Thomas K. Takayama

Characterization of the Precursor of Prostate-specific Antigen ACTIVATION BY TRYPSIN AND BY HUMAN GLANDULAR KALLIKREIN

The precursor or zymogen form of prostate-specific antigen (pro-PSA) is composed of 244 amino acid residues including an amino-terminal propiece of 7 amino acids. Recombinant pro-PSA was expressed in Escherichia coli, isolated from inclusion bodies, refolded, and purified. The zymogen was readily activated by trypsin at a weight ratio of 50:1 to generate PSA, a serine protease that cleaves the chromogenic chymotrypsin substrate 3-carbomethoxypropionyl-l-arginyl-l-prolyl-l-tyrosine-p-nitroaniline-HCl (S-2586). In this activation, the amino-terminal propiece Ala-Pro-Leu-Ile-Leu-Ser-Arg was released by cleavage at the Arg-Ile peptide bond. The recombinant pro-PSA was also activated by recombinant human glandular kallikrein, another prostate-specific serine protease, as well as by a partially purified protease(s) from seminal plasma. The recombinant PSA was inhibited by α1-antichymotrypsin, forming an equimolar complex with a molecular mass of approximately 100 kDa. The recombinant PSA failed to activate single chain urokinase-type plasminogen activator, in contrast to the recombinant hK2, which readily activated single chain urokinase-type plasminogen activator. These results indicate that pro-PSA is converted to an active serine protease by minor proteolysis analogous to the activation of many of the proteases present in blood, pancreas, and other tissues. Furthermore, PSA is probably generated by a cascade system involving a series of precursor proteins. These proteins may interact in a stepwise manner similar to the generation of plasmin during fibrinolysis or thrombin during blood coagulation.

Substrate-Guided Design of a Potent and Selective Kallikrein-Related Peptidase Inhibitor for Kallikrein 4

Human kallikrein-related peptidase 4 (KLK4/prostase), a trypsin-like serine protease, is a potential target for prostate cancer treatment because of its proteolytic ability to activate many tumorigenic and metastatic pathways including the protease activated receptors (PARs). Currently there are no KLK4-specific small-molecule inhibitors available for therapeutic development. Here we re-engineer the naturally occurring sunflower trypsin inhibitor to selectively block the proteolytic activity of KLK4 and prevent stimulation of PAR activity in a cell-based system. The re-engineered inhibitor was designed using a combination of molecular modeling and sparse matrix substrate screening.

Recurrent Prostate Cancer Despite Undetectable Prostate Specific Antigen

Serum prostate specific antigen (PSA) is a sensitive tumor marker following definitive therapy for adenocarcinoma of the prostate. Detectable levels of PSA after radical prostatectomy indicate residual and/or recurrent cancer. Therefore, we were surprised to document recurrent cancer following radical retropubic prostatectomy in a man who had undetectable serum PSA levels. This observation suggests that careful clinical monitoring remains essential after definitive therapy, since rare patients may have recurrent and/or residual carcinoma despite undetectable levels of PSA.

Kallikrein-related peptidase-4 initiates tumor-stroma interactions in prostate cancer through protease-activated receptor-1

In prostate cancer, the mechanism by which the stromal cells surrounding the cancer epithelium become reactive and overproduce growth factors is unclear. Furthermore, the precise process of how these stromal cells stimulate the cancer epithelium is not fully understood. We recently found that protease‐activated receptor‐1 (PAR‐1) in these reactive stromal cells is upregulated. To investigate the role of PAR‐1 in the stromal–epithelial interaction, WPMY‐1 stromal myofibroblasts were stimulated with PAR‐1 agonists including thrombin and PAR‐1 activating peptide. We show that WPMY‐1 cells have functional PAR‐1 by signaling through ERK1/2. Conditioned media (CM) from PAR‐1 agonists‐treated WPMY‐1 cells stimulate the epithelial LNCaP cells leading to ERK1/2 activation and cell proliferation. Cytokine array analysis of the CM demonstrates that PAR‐1 induces stromal cells to release numerous cytokines, of which interleukin 6 (IL‐6) is the major factor responsible for mitogenic signaling in LNCaP cells. CM further induces expression of prostate‐specific kallikrein‐related peptidase‐3 (KLK3/PSA) and KLK4 in LNCaP cells via the IL‐6 pathway. Moreover, KLK4 functions as a potent agonist of PAR‐1 by cleaving the receptor at the proper site on cell surface. KLK4 triggers transmembrane signaling and upregulates IL‐6 in WPMY‐1 cells through PAR‐1. Immunohistochemical analysis indicates that PAR‐1 is predominantly expressed in peritumoral stroma while KLK4 is produced exclusively by the epithelial cancer cells. These data provide evidence for a novel double‐paracrine mechanism whereby cancer epithelium produces KLK4 to activate PAR‐1 in the surrounding stroma, which in‐turn releases cytokines (IL‐6) that stimulate cancer cells to proliferate and increase production of KLKs.

Gene expression down-regulation in CD90+ prostate tumor-associated stromal cells involves potential organ-specific genes

BackgroundThe prostate stroma is a key mediator of epithelial differentiation and development, and potentially plays a role in the initiation and progression of prostate cancer. The tumor-associated stroma is marked by increased expression of CD90/THY1. Isolation and characterization of these stromal cells could provide valuable insight into the biology of the tumor microenvironment.MethodsProstate CD90+ stromal fibromuscular cells from tumor specimens were isolated by cell-sorting and analyzed by DNA microarray. Dataset analysis was used to compare gene expression between histologically normal and tumor-associated stromal cells. For comparison, stromal cells were also isolated and analyzed from the urinary bladder.ResultsThe tumor-associated stromal cells were found to have decreased expression of genes involved in smooth muscle differentiation, and those detected in prostate but not bladder. Other differential expression between the stromal cell types included that of the CXC-chemokine genes.ConclusionCD90+ prostate tumor-associated stromal cells differed from their normal counterpart in expression of multiple genes, some of which are potentially involved in organ development.

Protease-activated receptor-1 is upregulated in reactive stroma of primary prostate cancer and bone metastasis.

Prostate cancer progression is partly facilitated by tumor–stroma interactions. We recently reported that protease‐activated receptors (PAR‐1 and PAR‐2) are overexpressed in prostate cancer, and PAR‐1 expression in peritumoral stroma is associated with biochemical recurrence. However, the nature of PAR expression in prostate tumor microenvironment is not fully understood. We therefore evaluated PAR‐1 and PAR‐2 expression in primary prostate cancer and bone metastasis.

Extensive palliative surgery for advanced mesothelioma of the tunica vaginalis

Malignant mesothelioma of the tunica vaginalis is a rare tumor managed principally by radical surgical resection. Chemotherapy and radiotherapy have limited efficacy. We report on a 67-year-old man with severe debilitation from multiple scrotal and inguinal recurrences of a malignant mesothelioma originating in the right tunica vaginalis. Local pain from extensive tumor spread prevented ambulation. Aggressive surgical debridement (total penectomy and scrotectomy) and perineal urethrostomy afforded the patient significant improvement in his quality of life before he finally died of the disease 3 years after diagnosis.

Protease-activated receptor-1 upregulates fibroblast growth factor 7 in stroma of benign prostatic hyperplasia.

Benign prostatic hyperplasia (BPH) is characterized by abnormal epithelial and stromal proliferation causing urinary obstruction. Prostate growth is regulated by a variety of growth factors secreted from the stroma, including fibroblast growth factor 7 (FGF‐7), a potent epithelial‐specific growth factor which is increased in hyperplastic prostate. However, the mediator(s) of FGF‐7 over‐expression is unclear. Protease‐activated receptor‐1 (PAR‐1) is a G‐protein coupled receptor known to induce multiple biological processes, but its effect on BPH pathogenesis is mostly unknown. The aim of this study was to investigate the role of PAR‐1 as a mediator of BPH development.

Protease-activated receptor 2 signaling upregulates angiogenic growth factors in renal cell carcinoma

Renal cell carcinoma (RCC) is a highly vascular tumor associated with expression of various angiogenic growth factors. The precise process of how these growth factors are regulated in RCC is not fully understood. Recent evidence suggests that protease activated receptors (PARs), a new family of G-protein coupled receptors, play a crucial role in vascular development and tumor progression through a variety of mechanisms. However, the nature of PAR expression in human RCC tissues and its function in regulating angiogenesis in RCC are largely unknown. In this study, we investigated the expression and function of PAR-2 in RCC. RT-PCR and immunohistochemistry assays show that PAR-2 expression is significantly increased in human RCC tissue compared with the adjacent non-neoplastic kidney tissue. In RCC derived cells, PAR-2 is functional as evidenced by robust signaling through MAP kinases including ERK1/2 and JNK. Furthermore, activation of PAR-2 significantly upregulates several angiogenic cytokines, including interleukin-6 (IL-6), IL-8, monocytes chemotactic protein-1 (MCP-1) and growth-related oncogene (GRO). To our knowledge, this is the first report that characterized PAR-2 expression in RCC tissue and further demonstrated that PAR-2 has a critical role in regulating angiogenesis in RCC.

Preoperative planning for renal cell carcinoma: benefits of 64-slice CT imaging

Surgery is the primary form of treatment in localized renal cell carcinoma. Adrenal-sparing nephrectomy, laparoscopic nephrectomy and nephron-sparing partial nephrectomy are growing trends for more limited surgical resection. Accurate preoperative imaging is essential for planning the surgical approach. Multislice CT and MR are regarded as the most efficient modalities for imaging renal neoplasms. Development of faster CT systems like 64-slice CT with improved resolution and capability to achieve isotropic reformats have significantly enhanced the role of CT in imaging of renal neoplasms. This review article describes the present state, technique and benefits of 64-slice CT scanning in preoperative planning for RCC.