphatidylserine (DOPS) nanovesicles that are found to specifically target and destroy cancer and tumor-associated endothelial cells with little or no systemic toxicity (Qi et al. Clin. Cancer Res. 2009; Kaimal et al. Mol. Imaging Biol. 2011). Methods: We have developed a novel biotheragnositic agent, SapC-DOPS, which we assembled into cancer-selective nanovesicles. Surface exposed PS levels were analyzed by a flow cytometric method using fluorescently labeled annexin-V. Deparafinized tissue slides were stained using far-red fluorophore (CellVue Maroon, CVM) labeled SapC-DOPS nanovesicles, SapC-DOPS-CVM was used to determine cancer-selective targeting via PS-mediated membrane fusion in human cancer cells, patient-derived cancer tissue specimens, and preclinical animal tumor models, including brain, pancreatic, lung, and pediatric cancers. The cytotoxicity of SapC-DOPS was measured using the MTT assay. Live imaging for visualization of SapC-DOPS PS-targeting and survival curves were demonstrated in transgenic and xenograft mouse tumor models. Results: We demonstrated the specific targeting of SapC-DOPS in paraffin embedded patient-derived tumor tissue sections. Based on MTT assay and annexin-V labeled flow cytometry, we revealed that SapC-DOPS cytotoxic effects correlated to the level of surface exposed PS in a variety of human cancer cell lines. Kinetic studies indicated that SapC-DOPS fused with tumor cell membrane within a few minutes, followed by internalization into the cells. In vivo cancer cell targeting of SapC-DOPS was blocked by pretreatment with PS-specific binding proteins (Lact-C2 and beta-2 glycoprotin-1). Increased PS expression and externalization on cancer cell membrane surfaces after chemotherapy or extracellular stresses enhanced the SapC-DOPS response. SapC-DOPS showed cancer-selective targeting in numerous spontaneously formed and xenografted tumors in living mice using either a double-tracking optical or a contrast-enhanced magnetic resonance imaging system. Furthermore, we observed that tumor-bearing animals treated with intravenous SapC-DOPS had significant tumor reduction and increased survival. Tumor-free survivals were confirmed by tumor-luminescence imaging and at necropsy. Conclusion: We have demonstrated a novel biomarker for numerous human cancers that can be effectively targeted by using cancer-selective SapC-DOPS nanovesicles. The direct confirmation of tumor-targeting by SapC-DOPS provides powerful new evidence in support of our goal to develop PS-EMMs targeted nanovesicles as novel therapeutic agents for cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1840. doi:1538-7445.AM2012-1840