Hirak S. Basu

JunD Mediates Androgen-Induced Oxidative Stress in Androgen Dependent LNCaP Human Prostate Cancer Cells

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.

Androgen receptor requires JunD as a coactivator to switch on an oxidative stress generation pathway in prostate cancer cells

Relatively high oxidative stress levels in the prostate are postulated to be a major factor for prostate carcinogenesis and prostate cancer (CaP) progression. We focused on elucidating metabolic pathways of oxidative stress generation in CaP cells. Previously, we showed that the transcription factor JunD is essential for androgen-induced reactive oxygen species (ROS) production in androgen-dependent human CaP cells. We also recently showed that androgen induces the first and regulatory enzyme spermidine/spermine N1-acetyltransferase (SSAT) in a polyamine catabolic pathway that produces copious amounts of metabolic ROS. Here, we present coimmunoprecipitation and Gaussia luciferase reconstitution assay data that show that JunD forms a complex with androgen-activated androgen receptor (AR) in situ. Our chromatin immunoprecipitation assay data show that JunD binds directly to 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 essential for androgen-induced SSAT gene expression. The elucidation of JunD-AR complex inducing SSAT expression leading to polyamine oxidation establishes the mechanistic basis of androgen-induced ROS production in CaP cells and opens up a new prostate-specific target for CaP chemopreventive/chemotherapeutic drug development.

Theoretical and experimental characterization of polyamine/DNA interactions.

For several years, work in our laboratory has been directed toward an understanding of the interactions of polyamines and nucleic acids. The initial impetus to this research was the finding that the cytotoxicity of antineoplastic agents such as cis-platinum and the 2-chloroethylnitrosoureas are altered in tumor cells in which intracellular levels of polyamines had been depleted by pretreatment with α-difluoromethylornithine (1, 2). It is thought that these agents kill cells by forming DNA interstrand crosslinks that prevent replication and lead to cell death. One possible explanation for the effects of polyamine depletion on cytotoxicity is that the conformation of intracellular DNA is altered, which would affect the mechanism of crosslink formation.

Expression of spermidine/spermine N1‐acetyl transferase (SSAT) in human prostate tissues is related to prostate cancer progression and metastasis

Prostate cancer (PCa) in many patients remains indolent for the rest of their lives, but in some patients, it progresses to lethal metastatic disease. Gleason score is the current clinical method for PCa prognosis. It cannot reliably identify aggressive PCa, when GS is ≤ 7. It is shown that oxidative stress plays a key role in PCa progression. We have shown that in cultured human PCa cells, an activation of spermidine/spermine N1‐acetyl transferase (SSAT; EC 2.3.1.57) enzyme initiates a polyamine oxidation pathway and generates copious amounts of reactive oxygen species in polyamine‐rich PCa cells.

Two polyamine analogs (BE-4-4-4 and BE-4-4-4-4) directly affect growth, survival, and cell cycle progression in two human brain tumor cell lines

Abstract1,14-Bis-(ethyl)-amino-5,10-diazatetradecaneN1,N11-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 mechanism of polyamine analog-induced enhancement of cisplatin cytotoxicity in the U-251 MG human malignant glioma cell line

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.

Targeting androgen receptor and JunD interaction for prevention of prostate cancer progression

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.

Prostate-specific antigen: a diagnostic marker and a tool for targeted delivery of drugs to prostate tumours

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 compared with other polyamine analogues. The toxicity may be further decreased by attaching the novel polyamine to a monoclonal antibody raised against PSA for targeted delivery to prostate tumours.

Abstract 2165: Identification of a novel pathway of castrate resistant prostate cancer (CRPCa) growth

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL 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

CYTOTOXICITY OF POLYAMINE ANALOGS IS DIRECTLY RELATED TO THEIR DNA AFFINITY AS DETERMINED BY POLYACRYLAMIDE GEL COELECTROPHORESIS (PACE) METHOD

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.