Propylene Glycol Caprylate-Based Nanoemulsion Formulation of Plumbagin: Development and Characterization of Anticancer Activity.

Plumbagin, a bioactive naphthoquinone, has demonstrated potent antitumor potential. However, plumbagin is a sparingly water-soluble compound; therefore, clinical translation requires and will be facilitated by the development of a new pharmaceutical formulation. We have generated an oil-in-water nanoemulsion formulation of plumbagin using a low-energy spontaneous emulsification process with propylene glycol caprylate (Capryol 90) as an oil phase and Labrasol/Kolliphor RH40 as surfactant and cosurfactant excipients. Formulation studies using Capryol 90/Labrasol/Kolliphor RH40 components, based on pseudoternary diagram and analysis of particle size distribution and polydispersity determined by dynamic light scattering (DLS), identified an optimized composition of excipients for nanoparticle formulation. The nanoemulsion loaded with plumbagin as an active pharmaceutical ingredient had an average hydrodynamic diameter of 30.9 nm with narrow polydispersity. The nanoemulsion exhibited long-term stability, as well as good retention of particle size in simulated physiological environments. Furthermore, plumbagin-loaded nanoemulsion showed an augmented cytotoxicity against prostate cancer cells PTEN-P2 in comparison to free drug. In conclusion, we generated a formulation of plumbagin with high loading drug capacity, robust stability, and scalable production. Novel Capryol 90-based nanoemulsion formulation of plumbagin demonstrated antiproliferative activity against prostate cancer cells, warranting thus further pharmaceutical development.

Plumbagin-Serum Albumin Interaction: Spectral, Electrochemical, Structure-Binding Analysis, Antiproliferative and Cell Signaling Aspects with Implications for Anticancer Therapy.

Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) is a small molecule with potent anticancer activity. Like other 1,4-naphthoquinones, it exhibits electrophilic reactivity towards biological nucleophiles. We demonstrate that plumbagin and structurally related 1,4-naphthoquinones with at least one unsubstituted quinoid carbon (C2 or C3) bind to albumin, an ubiquitously present nucleophile, with minimum recovery of free drug. Extraction recovery of plumbagin from albumin in solution showed one-phase exponential decline with a half-live of 9.3 min at 10 µmol/L. In the presence of albumin, plumbagin exhibited instant changes in UV/Vis absorption bands. Electrochemical analysis using cyclic voltammetry showed a decrease in redox peak currents over time until electro-inactivity, thus suggesting the formation of a supramolecular adduct inaccessible for electron transfer. The adduct inhibited cell growth and caused cell-cycle arrest of prostate cancer cells, in part by decreasing levels of the cell-cycle regulator RBBP. The conjugate displayed similar cellular effects to those described for plumbagin, such as decreased levels of androgen receptor and protein kinase C epsilon. The effect of plumbagin-albumin on cancer cells was species-specific, suggesting a receptor-mediated mechanism. Furthermore, it was blocked by cathepsin inhibitor pepstatin A, indicating that lysosomal degradation is involved in the processing of plumbagin-albumin adduct. The spontaneously formed adduct of plumbagin with serum albumin is likely to mediate the biological activities of plumbagin in vivo and to fundamentally influence its pharmacodynamics.

Plumbagin-Loaded Nanoemulsion Drug Delivery Formulation and Evaluation of Antiproliferative Effect on Prostate Cancer Cells.

BACKGROUND: Plumbagin, a medicinal plant-derived 5-hydroxy-2-methyl-1,4-naphthoquinone, is an emerging drug with a variety of pharmacological effects, including potent anticancer activity. We have previously shown that plumbagin improves the efficacy of androgen deprivation therapy (ADT) in prostate cancer and it is now being evaluated in phase I clinical trial. However, the development of formulation of plumbagin as a compound with sparing solubility in water is challenging. METHODS: We have formulated plumbagin-loaded nanoemulsion using pneumatically controlled high pressure homogenization of oleic acid dispersions with polyoxyethylene (20) sorbitan monooleate as surfactant. Nanoemulsion formulations were characterized for particle size distribution by dynamic light scattering (DLS). The kinetics of in vitro drug release was determined by equilibrium dialysis. Anticancer activity toward prostate cancer cells PTEN-P2 was assessed by MTS (Owen's reagent) assay. RESULTS: Particle size distribution of nanoemulsions is tunable and depends on the surfactant concentration. Nanoemulsion formulations of plumbagin with 1-3.5% (w/w) of surfactant showed robust stability of size distribution over time. Plumbagin-loaded nanoemulsion with average hydrodynamic diameter of 135 nm showed exponential release of plumbagin with a half-life of 6.1 h in simulated gastric fluid, 7.0 h in simulated intestinal fluid, and displayed enhanced antiproliferative effect toward prostate cancer cells PTEN-P2 compared to free plumbagin. CONCLUSION: High drug-loading capacity, retention of nanoparticle size, kinetics of release under simulated physiological conditions, and increased antiproliferative activity indicate that oleic-acid based nanoemulsion formulation is a suitable delivery system of plumbagin.

Plumbagin improves the efficacy of androgen deprivation therapy in prostate cancer: A pre-clinical study.

BACKGROUND: Plumbagin is a candidate drug for the treatment of prostate cancer. Previous observations indicated that it may improve the efficacy of androgen deprivation therapy (ADT). This study evaluates the effectiveness of treatment with combinations of plumbagin and alternative strategies for ADT in mouse models of prostate cancer to support its clinical use. METHODS: Plumbagin was administered per oral in a new sesame oil formulation. Standard toxicology studies were performed in rats. For tumor growth studies, mouse prostate cancer cell spheroids were placed on top of grafted prostate tissue in a dorsal chamber and allowed to form tumors. Mice were separated in various treatment groups and tumor size was measured over time by intra-vital microscopy. Survival studies were done in mice after injection of prostate cancer cells in the prostate of male animals. Androgen receptor (AR) levels were analyzed by Western blot from prostate cancer cells treated with plumbagin. RESULTS: Plumbagin caused a decrease in AR levels in vitro. In mice, plumbagin at 1 mg/kg in sesame oil displayed low toxicity and caused a 50% tumor regression when combined with castration. The combination of plumbagin with various forms of chemical ADT including treatment with a GnRH receptor agonist, a GnRH receptor antagonist, or CYP17A1 inhibitors, outperformed ADT alone, increasing mouse survival compared to the standard regimen of castration alone. In contrast, the combination of plumbagin with AR antagonists, such as bicalutamide and enzalutamide, showed no improvement over AR antagonists alone. Thus, plumbagin is effective in combination with drugs that prevent the synthesis of testosterone or its conversion to dihydrotestosterone, but not with drugs that bind to AR. CONCLUSION: Plumbagin significantly improves the effect of ADT drugs currently used in the clinic, with few side effects in mice.

Effects of different tissue microenvironments on gene expression in breast cancer cells.

In metastasis, circulating tumor cells penetrate the walls of blood vessels and enter the metastatic target tissue, thereby becoming exposed to novel and relatively unsupportive microenvironments. In the new microenvironments, the tumor cells often remain in a dormant state indefinitely and must adapt before they are able to successfully colonize the tissue. Very little is known about this adaptive process. We studied temporal changes in gene expression when breast cancer cells adapt to survive and grow on brain, bone marrow, and lung tissue maintained in an in vivo culture system, as models of the metastatic colonization of these tissues. We observed the transient activation of genes typically associated with homeostasis and stress during the initial stages of adaptation, followed by the activation of genes that mediate more advanced functions, such as elaboration of cell morphology and cell division, as the cells adapted to thrive in the host tissue microenvironment. We also observed the temporary induction of genes characteristic of the host tissue, which was particularly evident when tumor cells were grown on brain tissue. These early transient gene expression events suggest potential points of therapeutic intervention that are not evident in data from well-established tumors.

Early growth response 3 (Egr3) is highly over-expressed in non-relapsing prostate cancer but not in relapsing prostate cancer.

Members of the early growth response (EGR) family of transcription factors play diverse functions in response to many cellular stimuli, including growth, stress, and inflammation. Egr3 has gone relatively unstudied, but here through use of the SPECS (Strategic Partners for the Evaluation of Predictive Signatures of Prostate Cancer) Affymetrix whole genome gene expression database we report that Egr3 mRNA is significantly over-expressed in prostate cancer compared to normal prostate tissue (5-fold). The Human Protein Atlas (http://www.proteinatlas.org), a database of tissue microarrays labeled with antibodies against over 11,000 human proteins, was utilized to quantify Egr3 protein expression in normal prostate and prostate cancer patients. In agreement with the SPECS data, we found that Egr3 protein is significantly increased in prostate cancer. The SPECS database has the benefit of extensive clinical follow up for the prostate cancer patients. Analysis of Egr3 mRNA expression in relation to the relapse status reveals that Egr3 mRNA expression is increased in tumor cells of non-relapsed samples (n = 63) compared to normal prostate cells, but is significantly lower in relapsed samples (n = 38) compared to non-relapse. The observations were confirmed using an independent data set. A list of genes correlating with this unique expression pattern was determined. These Egr3-correlated genes were enriched with Egr binding sites in their promoters. The gene list contains inflammatory genes such as IL-6, IL-8, IL1ß and COX-2, which have extensive connections to prostate cancer.

The combination of plumbagin with androgen withdrawal causes profound regression of prostate tumors in vivo.

BACKGROUND: Hormonal ablation is the standard treatment for disseminated androgen-dependent prostate cancer. Although tumor growth is controlled at first, the tumor invariably recurs in the form of castration-resistant prostate cancer. This study assessed the efficacy of a new therapeutic strategy that combines plumbagin, a naturally occurring naphthoquinone, with androgen ablation. METHODS: Viewing microscopy chambers were placed in the dorsal skinfold of mice. Syngeneic prostate tissue was grafted within the chambers and allowed to vascularize. H2B-GFP/PTEN-P2 prostate cancer cells were co-implanted on top of the grafted prostate tissue. Androgen ablation was achieved using surgical castration. Intact and castrated mice were administered plumbagin or sham treatment. Tumor growth, mitosis and apoptosis were monitored in real-time using fluorescent Intra-Vital Microscopy. The mechanism of action of plumbagin was explored using human and mouse prostate cancer cells. RESULTS: Whereas both plumbagin and castration alone impeded tumor growth, only the combination of plumbagin and castration caused profound tumor regression in vivo, mostly due to increased apoptosis of the tumor cells. The cytotoxicity of plumbagin was not affected by androgens in vitro, suggesting that microenvironmental factors not present in culture play a crucial role in the combination effect. Plumbagin-induced cell death was mediated, at least in part, by activation of ERK and was due to generation of reactive oxygen species, because it was abolished by the anti-oxidant N-acetyl-L-cysteine. CONCLUSION: Androgen deprivation in combination with plumbagin may provide a significant improvement over androgen deprivation alone and deserves further evaluation.

Intravital microscopy in the mouse dorsal chamber model for the study of solid tumors.

Intra-Vital Microscopy (IVM) is used to visualize tumors in animals and analyze various aspects of cancer physiology such as tumor vascularization, cell migration and metastasis. The main advantages of IVM include the real -time analysis of dynamic processes with single-cell resolution. The application of IVM, however, is limited by the availability of animal models that carry visually accessible tumors. These models have evolved over time to become more and more relevant to human tumors. The latest step is the development of a pseudo-orthotopic, syngeneic model for tumor growth and metastasis. In this model, tissue from a variety of mouse organs are grafted in a dorsal skinfold chamber and allowed to revascularize, whereupon tumor cell spheroids are implanted. These spheroids develop into tumors that bear a much closer resemblance to human tumors than xenografts. Unlike xenografts, the vasculature is well-ordered and, because the model is syngeneic, there are no cross-species host immune reactions. The use of fluorescence-tagged pseudo-organs and tumor cells allows IVM analysis and provides real-time access to the development of tumors that closely resemble the real disease. This model can be used to test therapeutics and to image tumor development and stroma-tumor interactions.

Regression of prostate tumors upon combination of hormone ablation therapy and celecoxib in vivo.

BACKGROUND: Hormonal ablation is the standard of treatment for advanced androgen-dependent prostate cancer. Although tumor regression is usually achieved at first, the cancer inevitably evolves toward androgen-independence, in part because of the development of mechanisms of resistance and in part because at the tissue level androgen withdrawal is not fully attained. Current research efforts are focused on new therapeutic strategies that will increase the effectiveness of androgen withdrawal and delay recurrence. We used a syngeneic pseudo-orthotropic mouse model of prostate cancer to test the efficacy of combining androgen withdrawal with FDA-approved COX-2 inhibitor celecoxib. METHODS: GFP-tagged TRAMP-C2 cells were co-implanted with prostate tissue in the dorsal chamber model and tumors were allowed to establish and vascularize. Tumor growth and angiogenesis were monitored in real-time using fluorescent intravital microscopy (IVM). Androgen withdrawal in mice was achieved using surgical castration or chemical hormonal ablation, alone or in combination with celecoxib (15 mg/kg, twice daily). RESULTS: Celecoxib alone decreased the growth of prostate tumors mostly by inducing mitotic failure, which resulted in increased apoptosis. Surprisingly, celecoxib did not possess significant angiostatic activity. Surgical or chemical castration prevented the growth of prostate tumors and this, on the other hand, was associated with disruption of the tumor vasculature. Finally, androgen withdrawal combined with celecoxib caused tumor regression through decreased angiogenesis and increased mitosis arrest and apoptosis. CONCLUSION: Celecoxib, a relatively safe COX-2-selective anti-inflammatory drug, significantly increases the efficacy of androgen withdrawal in vivo and warrants further investigation as a complement therapy for advanced prostate cancer.

Is EGR1 a potential target for prostate cancer therapy?

Prostate cancer is a major cause of cancer-related death in American men, for which finding new therapeutic strategies remains a challenge. Early growth response-1 (EGR1) is a transcription factor involved in cell proliferation and in the regulation of apoptosis. Although it has long been considered a tumor suppressor, a wealth of new evidence shows that EGR1 promotes the progression of prostate cancer. This review addresses the paradoxes of EGR1 function. While EGR1 mediates apoptosis in response to stress and DNA damage by regulating a tumor suppressor network, it also promotes the proliferation of prostate cancer cells by a mechanism that is not fully understood. Thus, EGR1 might be targeted for prostate cancer therapy either by ectopic expression in combination with radiotherapy or chemotherapy, or by direct inhibition for systemic treatment. Possible strategies to antagonize EGR1 function in a therapeutic setting are discussed.

PTEN regulation by Akt-EGR1-ARF-PTEN axis.

The PTEN tumour suppressor gene is induced by the early growth response 1 (EGR1) transcription factor, which also transactivates p53, p73, and p300/CBP as well as other proapoptotic and anti-cancer genes. Here, we describe a novel Akt-EGR1-alternate reading frame (ARF)-PTEN axis, in which PTEN activation in vivo requires p14ARF-mediated sumoylation of EGR1. This modification is dependent on the phosphorylation of EGR1 at S350 and T309 by Akt, which promotes interaction of EGR1 with ARF at K272 in its repressor domain by the ARF/Ubc9/SUMO system. EGR1 sumoylation is decreased by ARF reduction, and no EGR1 sumoylation is detected in ARF(-/-) mice, which also exhibit reduced amounts of PTEN. Our model predicts that perturbation of any of the clinically important tumour suppressors, PTEN, EGR1, and ARF, will cause some degree of dysfunction of the others. These results also explain the known negative feedback regulation by PTEN on its own synthesis through PI3 kinase inhibition.

Tumor-derived fibronectin is involved in melanoma cell invasion and regulated by V600E B-Raf signaling pathway.

Melanomas are malignant tumors of melanocytes that, if not detected early, are highly aggressive and poorly treatable. Activation of extracellular signal-regulated (ERK)/mitogen-activated protein (MAP) kinase signaling is commonly found in melanomas mainly through oncogenic mutations of B-Raf. We previously reported that activation of ERK/MAP kinase stimulates synthesis of fibronectin by upregulating the transcription factor early growth response-1 (Egr-1). To further analyze the link between ERK/MAP kinase pathway and fibronectin in melanoma, we have studied the regulation and role of fibronectin produced by melanoma cells bearing oncogenic B-Raf mutation. We show that fibronectin is expressed in situ during tumor progression and that high fibronectin and Egr-1 levels are found in cells expressing this mutation. Expression of active mutants of B-Raf induces fibronectin, whereas endogenous fibronectin is inhibited by small interfering RNA (siRNA)-mediated depletion of B-Raf or Egr-1. In contrast, stimulation of ERK pathway is insufficient to promote fibronectin upregulation in normal melanocytes. Finally, we show that suppression of fibronectin by siRNA leads to decreased melanoma cell invasiveness in vitro. These results reveal a tumor-specific regulation of fibronectin by constitutive ERK/MAP kinase signaling and indicate that self-production of fibronectin may play a role in melanoma tumorigenesis, by promoting tumor cell invasion.

Inhibition of Egr-1 expression reverses transformation of prostate cancer cells in vitro and in vivo.

Transcription factor early growth response-1 (Egr-1) is a crucial regulator of cell growth, differentiation and survival. Several observations suggest that Egr-1 is growth promoting in prostate cancer cells and that blocking its function may impede cancer progression. To test this hypothesis, we developed phosphorothioate antisense oligonucleotides that efficiently inhibit Egr-1 expression without altering the expression of other family members Egr-2, Egr-3 and Egr-4. In TRAMP mouse-derived prostate cancer cell lines, our optimal antisense oligonucleotide decreased the expression of the Egr-1 target gene transforming growth factor-beta1 whereas a control oligonucleotide had no effect, indicating that the antisense blocked Egr-1 function as a transcription factor. The antisense oligonucleotide deregulated cell cycle progression and decreased proliferation of the three TRAMP cell lines by an average of 54+/-3%. Both colony formation and growth in soft agar were inhibited by the antisense oligonucleotide. When TRAMP mice were treated systemically for 10 weeks, the incidence of palpable tumors at 32 weeks of age in untreated mice or mice injected with the control scramble oligonucleotide was 87%, whereas incidence of tumors in antisense-Egr-1-treated mice was significantly reduced to 37% (P=0.026). Thus, Egr-1 plays a functional role in the transformed phenotype and may represent a valid target for prostate cancer therapy.

Characterization of antigen-specific repertoire diversity following in vitro restimulation by a recombinant adenovirus expressing human cytomegalovirus pp65.

Human cytomegalovirus (HCMV) and adenovirus cause significant morbidity and mortality in immunocompromised hosts undergoing allogeneic stem cell transplantation. We have previously established a procedure for the generation of polyclonal CTL with specificity against adenovirus and HCMV using a recombinant adenovirus encoding the HCMV pp65 protein (RAdpp65). However, specific CTL expanded after in vitro culture steps were subjected to several in vitro restimulations and, depending on the protocol adopted, this could lead to a selection bias, compromising the clinical benefit. To determine which part of the memory repertoire is selected after in vitro restimulation, we have followed the specificity and clonal composition of pp65-peptide-specific CD8(+) T cells in HLA-A*201 individuals before and after repeated in vitro restimulation of cells with RAdpp65, combining HLA tetrameric complexes and immunoscope analysis. Tetramer staining showed that, after in vitro restimulation, up to 60% of CD8(+) T cells were virus-specific. Immunoscope analysis showed that the predominant TCRBV diversity of pp65-specific clones was conserved, demonstrating that the memory repertoire was preserved all along the procedure. Altogether, these results suggest that the use of RAdpp65 to induce CMV- and adenovirus-specific CTL maybe appropriate for immunotherapy.

The repertoires of circulating human CD8(+) central and effector memory T cell subsets are largely distinct.

Memory T cells are divided into central and effector subsets with distinct functions and homing capabilities. We analyzed the composition and dynamics of the CD8(+) T cell repertoire of these subsets within the peripheral blood of four healthy individuals. Both subsets had largely distinct and autonomous TCRbeta repertoires. Their composition remained stable over a 9 month period, during which no cell passage between these subsets was detected despite important size variation of several clones. In one donor, four out of six TCRbeta clonotypes specific for the influenza A virus were detected in the central subset only, while the two others were shared. Altogether, these observations suggest that most effector memory T cells may not have derived from the central memory subset.

Egr1 promotes growth and survival of prostate cancer cells. Identification of novel Egr1 target genes.

In the majority of aggressive tumorigenic prostate cancer cells, the transcription factor Egr1 is overexpressed. We provide new insights of Egr1 involvement in proliferation and survival of TRAMP C2 prostate cancer cells by the identification of several new target genes controlling growth, cell cycle progression, and apoptosis such as cyclin D2, P19ink4d, and Fas. Egr1 regulation of these genes, identified by Affymetrix microarray, was confirmed by real-time PCR, immunoblot, and chromatin immunoprecipitation assays. Furthermore we also showed that Egr1 is responsible for cyclin D2 overexpression in tumorigenic DU145 human prostate cells. The regulation of these genes by Egr1 was demonstrated using Egr1 antisense oligonucleotides that further implicated Egr1 in resistance to apoptotic signals. One mechanism was illustrated by the ability of Egr1 to inhibit CD95 (Fas/Apo) expression, leading to insensitivity to FasL. The results provide a mechanistic basis for the oncogenic role of Egr1 in TRAMP C2 prostate cancer cells.

Antisense to the early growth response-1 gene (Egr-1) inhibits prostate tumor development in TRAMP mice.

Egr-1 is a transcription factor induced by stress or injury, mitogens, and differentiation factors. Egr-1 regulates the expression of genes involved in growth control or survival. Expression of Egr-1 results in either promotion or regression of cell proliferation, depending on cell type and environment. Egr-1 acts as a tumor suppressor in many cell types and loss of Egr-1 has been proposed to contribute to cancer progression. There is strong new evidence however suggesting that Egr-1 overexpression is involved in prostate cancer progression. For example, Egr-1 expression levels are elevated in human prostate carcinomas in proportion to grade and stage. Furthermore, prostate cancer progression was significantly delayed in two models of prostate cancer mice lacking Egr-1. Our objective in the present study is to test whether inhibition of Egr-1 function would block cell proliferation and inhibit the transformed phenotype of prostate cancer cells in vitro and in vivo. We describe the development of high affinity and high specificity antisense oligonucleotides that efficiently inhibit Egr-1 expression. We show that inhibition of Egr-1 expression in mouse or human prostate cancer cells decreased proliferation and reduced the capacity of these cells to form colonies and to grow in soft agar. Conversely, stable expression of Egr-1 in normal human prostate epithelial 267B1 cells promoted transformation. In TRAMP mice, treatment with Egr-1 antisense oligonucleotides delayed the occurrence of prostate tumors. Importantly, Egr-1 antisense showed little or no toxicity when injected into animals. Finally, we identified a few genes such as cyclin D2, p19ink4d, and Fas that are directly regulated by Egr-1 in prostate cancer cells and that control cell cycle and survival.

Combination of MHC-peptide multimer-based T cell sorting with the Immunoscope permits sensitive ex vivo quantitation and follow-up of human CD8+ T cell immune responses.

Identification of MHC-restricted antigens and progress in the induction and control of adaptive cytotoxic immune responses have led to renewed interest in immunotherapy as a treatment for severe pathologies such as cancer and autoimmune diseases. Reliable procedures for detecting and monitoring T cell responses induced by the treatment throughout a clinical trial are needed in order to design rational protocols with increased efficiency. We have attempted to develop such a procedure by combining T cell sorting using HLA-peptide complexes multimerized on magnetic beads together with the quantitative Immunoscope approach. Once a recruited patient has been typed for HLA and target antigens, relevant HLA--peptide multimers can be selected and used for sorting specific peripheral T cells prior to any treatment and at the peak of the expected response to treatment. Clonotypic primers specific for the TCR rearrangements of the specific T cell clones can then be designed and used for measuring the frequency of their TCR transcripts by quantitative PCR on blood samples or T cell subsets throughout the trial. In reconstruction experiments as well as in samples from one rheumatoid arthritis patient, we were readily able to detect and follow several T cell clones with a frequency as low as 10(-5) among CD8+ T cells. The main advantages of this procedure over other currently available assays are that it does not require any assumptions on the functional status of the specific T cells and it permits the monitoring of individual T cell clones whose phenotypic shift can thus be evaluated.