Regina Gandour-Edwards

Arginine Deiminase as a Novel Therapy for Prostate Cancer Induces Autophagy and Caspase-Independent Apoptosis

Arginine deprivation as an anticancer therapy has historically been met with limited success. The development of pegylated arginine deiminase (ADI-PEG20) has renewed interest in arginine deprivation for the treatment of some cancers. The efficacy of ADI-PEG20 is directly correlated with argininosuccinate synthetase (ASS) deficiency. CWR22Rv1 prostate cancer cells do not express ASS, the rate-limiting enzyme in arginine synthesis, and are susceptible to ADI-PEG20 in vitro. Interestingly, apoptosis by 0.3 microg/mL ADI-PEG20 occurs 96 hours posttreatment and is caspase independent. The effect of ADI-PEG20 in vivo reveals reduced tumor activity by micropositron emission tomography as well as reduced tumor growth as a monotherapy and in combination with docetaxel against CWR22Rv1 mouse xenografts. In addition, we show autophagy is induced by single amino acid depletion by ADI-PEG20. Here, autophagy is an early event that is detected within 1 to 4 hours of 0.3 microg/mL ADI-PEG20 treatment and is an initial protective response to ADI-PEG20 in CWR22Rv1 cells. Significantly, the inhibition of autophagy by chloroquine and Beclin1 siRNA knockdown enhances and accelerates ADI-PEG20-induced cell death. PC3 cells, which express reduced ASS, also undergo autophagy and are responsive to autophagy inhibition and ADI-PEG20 treatment. In contrast, LNCaP cells highly express ASS and are therefore resistant to both ADI-PEG20 and autophagic inhibition. These data point to an interrelationship among ASS deficiency, autophagy, and cell death by ADI-PEG20. Finally, a tissue microarray of 88 prostate tumor samples lacked expression of ASS, indicating ADI-PEG20 is a potential novel therapy for the treatment of prostate cancer

Tumor suppressive miR-124 targets androgen receptor and inhibits proliferation of prostate cancer cells

Although prostate cancer (CaP) is the most frequently diagnosed malignant tumor in American men, the mechanisms underlying the development and progression of CaP remain largely unknown. Recent studies have shown that downregulation of the microRNA miR-124 occurs in several types of human cancer, suggesting a tumor suppressive function of miR-124. Until now, however, it has been unclear whether miR-124 is associated with CaP. In the present study, we completed a series of experiments to understand the functional role of miR-124 in CaP. We detected the expression level of miR-124 in clinical CaP tissues, evaluated the influence of miR-124 on the growth of CaP cells and investigated the mechanism underlying the dysregulation of miR-124. We found that (i) miR-124 directly targets the androgen receptor (AR) and subsequently induces an upregulation of p53; (ii) miR-124 is significantly downregulated in malignant prostatic cells compared to benign cells, and DNA methylation causes the reduced expression of miR-124; and (iii) miR-124 can inhibit the growth of CaP cells in vitro and in vivo. Data from this study revealed that loss of miR-124 expression is a common event in CaP, which may contribute to the pathogenesis of CaP. Our studies also suggest that miR-124 is a potential tumor suppressive gene in CaP, and restoration of miR-124 expression may represent a novel strategy for CaP therapy.

p53 and bcl-2 immunohistochemical alterations in prostate cancer treated with radiation therapy☆

OBJECTIVES Radiation therapy is definitive treatment for localized prostate cancer. It causes cellular deoxyribonucleic acid (DNA) damage, which, if irreparable, results in apoptosis or programmed cell death. Overexpression of mutant p53 and/or bcl-2 proteins prolongs cell survival despite exposure to damaging agents. We examined whether abnormal expression of either gene could help to explain radiation therapy failures in prostate cancer. METHODS Archival tissue from patients who had failed radiation therapy as treatment for prostate cancer was obtained before and after treatment. These cancer samples were examined immunohistochemically for accumulation of p53 and bcl-2 proteins. Comparison was made with specimens from patients who had no evidence of recurrent or persistent disease at least 3 years following radiation therapy. RESULTS High rates of p53 immunopositivity were found in the prostate tissue from all groups studied. More patients who had failed radiation therapy were found to have bcl-2 immunopositive specimens than were those without evidence for recurrent disease (41% preradiation and 61% postradiation versus 8%, P <0.05). More patients who failed radiation therapy had both p53 and bcl-2 immunopositive prostate tissue than did those who were treated successfully (32% preradiation and 48% postradiation versus 8%). CONCLUSIONS bcl-2 immunopositivity, with or without concomitant detection of p53, was found in significantly more cancers of patients who failed radiation therapy. Positive staining for bcl-2 may serve as a marker for determining the radiation sensitivity of a tumor and thus may help to guide treatment options. It is also notable that a high proportion of the prostate cancers examined were immunopositive for p53.

Neural cell adhesion molecule in adenoid cystic carcinoma invading the skull base

Neural cell adhesion molecules (N-CAMs) are expressed in neuromuscular tissues, neuroblastoma, and small cell lung carcinoma. Adenoid cystic carcinoma may invade the skull by either direct extension or neural involvement, particularly along the second and third divisions of the trigeminal nerve (V2 and V3). Eighteen patients with adenoid cystic carcinoma that invaded the skull base were studied. The tumors were graded into predominantly solid (3), cribriform (11), or tubular-trabecular (4) patterns, and neural involvement was evaluated histologically. Paraffin sections were examined by use of monoclonal antibodies for N-CAM and Ki-67, a proliferation marker, with the avidin-biotin-peroxidase method. Fifteen (83%) tumors showed perineural involvement; in the remaining three cases no nerves were present for histologic examination. Fourteen (93%) of 15 tumors with perineural involvement were reactive with N-CAM. Proliferation, measured by the presence of nuclear Ki-67, was markedly increased in tumors with predominantly solid patterns. We demonstrated that N-CAM is expressed in adenoid cystic carcinoma. The role of N-CAM as a neurodeterminant that facilitates the spread of adenoid cystic carcinoma along nerves, however, remains unanswered and warrants further study.

Immunolocalization of Low-Molecular-Weight Stress Protein HSP 27 in Normal Skin and Common Cutaneous Lesions

Stress proteins, which are found ubiquitously in mammalian cells, appear to be implicated in the regulation of cell growth and protection from environmental insult. Although we previously demonstrated the expression of low-molecular-weight stress protein, HSP 27, in cultured keratinocytes, HSP 27 has not yet been identified in human skin. Using standard immunohistochemistry on routinely processed paraffin sections, we examined specimens of common epidermal lesions and normal skin with a monoclonal antibody to HSP 27. Normal skin from the breast, foreskin, and lower extremity demonstrated strong cytoplasmic staining in the suprabasal epidermis. There was no change in the intensity of staining or cellular localization related to age, body location, or gender. Sections of actinic keratosis, superficial basal cell carcinoma, seborrheic keratosis, and psoriasis also exhibited strong cytoplasmic staining in the suprabasal layers of the epidermis. In contrast, cutaneous squamous cell carcinoma demonstrated only weak cytoplasmic staining throughout the infiltrating tumor. This is of particular interest, since other investigators have reported a loss of HSP 27 expression in oncogenically transformed cells that exhibit a tumorigenic phenotype. To our knowledge, this study provides the first demonstration of HSP 27 expression in human skin.

Near-infrared autofluorescence imaging for detection of cancer

Near-infrared autofluorescence imaging of tissues under long-wavelength laser excitation in the green and red spectral region complemented by cross-polarized elastic light scattering was explored for cancer detection. Various types of normal and malignant human tissue samples were utilized in this investigation. A set of images for each tissue sample was recorded that consisted of two autofluorescence images obtained under 532- and 632.8-nm excitation and light-scattering images obtained under linearly polarized illumination at 700, 850, and 1000 nm. These images were compared with the histopathology of the tissue sample. The experimental results indicated that for various tissue types, the intensity of the autofluorescence integrated over the 700 to 1000-nm spectral region was considerably different in cancer tissues than in that of the contiguous non-neoplastic tissues. This difference provided the basis for the detection of cancer and delineation of the tumor margins. Variations on the relative intensity were observed among different tissue types and excitation wavelengths.

Photodynamic Synovectomy Using Benzoporphyrin Derivative in an Antigen-induced Arthritis Model for Rheumatoid Arthritis

Experimental photodynamic therapy (PDT) has recently been adapted for the treatment of inflammatory and rheumatoid arthritis. The biodistribution of benzoporphyrin derivative monoacid ring A (BPD‐MA) and the effect of percutaneous light activation via intra‐articular bare cleaved optical fibers was investigated using a rabbit‐antigen‐induced arthritis model. Qualitative evaluation of intra‐articular photosensitizer clearance was performed with laser‐induced fluorescence from 0 to 6 h following intravenous injection. The compound was rapidly taken up within the joint and then cleared steadily over the 6 h interval. Biodistribution was determined by fluorescence microscopy and spectrofluoroscopic extraction techniques 3 h following intravenous injection of 2 mg/kg BPD‐MA. The biodistribution study demonstrated elevated levels of BPD‐MA in synovium (0.35 μg/g) and muscle (0.35μg/g). Fluorescence microscopy demonstrated presence of the compound within pathologic synovium but absence of the photosensitizer within meniscus, ligament, bone and articular cartilage. Tissue effects were evaluated histologically at 2 and 4 weeks posttreatment. BPD‐MA‐mediated PDT caused synovial necrosis in the region of light activation in 50% of treatment knees at 2 weeks and 43% at 4 weeks. No damage to nonpathologic tissues was observed. These studies indicate that selective destruction of synovium can be achieved by the light‐activated photosensitizing agent BPD‐MA without damage to articular cartilage or periarticular soft tissues. PDT needs to be further evaluated to optimize treatment parameters to provide for a new minimally invasive synovectomy technique.

The R273H p53 mutation can facilitate the androgen-independent growth of LNCaP by a mechanism that involves H2 relaxin and its cognate receptor LGR7

Mutations in p53 occur at a rate of approximately 70% in hormone-refractory prostate cancer (CaP), suggesting that p53 mutations facilitate the progression of CaP to androgen-independent (AI) growth. We have previously reported that transfection of p53 gain of function mutant alleles into LNCaP, an androgen-sensitive cell line, allows for AI growth of LNCaP in vitro. We herein confirm the in vivo relevance of those findings by demonstrating that the R273H p53 mutation (p53R273H) facilitates AI growth in castrated nude mice. In addition, we demonstrate that H2 relaxin is responsible for facilitating p53R273H-mediated AI CaP. H2 relaxin is overexpressed in the LNCaP-R273H subline. Downregulation of H2 relaxin expression results in significant inhibition of AI growth, whereas addition of recombinant human H2 relaxin to parental LNCaP promotes AI growth. Inhibition of AI growth was also achieved by blocking expression of LGR7, the cognate receptor of H2 relaxin. Chromatin immunoprecipitation analysis was used to demonstrate that p53R273H binds directly to the relaxin promoter, further confirming a role for H2 relaxin signaling in p53R273H-mediated AI CaP. Lastly, we used a reporter gene assay to demonstrate that H2 relaxin can induce the expression of prostate-specific antigen via an androgen receptor-mediated pathway.

IMMUNOHISTOCHEMICAL ANALYSIS OF BCL-2 PROTEIN EXPRESSION IN RENAL CELL CARCINOMA

PURPOSE To investigate the incidence, extent, and distribution of bcl-2 protein expression in human renal cell carcinomas. MATERIALS AND METHODS Using immunohistochemical tissue staining techniques, bcl-2 protein expression was analyzed in archival nephrectomy specimens removed for renal cell carcinoma or trauma and in 3 renal cell carcinoma cell lines. RESULTS Normal kidneys demonstrated bcl-2 immunopositivity primarily within the cytoplasm of distal tubule cells. Only rare and minor staining of the proximal tubular cells, thought to be the origin of renal cell carcinoma, was noted in histologically normal controls and areas adjacent to tumor. In contrast, bcl-2 protein expression was demonstrated in 70% of renal cell carcinomas and in all 3 experimental cell lines. CONCLUSIONS Bcl-2 is a proto-oncogene known to regulate apoptosis (programmed cell death). Bcl-2 protein is overexpressed in the majority of renal cell carcinomas examined. Bcl-2 overexpression may have a role in tumorigenesis and may explain the relative resistance of renal cell carcinoma to chemotherapeutic agents and to radiation therapy.

Midkine is a NF-κB-inducible gene that supports prostate cancer cell survival

BackgroundMidkine is a heparin-binding growth factor that is over-expressed in various human cancers and plays important roles in cell transformation, growth, survival, migration, and angiogenesis. However, little is known about the upstream factors and signaling mechanisms that regulate midkine gene expression.MethodsTwo prostate cancer cell lines LNCaP and PC3 were studied for their expression of midkine. Induction of midkine expression in LNCaP cells by serum, growth factors and cytokines was determined by Western blot analysis and/or real-time quantitative reverse-transcription – polymerase chain reaction (RT-PCR). The cell viability was determined by the trypan blue exclusion assay when the LNCaP cells were treated with tumor necrosis factor alpha (TNFα) and/or recombinant midkine. When the LNCaP cells were treated with recombinant midkine, activation of intracellular signalling pathways was determined by Western blot analysis. Prostate tissue microarray slides containing 129 cases (18 normal prostate tissues, 40 early stage cancers, and 71 late stage cancers) were assessed for midkine expression by immunohistochemical staining.ResultsWe identified that fetal bovine serum, some growth factors (epidermal growth factor, androgen, insulin-like growth factor-I, and hepatocyte growth factor) and cytokines (TNFα and interleukin-1beta) induced midkine expression in a human prostate cancer cell line LNCaP cells. TNFα also induced midkine expression in PC3 cells. TNFα was the strongest inducer of midkine expression via nuclear factor-kappa B pathway. Midkine partially inhibited TNFα-induced apoptosis in LNCaP cells. Knockdown of endogenous midkine expression by small interfering RNA enhanced TNFα-induced apoptosis in LNCaP cells. Midkine activated extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase pathways in LNCaP cells. Furthermore, midkine expression was significantly increased in late stage prostate cancer, which coincides with previously reported high serum levels of TNFα in advanced prostate cancer.ConclusionThese findings provide the first demonstration that midkine expression is induced by certain growth factors and cytokines, particularly TNFα, which offers new insight into understanding how midkine expression is increased in the late stage prostate cancer.