Monoamine oxidase inhibitors and methods for treatment and diagnosis of prostate cancer

Abstract

A mechanism of monoamine oxidases (MAOs) driven epithelium-to-mesenchymal transition (EMT) is disclosed. Also disclosed are methods for treating cancer by inhibiting or suppressing MAOs in cancer cells. Novel MAOs inhibitors, such as small molecules, siRNA, shRNA, antisense oligonucleotides, aptamers, decoys, and pharmaceutical compositions useful for treating cancer by disrupting the workings of MAOs are provided. In particular, a class of conjugates formed by covalently conjugating near infrared dye 783, IR-780, and MHI-148 to a MAO inhibitor, such as clorgyline, with and without encapsulation it in a nanoparticle is provided. Other aspects of the invention include methods for forming the nano-conjugates, method for monitoring treatment progress in a cancer patient by monitoring the changes in MAO activity, methods for screening patients who are at risk of cancer or differentiating different forms of cancer by assaying the level and location of MAO activity.

Claims

1. A compound of the formula: ##STR00023## or a pharmaceutically acceptable salt thereof.

2. A pharmaceutical composition useful for treating prostate cancer, comprising the compound of claim 1, and a pharmaceutically acceptable excipient.

3. A method for treating prostate cancer, comprising: administering to a subject having prostate cancer a therapeutically effective amount of the pharmaceutical composition of claim 2.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) “The file of this patent contains at least one drawing executed in color. Copies of this patent with color drawings will be provided by the Office upon request and payment of the necessary fee”.

(2) FIG. 1A-B shows the tumor xenografts growth rates were much reduced in mice injected with MCP3 cells (a mouse cell lines with PTEN and p53 double KO, see filled circles) with MAO-A knock down compared with WT MCP3 cells (open circles). There was no tumor growth when MAO-A knock down MCP3 prostate cancer cells (1×10.sup.6 cells) were injected in mice, whereas significant number of tumors were found in WT MCP3 cell injected mice. FIG. 1C shows that MAO-A expression is correlated with cell proliferation profiles in human and murine cancer cells. 2×10.sup.4 human or murine prostate cancer cells of manipulated MAO-A expression were seeded, and cell numbers were counted consecutively over a 6-day period. Experiments were performed in triplicate. shMAO-A, MAO-A knockdown by shRNA lentiviral infection.

(3) FIG. 2 shows the synthetic scheme for clorgyline-NIR dye conjugate and preparation of nano-clorgyline.

(4) FIG. 3 shows examples of novel MAO-A inhibitors according to the present invention.

(5) FIGS. 4A-4D show that MAO-A knockout in host impeded the growth of murine F9 teratocarcinoma xenograft. 1×10.sup.5 murine F9 teratocarcinoma cells were subcutaneously injected into WT (N=9) and MAO-A KO (N=9) mice. *, p<0.05; **, p<0.01.

(6) FIGS. 5A and 5B show that MAO-A knockout in host inhibited the growth of murine MCP3 prostatic carcinoma xenograft. 1×10.sup.5 murine MCP3 prostatic carcinoma cells were subcutaneously injected into WT (N=4) and MAO-A neo KO (N=5) mice. *, p<0.05; **, p<0.01.

(7) FIGS. 6A-6D show that MAO-A knockdown in murine MCP3 prostatic carcinoma cells inhibited the growth of tumor xenograft in vivo. 1×10.sup.6 WT and MAO-A-KD murine MCP3 prostatic carcinoma cells were subcutaneously injected into 6 (WT cells) and 4 (MAO-A-KD cells) C57BL/6 mice, respectively. *, p<0.05.

(8) FIG. 7A, 7B, 7C, and 7D show that immunohistochemical staining of MAO-A and MAO-B in a tissue microarray consists of prostate cancer tissues from 88 patients (2 cores from each patient). 9A shows MAO-A, a basal cell protein, expresses in cancer cells; minimal stromal reaction in the benign and cancerous areas of the specimens. 9B, in contrast, shows MAO-B, a mesenchymal cell protein, was only minimally expressed in normal and cancerous prostate epithelial cells, but with increased expression in prostate cancer-associated stromal cells. Since prostate stromal cells are known to induce prostate cancer epithelial growth and progression, and clonal evolution of prostate epithelium, MAO-B could also be considered as an effective stromal target for therapeutic intervention. 9C shows intense MAO-A positive stained prostate cancer cells in human bone, suggesting MAO-A may be an excellent target for prostate cancer bone metastasis. 9D shows that normal prostate epithelial cells also expressed MAO-A but not MAO-B (data not shown).

(9) FIG. 8 shows a brief synthetic scheme of IR-783, a NIR dye, -docetaxel conjugate.

(10) FIG. 9A-9E shows that IR-783-docetaxel was found to be uptaken into human prostate cancer cells (C4-2, PC-3), pancreatic cancer cells (MIA-PaCa2) and renal cancer cells (SN12C, see panel a); this NIR-docetaxel conjugate was found not uptaken into human normal prostate epithelial cells (P69) and a fetal human kidney 293 cells (see panel b). Cytotoxicity assays shows that this NIR-docetaxel conjugate exerted growth inhibitory effects on a panel of human cancer cell lines in vitro in a concentration dependent manner (see panel c). Using human renal cancer (SN12C) and normal fetal kidney cells (HFK293) as models, we observed that this dye-drug conjugate has equal effectiveness like the parental drug, docetaxel, in killing SN12C but not HFK293 cells, a result consistent with the suggestion that the dye-drug conjugate entered cancer but not normal cells.

(11) FIG. 10 shows an exemplary synthetic route leading to MHI-clorgyline.

(12) FIG. 11 shows exemplary confocal images of C4-2B prostate cancer cells treated with Mitotracker Green (top left), compound 10 (top right), DAPI (bottom left) and overlay (bottom center. The brightfield image is in the top center.

(13) FIG. 12 shows an exemplary MAO-A inhibition curve for MHI-clorgyline 10. Compound 10 was pre-incubated with 1×10.sup.6 prostate C4-2B cells at 37° C. for 20 min. Then, MAO A substrate C-14 serotonin was added to the incubation solution for 37° C. 20 min. At the end of the incubation, the reaction product was extracted and the radioactivity was counted. The MAO-A activity was expressed as 69.6 nM product formed/20 min/mg protein. The activity without the presence of inhibitor, compound 10, was taken as 100%.

(14) FIG. 13 shows representative immunohistochemical staining of normal (A), Gleason pattern 3 (B) and 5 (C), and bone-metastatic (D) human prostate adenocarcinoma clinical samples showed increased MAO-A expression in high grade and bone metastatic PCa. Magnifications are ×400 (A-C) and ×200 (D), respectively.

(15) FIG. 14 MAO-A determines the growth of human PCa tumor xenografts in viva Left panel (A-B), stable overexpression of MAO-A in human PC-3 cells, which exhibit limited MAO-A expression at baseline, enhanced the growth of tumor xenografts (A) and tumor weight (B) in athymic nude mice (N=8). Right panel (C-D), shMAO-A knockdown (KD) of MAO-A in human ARCaPM cells eliminated the growth of tumor xenografts (C-D) in athymic nude mice (N=5). shCon and shMAO-A, WT and MAO-A-KD cells. *, p<0.05, **, p<0.01.

(16) FIG. 15 shows effect of host MAO-A on prostate cancer growth. 1×10.sup.5 of murine prostate carcinoma TRAMPC-2 cells were subcutaneously injected into WT (N=6) and MAOA KO (N=4) mice, 3 injection sites per mouse. Murine prostate carcinoma TRAMPC-2 (neuroendocrine phenotype), subcutaneously injected into the MAO-A neo mice, showed significantly reduced growth rate of PCa, thereby suggesting a key role that host MAO-A plays in determining the rate of prostate cancer growth. Tumor incidence rate (A) and tumor volume (B) were determined along with tumor progression, and tumor weight (C).

(17) FIG. 16 Representative X-ray (A) and Micro-CT (B) of bone destruction (13-19 week) in mice intratibially injected with scramble/MAO-A-KD human ARCaPM or C4-2 Pea cells. White arrows point to osteolytic lesions.

(18) FIG. 17 MAO-A induces EMT in human PCa cells. Left panel (A-B), overexpression of MAO-A in PC-3 cells repressed E-cadherin and up-regulated Vimentin, N-cadherin and Twist1 (A), and increased cell migration and invasion (B). Right panel (C-E), shRNA knockdown of MAOA in ARCaPM cells increased E-cadherin and down-regulated N-cadherin and Twist1 (C), reduced cell migration and invasion (D), and changed cell morphology (E). **, p<0.01. Magnifications are 200×.

(19) FIG. 18 MAO-A enhances HIF1α expression in human PCa cells. (A) Overexpression of MAO-A increased HIF1α levels under hypoxia (0.5% O2), and (B) activated HIF1α-regulated VEGFA, glucose transporter 1 (Glut1), Snail2 and Twist1 mRNA expression in response to 24-h hypoxia in PC-3 cells. Relative mRNA expression was all normalized with control PC-3 cells under normoxia. **, p<0.01.

(20) FIG. 19 MAO-A enhanced NIR dye uptake in PC-3 tumor xenografts. (A) Representative in vivo MHI-148 NIR imaging (i.p. injection, 10 nmol/20 g) of nude mice subcutaneously implanted with control (left flank) and MAO-A-overexpressing (right flank) PC-3 cells. Arrows point to tumor xenografts. (B) Tumor tissues but not organs displayed strong signals by ex vivo NIR imaging. (C) Quantitation of tumor NIR intensity in (A) by determining total emission divided by tumor weight (5 mice). *, p<0.05.

(21) FIG. 20 (A) shows immunohistochemistry of E-cad, Vim and MAO-A in human patient samples of normal, G3 and G5. (B) shows Western blot of MAO-A, E-cad, Vim, N-cad and Twist1 in MAO-A overexpressing PC-3 cells. (C) shows Luc assay of E-cad promoter in control and MAO-A overexpressing PC-3 cells. (D) shows Western blot of MAO-A and E-cad in MAO-KD LNCaP cells, real-time PCR of Vim and N-cad in MAO-A-KD LNCaP cells. (E) shows migration assays of MAO-A-manipulated PC-3 and LNCaP cells. (F) shows invasion assays of MAO-A-manipulated PC-3 and LNCaP cells.

(22) FIG. 21 (A) shows Western blot of nuclear HIF1α in MAO-A overexpressing PC-3 cells. (B) shows Western blot of HIF1α in MAO-A overexpressing PC-3 cells in a time-dependent manner. (C) shows real-time RT-PCR of Snail2, Twist1, VEGFA, Glut1 and HIF1α in MAO-A overexpressing PC-3 cells. (D) Western blot of HIF1α, in MAO-A-KD LNCaP cells in a time-dependent manner. (E) shows real-time RE-PCR of Snail2, Twist1, VEGFA, Glut1 and HIF1α in MAO-A-KD LNCaP cells.

(23) FIG. 22 (A) Western blot of HIF1α-OH and HIF1α, in MAO-A overexpressing PC-3 cells with the treatment of MG-132. (B) real-time PCR of VEGFA and Glut1 in MAO-A overexpressing PC-3 cells of DMOG treatment. (C) FACS of ROS measurement in MAO-A expressing PC-3 cells under hypoxia. (D) Western blot of HIF1α in MAO-A overexpressing PC-3 cells of NAC treatment under hypoxia. (E) real-time RT-PCR of Twist1, VEGFA and Glut1 in MAO-A overexpressing PC-3 cells of NAC treatment under hypoxia. (F) shows Western blot of HIF1α in MAO-A overexpressing PC-3 cells of both NAC and DMOG treatment. (G) shows exemplary cells proliferation curves of MO-A overexpressing PC-3 cells under the treatment of NAC.

(24) FIG. 23 (A) shows real-time RT-PCR and ELISA of VEGF in MAO-A manipulated PC-3 and LNCaP cells. (B) shows Western blot of pAkt and pFoxO1 in PC-3 cells with VEGF treatment. (C) shows Western blot of pAkt and pFoxO1 in MAO-A manipulated PC-3 and LNCaP cells. (D) Western blot of NRP-1, pAkt and pFoxO1 in MAO-A overexpressing/NRP-1-KD PC-3 cells. (E) shows immunohistochemistry of H&E, VEGF and NRP-1 in MAO-A overexpressing PC-3 tumor xenografts. (F) shows migration and invasion assays of MAO-A overexpressing and NRP-1-KD PC-3 cells.

(25) (G) shows exemplary cell proliferation curves of MAO-A overexpressing PC-3 and NRP-1-KD PC-3 cells. (H) shows Western blot of nuclear FoxO1 in MAO-A overexpressing PC-3 cells, PC-3 cells with VEGF treatment, and NRP-1-KD PC-3 cells.

(26) FIG. 24 (A) real-time RT-PCR and luc assay of Twist1 mRNA or promoter in MAO-A manipulated PC-3 and LNCaP cells. (B) Western blot and real-time RT-PCR of Twist1. in FoxO1manipulated PC-3 cells. (C) shows luc assay of Twist1 promoter with WTIH215R AAA FoxO1 construct in PC-3 cells. (D) shows characterization of a FoxO1-binding site in Twist1 promoter across different species. (E) shows luc assay of WT/Mut Twist1 promoter with FoxO1 construct in PC-3 cells. C/GA/TAAAC/AA is SEQ ID NO:6. actgctgcccCCAAACTttccgcctgc is SEQ ID: NO:7. aaaatatagaCCAAACTctaaggttct is SEQ ID: NO:8. accgctgcceCCAAACTttccgcccgc is SEQ ID: NO:9. actgctgcccCCAAACTttccgcccgc is SEQ ID: NO:10. actgctgcccCCAAACTttccgcttgc is SEQ ID: NO:11. (F) shows luc assay of WT/Mut Twist1 promoter in MAO-A overexpressing PC-3 cells. (G) ChIP assay of the FoxO1-binding site in MAO-A overexpressing PC-3 cells. (H) shows a comparison of knock-down versus MOA-A inhibition.

(27) FIG. 25A-E (A) shows tumor incidence, tumor volume and tumor weight of MAO-A-KD LNCaP, C4-2, ARCaP.sub.m and MCP3 tumor xenograft. (B) shows MAO-A activity of MAO-A-KD LNCaP and C4-2 tumor xenografts. (C)-(E) shows immunohistochemistry of H&E, MAO-A, E-cad, Vim, HIF1α, and VEGF in MAO-A-KD LNCaP and C4-2 tumor xenografts. (D) shows tumor mitochondrial ROS measurement in MAO-A-KD LNCaP and C4-2 tumor xenografts.

(28) FIG. 26 (A) shows immunohistochemistry of MAO-A, HIF1α, VEGFA, FoxO1, pFoxO1 and Twist1 in human patient samples of G3 and G5. (B) shows statistical analysis of immunohistochemistry data.

(29) FIG. 27 shows a schematic representation of the MAO-A driven EMT mechanism.

DETAILED DESCRIPTION

(30) Definition

(31) Unless otherwise indicated herein, all terms used herein have the meanings that the Willis would have to those skilled in the art of the present invention. Practitioners are particularly directed to current textbooks for definitions and terms of the art. It is to be understood, however, that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary.

(32) “Treatment” refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented.

(33) A “therapeutically effective amount” of a monoamine inhibitor is an amount sufficient to carry out a specifically stated purpose. An “effective amount” may be determined empirically and in a routine manner in relation to the stated purpose.

(34) A “Carrier” or “Carriers” as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. The physiologically acceptable carrier may be a sterile aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants.

(35) Recent studies indicate that increased MAO-A is associated with prostate cancer progression [5]; conversely, pharmacological MAO-A inhibition or lentiviral shRNA-mediated silencing of MAO-A significantly reduced the growth of prostate cancer cells in vitro and tumor xenograft in vivo [6-8]. Furthermore, our data showed that MAO-A induced epithelial-to-mesenchymal transition (EMT) in human prostate cancer cells, by promoting the loss of E-cadherin (an epithelial marker) expression, up-regulation of vimentin (a mesenchymal marker) levels as well as increased invasion and migration of prostate cancer cells. These results suggest that MAO-A expression may be correlated with the metastatic potential of prostate cancer cells. Taken together, this evidence strongly supports the role of MAO-A as a potential novel target for the treatment of human prostate cancer.

(36) We have found that monoamine oxidase A (MAO-A) knock-down (KD) prostate cancer cells did not grow when injected into mice. This result was dramatically different from the WT prostate cancer cells (See FIG. 1). A total of 10 wild-type (WT) mice in C57BL/6 background were used. Six mice were injected with WT MCP3 (PTEN/p53 double knockout) prostate carcinoma cells of C57 mouse strain origin, 4 sites per mouse with a total of 24 sites. Four mice were injected with MAO-A knock-down (KD) MCP3 cells, 4 sites per mouse with a total 16 sites. The number of tumors was counted on the days as indicated. The tumor incidence rate is defined as the total number of detectable tumors divided by the total number of the sites injected. As shown in FIG. 1, the tumor growth rate in mouse tumor xenografts injected with MAO-A knock down MCP3 cells (filled circles) compared with WT MCP3 cells (open circles). There was no tumor growth when MAO-A knock down MCP3 (PTEN and p53 double KO) prostate cancer cells (1×10.sup.6 cells) were injected in mice, whereas significant number of tumors were found in WT MCP3 cell injected mice.

(37) One aspect of the present invention is a method in which a MAO-A inhibitor, clorgyline, can be delivered to cancer cells and tissues by chemically conjugating clorgyline to a NIR dye. The NIR-dye-clorgyline conjugate is expected to be uptaken by cancer but not normal cells thus avoiding systemic toxicity of this MAOI. Laser-scanning confocal microscopy can be used to determine cellular uptake and localization of the nano-clorgyline within cells (LNCaP, C4-2, and ARCaP.sub.M prostate cancer cell lines). This class of NIR dyes can be readily uptake into cancer cells via organic anion transporting peptides. FIGS. 10 and 11 show that NIR dye (IR-783) conjugate of docetaxel, IR-783-docetaxel, was found to be uptaken into human prostate cancer cells, pancreatic cancer cells and renal cancer cells but not human prostate epithelial cells or fetal human kidney cells, suggesting such NIR dye-chemotherapeutic agent conjugates enter cancer cells but not normal cells.

(38) To determine the ability of nano-clorgyline to inhibit activity of MAO-A. MAO-A inhibition curve is obtained for prostate cancer cells LNCaP, C4-2, and ARCaP.sub.M cell lines and compared with that clorgyline itself. These cell lines have moderate to high MAO-A activity. IC.sub.50 of nano-clorgyline is determined and compared to that of clorgyline.

(39) To study the effect of nano-clorgyline and NIR dyes (IR-783, IR-780 and MHI-148)-clorgyline on MAO-A and MAO-B inhibition. MAO-A and MAO-B inhibition curves is performed in mice. ICs.sub.50 are determined.

(40) To study the localization of nano-clorgyline, and NIR dyes-clorgyline conjugates and its effect on tumor growth, prostate cells are injected into mice. In one exemplary experiment, mice are divided into 3 groups separately injected with (a) nano-clorgyline of NIR dyes-clorgyline, (b) clorgyline itself, and (3) dye only, respectively. The location of the nano-clorgyline was imaged, the tumor's growth (size, number, and weight) was monitored, and the results from the 3 groups were compared.

(41) The result of the experiment demonstrated the effect of nano-clorgyline on tumor growth. Having established the effectiveness of the clorgyline nano-conjugate, it will be appreciated by those skilled in the art that other parameters such as the concentration required for inhibition of MAO-A activity and tumor growth may be readily determined via routine experimentation.

(42) The MAO-A inhibitors, including the NIR dyes-MAOI conjugates thereof, may be used alone or in combination with the existing treatments on tumor growth and metastasis such as (a) surgical castration; (b) radiation (c) docetaxel; (d) abiraterone.

(43) Furthermore, this clorgyline-dye conjugate may be used in connection with methods for treatment of prostate cancer and methods for diagnosis and monitoring of the progression of the prostate cancers by administering to a patient a composition comprising an effective amount of the clorgyline-dye conjugate.

(44) Design, Synthesis, Encapsulation and Testing of Clorgyline-dye Conjugate Nanoparticles (Nano-clorgyline) in Cells.

(45) 1. IR-783 Nanoparticle Dye Conjugate

(46) Preparation of clorgyline-dye conjugate. We have identified a class of near-infrared (NIR) fluorescent heptamethine cyanine dyes, IR-783 (1) (see FIG. 2), as a candidate for clorgyline conjugation. The near-infrared dye, IR-783, is commercially available and can be readily converted to precursor 3 in a single-step reaction with p-thioaniline 2. Based on our modeling studies with crystal structure of clorgyline-MAO-A complex [9], we determined that the amine nitrogen can be modified by the linker needed to conjugate this compound to precursor 3, but does not affects its inhibitory potency. The synthetic sequence for the preparation of the derivative clorgyline acid 9 is outlined in FIG. 2. The synthesis consists of a series of reductive amination reactions that yield from the commercially available propargylamine 4 and 3-(2,4-dichlorophenoxy)propanal 5. Conjugation of the two building blocks 3 and 9 can be accomplished by well-established sequence of synthetic steps. Similarly to IR-783, this conjugate is expected to have strong emission at 820-860 nm upon excitation at 750-780 mm. It can be easily read by NIR imaging.

(47) Encapsulation of the clorgyline-dye conjugates in nanoparticles. In order to achieve water solubility of the clorgyline-dye conjugates and enhance their delivery to tumors, we will encapsulate them in calcium phosphate/silicate nanoparticles of average size of 22 nm that are freely dispersible in water [10]. This encapsulation process results only in minor changes to the photophysical properties of the dyes. Such an encapsulation method has been reported by Adair as effective means of delivery of hydrophobic dyes to cells [11]. Calcium phosphate is an excellent matrix for nanoparticle encapsulation because moderate concentrations of Ca.sup.2+ ions are not toxic to cells and in vivo (found in human bone and teeth). It was shown that calcium phosphate dissolves below pH 5.5, liberating the cargo, but it is stable at pH 7.4 [12]. In addition, particles of this matrix disperse freely in aqueous media.

(48) Typically, such nanoparticles are prepared using aqueous co-precipitation of calcium chloride and disodium hydrogen phosphate in the presence of disodium silicate within water-in-oil microemulsions [11, 13]. The encapsulation of the clorgyline-dye conjugate in nanoparticles will be accomplished through its addition into the microemulsion during precipitation. This process will yield nano-clorgyline as a colloidal suspension of nanoparticles of 22-30 nm in size. Its characterization will be done through analysis of the size distribution, morphology, and colloidal state of dispersion of the nanoparticle suspensions using transmission electron microscopy (TEM).

(49) To determine the inhibition of MAO-A activity by nano-clorgyline. We will study the ability of nano-clorgyline to inhibit MAO-A activity by performing the inhibitory curves in human prostate cancel cell lines, LNCaP (non-metastatic cell line), C4-2 and ARCaPm, two metastatic cell lines. IC.sub.50 will be determined. All these cell lines have moderate to high MAO-A activity. Shih's lab routinely performs MAO-A inhibition assay in vitro and in vivo.

(50) To determine if nano-clorgyline is targeted to mitochondria of cancer cells. We will use laser-scanning confocal microscopy to determine cellular uptake and localization of the nano-clorgyline within cells. The near-infrared dye moiety within the conjugate will serve as a directing group, guiding the conjugate to mitochondria. It has been shown that redox potential of mitochondria is different between cancer and normal cells.

(51) Human cancer cell lines (LNCaP, non-metastastic cell line, C4-2, ARCaP.sub.M, two metastastic cell lines), will be tested. As negative controls, normal human prostate epithelial cells (P69 and NPE), normal human prostate fibroblasts (NPF), will be used.

(52) It is anticipated the following studies will show that the growth of prostate cancer will be significantly reduced after the treatment with nano-clorgyline. The outcome will be determined by reduced tumor growth in rate, size and weight. We expect nano-clorgyline will be more effective with fewer side effects than clorgyline itself. In addition, we will establish MAO-A as a biomarker for prostate cancer, with nano-clorgyline to be used for diagnostic tool; further, we will follow the progression of prostate cancer during treatment through imaging of the uptake of the nano-clorgyline.

(53) 2. MHI Dye Nanoparticle Conjugate

(54) Synthesis of MHI-corgyline: MHI-clorgyline has been prepared through a series of synthetic steps by conjugating dye MHI-148 and clorgyline as outlined in FIG. 10. A linker of proper length was designed to minimize negative impact of the dye on the inhibitory potency of clorgyline.

(55) Preparation of MHI-clorgyline started with commercially available 2,4-dichlorophenol 1. This compound was alkylated under standard conditions (NaOH, H.sub.2O) with 1,3-dibromopropane. The product 2 was then reacted with NaN.sub.3 in DMF to yield the azide 3, which was subjected to the next step as a solution in MTBE without further purification. The solution of 3 was hydrogenated under low H.sub.2 pressure using Pd on activated charcoal as a catalyst in the presence of Boc.sub.2O and resulting in the formation of the carbamate 4. This compound was alkylated with propargyl bromide using NaH in dry DMF, producing Boc-protected alkyne 5. The Boc protecting group was removed under acidic conditions using TFA in DCM. The product 6 was alkylated again with 1-bromo-3-thioacetylpropane 7, resulting in the formation of 8, albeit in low yield. Removal of the acetyl protective group was carried out in methanolic HCl and afforded intermediate 9. This intermediate was then coupled with MHI-148 dye using EDC and 4-DMAP to afford the product MHI-clorgyline 10. This product was purified using reverse-phase HPLC and its identity was confirmed by mass spectrometry.

(56) Imagining of MHI-clorgyline in live cells: Previous study showed that the near-IR hepatamethine cyanine dyes IR-783 and MHI-148 can be retained in cancer cells but not normal cells in tumor xenografts and in spontaneous tumors in transgenic mice. Moreover, the two dyes also have strong emission at 820-860 nm upon excitation at 750-780 nm, which can be easily detected and visualized by NIR imaging equipment and laser-scanning confocal microscopes. Here, we resorted to the use of Zeiss LSM 510 confocal microscope as the imaging equipment. Although cellular uptake of the conjugate was uncertain, due to the similarity in structure, MHI-clorgyline conjugates were expected to have similar fluorescence properties as the NIR dye itself. Therefore, NIR imaging with laser-scanning confocal microscope was used to examine the cellular uptake of MHI-clorgyline in human prostate epithelial cancer cells (C4-2B). This cell line was selected due to its high levels of the expressed MAO-A.

(57) In the preliminary study we found that IR-783 dye co-localized in mitochondria of the live cells. An imaging study with the newly synthesized MHI-clorgyline 10 was carried out (FIG. 11). This compound also showed rapid accumulation in C4-2B PCa cells and was localized in the mitochondria, as determined by the co-staining with mitochondria-specific dye.

(58) In order to test an inhibitory activity of the MHI-clorgyline 10, a standard MAO A inhibition assay was carried out in C4-2B cells using radiolabeled substrates. The results clearly indicate that our designed conjugate 10 inhibits MAO A activity with mean IC.sub.50 of 2.18×10.sup.−5 M (FIG. 12). The activity of the conjugate 10, while lesser than that of clorgyline itself (data not shown), is sufficient for the in vivo studies, which are currently ongoing.

(59) The Function of the Nano-clorgyline In Vivo—its Location and Inhibition of Tumor Growth in Mice

(60) To study the location of nano-clorgyline, and its effect on tumor progression in mice. We will inject human LNCAP non-metastatic prostate cancer cells (1×10.sup.6) into immunodeficient nude mice with (1) nano-clorgyline, concentration required to inhibit nearly 100% of MAO-A activity using results from Specific Aim 1b; (2) clorgyline itself (positive control, 10 mg/kg for 100% inhibition of MAO-A activity); (3) dye itself (negative controls, same concentration as nano-clorgyline). A total of 18 mice is required for this part of the study (6 per each group).

(61) Then, we will image the tumor location, monitor the tumor growth (size, number) every other day for one month. The tumor will be located by NIR imaging. The Olenyuk lab has an extensive experience in NIR imaging and the needed equipment is available at the Norris Cancer Center. If our hypothesis is correct, the nano-clorgyline will be located in prostate cancer, and the tumor growth will be reduced. At the end of 30 days, the mice will be sacrificed, the MAO-A activity will be determined in the tumor (if there is still tumor) and the normal prostate tissues. See FIG. 1 in prior work for details of this experiment). This study will demonstrate the potential useful of nano-clorgyline for diagnosis and therapy.

(62) To study the effect of nano-clorgyline on the metastasis of prostate cancer in mice. Human prostate cancer cells with metastatic potential will be injected to mice, human C4-2, ARCaPm, (1×10.sup.6 cells), same three group of mice will be used, the procedure and the experiments will be the same as described in Specific Aim 2a. The tumor growth rate and size and locations will be determined. The presence of absence of the tumor in the bone, will be examined and as an indication of metastasis. A total of 36 mice will be used (18 mice per each cell line with 3 groups of treatment; 2 metastatic cell lines).

(63) The effects of nano-clorgyline alone or in combination with the existing treatments on tumor growth and metastasis in mice.

(64) First line treatment for advanced prostate cancer is androgen ablation therapy (ADT). Unfortunately the duration of response to ADT is limited (about 18 months) and the patients eventually develop castration resistance. The first line treatment for patients with castration resistant PCA is usually chemotherapy with the microtubule inhibitor, docetaxel. Recently, FDA approved the specific CYP17 inhibitor, abiraterone, for the treatment of castration resistant patients who fail docetaxel therapy. This study will evaluate the effects of nano clorgyline alone or in combination with one of these treatment approaches on tumor growth. Since the nano clorgyline can be read non-invasively by INR imaging, the prognosis of each treatment can be easily determined.

(65) Human LNCAP cells (non-metastatic prostate cancer cell line). Alternatively, ARCaPm (with potential for metastasis) will be injected to immunodeficient nude. Next, mice will be divided to three groups as described in A. The tumor location, size, number of lesions will be determined every other day from day 1 to day 30. On day 31 mice will be sacrificed, tumor and host MAO Activity will be determined.

(66) TABLE-US-00005 A: (Group I) nano-chlorgyline (1 mg/kg*) with androgen ablation therapy (castration)** (Group II) nano-chlorgyline alone (1 mg/kg*) (Group III) castration alone** B: (Group I) docetaxel (daily, 15 mg/kg) (Group II) nano-chlorgyline alone 1 mg/kg*), (Group III) docetaxel (15 mg/kg), nano-chlorgyline (1 mg/kg*) C: (Group I) new drug (abiraterone, 180 mg/kg) daily (Group II) nano-chlorgyline alone (1 mg/kg*), (Group III) new drug (abiraterone, 180 mg/kg) and nano-clorgyline 1 mg/kg*) *The concentration of nano-clorgyline to be used will be adjusted based on the results obtained from specific aim 1b.

(67) For the castration group, trans-scrotal castration will be performed under isoflurane anesthesia with proper aseptic and antiseptic technique. A total of 108 mice will be used.

(68) Synthesis of Nanoparticle Conjugates with Other Novel MAO-A Inhibitors Obtained from High Throughput Screening.

(69) Optionally, other novel MAO-A inhibitors, disclosed herein may be conjugated for use in the methods and treatments of the present invention. Specifically, below are examples of four high affinity novel MAO-A inhibitors 11-14. They may be conjugated with the near-infrared dye, such as IR-783. The phenol functionality (—OH) presents a viable choice for linker attachment and the subsequent derivatization of these molecules with fluorescent precursor 3 in order to generate novel nanoparticle based MAO-A inhibitors.

(70) MAO-A Confers Prostate Cancer EMT by Stabilizing H1F1α and Enhancing VEGF-mediated Twist1 Activation

(71) High Gleason grade prostate carcinomas are aggressive, poorly differentiated tumors that exhibit elevated MAO-A expression. We have found that a key function of MAO-A is to promote an epithelial-to-mesenchymal transition (EMT). EMT is the process of cellular development characterized by loss of cell adhesion, repression of E-cadherin expression, and increased cell mobility. In the context of cancer, promotion of EMT correlates with the increased cell invasion, migration and metastatic potential, hence, the EMT-promoting effect of MAO-A connects MAO-A activity to cancer. More specifically, we have found that overexpression of MAO-A in human prostate cancer cells induces the loss of E-cadherin (an epithelial marker), up-regulates Vimentin/N-cadherin (mesenchymal markers) and increases cell migration and invasion Conversely, knockdown of MAO-A impedes EMT in human prostate cancer cells.

(72) Without being bound to any particular theory, we offer the following experimental observations (FIGS. 20-26). to explain in mechanistic terms the corrections between MAO-A activity and its various cancer promoting effects.

(73) First, we found that MAO-A enhances HIF1α stability by reducing prolyl hydroxylase (PHD) activities and increasing intracellular ROS levels. We then found that by treating prostate cancer cells with a ROS scavenger (N-acetylcysteine), MAO-A-induced HIF1α expression is diminished, which in turn, also decreased MAO-A-enhanced cell proliferation. Moreover, we also found that MAO-A mediated the activation of VEGF and its receptor Neuropilin-1 (NRP1) in response to hypoxia, which in turn stimulated the Akt/FoxO1 signaling pathway and reduced FoxO1 activity by promoting its phosphorylation followed by nuclear export. We further discovered that FoxO1 acts as a transcriptional repressor of Twist1 and binds to a response element in the proximal region of Twist1 promoter. Twist1 is known to be an oncogene in several cancers and is involved in tumor metastasis. FIG. 27 summarizes the mechanism.

(74) Importantly, this mechanism is manifested in high Gleason grade cancers, which exhibit significantly more HIF1α, VEGF and Twist1 expression, but less FoxO1 nuclear localization compared to low Gleason grade cancers. Therefore, expression levels of MAO-A, HIF1α, VEGF and Twist1 serve as a biomarker for objectively differentiating high Gleason grade cancers from low Gleason grade cancers.

EXAMPLES

(75) The following examples are provided in order to demonstrate and further illustrate certain embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof. While such examples are typical of those that might be used, other procedures known to those skilled in the art may alternatively be utilized. Indeed, those of ordinary skill in the art can readily envision and produce further embodiments, based on the teachings herein, without undue experimentation.

Example 1

MAO-A KO in Host Experiment 1: Murine F9 Teratocarcinoma Tumor Xenograft in WT and MAO-A KO Mice

(76) Cell # injected: 1×10.sup.5 Mice #: WT (N=9) and MAO-A KO (N=9) Tumor injection site #: WT (2×9=18) and MAO-A KO (2×9=18) Tumor incidence rate: WT (11/18=61.1%) and MAO-A KO (3/18=16.7%) Tumor growth: WT>MAO-A KO (p<0.05) Tumor weight: WT>MAO-A KO (p<0.05)

Example 2

MAO-A KO in Host Experiment 2: Murine MCP3 (pten/p53 Double KO) Prostatic Tumor Xenograft in WT and MAO-A KO Mice

(77) Cell # injected: 1×10.sup.6 Mice #: WT (N=3) and MAO-A KO (N=3) Tumor injection site #: WT (4×3=12) and MAO-A KO (4×3=12) Tumor incidence rate: WT (11/12=91.7%) and MAO-A KO (10/12=83.3%) Tumor growth: WT>MAO-A KO (p<0.05) Tumor weight: WT>MAO-A KO (p=0.25)

Example 3

MAO-A KO in Host Experiment 3: Murine MCP3 Prostatic Tumor Xenograft in WT and MAOA KO Mice

(78) Cell # injected: 1×10.sup.5 Mice #: WT (N=4) and MAO-A KO (N=5) Tumor injection site #: WT (3×4=12) and MAO-A KO (3×5=15) Tumor incidence rate: WT (10/12=83.3%) and MAO-A KO (0/15=0) Tumor growth: No MCP3 tumor growth in MAO-A KO mice

Example 4

MAO-A KD in Tumor Experiment 1: Murine WT and MAO-A-Kb MCP3 Prostatic Tumor Xenograft in C57BL/6 Mice

(79) Cell # injected: 1×10.sup.6 Mice #: Mice for WT MCP3 cells (N=6) and mice for MAO-A-KD MCP3 cells (N=4) Tumor injection site #: WT MCP3 cells (4×6-24) and MAO-A-KD MCP3 cells (4×4=16) Tumor incidence rate: WT MCP3 cells (21/24=87.5%) and MAO-A-KD MCP3 cells (0/16=0) Tumor growth: With MAO-A KD in tumor, there is no tumor growth.

Example 5

MAO-A KD in Tumor Experiment 2: Murine WT and MAO-A-KD MCP3 Prostatic Tumor Xenograft in C57BL/6 Mice

(80) Cell # injected: 1×10.sup.6 Mice #: Mice for WT MCP3 cells (N=6) and mice for MAO-A-KD MCP3 cells (N=6) Tumor injection site #: WT MCP3 cells (3×6=18) and MAO-A-KD MCP3 cells (3×6=18) Tumor incidence rate: As of July 16, WT MCP3 cells (15/18=83.33%) and MAO-A-KD MCP3 cells (0/18=0).

Example 6

Synthesis of MHI-clorgyline and the Role of MAO-A in Prostate Cancer Progression

(81) General Synthesis: All reagents and solvents were obtained from commercial sources and were used as received unless otherwise stated. All reactions involving moisture-sensitive reagents were conducted under argon atmosphere with anhydrous solvents and flame-dried glassware. Hygroscopic liquids were transferred via a syringe and were introduced into reaction vessels through rubber septa. Reaction product solutions were concentrated using a rotary evaporator at 30-150 mm Hg. Column chromatography was performed on silica gel (230-400 mesh) using reagent grade solvents. Analytical thin-layer chromatography (TLC) was performed on glass-backed, pre-coated plates (0.25 mm, silica gel 60, F-254, EM Science). Analytical HPLC were performed on Microsorb-MV C.sub.8 reverse-phase column (250×4.6 mm, Varian) using Shimadzu LC-10A VP pump and Shimadzu SPD 10A VP UV-vis variable-wavelength detector. Preparative HPLC purifications were carried out with C.sub.8 reverse phase preparative column (Grace Davison). The flow rate for preparative reverse-phase HPLC was 4 mL/min. In all cases, 5%-95% gradients of acetonitrile in 0.1% aqueous trifluoroacetic acid (TEA) were used as eluents. Water (18 MΩ) was obtained from a Barnstead water purification system, and all buffers were 0.2 μm filtered. Nuclear magnetic resonance (NMR) spectra were collected on Varian 400 MHz instruments in the indicated solvents. The peak positions are reported with chemical shifts (δ) in parts per million (ppm) downfield from the signal for tetramethylsilane (0 ppm) and referenced to the signal resulting from the incomplete deuteration of a solvent used in the experiment (CDCl.sub.3: 7.26 ppm, or the center line of the multiplet of DMSO-D.sub.6: 2.50 ppm). Carbon-13 chemical shifts are reported as δ values in ppm and referenced to the carbon-13 signal of a solvent used in the experiment (CDCl.sub.3: 77.0 ppm, or the center line of the multiplet DMSO-D.sub.6: 39.51 ppm). The coupling constants (J) are reported in Hertz (Hz). The following abbreviations are used: singlet (s), doublet (d), triplet (t), doublet of doublets (dd), multiplet (m). Mass spectra were obtained from the Agilent 6520 time-of-flight mass spectrometer.

(1) Synthesis of MHI-Clorgyline

Synthesis of 1-(3-bromopropoxy)-2,4-dichlorobenzen (2)

(82) ##STR00014##

(83) A mixture of 2,4-dichlorophenol 1 (4.1 g, 25 mmol), 1,3-dibromopropane (10 g, 50 mmol) and a solution of sodium hydroxide (1.0 g) in water (4 mL) was stirred at reflux for 1.5 h. A solution of sodium hydroxide (1.0 g) in water (6 mL) was added and the mixture was refluxed for an additional 1.5 h. After cooling, the reaction mixture was extracted with chloroform (50 mL) and washed with water (30 mL×3). The organic layer was dried over sodium sulfate and evaporated in vacuo. Crude product was obtained (10.49 g) and then purified by silica gel column (79.16 g). Yield 10.7% (0.794 g).

Synthesis of 1-(3-azidopropoxy)-2,4-dichlorobenzene (3)

(84) ##STR00015##

(85) To a solution of 1-(3-bromopropoxy)-2,4-dichlorobenzene (600 mg, 2.11 mmol) in 6.0 mL DMF in a 25 mL round-bottom flask equipped with a stir bar, a thermocouple in a thermowell and a rubber septum stopper with sleeve, 234.0 mg (3.60 mmol) of NaN.sub.3 was added at room temperature. Under N.sub.2 pressure, the mixture was stirred overnight. The formation of off-white suspension was observed. 20 μL, of the reaction mixture was partitioned with 0.5 mL MTBE and 0.5 mL water, and the MTBE layer was used for TLC (silica gel, 100% hexane). The rest of the reaction mixture was partitioned with MTBE and water. The water layer was washed by MTBE. The MTBE layer were washed sequentially with water and NaHCO.sub.3, and then used in the next step without further purification.

Synthesis of tert-butyl 3-(2,4-dichlorophenoxy)propylcarbamate (4)

(86) ##STR00016##

(87) The MTBE layer obtained from the previous step was transferred into a 500 mL round-bottom flask equipped with a stir bar and a rubber septum stopper with sleeve. In N.sub.2 atmosphere, Boc.sub.2 (571.0 mg, 2.616 mmol) and Pd/C (518 mg) was added. N.sub.2 was carefully replaced by H.sub.2. A rubber balloon was used to keep the system under positive gas pressure. TLC (silica gel, 100% hexane to detect starting material and MTBE: hexane=1:1 to detect the product, ninhydrin stain) was used to follow the process of the reaction. Under N.sub.2 pressure, the mixture was stirred for 21 h. The reaction mixture was filtered under vacuum through glass microfiber and Celite 545. The filtrate was partition by MTBE and water. The water layer was washed by MTBE. The MTBE layers were washed sequentially with water, saline and NaHCO.sub.3, dried by MgSO.sub.4, filtered and evaporated. Crude product (991.2 mg) was obtained after work up and purified by silica gel column. Yield 11.4% (77.3 mg).

Synthesis of tert-butyl 3-(2,4-dichlorophenoxy)propyl(prop-2-ynyl)carbamate (5)

(88) ##STR00017##

(89) To a solution of tert-butyl 3-(2,4-dichlorophenoxy)propylcarbamate (77.3 mg, 0.24 mmol) in 0.8 mL DMF in a 20 mL vial equipped with a stir bar and a rubber septum stopper with sleeve and under Ar pressure, NaH (11.9 mg, 0.30 mmol) was added with cooling by ice bath. The reaction mixture was kept under Ar atmosphere at all times. Propargyl bromide in toluene (44.5 mg, 0.30 mmol) was added. The reaction mixture was stirred at room temperature. Next, a small portion of the reaction mixture (10-15 μL) was partitioned with 350 μL MTBE and 350 μL water, and the MTBE layer was used for TLC (silica gel, hexane:MTBE=1:1). The rest of the reaction mixture was partitioned with 20 mL MTBE and 20 mL water. The water layer was washed by 20 mL MTBE. The MTBE layers were washed sequentially with water, saline and NaHCO.sub.3, dried by MgSO.sub.4, filtered and evaporated. Crude product was purified by silica gel column (1.23 g). Yield 27.4% (23.7 mg).

Synthesis of N-(3-(2,4-dichlorophenoxy)propyl)prop-2-yn-1-aminium 2,2,2-trifluoroacetate (6)

(90) ##STR00018##

(91) To a solution of tert-butyl 3-(2,4-dichlorophenoxy)propyl(prop-2-ynyl)carbamate (23.7 mg, 66.1 μmol) in 600 μL DMF in a 20 mL vial equipped with a stir bar, 600 μL TFA was added at room temperature while stirring. In 0.5 h, TLC (MTBE:hexane=1:1, ninhidrin stain) indicated the completion of the reaction. The volatiles were evaporated. The residue was co-evaporated with ACN for 3 times and then used in the next step without further purification.

(92) Structure of the product was proved by NMR and LC-MS.

Synthesis of S-3-bromopropyl ethanethioate (7)

(93) ##STR00019##

(94) A 250 mL three-neck round-bottom flask equipped with a thermocouple in a glass sleeve, a magnetic stirrer, a vigreux column with an Argon inlet (middle stem) and a sleeved rubber septum stopper was assembled and dried with a heat gun under flow of Ar. Approximately 110-120 mL of anhydrous DMF was added via cannula under Ar. AcSK (11.68 g, 102.3 mmol) was added by portions into the flask while cooled with ice-MeOH bath. The reaction went on for 7 h at about −10° C. The ice-MeOH bath was removed after quenching the reaction by adding 165 mL water. The reaction mixture was partitioned with 300 mL MTBE and 700 mL water. The water layer was washed by 200 mL MTBE. The MTBE layers were washed sequentially with water, saline and NaHCO.sub.3, dried by MgSO.sub.4, filtered and evaporated. Yield 98.7% (19.1 g).

Synthesis of S-343-(2,4-dichlorophenoxy)propyl)(prop-2-ynyl)amino)propyl ethanethioate (8)

(95) ##STR00020##

(96) To a solution of N-(3-(2,4-dichlorophenoxy)propyl)prop-2-yn-1-aminium 2,2,2-trifluoroacetate (2.14 mg, 0.05 mmol) in 100 μL ACN in a 5 mL vial equipped with a stir bar, 12.1 mg (0.09 mmol) of K.sub.2CO.sub.3 and 142.4 mg (0.720 mmol) S-3-bromopropyl ethanethioate was added. The mixture was stirred while heated to 50° C. in an oil bath. TLC was performed (silica gel, MTBE:hexane=9:1 to detect starting material and MTBE:hexane=1:9 to detect the consumption of the thioacetate reagent) to follow the process of the reaction. To a solution of N-(3-(2,4-dichlorophenoxy)propyl)prop-2-yn-1-aminium 2,2,2-trifluoroacetate (17.1 mg, 0.05 mmol) in 800 μL ACN in a 20 mL vial equipped with a stir bar, 120.0 mg (0.870 mmol) of K.sub.2CO.sub.3 and 95.2 mg (0.48 mmol) S-3-bromopropyl ethanethioate was added. The mixture was stirred while heated by 50° C. oil bath for 5 h. The reaction mixtures of the two reactions were combined, filtered and evaporated. Crude product (168.4 mg) was obtained and co-evaporated with hexane for 3 times to remove ACN. Silica gel column (1.25 g) was used to purify the crude product. Yield 14% (2.6 mg).

(97) Structure of the Product was Proved by NMR and LC-MS

Synthesis of 3-((3-(2,4-dichlorophenoxy)propyl)(prop-2-ynyl)amino)propane-1-thiol (9)

(98) ##STR00021##

(99) A solution of S-3-((3-(2,4-dichlorophenoxy)propyl)(prop-2-ynyl)amino)propyl ethanethioate (1.17 mg, 3.10 μmol) in 200 μL ACN was added into a 20 mL vial equipped with a stir bar, evaporated and then co-evaporated with MeOH for 3 times to remove ACN. MeOH/HCl (200 μL) was added into the vial and then the vial was heated by 85° C. oil bath for 6 h. The reaction mixture was evaporated, co-evaporated sequentially by MeOH for 3 times and ACN for 3 times, and then used in the next step without further purification.

Synthesis of MHI 148-clorgyline Conjugate (10)

(100) ##STR00022##

(101) MHI-148 (4.7 mg, 6.2 mmol) and EDC (1.5-2.4 mg, 7.8-12 mmol), followed by 1.5 mg of DMAP (12 mmol) were added into a 20 mL vial equipped with a stir bar. ACN (400 μL) was added to make solution. The reaction mixture of the previous step was transferred dropwise to the vial with 200 μL it ACN at room temperature. The reaction mixture was purified by HPLC (GRACE Davison Apollo C.sub.8 5 u column, 250 mm×10 mm).

Mechanistic Investigation of the Role of MAOA in PCa Progression

(102) Our preliminary data suggest that MAO-A is closely related to prostate cancer (PCa) metastasis to bone and for the first time demonstrate that MAO-A protein expression was elevated in PCa bone metastasis relative to normal and low Gleason grade cancerous epithelium (FIG. 13).

(103) Manipulation of levels of MAO-A expression in human bone-metastatic PC-3 and ARCaP.sub.M PCa cells resulted in altered tumor growth in mice. PC-3 cells overexpressing wild-type MAO-A enhanced its growth whereas ARCaP.sub.M cells with specific lentiviral shRNA-mediated silencing completely abrogated the growth of this invasive and bone-metastatic PCa tumor in mice (FIG. 14). These results raise the possibility that MAO-A is an ideal therapeutic target for the treatment of PCa tumors with high propensity for bone and visceral organ metastases.

(104) Our experiments with murine prostate carcinoma TRAMPC-2 (neuroendocrine phenotype), subcutaneously injected into the MAOA neo mice, showed significantly reduced growth rate of PCa (FIG. 15), thereby suggesting a key role that host MAOA plays in determining the rate of prostate cancer growth.

(105) Specifically in the bone microenvironment, knockdown of MAO-A in two castration-resistant human PCa cell lines, ARCaP.sub.M and C4-2, also significantly reduced cancer-induced local bone destruction by osteolytic lesions (FIG. 16).

(106) Mechanistically, MAO-A was found to induce epithelial-to-mesenchymal transition (EMT) in human PCa cells, by promoting the loss of E-cadherin expression (an epithelial marker), up-regulation of Vimentin-N-cadherin (mesenchymal markers) and increased migration and invasion in PC-3 cells (FIG. 17A-B); conversely, MAOA knockdown impeded EMT in human ARCaP.sub.M cells (FIG. 17C-E). Activation of the EMT program can direct the local growth and distant dissemination of PCa cells to skeletons and soft tissues. These data suggest that MAO-A expression and its downstream signaling axes might be highly relevant to the development of metastatic PCa and its associated EMT phenotypes.

(107) We also observed that overexpression of MAO-A enhanced hypoxia-inducible factor 1α (HIF1α) expression, and select HIF1α target genes known to promote PCa progression and metastasis, such as VEGF and EMT-promoting genes (Snail2 and Twist1), are also influenced by MAO-A in PCa cells (FIG. 18). Hypoxia increases tumor angiogenesis and survival responses as well as invasion and metastasis through the up-regulation of HIF1α-dependent relevant genes. Chronic hypoxia, a hallmark of many solid tumors, often in conjugation with elevated levels of reactive oxygen species, has been suggested to affect each step of the metastasis process, from the initial EMT to the ultimate organotropic colonization. Thus, this data further provides important mechanistic insights into the roles of MAO-A in mediating human PCa metastasis to bone and other soft tissues.

(108) A class of fluorescent heptamethine cyanines with near-infrared (NIR) emission maxima, such as MHI-148 dye, which has been identified recently, are non-toxic and have dual functions as tumor-specific targeting and imaging modalities. These dyes, partially mediated by hypoxia, are specifically retained in cancer but not normal cells, and also in tumor xenografts as well as spontaneous tumors in transgenic mice. We have shown enhanced uptake of MHI-148 NIR dye in MAO-A-overexpressing PC-3 tumor xenografts (FIG. 19). This would allow the development and validation of novel PCa-seeking MAO-A inhibitors with acquired synergistic tumor-targeting abilities as new agents for PCa therapies with minimal systemic host toxicity.

(109) Although the present invention has been described in terms of specific exemplary embodiments and examples, it will be appreciated that the embodiments disclosed herein are for illustrative purposes only and various modifications and alterations might be made by those skilled in the art without departing from the spirit and scope of the invention as set forth in the following claims.

REFERENCES

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