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Prostate cancer (PCa) typically spreads to the bone, and this distribution is attributed to the central role of the microenvironment in progression. However, metastasis to the adrenal glands, while not as common, does occur. The biology that accounts for adrenal metastases may be attributed to the unique local steroid metabolome and co-clinical characterization may elucidate the role steroid biosynthesis plays in PCa progression.Three patients with metastatic PCa who had archived tumor tissue from an adrenalectomy were retrospectively identified, and one adrenal metastasis was developed into a xenograft (MDA-PCa-250). The adrenal metastases were characterized by performing somatic DNA whole exome sequencing (WES), RNA-Seq, immunohistochemistry (IHC), and steroid metabolite quantitation. The influence of steroid metabolites on adrenal metastasis cells and tumor growth was tested in vitro and in vivo.Clinically, adrenalectomy was performed during castration-resistant oligometastatic disease, and two men experienced resensitization to leuprolide. Somatic DNA WES revealed heterogeneous alterations in tumor suppressor and DNA damage repair pathway genes. Adrenal metastases had active androgen receptor (AR) signaling by IHC, and RNA-Seq supported a potential role for adrenal androgen precursor metabolism in activating the AR. Steroid quantitation suggested the adrenal androgen precursors were converted into testosterone in these metastases, and stable isotope tracing of an organoid from MDA-PCa-250 confirmed the capability of adrenal metastases to biosynthesize testosterone from adrenal precursors. In vitro testing of a cell line derived from MDA-PCa-250 showed that testosterone and cortisol stimulated tumor cell growth. In vivo experiments demonstrated that MDA-PCa-250 grew in intact mice with circulating testosterone, but not in castrated mice.PCa adrenal metastases depend upon AR signaling driven by androgen precursors, androstenedione and dehydroepiandrosterone, available in the microenvironment, despite the presence of heterogeneous somatic DNA alterations. Moreover, MDA-PCa-250 provides a preclinical model that can recapitulate the unique androgen-dependence of adrenal metastases.This study does not report the clinical results of a clinical trial, but it does use samples from a completed clinical trial that is registered with clinicaltrials.gov (NCT01254864).

Innate immune cell infiltration into neoplastic tissue is the first line of defense against cancer and can play a deterministic role in tumor progression. Here, we describe a series of assays, using a reconfigurable microscale assay platform (i.e. Stacks), which allows the study of immune cell infiltration in vitro with spatiotemporal manipulations. We assembled Stacks assays to investigate tumor–monocyte interactions, re-education of activated macrophages, and neutrophil infiltration. For the first time in vitro, the Stacks infiltration assays reveal that primary tumor-associated fibroblasts from specific patients differ from that associated with the benign region of the prostate in their ability to limit neutrophil infiltration as well as facilitate monocyte adhesion and anti-inflammatory monocyte polarization. These results show that fibroblasts play a regulatory role in immune cell infiltration and that Stacks has the potential to predict individual patients’ cancer-immune response.

Background: Most cancer cells depend more on glycolysis for their energy need compared with their normal counterparts incubated under identical condition. In addition to glycolysis, tumor cells also engage mitochondrial ox-phos to support growth. Here, we show that PCa cells surviving under anti-androgen enzalutamide (ENZA) treatment switch from glycolysis to ox-phos for their energy need and are more vulnerable to mitochondria-targeted metabolic inhibitors. We have also standardized a high-resolution quantitative fluorescence microscopic method to detect this metabolic switch in patient circulating tumor cells (CTCs). Methods: Seahorse assay has been used to compare mitochondrial metabolism in ENZA treated anti-androgen-sensitive LNCaP and -resistant C4-2 and PCa2b cells. The Seahorse data were supported by mass spectroscopic analysis of corresponding metabolites and metabolic fluxes. An ex vivo fluorescence staining for the CTC mitochondria with high ox-phos followed by high-resolution quantitative microscopic image analysis method has been standardized to follow such changes in cultured cells and patient CTCs. Results: Seahorse, mass spectroscopy and high-resolution microscopy of mitochondrial ox-phos showed that ENZA significantly decreases glycolysis and increases ox-phos in all surviving PCa cells within 24h of treatment. These cells are more vulnerable to treatment with mitochondrial ox-phos inhibitor IACS-010759 and a glutaminase inhibitor CB-839. High-resolution microscopic analysis of CTCs has thus far been performed in 18 patient blood samples. Six out of the 18 patients developed resistance to anti-androgen therapy within 0-6 months of sample collection. CTCs from all six patients showed a relatively higher average fluorescence due to high mitochondrial ox-phos as compared with the rest of the patients. Discussion: The data presented here may lead to informed combination therapy for selected PCa patients developing resistance to anti-androgen therapy for better clinical outcome. Citation Format: Hirak S. Basu, Nathaniel Wilganowski, Samantha Robertson, Sumankalai Ramachandran, Amado Zurita-Saavedra, Mark Titus, Evan Cohen, James Reuben, George Wilding. Metabolic switch from glycolysis to oxidative phosphorylation (ox-phos) provides survival advantage to anti-androgen-treated prostate cancer cells and make them vulnerable to mitochondrial metabolism inhibitors IACS-010759 and CB-839 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4791.

Accumulating evidence shows that androgen receptor (AR) activation and signaling plays a key role in growth and progression in all stages of prostate cancer, even under low androgen levels or in the absence of androgen in the castration-resistant prostate cancer. Sustained activation of AR under androgen-deprived conditions may be due to its interaction with co-activators, such as p52 NF-κB subunit, and/or an increase in its stability by phosphorylation that delays its degradation. Here we identified a specific inhibitor of AR/p52 interaction, AR/p52-02, via a high throughput screen based on the reconstitution of Gaussia Luciferase. We found that AR/p52-02 markedly inhibited growth of both castration-resistant C4-2 (IC50 ∼6 μM) and parental androgen-dependent LNCaP (IC50 ∼4 μM) human prostate cancer cells under low androgen conditions. Growth inhibition was associated with significantly reduced nuclear p52 levels and DNA binding activity, as well as decreased phosphorylation of AR at serine 81, increased AR ubiquitination, and decreased AR transcriptional activity as indicated by decreased prostate-specific antigen (PSA) mRNA levels in both cell lines. AR/p52-02 also caused a reduction in levels of p21(WAF/CIP1), which is a direct AR targeted gene in that its expression correlates with androgen stimulation and mitogenic proliferation in prostate cancer under physiologic levels of androgen, likely by disrupting the AR signaling axis. The reduced level of cyclinD1 reported previously for this compound may be due to the reduction in nuclear presence and activity of p52, which directly regulates cyclinD1 expression, as well as the reduction in p21(WAF/CIP1), since p21(WAF/CIP1) is reported to stabilize nuclear cyclinD1 in prostate cancer. Overall, the data suggest that specifically inhibiting the interaction of AR with p52 and blocking activity of p52 and pARser81 may be an effective means of reducing castration-resistant prostate cancer cell growth.

BACKGROUND Multiple studies show that reactive oxygen species (ROS) play a major role in prostate cancer (PCa) development and progression. Previously, we reported an induction of Spermidine/Spermine N1-Acetyl Transferase (SSAT) by androgen-activated androgen receptor (AR)-JunD protein complex that leads to over-production of ROS in PCa cells. In our current research, we identify small molecules that specifically block AR-JunD in this ROS-generating metabolic pathway.

Introduction: The mechanism of prostate cancer (PCa) progression to the mostly lethal, metastatic, castrate-resistant stage (mCRPC) remains generally unknown. This prevents identifying patients likely to progress to mCRPC and designing new therapies for their treatment. A transcription factor JunD is overexpressed in PCa, but not in normal prostate epithelial cells. It complexes with androgen receptor (AR) to induce spermidine/spermine acetyl transferase (SSAT) [3]. SSAT initiates polyamine oxidation that generates copious amounts of reactive oxygen species (ROS) production in polyamine-rich PCa cells. ROS activate NF-κB. NF-κB can also induce SSAT and activate AR. This sets up a feed-forward loop for PCa growth at low androgen. Here, we present one mechanism of PCa progression to CRPC as well as its invasion and metastasis that may provide predictive biomarkers to identify patients with potentially lethal PCa at an early stage. Effects of an agent that can block both AR-JunD and AR-NF-κB interactions and prevent growth of PCa cells in vitro as well as in vivo xenografts will also be presented. Method: We have used a novel microscale device to separate invading from noninvading PCa cells ex vivo in a bone microenvironment. Immunocytochemistry (ICC), proteomic and metabolomic techniques have been employed to analyze the separated cells to understand the mechanism of invasion. Gaussia luciferase reconstitution assay has been used in a high-throughput screen (HTS) to identify inhibitors of AR-JunD and AR-NF-κB interactions. State-of-the-art in vitro cell growth assay along with in vivo pharmacokinetic (PK), maximum tolerated dose (MTD) and enzalutamide-resistant CRPC cell xenograft growth in nude mice are used to optimize the lead. Summary of Unpublished Results: Immunocytochemistry (ICC) data show that in cultured enzalutamide-resistant C4-2 and -sensitive LNCaP cells as well as in some patient PCa cells, more SSAT-positive cells are in the migratory than in the stationary section of the microscale device. Metabolomic analysis show a decrease in 3-phospho-glycerate levels in C4-2 cells, suggesting a decrease in GAPDH enzymatic activity that is related to an enhanced oxidation of GAPDH protein. Our published data of screening of 27,000+ compounds from two different chemical libraries detected a single compound that specifically inhibits both AR-JunD and AR-NF-κB2 (p52) interactions. We have synthesized several of its analogs. Lead analogs inhibit growth of AR-positive C4-2 and LNCaP cells, but have little effect on the growth of AR-negative PC-3 and SSAT-silent LNCaP siSSAT cells in culture. Co-immunoprecipitation (co-IP) assay shows its efficacy in inhibiting AR-JunD and AR-p52 interactions in LNCaP cells. It has been formulated in 25% ethanol:water and administered to nude mice orally. It is well tolerated at 50 mg/kg p.o. daily for more than 28 days and has a serum Cmax of ~ 3 μM within 30 minutes and a plasma t1/2 of ~1 h after a single oral dose of 100 mg/kg. Twenty-four hours after dosing it is found in the flank tumor (1.5 ng/mg tissue; ~450 nM) and in the prostate tissues (10 ng/mg tissue; ~3 μM) in addition to liver and kidney. Its effects on the growth of C4-2 xenografts in nude mice will be presented. Conclusion: A mechanism of PCa progression to CRPC and then to mCRPC provides a rational basis for targeted drug design that prevents PCa progression and metastasis. This should open up a new avenue of precision therapy of potentially lethal PCa at an early stage. Citation Format: Hirak Basu, Nathaniel Wilganowski, Jessica Lieblich, Grace Wu, Izabela Fokt, Sumankalai Ramachandran, Jiaquin Yu, Mark Titus, Waldemar Priebe, David J. Beebe, George Wilding. Small-molecule inhibitors targeting a specific metabolic pathway for precision therapy of advanced castrate-resistant prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B091.

Background: We have previously reported that an AP-1 factor JunD is overexpressed in PCa. It complexes with androgen receptor (AR) to induce spermidine/spermine acetyl transferase (SSAT). SSAT initiates polyamine oxidation that generates copious amounts of reactive oxygen species (ROS) production in polyamine-rich PCa cells. ROS activate NF-κB. NF-κB also induces SSAT and activates AR. This sets-up a feed-forward loop for PCa growth at low androgen. An IHC assay for SSAT can be utilized to identify PCa patients with this loop activated. AR-N-terminal targeted inhibitors that block AR interactions with JunD and NF-κB can be developed as a precision therapy for these patients. Method: We applied Immunocytochemistry (ICC), proteomic and metabolomic techniques to analyze stationary and invading cells that are separated in a novel micro-scale dvice to understand the mechanism of invasion. Gaussia luciferase reconstitution assay in a high throughput screen (HTS) to identify inhibitors of AR-JunD and AR-NF-κB interactions. In vitro cell growth assay along with in vivo pharmacokinetic (PK), maximum tolerated dose (MTD) and enzalutamide-resistant CRPC cell xenograft growth in nude mice are used to optimize the lead. Unpublished data: Immunocytochemistry (ICC) data show that in cultured enzalutamide-resistant C4-2 and -sensitive LNCaP cells as well as in some patient PCa cells, more SSAT positive cells are in the migratory than in the stationary section of the microscale device. Screening of 27,000+ compounds from 2 different chemical libraries detected a single compound that specifically inhibits both AR-JunD and AR-NF-κB2 (p52) interactions. We have synthesized several of its analogs. Lead analogs inhibit growth of AR-positive C4-2 and LNCaP cells at nanomolar concentration, but have little effect on the growth of AR-negative PC-3 and SSAT silent siSSAT cells in culture. Co-immunoprecipitation (co-IP) assay shows its efficacy in inhibiting AR-JunD and AR-p52 interactions in situ. It has been formulated in 25% ethanol:water and administered orally. PCa tumor bearing nude mice tolerated it at 50 mg/kg p.o. daily for more than 28 days and has a serum Cmax of ~ 3 μM within 30 minutes and a plasma t1/2 of ~1 h after a single oral dose of 100 mg/kg. Twenty four hours after dosing it is found in the flank tumor (1.5 ng/mg tissue; ~450 nM) and in the prostate tissues (10 ng/mg tissue; ~3 μM) in addition to liver and kidney and inhibited growth of C4-2 xenografts in nude mice. Citation Format: Hirak S. Basu, Nathaniel L. Wilgonowski, Jessica L. Lieblich, Grace T. Wu, Izabela Fokt, Sumankalai Ramachandran, Jiaquin Yu, Mark Titus, Waldemar Priebe, David J. Beebe, George Wilding. A metabolic pathway targeted inhibitor for precision therapy of castrate-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3846.

Most prostate, colon and breast cancer cells are resistant to growth inhibitory effects of suberoylanilide hydroxamic acid (SAHA). We have examined whether the high oxidative stress in these cells causes a loss of SAHA activity and if so, whether pretreatment with an anti-oxidant can sensitize these cells to SAHA.A DNA-Hoechst dye fluorescence measured cell growth and dichlorfluorescein-diacetate (DCF-DA) dye fluorescence measured reactive oxygen species (ROS). Growth inhibitory and ROS-generating activities of SAHA in androgen-treated or untreated LNCaP cells and PC-3 prostate cancer cells, HT-29 and HCT-115 colon cancer cells, MDA-MB231 breast cancer cells and A549 and NCI-H460 lung cancer cells with or without pretreatment with an anti-oxidant Vitamin E was determined. SAHA activity against LNCaP cells treated with another anti-oxidant N-acetyl cysteine (NAC) was also determined. Liquid chromatography-mass spectrometry (LC-MS) was used to determine intracellular SAHA level.SAHA treatment markedly inhibits LNCaP cell growth, when the cells are at a low ROS level. SAHA is, however, inactive against the same cell line, when the cells are at a high ROS level. A significant decrease in SAHA level was observed in LNCaP cells with high ROS after 24- and 72-h treatment when compared to cells with low ROS. Vitamin E pretreatment that reduces cellular ROS, synergistically sensitizes oxidatively stressed LNCaP, PC-3, HT-29, HCT-115 and MDA-MB231 cells, but not the A-549 and NCI-H460 cells with low ROS to SAHA. NAC treatment also sensitized androgen-treated LNCaP cells to the growth inhibitory effects of SAHA.Response to SAHA could be improved by combining anti-oxidants such as Vitamin E with SAHA for the treatment of oxidatively stressed human malignancies that are otherwise resistant to SAHA.

BACKGROUND. Numerous and compelling evidence shows that high level of reactive oxygen species (ROS) plays a key role in prostate cancer occurrence, recurrence and progression. The molecular mechanism of ROS overproduction in the prostate gland, however, remains mostly unknown. Unique AP-1 transcription factor JunD has been shown to inhibit cell proliferation, promote differentiation and mediate stress responses in a variety of eukaryotic cells. We previously reported that androgen–androgen receptor induced ROS production in androgen-dependent LNCaP human prostate cancer cells is associated with increased JunD level/AP-1 transcriptional activity. METHODS. LNCaP cells constitutively overexpressing a functionally inactive form of JunD (JunDDTA) or stably transfected with JunD siRNA (siJunD) to suppress JunD protein expression were established. Overexpression of JunD in LNCaP cells using transient transfection method was applied to assess the induction of ROS production in LNCaP cells. DCF assay was used to measure the ROS concentrations in the transfected as well as nontransfected control cells. RT-PCR and Western blot analyses were used to confirm silencing or overexpression of JunD in the transfected cells. RESULTS. In the absence of androgen, LNCaP cells transiently transfected with a JunD overexpressing vector have relatively enhanced cellular ROS levels as compared to LNCaP cells transfected with a vector control. LNCaP cells that fail to express functional JunD (JunDDTA or siJunD) do not exhibit any increase in ROS production in response to androgen. CONCLUSION. Based on these data, we conclude that JunD is an essential mediator of the androgen-induced increase in ROS levels in LNCaP cells. Prostate 68: 924–934, 2008. # 2008 Wiley-Liss, Inc.

Leinamycin (LNM) is a potent antitumor antibiotic produced by Streptomyces atroolivaceus S-140, featuring an unusual 1,3-dioxo-1,2-dithiolane moiety that is spiro-fused to a thiazole-containing 18-membered lactam ring. Upon reductive activation in the presence of cellular thiols, LNM exerts its antitumor activity by an episulfonium ion-mediated DNA alkylation. Previously, we have cloned the lnm gene cluster from S. atroolivaceus S-140 and characterized the biosynthetic machinery responsible for the 18-membered lactam backbone and the alkyl branch at C3 of LNM. We now report the isolation and characterization of leinamycin E1 (LNM E1) from S. atroolivacues SB3033, a ΔlnmE mutant strain of S. atroolivaceus S-140. Complementary to the reductive activation of LNM by cellular thiols, LNM E1 can be oxidatively activated by cellular reactive oxygen species (ROS) to generate a similar episulfonium ion intermediate, thereby alkylating DNA and leading to eventual cell death. The feasibility of exploiting LNM E1 as an anticancer prodrug activated by ROS was demonstrated in two prostate cancer cell lines, LNCaP and DU-145. Because many cancer cells are under higher cellular oxidative stress with increased levels of ROS than normal cells, these findings support the idea of exploiting ROS as a means to target cancer cells and highlight LNM E1 as a novel lead for the development of anticancer prodrugs activated by ROS. The structure of LNM E1 also reveals critical new insights into LNM biosynthesis, setting the stage to investigate sulfur incorporation, as well as the tailoring steps that convert the nascent hybrid peptide-polyketide biosynthetic intermediate into LNM.

Polyamines are essential for cell growth. Polyamine analogs that can replace intracellular polyamines inhibit tumor cell proliferation both in culture as well as in animal models. The positively charged polyamines interact with the negatively charged DNA backbone both in a nonspecific manner, as well as sequence specifically through direct or water mediated hydrogen bonds. Therefore, it is difficult to ascertain the exact interactions that regulate the biological functions of polyamines. Several attempts have been made to determine the thermodynamic parameters of polyamine-DNA interactions with conflicting results. Here, we report a simple method of determining the apparent association constants for polyamine-DNA interaction by using polyacrylamide gel coelectrophoresis (PACE). We have used several cytotoxic polyamine analogs of different conformations and chain lengths. We observed that polyamine analogs with higher charge density or with conformational restrictions, which are absent in the naturally occurring polyamines, interact with DNA more strongly than do natural polyamines. A comparison of the cytotoxicities of the polyamine analogs against human tumor cell lines with their DNA affinities revealed that the higher the DNA affinity the more the cytoxicity of the analogs. The direct correlation between DNA affinities and cytotoxities provides a novel method for a rational design of therapeutically effective cytotoxic polyamine analogs.

In solid tumors, when O(2) partial pressure drops below 10 mmHg, ATP levels rapidly decrease due to the Warburg effect. It is known that certain macrocyclic polyamines catalyze the chemical hydrolysis of ATP with release of inorganic phosphate. Since tumor cells have diminished ATP levels as compared to normal cells, we attempted to deplete cellular ATP with macrocyclic polyamines in an effort to inhibit tumor cell proliferation. Five macrocyclic polyamines, related to the budmunchamine family of alkaloids, were prepared by total synthesis. They were the [17]-N(4) macrocycle 1, the [16]-N(4) macrocycle 20, the [18]-N(4) macrocycle 13, the [20]-N(5) macrocycle 8, and the [13]-N(3) macrocycle 17. Each one of them hydrolyzed ATP in vitro with release of P(i); the largest ring macrocycle 8 was the most efficient catalyst, while the smallest ring macrocycle 17 was the least efficient (P(i) released in these runs was on the order of 40-100 microM). The linear polyamine spermine had no hydrolytic effect on ATP. The macrocycles were found to be cytotoxic when assessed by means of a MTT assay against two human prostate cell lines, DuPro and PC-3, with resultant ID(50) values ranging between 0.5 and 1.8 microM. Colony forming efficiency (CFE) assays performed on DuPro cells, where the macrocycles were used in a concentration range of 1-8 microM, confirmed the cytotoxic effect of each macrocycle. Each killed 3-4 log of DuPro cells. The smallest ring 17 was the least cytotoxic after 24 h of incubation, although after 144 h of incubation it showed significant cytotoxicity at 8 microM. The macrocycles were equally efficient in depleting the intracellular ATP pools; after a 24 h incubation with each macrocycle other than 17 at 1-8 microM concentrations, cellular ATP concentrations were decreased by 3 orders of magnitude. The decrease in ATP levels was more pronounced after a 72 h incubation, when even 17 reduced ATP by 2 orders of magnitude. A linear pentamine of established cytotoxicity was without effect on the ATP pools. The macrocycles depleted almost entirely the intracellular pools of polyamines and were efficiently taken up by cells. A rough correlation could be established between the cytotoxic effect of the macrocyclic polyamines and their ATP-ase like activity in the DuPro cell line. As ATP is a scarce metabolite in cancer cells, where it can only be replenished through the very ATP-inefficient glycolytic pathway; macrocyclic polyamines appear to be promising new anticancer agents.

α N , ω N -bis(ethyl) octamine SL-11160, decamine SL-11159, dodecamine SL-11226, and tetradecamine SL-11175 were chemically synthesized. We called this class of compounds ‘oligoamines’. In these compounds, each –NH 2 + residue is separated by four CH 2 residues. trans -Unsaturation was also introduced into the center of the oligoamine chain resulting in the trans -octamine SL-11158, trans -decamine SL-11144, trans -dodecamine SL-11172 and trans -tetradecamine SL-11227. cis -Unsaturation gave the cis -octamine SL-11157 and cis -decamine SL-11150. When assayed for their growth inhibitory effect against four human prostate cancer cell lines LnCap, DU-145, DuPro, and PC-3 by a MTT assay, the ID 50 values were less than 1 μM in all four cell lines. On day 6 of treatment, 2 μM SL-11159, SL-11144 and SL-11175 killed over five logs of DuPro cells while SL-11172 killed over four logs as determined by a colony forming efficiency (CFE) assay. In addition, SL-11159, SL-11226 and SL-11227 killed four logs of PC-3 cells. PC-3 cells are generally resistant to shorter chain polyamine analogues. Such a level of cytotoxicity in any of the prostate tumor cell lines has not been observed for any other polyamine analogues tested thus far. The DU-145 cell line was too sensitive to oligoamines to perform a CFE analysis and the DuPro cell line was too sensitive to SL-11227 treatment to obtain reproducible CFE data. Interestingly, all 10 oligoamines were efficient DNA aggregators in a cell-free system and their cytotoxicities generally parallel their capacities to aggregate DNA.

We tested the polyamine analog SL-11093 (3,8,13,18-tetraaza-10,11-[(E)-1,2-cyclopropyl]eicosane tetrahydrochloride) as an effective chemotherapeutic agent against human prostate cancer grown in nude mice.NCr-nu mice grafted with DU-145 human prostate tumor cells were treated i.p. with SL-11093 at 50 mg/kg q1dx5 for either three or five cycles separated by intervals of about 10-15 days.In treated animals, tumor growth remained arrested for up to 100 days with minimal animal weight loss. None of the animals died during the treatment and in one experiment two out of six animals showed no palpable tumor. SL-11093 was readily taken up by the tumors, where its levels remained elevated for about 48 h after the end of drug administration. In liver and in kidney, SL-11093 (a (alpha)N,(omega)N-bisethyl derivative) was oxidatively N-deethylated predominantly to its monoethyl and di-deethyl derivatives. In time, the monoethyl derivative was further dealkylated, with a loss of an aminobutyl chain to form an aminomethyl cyclopropyl derivative. In tumor (and in lung), N-dealkylation reactions were less evident.SL-11093 is an effective chemotherapeutic agent against a human prostate tumor xenograft grown in nude mice. The drug accumulation and slow metabolism in tumor compared to other tissues would most likely reduce systemic toxicity of the drug and contribute to a larger therapeutic window for SL-11093 as compared to other cytotoxic polyamine analogs.

Prostatic carcinoma is the second most common malignancy in the USA and the second highest cause of cancer related deaths in men. A prostate-specific antigen (PSA) isolated from prostate tissue and seminal fluid, and also detected in blood serum of prostate cancer patients, led to the development of a serum PSA determination method as a diagnostic and prognostic marker for prostate cancer. PSA is an active protease when released by prostatic tissue, either normal or malignant. In serum it is present as an immunologically reactive but enzymatically inactive complex. Both the antigenic and the proteolytic activities of PSA were exploited to deliver cytotoxic agents at or near the prostate tumour tissues. The enzymatic activity released the agent from short peptide conjugates that are specific substrates of PSA. A new polyamine analogue (SL-11093) was found to inhibit the growth of relatively large (200 mm3) human prostate tumours grafted in athymic mice by ~ 90%. It has greatly reduced systemic toxicity com...

In prostate cancer, bone is a frequent site of metastasis; however, the molecular mechanisms of this tumor tropism remain unclear. Here, we integrate a microfluidic coculture platform with multi-photon imaging based techniques to assess both phenotypic cell behavior and FAD fluorescence intensity and fluorescence lifetime in the same cell. This platform combines two independent assays normally performed with two different cell populations into a single device, allowing us to simultaneously assess both phenotypic cell behavior and enzyme activity. We observed that the osteotropic prostate cancer cell line (C4-2B), when in a coculture with bone marrow stromal cells (MC3T3-E1), has increased protrusive phenotype and increased total and protein-bound FAD compared to its parent cell line (LNCaP). We hypothesized that an increase in ROS-generating APAO activity may be responsible for these effects, and found that the effects were decreased in the presence of the antioxidant N-Acetyl Cysteine (NAC). This suggests that an ROS-related signaling mechanism at the bone metastatic site may be correlated with and play a role in increased invasion of metastasizing prostate cancer cells. The studies performed using this combined platform will lead to new insights into the mechanisms that drive prostate cancer metastasis.

Twelve analogues of 1N,14N-bisethylhomospermine (BE-4-4-4) with restricted conformations were synthesized in the search for cancer chemotherapeutic agents with higher cytotoxic activities and lower systemic toxicities than BE-4-4-4. The central butane segment of BE-4-4-4 was replaced with a 1,2-substituted cyclopropane ring, a 1,2-substituted cyclobutane ring, and a 2-butene residue. In each case, the cis/trans-isomeric pair was synthesized. Cis-monounsaturation(s) was also introduced at the outer butane segment(s) of BE-4-4-4. The two possible cis-dienes and a cis-triene formally derived from the tetraazaeicosane skeleton of BE-4-4-4 were also prepared. Four cultured human prostate cancer cell lines (LnCap, DU145, DuPro, and PC-3) were treated with the new tetramines to examine their effects on cell growth with a MTT assay. One representative cell line (DuPro) was selected to further study the cellular uptake of the novel tetramines, their effects on intracellular polyamine pools, and their cytotoxicity. All tetramines entered the cells, reduced cellular putrescine and spermidine pools while exerting only a small effect on the spermine pool, inhibited cell growth, and killed 2-3 logs of cells after 6 days of treatment at 10 microM. Four new tetramines, the two cyclopropyl isomers, the trans-cyclobutyl isomer, and the (5Z)-tetraazaeicosene, were more cytotoxic than their saturated counterpart (BE-4-4-4). Their cytotoxicity, however, could not be correlated either with their cellular uptake or with their ability to deplete intracellular polyamine pools. We attribute their cytotoxicity to their specific molecular structures. The cytotoxicity was markedly reduced when the central butane segment was deprived of its rotational freedom by replacing it with a double bond. Introduction of a triple bond or a benzene-1,2-dimethyl residue at the central segment of the polyamine chain, led to complete loss of biological activity. The conformationally restricted alicyclic derivatives were not only more cytotoxic than was the freely rotating BE-4-4-4 by several orders of magnitude but also had much lower systemic toxicities than the latter. Thus, we obtained new tetramines with a wider therapeutic window than BE-4-4-4.

From the results of our previous physicochemical studies of polyamine-nucleic acid interactions, we concluded that polyamine analogues in cisoidal conformation are capable of wrapping around the major groove of the double helix, of displacing natural polyamines from their nucleic acid binding sites, and of inhibiting cell division. On the basis of this hypothesis, nine unsaturated pentamines, formally derived from the cytotoxic pentamine 3,8,13,18,23-pentaazapentacosane (BE-4-4-4-4), were prepared in an attempt to increase antineoplastic activity. Cis-double bonds were introduced in all possible sites in the saturated pentaazapentacosane structure of BE-4-4-4-4 to yield two pentacosenes, four pentacosadienes, two pentacosatrienes, and one pentacosatetraene. Cis-double bonds should also provide good targets for mixed-function oxidases that might eliminate the accumulation of unsaturated pentamines in serum, thereby reducing systemic toxicity in animals. We determined the ability of these new pentamines to inhibit growth in four cultured human prostate cancer cell lines (LnCap, DU145, PC-3, and DuPro) using a MTT assay. LnCap and DU145 cells were very sensitive, PC-3 cells were relatively resistant, and DuPro cells were intermediate in sensitivity to most of these synthetic pentamines. In all cell lines, pentamines that had unsaturation(s) at the end of the chain showed the highest cell growth inhibitory effects. The cellular uptake, effects on cellular polyamine levels, and cytotoxicity of these pentamines on one representative prostate cancer cell line (DuPro) were further examined with a colony-forming efficiency (CFE) assay. The pentamines with unsaturation(s) at the end of the chain were once again the most cytotoxic among both the saturated (BE-4-4-4-4) and unsaturated analogues. Appreciable amounts of all pentamines entered DuPro cells and depleted cellular polyamine pools by day 6 of treatment. For most pentamines, however, cell growth inhibitory and cytotoxic effects could not be directly correlated either with their cellular uptake or with their ability to deplete cellular polyamine pools. The position of the double bonds in the aliphatic backbone seems to be the most important determinant of cytotoxicity. For some pentamines, however, depletion of cellular polyamines may add to their efficacy.

Eight analogues of 1N,12N-bisethylspermine (BES) with restricted conformations were synthesized in the search for new spermine mimetics with cytotoxic activities. By replacing the central butane segment of BES with a 1,2-disubstituted cyclopropane ring, a pair of cis/trans-isomers was obtained that introduced a spatial constraint in the otherwise freely mobile butane chain. An analogous pair of isomers was obtained when the butane segment was replaced with a 1, 2-disubstituted cyclobutane ring or with a 2-butene residue. The six new BES analogues thus obtained (three pairs of cis/trans-isomers) were growth inhibitory at low-micromolar concentrations against four human tumor cell lines (A549, HT-29, U251MG, and DU145) but were less growth inhibitory against two other human tumor cell lines (PC-3 and MCF7). 1N,12N-Bisethylspermyne, where the central butane segment of BES was replaced by the rigid 2-butyne segment, was devoid of growth inhibitory activity against five of the six human cell lines studied (DU145 being the only exception), a clear indication of the importance of conformational mobility at the 4N, 9N-butane segment of BES for its biological activity. When the butane segment was replaced by a benzene-1,2-dimethyl residue, the resulting BES analogue was devoid of growth inhibitory activity despite its cisoid conformation. The cytotoxicity of the analogues does not seem to be directly related to their uptake by the cells or to their effects on cellular polyamine levels. BES analogues with restricted conformations but which contained the equivalent of a two-carbon unit, rather than the natural four-carbon unit, at the central segment, such as 1,2-diaminocyclopropyl or 1, 2-diaminocyclobutyl derivatives, were devoid of growth inhibitory effects at the concentrations studied. The development of conformationally restricted polyamine analogues appears to show promise in the further quest for polyamine-related therapeutic agents with specificity of action.

We investigated the effects of the polyamine spermine and two of its cytotoxic analogs 1,11-bis(ethyl-amino)-4,8-diazaundecane (BE-3-3-3) and 1,19-bis(ethylamino)-S,10,15-tirazanonadecane (BE-4-4-4-4) on the formation of nucleosomes on negatively and positively supercoiled DNA in vitro. Histones H2A, H2B, H3 and H4 were reconstituted onto DNA to form nucleosomes and the polyamines were added either before or after histone addition. The structural state of the nucleosome was monitored by analyzing the DNA topoisomers that were present after topoisomerase I treatment. Although polyamines induced DNA aggregation to various degrees, high concentrations of topoisomerase I were able to relax the aggregated DNA and the helical pitch was found to be unaltered in the aggregates. When histones were associated with negatively coiled DNA, the polyamine-induced aggregation did not alter nucleosome structure. The induced aggregate did inhibit nucleosomal transitions when examined on positively coiled DNA. BE-4-4-4-4 was most effective and BE-3-3-3 least effective. These analogs were also extremely effective in inhibiting histone deposition onto DNA. A potential mechanism for the action of these analogs is both to inhibit histone deposition during DNA replication and also disrupt nucleosomal dynamics due to aberrant chromatin condensation. These results also suggest that BE-4-4-4-4 and BE-3-3-3 may produce their cytotoxic effect through slightly different mechanisms.

Ornithine decarboxylase (ODC) is a rate-determining enzyme of the polyamine-biosynthetic pathway. We sought to produce cells with impaired ODC function in order to study the biological functions of polyamines. Saccharomyces cerevisiae strains were obtained by one-step gene replacement of a 900 bp fragment of the yeast ODC gene (SPE1) with the yeast URA3 gene. Spores derived from SPE1/spe1 cells germinated at reduced efficiency relative to SPE1/SPE1. Sustained growth of spe1 haploid mutants in polyamine-free medium led to intracellular polyamine depletion, reduction in budding index, G1 arrest and cessation of growth, and cells that were large and misshapen. All of these effects were completely reversed by adding polyamines to the medium, even after 5 days of polyamine starvation. A diploid yeast strain bearing two copies of disrupted spe1 lost heterozygosity at the mating-type locus more often when grown in the absence of polyamines than when grown in their presence, indicating that polyamine deficiency leads to either chromosome loss or to mitotic recombination.

The naturally occurring polyamine spermine induces haemoglobin synthesis in murine erythroleukaemia (MEL) cells. Haemoglobin production was accompanied by accumulation of cytoplasmic beta-globin mRNA and growth inhibition, but not by cell-cycle block or changes in cell volume. Hexamethylene-bisacetamide (HMBA), a well known differentiating agent, also induces haemoglobin production, but causes a G1 block and decreases cell volume. These findings indicate that HMBA and spermine affect MEL cells differently, even though both induce haemoglobin production.

The naturally occurring polyamine spermine induces haemoglobin synthesis in murine erythroleukaemia (MEL) cells. We have studied the ability of various polyamine analogues to inhibit cell growth and induce haemoglobin production. Polyamine analogues with free terminal amino groups were good inducers of haemoglobin production in MEL cells. Haemoglobin levels correlated with the number of positive charges: pentamines (five positive charges) were stronger inducers than tetramines (four positive charges). Compounds ethylated at their terminal amines were poor inducers of haemoglobin production but good inhibitors of MEL cell growth. These results provide evidence that polyamine analogues support specific biological functions of polyamines in MEL cells and suggest relationships between polyamine structure and function.

PURPOSE These preclinical studies were carried out to examine the potential of the antiproliferative polyamine analog 1,19-bis-(ethylamino)-5,10,15-triazanonadecane (BE-4-4-4-4) to serve as a therapy adjuvant to radiation for patients with rapidly dividing tumors of the head and neck (H&N). METHODS AND MATERIALS Cytostatic and cytotoxic effects of this polyamine analog were investigated in three squamous cell carcinoma (SCC) cell lines derived from human H&N tumors. RESULTS Growth inhibition was achieved in all cell lines within 3-4 days of continuous 10 microM drug exposure, and inhibition of cell cycle proliferation kinetics was confirmed via flow cytometry. Cytotoxicity was pronounced (3-4 log cell kill) in the SCC-38 and SCC-4Y cell lines with continuous 10 microM analog exposure over 5 days, and was minimal in the SCC-13Y cell line. No demonstrable effect of BE-4-4-4-4 on single dose radiation survival was identified in any SCC cell line. Ornithine decarboxylase (ODC) activity was rapidly inhibited (1-2 h) following 10 microM BE-4-4-4-4 exposure in all SCC cell lines (approximately 90%), whereas identical exposure to 10 microM difluoromethylornithine (DFMO) induced animal ODC inhibition (approximately 10%). Dose-dependent depletion of endogenous polyamines (putrescine, spermidine, spermine) was achieved in all SCC cell lines following 1 microM and 10 microM BE-4-4-4-4 exposures. Difluoromethylornithine was significantly less potent than BE-4-4-4-4 in its capacity to deplete endogenous polyamines, with no measureable depletion of spermine pools even with 5 mM x 48 h DFMO exposures. CONCLUSIONS These data evaluate cytostatic and cytotoxic properties of the polyamine analog BE-4-4-4-4 in human SCCs, and suggest a role for investigation of such agents as an adjuvant to radiation in the therapeutic approach to rapidly dividing human tumors such as those that occur in the H&N.

Background: Castrate-resistant prostate cancer (CRPC) is the second leading cause of cancer deaths among US men. Currently, a large number of small and low grade prostate cancers (PCa) are being diagnosed. Only a few of them will metastasize and become lethal. A clinical method to distinguish aggressive from the indolent tumors is warranted. An enhanced glucose metabolism (Warbürg effect) and invasion of cells from their organs of origin and metastasis to distant organs are two characteristics that are ubiquitous in most solid tumors. In the last few years, the non-glycolytic activities (“moonlighting functions”) of glycolytic enzymes have been investigated in relation to cancer cell invasion. We propose that oxidatively stressed PCa cells, as a countermeasure, redirect some of the glycolytic enzymes to their moonlighting functions. Methods: We used a novel microfluidic device that separates the invading from the non-invading PCa cells based on their migratory characteristics in a 3D collagen matrix. We performed ICC analysis of the separated cells for oxidative stress generating enzyme spermidine/spermine N1 acetyl transferase (SSAT) and glycolytic enzymes aldolase, GAPDH and F-actin levels. We also carried out proteomic analysis of aldolase and GAPDH levels and oxidation state and metabolomic analysis for enzymatic activities of those two enzymes. Results: Under identical condition, LNCaP cells show minimum invasion, whereas between 30-40% of C4-2 cells invade more than 0.8 mm in 16 hours. ICC assay of C4-2 cells shows that most migratory C4-2 cells have SSAT overexpression, but less than 5% stationary cells show this effect. Proteomic analysis shows that androgen treatment that increases overall oxidative stress in both LNCaP and C4-2 cells actually decreases oxidation status of GAPDH in LNCaP cells and increases that in C4-2 cells. Anti-androgen enzalutamide reverses the androgen effect in both cell lines. The glycolytic activity of GAPDH decreases with an increase in protein oxidation. Our metabolomic data further confirmed an increase in GAPDH activity in LNCaP and a decrease in C4-2 cells after androgen treatment and the reversal with enzalutamide as expected from its oxidation status. Discussion: SSAT expression and a consequent increase in oxidative stress are related to invasion of CRPC. Oxidation of certain glycolytic enzymes in CRPC cells may divert them to their moonlighting function related to cellular invasion and metastasis. These functions may be monitored in patient biopsies and/or prostatectomy tissues for PCa prognosis. Application of the microfluidic method for the separation of invading from the non-invading cells and proteogenomic and metabolomic analysis of these cells isolated from patient prostatectomy tissues are currently being standardized. Citation Format: Jiaquan Yu, Ashley M. Weichmann, Alexandria Craig, Wei Huang, Dawn R. Church, Farideh Mehraein, Laurie L. Parker, David J. Beebe, George Wilding, Hirak S. Basu. “Moonlighting Functions” of glycolytic enzymes relate to human prostate cancer invasion. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4968.

1,14-Bis-(ethyl)-amino-5,10-diazatetradecaneN 1,N 11-bis(ethyl)norspermine (BE-4-4-4) and 1,19-bis-(ethylamino)-5,10,15 triazanonadecane (BE-4-4-4-4) are two relatively new polyamine analogs synthesized for use as antineoplastic agents. In human brain tumor cell lines U-251 MG and SF-767, both agents inhibited cell growth, were cytotoxic, induced a variable G1/S block, and depleted intracellular polyamines. Since intracellular polyamine depletion did not always correlate with growth inhibition, cell survival, or cell cycle progression, it cannot completely explain the effects of these agents on growth, survival, and cell cycle progression in U-251 MG and SF-767 cells.

The polyamine analog 1,14-bis(ethylamino)-5,10-diazatetradecane (BE-4-4-4) depletes polyamines and inhibits the growth of tumor cells in tissue culture. We treated HeLa cells in culture with BE-4-4-4 for different time periods to produce different degrees of polyamine depletion. The cells were lysed and dehistonized to obtain nucleoids containing DNA attached to the nuclear matrix. Titration of the nucleoids with propidium iodide caused an uncoiling of negatively supercoiled DNA, resulting in the formation of a halo surrounding the nucleoid periphery. The halo diameters in both the BE-4-4-4-treated cells and the untreated control cells were measured using a fluorescence image analysis system. As compared to the control cells, the BE-4-4-4-treated cells showed a 20-25% decrease in halo diameter, indicating that there was less relaxation of the negative supercoils in the nuclear DNA of the BE-4-4-4-treated cells than in the controls.

The interactions of spermine and polyamine analogues with synthetic polynucleotides of various base sequences complexed with ethidium bromide (EB) were investigated using measurements of fluorescence intensity and steady-state fluorescence polarization. Spermine and polyamine analogues displaced some but not all of the EB bound to poly(dA-dT).poly(dA-dT) or poly(dG-dC).poly(dG-dC), suggesting that polyamines may stabilize these polynucleotides in a conformation with reduced affinity for EB. Modifications of the aliphatic backbone of spermine have pronounced effects on its ability to displace EB from poly(dA-dT).poly(dA-dT) but not from poly-(dG-dC).poly(dG-dC). Spermine and some but not all of the polyamine analogues caused fluorescence depolarization when they interacted with the complex of EB and poly(dA-dT).poly-(dA-dT). Neither spermine nor any of the analogues, however, induced fluorescence depolarization in the complex of EB with poly(dG-dC).poly(dG-dC) or poly(dA).poly(dT). This suggests that spermine and some spermine analogues induce structural changes specific to alternating A-T sequences.

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The relatively high oxidative stress in the prostate is postulated to be one of the major factors for prostate carcinogenesis and prostate cancer (CaP) progression. We focused on elucidating metabolic pathways of oxidative stress generation in CaP cells. Previously, we have shown that the transcription factor JunD is essential for the androgen-induced reactive oxygen species (ROS) production in androgen-dependent LNCaP human prostate cancer cells. We have also recently demonstrated that androgen induces the first and the regulatory enzyme spermidine/spermine N1-acetyl transferase (SSAT) in a polyamine catabolic pathway that produces copious amounts of metabolic ROS. Here, we present co-immunoprecipitation and Gaussia luciferase reconstitution assay data that show that JunD forms a complex with activated AR in situ. Our chromatin immnoprecipitation (ChIP) assay data demonstrate that JunD binds directly with a specific SSAT promoter sequence only in androgen-treated LNCaP cells. Using a vector containing a luciferase reporter gene connected to the SSAT promoter and a JunD silenced LNCaP cell line, we show that JunD is an essential co-activator of androgen-induction of SSAT gene expression. As there is no Androgen Response Element (ARE) in the SSAT promoter, we hypothesize that JunD mediates binding of androgen-activated AR to the SSAT promoter for the induction of SSAT that leads to polyamine oxidation and ROS production in prostate cells. Elucidation of this pathway may help the design of inhibitors of oxidative stress generation specifically in prostate cells to prevent prostate cancer occurrence and progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4386.

High levels of reactive oxygen species (ROS) present in human prostate epithelia are an important etiologic factor in prostate cancer (CaP) occurrence, recurrence, and progression. Androgen induces ROS production in the prostate by a yet unknown mechanism. Here, to the best of our knowledge, we report for the first time that androgen induces an overexpression of spermidine/spermine N1-acetyltransferase, the rate-limiting enzyme in the polyamine oxidation pathway. As prostatic epithelia produce a large excess of polyamines, the androgen-induced polyamine oxidation that produces H2O2 could be a major reason for the high ROS levels in the prostate epithelia. A small molecule polyamine oxidase inhibitor N,N'-butanedienyl butanediamine (MDL 72,527 or CPC-200) effectively blocks androgen-induced ROS production in human CaP cells, as well as significantly delays CaP progression and death in animals developing spontaneous CaP. These data show that polyamine oxidation is not only a major pathway for ROS production in prostate, but inhibiting this pathway also successfully delays CaP progression. [Cancer Res 2009;69(19):7689–95]

Modeling, x-ray diffraction, and solution studies have contributed to the understanding of interactions between polyamines and nucleic acids. Polyamines stabilize a variety of unusual DNA structures and conformations in vitro, including both the left-handed Z and the right-handed A DNA. In addition, polyamines condense DNA and may be important in bending specific sequences. Investigations into the mechanisms of these effects provide support for both specific and nonspecific interactions between polyamines and DNA. Although exact relationships between the binding of polyamines and conformational changes in nucleic acids are still being clarified, polyamines remain important candidates for regulators of DNA conformation in vivo.

Two new photoaffinity derivatives of polyamines have been synthesized by the reaction of spermine or spermidine with methyl 4-azidobenzimidate. The new compounds were purified chromatographically and characterized by several methods including proton magnetic resonance spectroscopy. The spermine derivative is N1-ABA-spermine [(azidobenzamidino)spermine], and the spermidine derivative is a mixture of N1- and N8-ABA-spermidine. ABA-spermine stabilizes nucleosome core particles in thermal denaturation experiments, with similar but not identical effects when compared with the parent polyamine, spermine. In circular dichroism experiments, ABA-spermine was capable of producing a B----Z transition in poly(dG-m5dC) at a concentration of 30 microM, compared with 5 microM required to produce the same effect with spermine. On the other hand, ANB-spermine [(azidonitrobenzoyl)spermine; Morgan, J. E., Calkins, C. C., & Matthews, H. R. (1989) Biochemistry 28, 5095-5106] stabilized the B form of poly(dG-br5dC). ABA-spermine is a potent inhibitor of ornithine decarboxylase from Escherichia coli, giving 50% inhibition at 0.12 mM, while ANB-spermine is a modest inhibitor, comparable to spermine or spermidine. Under conditions of nitrogen-limited growth, yeast take up ABA-spermine and ABA-spermidine at approximately one-third to half the rate of spermidine or spermine. In contrast, ANB-spermine was not significantly taken up. The photoaffinity polyamines were used to photoaffinity label the DNA in nucleosome core particles, and the sites of labeling were determined by exonuclease protection. All photoaffinity reagents showed both nonspecific labeling and specific sites of higher occupancy. However, the positions of the sites varied: the ANB-spermine sites confirmed those previously reported (Morgan et al., 1989); the ABA-spermine and ABA-spermidine sites were spaced at 9.8 bp intervals from the 3' end of each DNA strand. This observation, together with the effect of spermine on the circular dichroism of DNA in nucleosome core particles, implies that polyamines alter the helical twist of DNA in nucleosome core particles. The ABA-polyamines are offered as general-purpose photoaffinity polyamine reagents.

The effects of spermine and spermine analogues on the B-Z transition of poly(dG-me5dC) and on the aggregation and ‘melting’ temperature of calf thymus DNA were studied by spectroscopic methods. The association constants of these polyamines with double- and single-stranded calf thymus DNA were calculated from their effects on the melting temperature. The effect of these compounds on the release of ethidium bromide (EB) from an EB-DNA complex were measured by a spectrofluorimetric method. This efficiency of the polyamine-induced B-Z transition strongly depended on the length of the central carbon chains of the compounds and on the functional groups attached to the carbon chains. Both the terminal primary amino groups and the length of the central carbon chain affected the aggregation of DNA. The affinity of the analogues for DNA increased as the number of n-butyl groups increased, but decreased with either an increase or a decrease in the length of the central carbon chain. The effect of spermine and spermine analogues on the release of EB from an EB-DNA complex did not always correlate with the affinities of analogues for calf thymus DNA. In particular, tetra-amines with more than one n-butyl group bound better to DNA than did spermine, but released bound EB and induced aggregation of DNA less well than did spermine. We postulate that either a bend and/or other localized conformational changes of DNA are responsible for the spermine-induced aggregation of DNA and the release of EB from the EB-DNA complex.

Purpose: Anti-androgens are widely used in androgen deprivation therapy (ADT), a standard-of-care for patients with recurrent prostate cancer (PCa). Unfortunately, most patients ultimately develop resistance to ADT and progress to castrate-resistant prostate cancer (CRPC). Recently, two agents that block androgen receptor activation [abiraterone acetate (Zytiga, J&J) and enzalutamide (Xtandi, Medivation/Astellas)] have been approved for CRPC therapy. While some patients respond to these therapies, many fail. We investigated if sequestration of metabolically aberrant mitochondria in the autophagosomes (mitophagy) imparts anti-androgen resistance. Method: We studied the effects of anti-androgen enzalutamide on the autophagy of androgen-dependent LNCaP and -independent C4-2 cells. Autophagy was monitored by cellular fluorescence in cells treated with monodansylcadavarine (MDC). Cellular fluorescence due to Mitosox dye oxidation was used to identify mitochondria producing high superoxide (O2-). Mitophagy of O2- producing mitochondria was monitored using fluorescence resonance energy transfer (FRET) between MDC and Mitosox in a 96-well plate based high throughput (HTS) assay and by visualization of FRET images and quantitation of FRET image intensities using a Nikon A1 fluorescence confocal microscope and associated software. Results: Our data show that in low androgen media, the degree of autophagy is less in C4-2 cells than in LNCaP cells. Enzalutamide treatment induces autophagy in both cell lines, but the increase in autophagy is more pronounced in the androgen-independent C4-2 than in the -dependent LNCaP cells. FRET data from both HTS and fluorescence microscopy show that while enzalutamide increases mitophagy of O2- producing mitochondria in C4-2 cells in a dose dependent manner, it decreases after an initial increase in LNCaP cells. Mitophagy of such mitochondria has also been observed by FRET based fluorescence microscopy in live circulating tumor cells (CTCs) isolated from blood sample of a patient undergoing enzalutamide therapy. Conclusion: Our data demonstrate that PCa cells resistant to enzalutamide show high degree of mitophagy of O2- mitochondria in autophagosomes. If this effect correlates with CTCs from blood collected from CRPC patients undergoing enzalutamide therapy, it can become a clinically useful method of identifying patients who are most likely to benefit from enzalutamide treatment. Citation Format: Hirak S. Basu, Cynthia L. Schrieber, Jamie M. Sperger, Maryanne Naundorf, Ashley M. Weichman, Farideh Mehraein-Ghomi, Dawn R. Church, Joshua M. Lang, George Wilding. Mitophagy imparts enzalutamide resistance in prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2899. doi:10.1158/1538-7445.AM2015-2899

BACKGROUND. The androgen receptor and activator protein-1 (AP-1) transcription factors affect growth regulation in normal and cancerous prostate cells. Effects of androgen-activated androgen receptor on AP-1 activity were determined in the LNCaP human prostate carcinoma cell model. METHODS. Cells were exposed to 1 nM androgenantiandrogen bicalutamide. Cellular growth and cell cycle effects were determined by DNA, viability, and bromodeoxyuridine (BrdU)fluorescenceactivatedcellsorter(FACS)assays.AP-1effectsweredeterminedbyanAP- 1-luciferase enzyme reporter vector for transcriptional activity, electrophoretic mobility shift assay (EMSA)/antibody supershift for DNA-binding, quantitative RT-PCR for mRNA, and immunoblot for protein. RESULTS. Androgen induced G1 growth arrest. This growth arrest was abrogated by treatment with bicalutamide, demonstrating that growth arrest by androgen was due to andro- gen receptor activation. Concurrently, AP-1 DNA-binding and transcriptional activity was inducedover96hrandrogenexposure,whichwasalsoinhibitedbybicalutamide.Interestingly, although no change in AP-1 transcriptional activity was observed 24 hr after androgen exposure, there was an increase in Fra-2 expression and AP-1 DNA-binding. Paradoxically, while Fra-2 mRNA and protein levels continued to increase, binding of Fra-2 to the AP-1 site decreased over 96 hr,with a concomitant increase in JunD AP-1-binding and amarked increase in expression of the 35 kDa form of JunD. Enhanced expression of this short form of JunD is a novel effect of androgen exposure that occurred during the 24-96 hr time period, as growth effects emerged. CONCLUSION. Activation of androgen receptor by androgen induces changes in AP-1 activity and AP-1 factor DNA-binding that may contribute significantly to androgen-induced changes in prostate cancer cell growth. Prostate 63: 155-168, 2005. # 2004 Wiley-Liss, Inc.

Polyamine analogues 7, 10, 18, 27, and 32 containing cyclopropane rings were obtained by chemical synthesis. Their antineoplastic activities were assessed against the cultured human prostate tumor cell lines DU-145, DuPro, and PC-3. Decamines 32 and 27 exhibited variable levels of cytotoxicity against all three cell lines, while 7, 10, and 18 were efficacious against DU-145 and DuPro. Maximum tolerated doses (MTD) for all five compounds in a NCr-nu mouse model were determined at dosing schedules of q1d x 5 (ip) in two cycles with a break of 10 days between cycles. Their antitumor efficacies were then tested against DU-145 tumor xenografts in mice treated with all five agents at their respective MTDs. In addition, the efficacies of 7 and 10 against the same tumor xenograft were assessed at doses below their respective MTDs. In all experiments, administration began two weeks after tumor implantation. All compounds efficiently inhibited tumor growth for up to 50 days postimplantation, with negligible animal body weight loss. Tetramine 10 and hexamine 18 were the most efficient among the five analogues in arresting tumor growth. Tetramine 10 containing two cyclopropane rings had the lowest systemic toxicity as reflected in animal body weight loss. It was further assessed at a weekly administration regimen of (q1w x 4) in two cycles with a four-week break between the cycles. At this dosing schedule, 10 again efficiently arrested tumor growth with negligible effect on animal body weight. Tetramine 10 also arrested the growth of large tumors (ca. 2000 mm(3)) treated 66 days postimplantation. Studies on the metabolism of 10 showed that it accumulates in tumor within 6 h after the end of administration and reached a maximum level 72 h after cessation of dosing. Intracellular concentrations of 10 in liver and kidney were much smaller when compared to those in the tumor when measured 72 h after cessation of dosing. In liver and kidney, the deethyl metabolites of 10 accumulated over a 96 h period after cessation of dosing.

Purpose: The polyamine analog bis(ethylamino)-5,10,15-triazanonadecane (BE-4-4-4-4) depletes cellular polyamines and inhibits malignant cell growth. We have previously shown that BE-4-4-4-4 inhibits nucleosome condensation on supercoiled DNA in a cell-free system. Here we sought to determine whether BE-4-4-4-4 inhibits nucleosome condensation in cells, and whether that effect alters the expression of specific genes. Methods: We used the simian virus 40 (SV-40) minichromosome as a model system and studied the expression of the viral late genes. It is known that the SV-40 late genes are regulated by the steroid receptor elements that, in turn, control gene expression by altering nucleosomal organization. Results: We observed a more than six fold increase in SV-40 late gene expression in cells pretreated with BE-4-4-4-4 for 18 h. The polyamine analog bisethyl norspermine (BE-3-3-3), that does not affect nucleosomal condensation in cell free systems and has little effect on chromatin structure in cultured human tumor cells, had a negligible effect on SV-40 late gene expression under treatment conditions identical to those used with BE-4-4-4-4. Conclusion: Similar to the findings in the cell-free system, the polyamine analog BE-4-4-4-4 inhibited nucleosome formation and, thereby, altered the expression of specific genes in a cellular system.

Purpose: During the last decade, several polyamine analogs have been developed as antineoplastic agents that replace intracellular polyamines but cannot mimic the biological functions of polyamines related to cell growth. It has been shown that pretreatment of several human brain tumor cell lines with some of these polyamine analogs increases the cytotoxicity of cis-diamminedichloroplatinum (CDDP). It has also been established that some of these polyamine analogs affect chromatin organization. In the study reported here we attempted to elucidate the mechanism by which polyamine analog-induced changes in DNA and chromatin structure may increase CDDP cytotoxicity. Methods: We studied the micrococcal nuclease sensitivity of the nuclei and measured the amount of platinum incorporated into the nucleosomal and linker regions of chromatin isolated from CDDP-treated U-251 MG human malignant brain tumor cells with or without pretreatment with two cytotoxic polyamine analogs 1,11-bis(ethylamino)-4,8-diazaundecane (BE-3-3-3) and 1,19-bis(ethylamino)-5,10,15-diazanonadecane (BE-4-4-4-4). Results: The pretreatment with the polyamine analogs decreased the MNase sensitivity and increased the incorporation of CDDP preferentially into the linker region of the chromatin. Conclusions: Pretreatment of cells with polyamine analogs probably alters the structure and/or the organization of the linker region such that more CDDP incorporates into the linker DNA. This is probably the reason for the observed enhancement of CDDP cytotoxicity in the polyamine analog-pretreated cells.

Purpose: Polyamines are biologic cations necessary for normal cell growth. Polyamine analogues have been shown to be effective inhibitors of tumor growth. We tested the effect of the polyamine analogues 1,19-bis(ethylamino)-5,10,15-triazanonadecane (BE-4-4-4-4), N1,N11-bis(ethyl)norspermine (BE-3-3-3) and 1,15-bis(ethylamino)-4,12-diazapentadecane (BE-3-7-3) on the growth of the prostate cancer cell lines DU145, LNCaP and PC-3 in vitro. We also tested the effect of␣BE-4-4-4-4 on androgen-independent DU145 cells in␣vivo via a nude mouse xenograft model.Methods: In␣vitro, cell proliferation was measured using a DNA assay or a colony-formation assay. In vivo, mice were given saline or BE-4-4-4-4 3 mg/kg or 5 mg/kg intraperitoneally twice daily on days 7–10 and 14–17 (cycle 1), days 49–52 and 56–59 (cycle 2) and days 91–94 and 98–101 (cycle 3).Results: The proliferation of DU145, LNCaP and PC-3 prostate cancer cell lines was inhibited in a dose-dependent manner by BE-4-4-4-4. Intracellular putrescine, spermidine and spermine levels in all three cell lines declined after only 24 h exposure to BE-4-4-4-4 in vitro. Animals receiving BE-4-4-4-4 showed inhibition of tumor growth which continued throughout the experiment with 74% (3 mg/kg) and 81% (5 mg/kg) growth inhibition seen on day 101. No overt toxic reactions besides weight loss were observed in BE-4-4-4-4-treated animals. Tumor tissue from animals treated with BE-4-4-4-4 showed a dose-dependent decrease in spermidine and spermine levels but no decline in putrescine levels as compared with control. BE-4-4-4-4 levels were highest in tumors on day 63 with levels reaching 0.33 and 1.45 nmol/mg protein from animals treated at the 3 mg/kg and 5 mg/kg doses, respectively. Conclusion: These results show the polyamine analogues BE-4-4-4-4, BE-3-3-3 and BE-3-7-3 to be effective inhibitors of prostate cancer cell growth in vitro and BE-4-4-4-4 to be an effective inhibitor of DU145 cells in vivo with minimal toxicity.

Nucleoid halo diameters were measured to assay changes in DNA supercoiling in human brain tumor cell line SF-126 after irradiation under aerobic or hypoxic conditions. In unirradiated aerobic cells, a typical propidium iodide titration curve showed that with increasing concentrations of propidium iodide, the halo diameter increased and then decreased with the unwinding and subsequent rewinding of DNA supercoils. In irradiated cells, the rewinding of DNA supercoils was inhibited, resulting in an increased halo diameter, in a radiation dose-dependent manner. To produce equal increases in halo diameter required about a threefold higher radiation dose in hypoxic cells than in aerobic cells. Quantitatively similar differences in the radiation sensitivities of hypoxic and aerobic cells were demonstrated by a colony-forming efficiency assay. These findings suggest that the nucleoid halo assay may be used as a rapid measure of the inherent radiation sensitivity of human tumors.

Development of an effective therapy to prevent prostate cancer (PCa) progression to castrate-resistant PCa (CRPCa) remains an unmet medical need, mainly due to a poor understanding of the mechanism of PCa progression. Reactive oxygen species (ROS) are produced in high amounts in PCa cells. ROS play a major role in the development and progression of PCa. We have published that the activated androgen receptor (AR)-JunD complex induces spermidine/spermine N1-acetyl transferase (SSAT). SSAT is the first, and a regulatory, enzyme in a major polyamine catabolism pathway that leads to an over-production of ROS, specifically in the polyamine-rich PCa cells. A focus of our current research is to identify compounds that specifically target and block steps in this newly discovered ROS production pathway downstream to AR activation. Such compounds have the potential to become new targeted therapeutic agents offering minimal side effects for preventing progression to CRPCa in patients with early stage progressing PCa. Here we present data on drug-like small molecule inhibitors of the AR-JunD interaction that initiates this ROS-generating pathway in PCa. A novel high throughput assay based on Gaussia Luciferase enzyme reconstitution via protein-protein interaction was used to screen NCI Diversity Set and Life Chemicals libraries of drug-like small molecules to identify inhibitors of the AR-JunD interaction. The 27 selected hits were categorized as antiandrogens or non-antiandrogens through the use of a published fluorescence polarization assay. Sixteen non-antiandrogens, acting downstream of AR activation, were chosen for further study. In vitro studies entailed the measurement of the ability of the compounds to inhibit androgen-independent and dependent growth as well as androgen-induced ROS in human PCa cells. The top four compounds exhibited significant inhibition of growth and androgen-induced ROS at concentrations of 5 uM or less. Immunocytochemistry (ICC) studies were performed to determine the ability of the selected compounds to specifically inhibit the AR-JunD co-translocation to the nuclei. Based on the cell culture and ICC data, lead compounds were selected for pharmacokinetics studies to determine oral bioavailability. Detailed pharmacokinetic data of selected top compounds showing their bioavailability after intra venous and oral administration will be presented. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2844. doi:1538-7445.AM2012-2844

Development of an effective therapy to prevent PCa occurrence, recurrence and/or progression remains an unmet medical need. This is mainly due to a poor understanding of the mechanism of PCa occurrence and progression. There is accumulating evidence that ROS (Reactive Oxygen Species) such as hydrogen peroxide, superoxide, hydroxyl radical, etc. that are produced in high amounts in PCa cells may be a major factor in PCa occurrence and its progression to androgen-independence. One of the ROS (H2O2) plays a key role in triggering growth signals and expression of transcription factors to sustain growth of androgen-dependent PCa cells in the absence of androgen. One such factor is Nuclear Factor κB (NF-κB) that may sustain cell survival by preventing apoptosis and inducing cell proliferation. NF-κB activation kinetically correlates with androgen-induced ROS production in cultured LNCaP human PCa cells. This activation is also seen in resected PCa tissues from patients, who subsequently progressed to CRPCa. Our published data show that androgen induces an up-regulation of spermidine/spermine acetyl transferase (SSAT) mRNA as well as enzyme activity that initiates polyamine oxidation in LNCaP cells after 72 h exposure. Because of the unusually high polyamine levels in the PCa cells, polyamine oxidation causes a large increase in ROS production that is abrogated by silencing SSAT expression in PCa cells stably transfected with shRNA against SSAT (siSSAT). To determine whether SSAT is required for NF-κB activation following prolonged androgen stimulus, we transfected 3xκB-luciferase vector containing NF-κB consensus binding sites-luciferase reporter construct along with a β-gal expression vector (to control for the transfection efficiency) into both wild type LNCaP cells and its siSSAT clones. While 72 h androgen exposure causes an over 30-fold induction of luciferase activity in wild type LNCaP cells, no activation of NF-κB was observed in the siSSAT clones. These data clearly show a strong SSAT induction is required for a robust activation of NF-κB in androgen-treated PCa cells. Multiple published reports show that NF-κB can also induce SSAT in several human cancer cells. Thus, our data suggest an intriguing mechanism of CRPCa progression, where androgen induced SSAT expression and consequent upregulation of NF-κB can set up an autocrine feed forward loop of SSAT-ROS-NFκB-SSAT that can sustain PCa cell proliferation in the absence of androgen that can lead to CRPCa growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2165. doi:10.1158/1538-7445.AM2011-2165

Development of an effective therapy to prevent prostate cancer (PCa) progression to castrate-resistant PCa (CRPCa) remains an unmet medical need, mainly due to a poor understanding of the mechanism of PCa progression. Reactive oxygen species (ROS) are produced in high amounts in PCa cells and play a major role in PCa development and progression. We have published that activated androgen receptor (AR)-JunD complex induces spermidine/spermine N1-acetyl transferase (SSAT), the first and regulatory enzyme in a major polyamine catabolism pathway that yields over-production of ROS specifically in the polyamine-rich PCa cells. Our recent data further suggest an intriguing mechanism of PCa progression, where AR-JunD induced SSAT expression and consequent upregulation of the transcription factor NF-κB may set up an autocrine feed forward loop of SSAT-ROS-NFκB-SSAT that can sustain ROS production and PCa cell proliferation in the absence of androgen. A focus of our current research is to identify compounds that specifically target and block steps in this novel pathway downstream to AR activation and thereby have potential to be new targeted therapeutic agents to prevent PCa progression to CRPCa in early stage progressing PCa patients with minimal side effects. In the research presented here we utilized a novel high throughput screen (HTS) assay to find compounds that prevent the initiating AR-JunD interaction step in this ROS generating pathway and tested their efficacy in PCa cells. A high throughput assay based on Gaussia Luciferase enzyme reconstitution via protein-protein interaction was used to screen the NCI diversity set library of drug like molecules to identify inhibitors of the AR-JunD interaction. Selected hits were further screened to determine their ability to bind to the AR using a published fluorescence polarization assay in order to categorize the molecules as non-antiandrogens or antiandrogens. As we intend to target the pathway downstream of AR activation, we focused on non-antiandrogens, further testing them for efficacy against androgen induced ROS generation and effect on cell growth in PCa cells in vitro by our published DCFH dye oxidation and DNA fluorescence assays. Of the 14 hits from the HTS assay of the NCI diversity set library, nine small molecules were chosen based on their drug-like chemical characteristics for further studies along with two synthesized analogs of one of the selected small molecules. We have categorized six agents as non-antiandrogens and five as having some antiandrogenic activity. Our ROS assay identified a lead non-antiandrogen compound that blocks androgen induced ROS production in PCA cells at less than 1uM concentration. This agent also significantly inhibited androgen-independent growth of PCa cells at 4uM. Data for all compounds will be presented. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2583. doi:10.1158/1538-7445.AM2011-2583

The relatively high oxidative stress in the prostate is postulated to be one of the major factors for prostate carcinogenesis and prostate cancer (CaP) progression. We focused on elucidating metabolic pathways of oxidative stress generation in CaP cells. Previously, we have shown that the transcription factor JunD is essential for the androgen-induced reactive oxygen species (ROS) production in androgen-dependent LNCaP human prostate cancer cells. We have also recently demonstrated that androgen induces the first and the regulatory enzyme spermidine/spermine N1-acetyl transferase (SSAT) in a polyamine catabolic pathway that produces copious amounts of metabolic ROS. Here, we present co-immunoprecipitation and Gaussia luciferase reconstitution assay data that show that JunD forms a complex with activated AR in situ. Our chromatin immnoprecipitation (ChIP) assay data demonstrate that JunD binds directly with a specific SSAT promoter sequence only in androgen-treated LNCaP cells. Using a vector containing a luciferase reporter gene connected to the SSAT promoter and a JunD silenced LNCaP cell line, we show that JunD is an essential co-activator of androgen-induction of SSAT gene expression. As there is no Androgen Response Element (ARE) in the SSAT promoter, we hypothesize that JunD mediates binding of androgen-activated AR to the SSAT promoter for the induction of SSAT that leads to polyamine oxidation and ROS production in prostate cells. Elucidation of this pathway may help the design of inhibitors of oxidative stress generation specifically in prostate cells to prevent prostate cancer occurrence and progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4386.

The chromatin structure of polyamine-depleted U-87 MG human brain tumour cells was studied by following the kinetics of digestion of cell nuclei by micrococcal nuclease and bovine pancreatic DNAase I. Cells growing in monolayers were treated with either alpha-difluoromethylornithine (DFMO), to deplete putrescine and spermidine, or N1,N14-bis(ethyl)homospermine (BE-4-4-4), to deplete putrescine, spermidine and spermine. BE-4-4-4 increased the initial rates of digestion and the magnitudes of limit digest by both enzymes; DFMO increased the limit digests without affecting initial digestion rates. Addition of 1 mM-putrescine 1 day after addition of DFMO reversed the effect of DFMO on limit digests. (Because polyamine uptake is low in cells treated with BE-4-4-4, and because putrescine does not reverse the growth-inhibitory effects of BE-4-4-4, reversal of the effects of BE-4-4-4 with putrescine was not attempted.) The increases in initial rates and limit digests did not result from changes in the lengths of nucleosomal or linker DNA, from blocks in cell-cycle progression, or from growth inhibition caused by DFMO or BE-4-4-4. Thus, because the limit digest is highest in cells with the lowest polyamine levels, it seems clear that the enhanced enzymic digestion of nuclei is caused by polyamine depletion and its possible effect on chromatin structure.

Many human malignancies such as prostate cancer (CaP) and primary brain tumor (GBM) exhibit high oxidative stress. Developing a successful therapeutic drug to treat advanced metastatic CaP and GBM is a major unmet medical need. It has been demonstrated that in certain chemo- and/or radiation resistant cancer cells with high oxidative stress, cells depend on reactive oxygen species (ROS) for survival and proliferation. Therefore, reducing ROS can be a successful strategy for developing chemotherapeutic drugs for use against chemo- and/or radiation-resistant tumors. The mitochondria are a major source of superoxide and other ROS. We focused on targeting spin-trapping nitroxide analogs at the mitochondrial interstitial space with appropriate linker length in order to remove the superoxide in order to reduce the cellular ROS levels without interfering with the mitochondrial electron transport chain. Our lead compound in this new class of drugs is Mito-tempol-C10. The drug successfully blocked ROS production in human CaP and GBM cell lines as determined by DCF dye oxidation assay. It exhibited marked growth inhibitory effect against androgen-dependent as well as androgen-independent cultured human CaP cells (IC50 < 1 µM) and similar growth inhibitory activity against several human GBM cells lines as well as cultured primary brain tumor stem cells as determined by DNA fluorescence measurement assay. Preliminary pharmacokinetic (PK) study show that the drug has serum half-life of approximately 45 minutes, but is retained by the tumor and other animal tissue for more than 24 hours. The drug is tolerated by ICR white mice, nude mice and NOD-SCID mice at a dose of 15 mg/kg i.p. given once every week for four weeks without any overt systemic toxicity. Preliminary efficacy study at this treatment condition showed marked effect on the growth of intracranially implanted, radiation-resistant U-87 MG human GBM cells in NOD-SCID mice and a small, but significant effect on the growth of androgen-independent PC-3 human prostate cancer cell xenografts growing on the flanks of nude mice. These results confirm that mitochondria-targeted spin-traps that reduce cellular ROS can be therapeutically effective in treatment of advanced, hard-to-treat human tumors with high oxidative stress. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B203.

We studied the effects of spermine, two naturally-occurring pentamines isolated from the thermophile Thermus thermophilus and one synthetic pentamine on the aggregation and ‘melting’ temperature of calf-thymus DNA and on the B-to-Z transition of poly(dG-me5dC). All pentamines caused aggregation of DNA at much lower concentrations than that of spermine. Concentrations that increased the melting temperature of DNA and induced the B-to-Z transition in poly(dG-me5dC) were different for each pentamine, but were comparable with the concentration of spermine needed to cause these effects. Our results suggest that both the total charge and the distance separating the charge, which is a function of the length of the carbon chains between amino groups, are important for the induction of conformational changes in DNA. The biological role of pentamines in T. thermophilus appears to be related to their ability to promote DNA condensation at high temperature.

Protonated polyamines are among the most efficient cations that induce the left-handed Z-form in certain polynucleotides. It is not known, however, whether these cations bind to specific sites on Z-sequences in solution. We have studied potential polyamine binding sites by measuring the effects of polyamines on the binding of purified immunoglobulins (IgGs) to different regions of the Z-helix and by molecular mechanics modeling. The specific binding of anti-Z-DNA and anti-Z-RNA IgGs to Z-helices was studied as a function of spermidine or spermine concentration. The effect of polyamines on the antibody-nucleic acid interaction was different for IgGs with different specificities for various determinants on the Z-helix. Polyamines inhibit the binding of certain anti-Z IgGs directed against specific sites probably at or near the interface between the major convex surface and the phosphate backbone, most likely by competing with the antibody binding site(s). In contrast, polyamines have no effect on other anti-Z IgGs directed against sites determined by the phosphate backbone. Furthermore, these cations can enhance the binding of anti-Z IgG directed against bulky groups at the C-5 position on the major convex surface of the helix; the enhancement may be related to charge neutralization. Under these conditions, no direct binding of antibodies with polyamines was observed. These data suggest the existence of a specific binding site(s) for polyamines on both Z-DNA and Z-RNA in solution. These binding sites have some similarity to those observed in oligonucleotide crystals by Quigley (in "Molecular Structure and Biological Activity," J.F. Griffin and W.L. Duax, eds., Elsevier, Amsterdam (1982), pp. 317-331). The experimental evidence for specific spermine binding sites on the helical surface was supported by molecular mechanics modeling of the interaction of spermine with the major groove of (dG-dC)5.(dG-dC)5 in both the Z- and B-forms. The crystal coordinates of spermine-containing oligonucleotides in both the B- and Z-forms were used as the starting points for modeling studies. The potential energy of spermine bound to the major convex surface of the Z-form was much less favorable than that of spermine bound to the major groove of the B-form. In the presence of sodium ions, however, the Z-form-spermine complexes were favored over the B-form. Thus, both theoretical and experimental studies indicate that polyamines can specifically recognize Z-helical determinants in solution as well as in crystals.

The rates of H-D exchange for imino and amino protons in adenosine, calf thymus DNA, poly (dA-dT), poly(dG-dC), and poly (dG-me5dC) were determined using stopped flow kinetic methods in the presence of various concentrations of Tris, imidazole, Mg2+, and spermine in citrate buffer (pH 7, 25 degrees C). CD spectroscopic studies showed that all polynucleotides always remain in the B-form under these conditions. An increase in the concentration of Tris and imidazole from 5 mu M to 20 mM caused an increase in the rates of exchange of both fast-exchanging imino and slow-exchanging amino protons. The limiting rates of exchange at infinite concentrations of catalysts were found to be different for fast (31-57 sec-1) and slow (1-2 sec-1) exchanging protons. These results indicate that imino and amino protons of B-DNA exchange asymmetrically from two different open states as observed for Z-DNA. An increase in the concentration of spermine from a ratio of 1:50 to 1:2 of positive charge/phosphate decreased the rate of exchange of imino protons of calf-thymus DNA, poly(dG-dC), and poly(dG-me5dC), but increased the rate of exchange of the imino protons of poly(dA-dT) without affecting the exchange rate of the amino protons of any of the polynucleotides. These results are interpreted in terms of possible spermine-induced change of conformations of oligonucleotides of specific sequence that has been suggested by theoretical model building studies.