J Fluoresc (2010) 20:1159–1165 DOI 10.1007/s10895-010-0662-9 ORIGINAL PAPER Intrinsic Fluorescence of Protoporphyrin IX from Blood Samples Can Yield Information on the Growth of Prostate Tumours Flávia Rodrigues de Oliveira Silva & Maria Helena Bellini & Vivian Regina Tristão & Nestor Schor & Nilson Dias Vieira Jr. & Lilia Coronato Courrol Received: 12 November 2009 /Accepted: 30 March 2010 /Published online: 24 April 2010 # Springer Science+Business Media, LLC 2010 Abstract Prostate cancer is one of the most common types in those with prostate cancer induced by inoculation of of cancer in men, and unfortunately many prostate tumours DU145 cells. A significant contrast between the blood of remain asymptomatic until they reach advanced stages. normal and cancer subjects could be established. Blood Diagnosis is typically performed through Prostate-Specific porphyrin fluorophore showed an enhancement on the Antigen (PSA) quantification, Digital Rectal Examination fluorescence band around 632 nm following tumour (DRE) and Transrectal Ultrasonography (TU). The antigen growth. Fluorescence detection has advantages over other (PSA) is secreted by all prostatic epithelial cells and not light-based investigation methods: high sensitivity, high exclusively by cancerous ones, so its concentration also speed and safety. However it does carry the drawback of increases in the presence of other prostatic diseases. DRE low specificity of detection. The extraction of blood and TU are not reliable for early detection, when porphyrin using acetone can solve this problem, since histological analysis of prostate tissue obtained from a optical excitation of further molecular species can be biopsy is necessary. In this context, fluorescence techniques excluded, and light scattering from blood samples is are very important for the diagnosis of cancer. In this paper negligible. we explore the potential of using endogenous phorphyrin blood fluorescence as tumour marker for prostate cancer. Keywords Porphyrin . Tumour stage . Prostate cancer . Substances such as porphyrin derivatives accumulate Fluorescence . Spectroscopy substantially more in tumours than in normal tissues; thus, measuring blood porphyrin concentration by autofluores- cence intensity may provide a good parameter for deter- Introduction mining tumour stage. In this study, the autofluorescence of blood porphyrin was analyzed using fluorescence and Prostate cancer is the most frequently diagnosed neoplasm excitation spectroscopy on healthy male NUDE mice and in men; it can present as a localized disease within the prostate or may become highly invasive and metastasize to : : : : regional lymph nodes and bone. When prostate tumour F. R. de Oliveira Silva M. H. Bellini V. R. Tristão N. Schor cells metastasize beyond the confines of the gland, the L. C. Courrol Departamento de Nefrologia, UNIFESP, associated morbidity and mortality of prostate cancer are São Paulo, SP, Brazil significant. Curative therapeutic options are limited to localized disease detected early, which is initially done N. D. Vieira Jr. through digital rectal examination and measurement of Centro de Lasers e Aplicações, IPEN/CNEN-SP, São Paulo, SP, Brazil serum prostate-specific antigen (PSA) [1, 2]. PSA is produced and secreted by glandular cells in the epithelial L. C. Courrol (*) layer, and the quantity present in the prostate gland UNIFESP, increases with the occurrence of prostatic disease. Although Campus Diadema, Diadema, SP, Brazil regarded as the best conventional serum tumour marker e-mail: [email protected] currently available, PSA measurement is not specific 1160 J Fluoresc (2010) 20:1159–1165 enough for a definite diagnosis of prostate cancer to be synthase and uroporphyrinogen III cosynthase. Uroporphyri- established because it also increases in the case of benign nogen III is decarboxylated into coproporphyrinogen by the growth of the prostate (BPH) or prostate inflammation [3]. enzyme uroporphyrinogen decarboxylase. Coproporphyrino- The diagnosis of prostate cancer has a high rate false- gen III is then transported to the interior of the mitochondria, positive results because of normal cell growth in the where coproporphyrinogen III oxidase catalises the oxidative prostate. BPH can be histologically observed in approxi- decarboxylation step from coproporphyrinogen III to proto- mately 20% of men aged 40 years and older, and this porphyrinogen IX. Protoporphyrinogen IX oxidase converts percentage increases to 70% in men aged 60 and older and protoporphyrinogen IX to protoporphyrin IX, which is to as much as 90% in men aged 70 and older. Only 25% of responsible for the characteristic red color of heme. The final them develop clinical symptoms. reaction in heme synthesis involves the insertion of the iron A commonly used method to classify the stage of atom into PpIX, generating heme. The enzyme that catalyses prostate cancer is the Gleason grade [4], which is based this reaction is known as ferrochelatase [11]. on the patterns of prostatic glands. Lower grades indicate While in nonerythroid cells the rate of heme synthesis more differentiated carcinomas and therefore less severe depends on the rate of ALA production by the first and tumours. Another method used for diagnosing prostate rate-limiting porphyrin biosynthetic enzyme, ALA-S, in cancer is Transrectal Ultrasonography [2]. Nevertheless, erythroid cells it is determined by the availability of iron for histological analysis of prostate tissue is necessary for early ferrochelatase [12]. detection. These traditional techniques rely on tissue being Protoporphyrin IX is the predominant porphyrin in blood removed and then examined away from the patient. [13]. Abnormal metabolism of protoporphyrin IX has been Fluorescence techniques are very important for the observed in total blood, plasma, serum and tissues of diagnosis of cancer. Fluorescence detection has advantages cancerous patients, which indicates that protoporphyrin IX over other light-based investigation methods: high sensitiv- accumulates substantially more in cancer cells than in ity, high speed, and safety [5, 6]. Non-invasive autofluor- normal cells and tissues [14–20]. escence of blood components has the potential to provide in Through the analysis of PpIX spectroscopic properties it vivo diagnosis of tumour stage, because cancerous tissues is possible to monitor its concentration in tissues and show enhanced fluorescence [7–10] of endogenous por- biological fluids. The absorption spectra of porphyrin phyrins as a consequence of tumour-specific metabolic exhibit five bands: the Soret Band at around 400 nm and alterations, with a high concentration of porphyrins synthe- four bands, known as Q bands, in the region between 450 sized and tumour hypervascularity. and 700 nm [6, 21]. Protoporphyrin IX is a porphyrin derivative that com- Several studies can be found in the literature on the use bines with ferrous iron to form the heme group [10]. Most of endogenous fluorescence for disease diagnosis [7–9, 14– iron in mammalian systems is routed to mitochondria to 20]. In this work we propose that the analysis of the serve as a substrate for ferrochelatase. Ferrochelatase inserts intrinsic fluorescence of protoporphyrin IX from blood iron into protoporphyrin IX to form heme, which is samples can yield information on the progression of incorporated into hemoglobin (involved in the transport of prostate tumours. The aim of the current study is to search oxygen). A number of non-heme iron-containing proteins for a possible method to distinguish between normal are also known, such as the iron-sulphur proteins of specimens and those associated with prostate cancer in oxidative phosphorylation and the iron transport and order to estimate the stages of induced tumour development storage proteins transferrin and ferritin, respectively. in male NUDE mice, by examination with ultraviolet Tissue-specific regulatory features characterize the bio- 405 nm irradiation. synthetic pathway of heme. In erythroid cells, regulation is mediated by erythroid-specific transcription factors and by the availability of iron in the form of Fe/S clusters. In non- Materials and methods erythroid cells, the pathway is regulated by heme-mediated feedback inhibition [10]. Heme biosynthesis starts in the Cell line and cell culture conditions mitochondria with the formation of δ-aminolevulinic acid (ALA), which involves the enzymatic condensation of “DU145 (DU-145) is a “classical” cell lines of prostatic glycine with succinyl-Coa. In the cytosol, two molecules cancer. The DU-145 is a prostatic human tumor cell line, of ALA form monopyrrole porphobilinogen through a established from the removed metastatic central nervous condensation reaction catalyzed by aminolevulinate dehy- system tumour of an old man with prostate carcinoma in dratase (ALAD). The following step consists of the 1975 [22]. Du-145 cells were cultured in DMEM contain- condensation of porphobilinogen into uroporphyrinogen ing high glucose concentration (4.5 g/liter at 25 mM) and III by the action of two enzymes, uroporohyrinogen I supplemented with 100 units/ml of penicillin, 50 mg/ml of J Fluoresc (2010) 20:1159–1165 1161 streptomycin, and 10% of FBS. The cells were maintained at 7, 14, 21, 28 and 35 days after cells inoculation. in a humid chamber at 37 °C in an atmosphere of 5% CO2” Approximately 400 µL of blood was collected from the retro-orbital plexus of each animal with a glass capillary, Animals and tumour induction using heparin as an anticoagulant. All