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.

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.

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.

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.

Differential gene expression induced by anti-cancer agent plumbagin is mediated by androgen receptor in prostate cancer cells.

Treatment of mice harboring PTEN-P2 tumors in the prostate or on prostate tissue in vivo with 5-hydroxy-2-methyl-1,4-naphthoquinone, also known as plumbagin, results in tumor regression in castrated mice, but not in intact mice. This suggested that dihydrotestosterone (DHT) production in the testes may prevent cell death due to plumbagin treatment, but the underlying mechanism is not understood. We performed RNA-seq analysis on cells treated with combinations of plumbagin and DHT, and analyzed differential gene expression, to gain insight into the interactions between androgen and plumbgin. DHT and plumbagin synergize to alter the expression of many genes that are not differentially regulated by either single agent when used alone. These experiments revealed that, for many genes, increases in mRNAs caused by DHT are sharply down-regulated by plumbagin, and that many transcripts change in response to plumbagin in a DHT-dependent manner. This suggests that androgen receptor mediates some of the effects of plumbagin on gene expression.

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.

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.

Early Metabolic Flare in Squamous Cell Carcinoma after Chemotherapy is a Marker of Treatment Sensitivity In Vitro.

PURPOSE: Early metabolic response with a decrease in glucose demand after cytotoxic treatment has been reported to precede tumor volume shrinkage. However, preclinical studies report of a very early rise in metabolism, a flare, following treatment. To elucidate these observations, we performed an experimental study on early metabolic response with sequential analysis of metabolic changes. METHODS: Three squamous cell carcinoma cell lines and one nontumorigenic cell line were exposed to cisplatin. The uptake of the fluorescent glucose analogue 2-NBDG was examined at days 1-6 using fluorescence microscopy. The relation between 2-NBDG-uptake and cell survival was evaluated. RESULTS: The tumor cells exhibited a high uptake of 2-NBDG, whereas the uptake in the nonmalignant cells was low. The more cisplatin sensitive cell lines exhibited a more pronounced metabolic flare than the less sensitive cell line. CONCLUSION: A metabolic flare was a very early sign of treatment response and potentially it could be used as an early marker of treatment sensitivity.

Isolation of a caveolae-enriched fraction from rat lung by affinity partitioning and sucrose gradient centrifugation.

Caveolae were isolated from rat lungs by a combination of affinity partitioning and sucrose gradient centrifugation. After homogenization of the lungs directly in a polyethylene glycol-dextran two-phase system and conventional phase partitioning, the polyethylene glycol-rich top phase was affinity partitioned with fresh bottom phase containing dextran-linked wheat-germ agglutinin. The lectin selectively attracted plasma membranes to the bottom phase. The isolated plasma membrane fraction was treated with Triton X-100 or, alternatively, sonicated before centrifugation in a stepwise sucrose gradient. Caveolin-enriched material collected at the 5/24% sucrose boundary. This material also contained 5'-nucleotidase activity and actin. Electron microscopy showed the material to consist of a homogeneous population of 50- to 100-nm vesicles. This purification protocol should allow the facile purification of caveolae also from other tissues, facilitating structural and functional studies.

Purification of caveolae by affinity two-phase partitioning using biotinylated antibodies and NeutrAvidin-dextran.

A new concept for affinity two-phase partitioning was tested. The partitioning was based on the interaction of target membranes with a primary antibody which, in turn, interacted with a biotinylated secondary antibody and NeutrAvidin-dextran in a poly(ethylene glycol)/dextran two-phase system. Caveolae selectively redistributed from the top phase to the NeutrAvidin-dextran-containing bottom phase by employing anti-caveolin as the primary antibody. This immunoaffinity approach was more selective than the established sucrose gradient centrifugation method and resulted in highly purified caveolae from Triton X-100-treated liver and lung plasma membranes. The same approach, employing other selective primary antibodies, should facilitate the purification also of other membrane fractions.