Longzhu Piao

Lipid-based Nanoparticle Delivery of Pre-miR-107 Inhibits the Tumorigenicity of Head and Neck Squamous Cell Carcinoma

Head and neck squamous cell carcinoma (HNSCC) is the sixth most prevalent cancer worldwide with about 600,000 new cases diagnosed in the last year. Our laboratory showed that miR-107 expression is reduced and functions as a tumor suppressor gene in HNSCC suggesting the potential application of miR-107 as a novel anticancer therapeutic. In this study, we determined the efficiency and efficacy of cationic lipid nanoparticles to deliver pre-miR-107 (NP/pre-miR-107) in HNSCC cells in vitro and in vivo. NP/pre-miR-107 increased delivery of miR-107 into HNSCC cells by greater than 80,000-fold compared to free pre-miR-107. Levels of known miR-107 targets, protein kinase Cε (PKCε), cyclin-dependent kinase 6 (CDK6), and hypoxia-inducible factor 1-β (HIF1-β), decreased following NP/pre-miR-107 treatment. Clonogenic survival, cell invasion, and cell migration of HNSCC cells was inhibited with NP/pre-miR-107. Moreover, NP/pre-miR-107 reduced the cancer-initiating cell (CIC) population and dampened the expression of the core embryonic stem cell transcription factors, Nanog, Oct3/4, and Sox2. In a preclinical mouse model of HNSCC, systemic administration of NP/pre-miR-107 significantly retarded tumor growth by 45.2% compared to NP/pre-miR-control (P < 0.005, n = 7). Kaplan-Meier analysis showed a survival advantage for the NP/pre-miR-107 treatment group (P = 0.017). Our results demonstrate that cationic lipid nanoparticles are an effective carrier approach to deliver therapeutic miRs to HNSCC.

Targeting HPV16 E6-p300 interaction reactivates p53 and inhibits the tumorigenicity of HPV-positive head and neck squamous cell carcinoma.

The incidence of human papillomavirus (HPV)-positive head and neck squamous cell carcinoma (HNSCC) has rapidly increased over the past 30 years, prompting the suggestion that an epidemic maybe on the horizon. Therefore, there is a clinical need to develop alternate therapeutic strategies to manage the growing number of HPV-positive HNSCC patients. High-risk HPV E6 inactivates p53 through two distinct mechanisms; association with E6AP to degrade p53 and association with p300 to block p300-mediated p53 acetylation and activation. In this study, we determined if targeting the E6-p300 interaction is an effective approach to reactivate p53 in HPV-positive HNSCC. Ectopic expression of the CH1 domain of p300 in HPV-positive HNSCC blocks the association between E6 and p300, increases total and acetylated p53 levels and enhances p53 transcriptional activity. Moreover, expression of p21, miR-34a and miR-200c are increased, demonstrating functional p53 reactivation. CH1 overexpression in HPV-positive HNSCC has a global anticancer effect resulting in a decrease in cell proliferation and clonogenic survival and an increase in apoptosis. The in vivo tumor-initiating ability of HPV-positive HNSCC is severely compromised with CH1 overexpression, in part through a reduction in the cancer-initiating cell population. A novel small-molecule CH1 inhibitor, CH1iB, reactivates p53 and potentiates the anticancer activity of cis-platinum in HPV-positive HNSCC cells. Our work shows that CH1-domain inhibitors represent a novel class of p53-reactivation therapeutics for managing HPV-positive HNSCC patients.

A novel liposomal formulation of flavopiridol

Flavopiridol has shown promising activities in hematologic and solid tumor models, as well as in clinical trials in chronic lymphocytic leukemia patients. Flavopiridol has relatively low solubility and high plasma protein-binding. To address these issues and to provide an alternative strategy to achieve clinical efficacy, we encapsulated flavopiridol into a liposomal carrier and characterized its physicochemical and pharmacokinetic properties. The liposomes, comprising hydrogenated soy phosphatidylcholine (HSPC), cholesterol and poly (ethylene glycol) 2000-distearoyl phosphatidylethanolamine (PEG-DSPE), were prepared by polycarbonate membrane extrusion and then loaded with flavopiridol by a pH-gradient driven remote loading procedure. The liposomes had a mean diameter of 120.7 nm and a flavopiridol entrapment efficiency of 70.4%. Pharmacokinetic study in mice after i.v. bolus injection showed that the liposomal flavopiridol had an increased elimination phase half-life (T((1/2)beta), 339.7 min vs. 57.0 min), decreased clearance (CL, 0.012 L/min vs. 0.036 L/min), and increased area under the plasma concentration-time curve (AUC, 10.8 min micromol/L vs. 3.4 min micromol/L) compared to the free drug. This indicates a significant and potentially beneficial change in flavopiridol pharmacokinetics for the liposomal formulation. Further preclinical studies are warranted to define the toxicity and therapeutic efficacy of this novel formulation.

Transferrin Receptor Targeted Lipopolyplexes for Delivery of Antisense Oligonucleotide G3139 in a Murine K562 Xenograft Model

PurposeTransferrin (Tf) conjugated lipopolyplexes (LPs) carrying G3139, an antisense oligonucleotide for Bcl-2, were synthesized and evaluated in Tf receptor positive K562 erythroleukemia cells and then in a murine K562 xenograft model.Materials and MethodsParticle size and Zeta potentials of transferrin conjugated lipopolyplexs containing G3139 (Tf-LP-G3139) were measured by Dynamic Light Scattering and ZetaPALS. In vitro and in vivo sample’s Bcl-2 downregulation was analyzed using Western blot and tumor tissue samples also exhibited by immunohistochemistry method. For athymic mice bearing with K562 xenograft tumors, tumor growth inhibition and survival rate were investigated. Nanoparticle distribution in 3-D cell cluster was observed by Laser scan confocal microscopy. IL-12 production in the plasma was measured by ELISA kit.ResultsIn vitro, Tf-LP-G3139 was more effective in inducing down regulation of Bcl-2 in K562 cells than non-targeted LP-G3139, free G3139 and mismatched control ODN-G4126 in the same formulation. In vivo Tf-LP-G3139 was less effective than free G3139 in Bcl-2 down regulation. 3-D cell cluster model diffusion results indeed indicated limited penetration of the LPs into the cell cluster. Finally, the therapeutic efficacies of Tf-LP-G3139 and free G3139 were determined in the K562 xenograft model. Tf-LP-G3139 showed slower plasma clearance, higher AUC, and greater accumulation in the tumor compared to free G3139. In addition, Tf-LP-G3139 was found to be more effective in tumor growth inhibition and prolonging mouse survival than free G3139. This was associated with increased spleen weight and IL-12 production in the plasma.ConclusionThe role of the immune system in the therapeutic response obtained with the Tf-LPs is necessary and in vitro 3-D cell cluster model can be a potential tool to evaluate the nanoparticle distribution.

Phosphorylation of Nanog is essential to regulate Bmi1 and promote tumorigenesis.

Emerging evidence indicates that Nanog is intimately involved in tumorigenesis, in part, through regulation of the cancer-initiating cell (CIC) population. However, the regulation and role of Nanog in tumorigenesis are still poorly understood. In this study, human Nanog was identified to be phosphorylated by human protein kinase Cɛ at multiple residues, including T200 and T280. Our work indicated that phosphorylation at T200 and T280 modulates Nanog function through several regulatory mechanisms. Results with phosphorylation-insensitive and phosphorylation-mimetic mutant Nanog revealed that phosphorylation at T200 and T280 enhance Nanog protein stability. Moreover, phosphorylation-insensitive T200A and T280A mutant Nanog had a dominant-negative function to inhibit endogenous Nanog transcriptional activity. Inactivation of Nanog was due to impaired homodimerization, DNA binding, promoter occupancy and p300, a transcriptional co-activator, recruitment resulting in a defect in target gene-promoter activation. Ectopic expression of phosphorylation-insensitive T200A or T280A mutant Nanog reduced cell proliferation, colony formation, invasion, migration and the CIC population in head and neck squamous cell carcinoma (HNSCC) cells. The in vivo cancer-initiating ability was severely compromised in HNSCC cells expressing phosphorylation-insensitive T200A or T280A mutant Nanog; 87.5% (14/16), 12.5% (1/8), and 0% (0/8) for control, T200A, and T280A, respectively. Nanog occupied the Bmi1 promoter to directly transactivate and regulate Bmi1. Genetic ablation and rescue experiments demonstrated that Bmi1 is a critical downstream signaling node for the pleiotropic, pro-oncogenic effects of Nanog. Taken together, our study revealed, for the first time, that post-translational phosphorylation of Nanog is essential to regulate Bmi1 and promote tumorigenesis.

Elevated intrinsic cancer stem cell population in human papillomavirus-associated head and neck squamous cell carcinoma.

Human papillomavirus 16 (HPV16) is a major risk factor for the development of head and neck squamous cell carcinoma (HNSCC), particularly the development of oropharyngeal squamous cell carcinoma (OPSCC). Cancer stem cells (CSCs) are resistant to conventional therapies, and it is postulated that they are responsible for disease recurrence and/or progression. Because the prognoses of patients with HPV16‐positive and HPV‐negative HNSCC are distinct, the authors sought to determine whether differences in the number of CSCs could account for this clinical observation.

Targeting human clonogenic acute myelogenous leukemia cells via folate conjugated liposomes combined with receptor modulation by all-trans retinoic acid.

Our previous data demonstrated that folate receptor β (FR-β) targeted liposomal doxorubicin (FT-L-DOX) showed enhanced cytotoxicity relative to non-targeted liposomal doxorubicin (CON-L-DOX), and the effect was enhanced by selective FR-β upregulation by all-trans retinoic acid (ATRA) in AML blast cells. In this study, the enhanced cytotoxicity was investigated in the proliferating human AML clonogenic cells by combining FT-L-DOX with ATRA. Also, pharmacokinetic properties by pretreatment of ATRA were evaluated using FR-targeted liposomal calcein (FT-L-Calcein). Pharmacokinetic study showed that the area under the concentration curve (AUC) of FT-L-Calcein was decreased and total clearance was increased by pretreatment with ATRA. Meanwhile, the volume of distribution was significantly increased by pretreatment of ATRA. Moreover, calcein level in the liver, spleen and kidney was increased following intravenous administration of FT-L-Calcein by pretreatment of ATRA. In vitro cytotoxicity of FT-L-DOX was higher than that of CON-L-DOX and was increased by pretreatment with ATRA. Colony formation in AML cells was lower due to treatment with FT-L-DOX compared with CON-L-DOX and colony formation further decreased upon pretreatment with ATRA. Moreover, FT-L-DOX was more toxic to AML clonogenic cells than to AML blast cells. The results demonstrate that the efficiency of FR-mediated targeting of FT-L-DOX was preferentially enhanced by ATRA induced FR-β upregulation in AML clonogenic cells.

Human serum albumin-coated lipid nanoparticles for delivery of siRNA to breast cancer.

UNLABELLED Human serum albumin (HSA)-coated lipid nanoparticles (HSA-LNPs) loaded with phrGFP-targeted siRNA (HSA-LNPs-siRNA) were prepared and evaluated for gene downregulation effect in phrGFP-transfected breast cancer cells and the corresponding xenograft tumor model. HSA-LNPs-siRNA were successfully prepared with a particle size of 79.5±5.5 nm. In phrGFP-transfected MCF-7 cells, HSA-LNPs-siRNA significantly decreased cell fluorescence even in the presence of fetal bovine serum (FBS). Moreover, cell fluorescence and phrGFP mRNA expression were significantly downregulated by HSA-LNPs-siRNA in phrGFP-transfected MCF-7, MDA-MB-231, and SK-BR-3 cells in comparison with control or HSA-LNPs-siRNA (scrambled). In phrGFP-transfected MCF-7 xenograft tumor model, tumor fluorescence was significantly decreased after three IV administrations of HSA-LNPs-siRNA at a dose of 3 mg/kg in comparison with siRNA alone. HSA-LNPs-siRNA demonstrated a superior pharmacokinetic profile in comparison with siRNA at a dose of 1mg/kg. These results show that the novel nonviral carrier, HSA-LNPs, may be used for the delivery of siRNA to breast cancer cells. FROM THE CLINICAL EDITOR Targeted delivery of siRNA to cancer cells may be a viable anti-cancer strategy with low toxicity. In this study the novel nonviral carrier, human serum albumin-coated lipid nanoparticles (HSA-LNP) were demonstrated as an efficient delivery agent of siRNA to breast cancer cells.

Delivery of calf thymus DNA to tumor by folate receptor targeted cationic liposomes

Calf thymus DNA (ctDNA) has been shown to stimulate macrophages to produce cytokines both in vitro and in vivo when complexed with cationic liposomes. In addition, direct cytotoxicity of ctDNA has been found in tissue culture and in mice. In this study, ctDNA and folate receptor targeted cationic liposome complexes (ctDNA-F-CLs) were prepared and evaluated in FR (+) tumors. In addition, the underlying mechanism for the anti-cancer activity of ctDNA-F-CLs was investigated. Selective uptake of ctDNA-F-CLs was observed in FR (+) KB and L1210JF cells using flow cytometry. In RAW264.7 cells and DBA/2 mice, ctDNA-F-CLs and ctDNA-N-CLs significantly induced TNF-α and IL-6 production compared to free ctDNA. However, no significant difference in cytokine production was observed between ctDNA-N-CLs and ctDNA-F-CLs. In tumor bearing DBA/2 mice, ctDNA-F-CLs significantly increased INF-γ and IL-6 production compared to ctDNA-N-CLs. Furthermore in L1210JF cells, ctDNA-F-CLs had significantly increased cytotoxicity compared to ctDNA-N-CLs. Tumor cell apoptosis was also found in co-culture of RAW264.7 cells and ctDNA-F-CLs treated L1210JF cells. In L1210JF tumor bearing mice, ctDNA-F-CLs were found to significantly inhibit tumor growth and prolong the median survival time (MeST). In contrast, ctDNA-N-CLs and free ctDNA showed similar activities for tumor inhibition and animal survival. Moreover, the anti-cancer effect of ctDNA-F-CL was further enhanced by combination with anti-cancer drug doxorubicin. These results suggest that ctDNA-F-CLs are a promising agent for treatment of FR-positive tumors.

Folate-Immunoglobulin G as an Anticancer Therapeutic Antibody

Folate receptor-alpha (FR) is a promising cellular marker for tumor-specific drug delivery. Conjugation of folic acid to therapeutic and imaging agents has been shown to enhance their delivery to FR (+) cancer cells in vitro and in tumor-bearing mice via an FR-mediated cellular uptake mechanism. In this study, immunoglobulin G (IgG) was conjugated to folate and evaluated as a therapeutic antibody against folate receptor (FR)-positive tumors. Murine IgG (mIgG) was conjugated to folate via an amide bond to yield folate-conjugated mIgG (f-mIgG) that contained an average of approximately 2.6 folates per molecule. Selective uptake of f-IgG by FR (+) tumor cells was determined by fluorescence microscopy and by flow cytometry. Lysis of L1210JF cells by NK cells from murine donors was increased 1.4-9.0-fold at the effector:target (E:T) ratio of 25:1, relative to control mIgG. In mice bearing L1210JF tumors, f-mIgG was found to significantly inhibit tumor growth and to have prolonged the median survival time (MeST). Significantly, the antitumor efficacy of f-mIgG was greatly increased when combined with liposomal G3139, an 18-mer phosphorothioate oligonucleotide. In fact, the combination resulted in a 100% cure rate among the tumor-bearing mice. Injection of f-mIgG significantly increased serum INF-gamma and IL-6 level in mice compared with mIgG and dramatically increased serum INF-gamma and IL-6 level when combined with liposomal G3139. These results suggested that f-IgG, a novel immunotherapy agent, has potent activity as a therapeutic antibody to the FR-positive cancer, and the therapeutic activity is enhanced by immunomodulatory agents.