Development and use of ceramide nanoliposomes in cancer.

Integration of C6-ceramide into stealth pegylated nanoliposomes has led to the development of a promising preclinical therapeutic alone and in combination with other agents for treatment of cancer. Ceramide itself has been implicated as a bioactive lipid second messenger mediating cell senescence, cell cycle arrest, and apoptosis. Recent lipidomic analyses have demonstrated that specific ceramide species are differentially metabolized in individual cancers. Therapeutics that increase ceramide levels in cancer tissues have shown increased cell death and tumor inhibition. However, the use of ceramide itself as therapeutic has been problematic due to its inherent hydrophobicity and insolubility, therefore limiting the application for intravenous administration. Pegylated nanoliposomes eliminate this issue and are able to enhance the intracellular delivery of ceramide to cancer cells.

Combinatorial therapies improve the therapeutic efficacy of nanoliposomal ceramide for pancreatic cancer.

Poor prognosis cancers, such as pancreatic cancer, represent inherent challenges for ceramide-based nanotherapeutics due to metabolic pathways, which neutralize ceramide to less toxic or pro-oncogenic metabolites. We have recently developed a novel 80 nanometer diameter liposomal formulation that incorporates 30 molar percent C6-ceramide, a bioactive lipid that is pro-apoptotic to many cancer cells, but not to normal cells. In this manuscript, we evaluated the efficacy of combining nanoliposomal C6-ceramide (Lip-C6) with either gemcitabine or an inhibitor of glucosylceramide synthase. We first assessed the biological effect of Lip-C6 in PANC-1 cells, a gemcitabine-resistant human pancreatic cancer cell line, and found that low doses alone did not induce cell toxicity. However, cytotoxicity was achieved by combining Lip-C6 with either non-toxic sub-therapeutic concentrations of gemcitabine or with the glucosylceramide synthase inhibitor D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP). Furthermore, these combinations with Lip-C6 cooperatively inhibited PANC-1 tumor growth in vivo. Mechanistically, Lip-C6 inhibited pro-survival Akt and Erk signaling, whereas the nucleoside analog gemcitabine did not. Furthermore, by including PDMP within the nanoliposomes, which halted ceramide neutralization as evidenced by LC-MS3, the cytotoxic effects of Lip-C6 were enhanced. Collectively, we have demonstrated that nanoliposomal ceramide can be an effective anti-pancreatic cancer therapeutic in combination with gemcitabine or an inhibitor of ceramide neutralization.

Identification of novel small molecule antimicrobials targeting Mycoplasma bovis.

OBJECTIVES: To screen novel small molecule compounds for inhibition of Mycoplasma bovis growth and to characterize their activity in terms of dose-dependency and ability to function in milk. METHODS: Using a tetrazolium salt cytotoxicity assay, 480 natural compounds were screened to determine which of the small molecules have the potential to become therapeutic options for M. bovis prevention and treatment. The dose response was determined in broth culture and in fresh quarter milk for a subset of compounds shown to be capable of inhibiting M. bovis growth. RESULTS: Data suggest that 32 of the 480 compounds tested were able to inhibit growth of M. bovis using a tetrazolium salt assay. Methanesulphonic acid, 3-[(2E)-3-(3,4-dihydroxyphenyl)prop-2-enoyloxy](1S,3R,4R,5R)-1,4,5-trihydroxycyclohexane carboxylic acid, S-carboxymethyl-l-cysteine, l-aspartic acid, dihydrotachysterol, eriodictyol and (+)-α-tocopherol acid succinate were selected for further concentration-dependent studies and testing in fresh quarter milk. Each compound demonstrated a dose response in broth culture and at 3 h and 24 h in fresh quarter milk. CONCLUSIONS: Small molecule natural compounds are capable of inhibiting the growth of M. bovis in both a pleuropneumonia-like organism (PPLO) medium and in fresh quarter milk. Results suggest that the compounds are mycoplasmastatic in a dose-dependent manner. By inhibiting M. bovis, small molecule natural compounds offer the potential for prophylactic or therapeutic use on organic and natural farms as a viable alternative to traditional antimicrobial agents.

Use of liposomes as drug delivery vehicles for treatment of melanoma.

Melanoma is a progressive disease that claims many lives each year due to lack of therapeutics effective for the long-term treatment of patients. Currently, the best treatment option is early detection followed by surgical removal. Better melanoma therapies that are effectively delivered to tumors with minimal toxicity for patients are urgently needed. Nanotechnologies provide one approach to encapsulate therapeutic agents leading to improvements in circulation time, enhanced tumor uptake, avoidance of the reticulo-endothelial system, and minimization of toxicity. Liposomes in particular are a promising nanotechnology that can be used for more effective delivery of therapeutic agents to treat melanoma. Liposomes delivering chemotherapies, siRNA, asODNs, DNA, and radioactive particles are just some of the promising new nanotechnology based therapies under development for the treatment of melanoma that are discussed in this review.

Targeting V600EB-Raf and Akt3 using nanoliposomal-small interfering RNA inhibits cutaneous melanocytic lesion development.

Most events promoting early melanoma development are yet to be identified, but deregulation of the B-Raf and Akt3 signaling cascades is an important regulator of this process. Approximately 90% of normal moles and approximately 60% of early invasive cutaneous melanomas contain a T1799A B-Raf mutation ((V600E)B-Raf), leading to 10 times higher enzyme activity and constitutive activation of the mitogen-activated protein kinase pathway. Furthermore, approximately 70% of melanomas have elevated Akt3 signaling due to increased gene copy number and PTEN loss. Therefore, targeting (V600E)B-Raf and Akt3 signaling is necessary to prevent or treat cutaneous melanocytic lesions. Agents specifically targeting these proteins are needed, having fewer side effects than those inhibiting both normal and mutant B-Raf protein or targeting all three Akt isoforms. In this study, a unique nanoliposomal-ultrasound-mediated approach has been developed for delivering small interfering RNA (siRNA) specifically targeting (V600E)B-Raf and Akt3 into melanocytic tumors present in skin to retard melanoma development. Novel cationic nanoliposomes stably encapsulate siRNA targeting (V600E)B-Raf or Akt3, providing protection from degradation and facilitating entry into melanoma cells to decrease expression of these proteins. Low-frequency ultrasound using a lightweight four-cymbal transducer array enables penetration of nanoliposomal-siRNA complex throughout the epidermal and dermal layers of laboratory-generated or animal skin. Nanoliposomal-mediated siRNA targeting of (V600E)B-Raf and Akt3 led to a cooperatively acting approximately 65% decrease in early or invasive cutaneous melanoma compared with inhibition of each singly with negligible associated systemic toxicity. Thus, cationic nanoliposomes loaded with siRNA targeting (V600E)B-Raf and Akt3 provide an effective approach for targeted inhibition of early or invasive cutaneous melanomas.

Combining nanoliposomal ceramide with sorafenib synergistically inhibits melanoma and breast cancer cell survival to decrease tumor development.

PURPOSE: Deregulation of phosphatidylinositol 3-kinase/Akt and Ras/Raf/mitogen-activated protein kinase/extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase pathways occurs in melanoma and breast cancer, deregulating normal cellular apoptosis and proliferation. Therapeutic cocktails simultaneously targeting these pathways could promote synergistically acting tumor inhibition. However, agents with manageable toxicity and mechanistic basis for synergy need identification. The purpose of this study is to evaluate the preclinical therapeutic efficacy and associated toxicity of combining sorafenib with nanoliposomal ceramide. EXPERIMENTAL DESIGN: Effects of sorafenib and nanoliposomal ceramide as single and combinatorial agents were examined on cultured cells using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt assays and CalcuSyn software used to assess synergistic or additive inhibition. Western blotting measured cooperative effects on signaling pathways. Rates of proliferation, apoptosis, and angiogenesis were measured in size- and time-matched tumors to identify mechanistic basis for inhibition. Toxicity was evaluated measuring animal weight, blood toxicity parameters, and changes in liver histology. RESULTS: Sorafenib and nanoliposomal ceramide synergistically inhibited cultured cells by cooperatively targeting mitogen-activated protein kinase and phosphatidylinositol 3-kinase signaling. A 1- to 2-fold increase in cellular apoptosis and 3- to 4-fold decrease in cellular proliferation were observed following combination treatment compared with single agents, which caused synergistically acting inhibition. In vivo, an approximately 30% increase in tumor inhibition compared with sorafenib treatment alone and an approximately 58% reduction in tumor size compared with nanoliposomal ceramide monotherapy occurred by doubling apoptosis rates with negligible systemic toxicity. CONCLUSIONS: This study shows that nanoliposomal ceramide enhances effectiveness of sorafenib causing synergistic inhibition. Thus, a foundation is established for clinical trials evaluating the efficacy of combining sorafenib with nanoliposomal ceramide for treatment of cancers.

Targeting mitogen-activated protein kinase/extracellular signal-regulated kinase kinase in the mutant (V600E) B-Raf signaling cascade effectively inhibits melanoma lung metastases.

Malignant melanoma has a high propensity for metastatic spread, making it the most deadly form of skin cancer. B-RAF has been identified as the most mutated gene in these invasive cells and therefore an attractive therapeutic target. However, for uncertain reasons, chemotherapy inhibiting B-Raf has not been clinically effective. This has raised questions whether this pathway is important in melanoma metastasis or whether targeting a protein other than B-Raf in the signaling cascade could more effectively inhibit this pathway to reduce lung metastases. Here, we investigated the role played by (V600E)B-Raf in melanoma metastasis and showed that targeting this signaling cascade significantly reduces lung metastases. Small interfering RNA (siRNA)-mediated inhibition was used in mice to reduce expression (activity) of each member of the signaling cascade and effects on metastasis development were measured. Targeting any member of the signaling cascade reduced metastasis but inhibition of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (Mek) 1 and Mek 2 almost completely prevented lung tumor development. Mechanistically, metastatic inhibition was mediated through reduction of melanoma cell extravasation through the endothelium and decreased proliferative capacity. Targeting B-Raf with the pharmacologic inhibitor BAY 43-9006, which was found ineffective in clinical trials and seems to act primarily as an angiogenesis inhibitor, did not decrease metastasis, whereas inhibition of Mek using U0126 decreased cellular proliferative capacity, thereby effectively reducing number and size of lung metastases. In summary, this study provides a mechanistic basis for targeting Mek and not B-Raf in the mutant (V600E)B-Raf signaling cascade to inhibit melanoma metastases.