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Silencing of human ferrochelatase causes abundant protoporphyrin-IX accumulation in colon cancer

Wolfgang Kemmner^*,1, Kayiu Wan^*, Steffen Rüttinger, Bernd Ebert, Rainer Macdonald, Ursula Klamm and K. Thomas Moesta

^* Max Delbrueck Center for Molecular Medicine, Berlin, Germany;

Physikalisch-Technische Bundesanstalt, Department of Biomedical Optics, Berlin, Germany; and

Robert Roessle Clinic, Clinic for Surgical Oncology, Charite–Universitätsmedizin Berlin, Berlin, Germany

¹ Correspondence: Max Delbrueck Center for Molecular Medicine, Robert Roessle Str 10, 13125 Berlin, Germany. E-mail: wkemmner{at}mdc-berlin.de

Hemes and heme proteins are vital components of essentially every cell of virtually every eukaryote organism. Previously, we demonstrated accumulation of the heme precursor protoporphyrin-IX (PpIX) in gastrointestinal tumor tissues. To elucidate the mechanisms of PpIX accumulation by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), we studied expression of the relevant enzymes of the heme synthetic pathway. Here, we describe a significant down-regulation of ferrochelatase (FECH) mRNA expression in gastric, colonic, and rectal carcinomas. Accordingly, in an in vitro model of several carcinoma cell lines, ferrochelatase down-regulation and loss of enzymatic activity corresponded with an enhanced PpIX-dependent fluorescence. Direct detection of PpIX in minute amounts was achieved by a specifically developed pulsed solid-state laser dual delay fluorimetry setup. Silencing of FECH using small interfering RNA (siRNA) technology led to a maximum 50-fold increased PpIX accumulation, imageable by a specifically adapted two-photon microscopy unit. Our results show that in malignant tissue a transcriptional down-regulation of FECH occurs, which causes endogenous PpIX accumulation. Furthermore, accumulation of intracellular PpIX because of FECH siRNA silencing provides a small-molecule-based approach to molecular imaging and molecular therapy—Kemmner, W., Wan, K., Rüttinger, S., Ebert, B., Macdonald, R., Klamm, U., Moesta, K. T. Silencing of human ferrochelatase causes abundant protoporphyrin-IX accumulation in colon cancer.

Key Words: heme metabolism • gastrointestinal carcinomas • siRNA silencing, • cytoplasmatic red fluorescence