Phytoestrogen Suppresses Efflux of the Diagnostic Marker Protoporphyrin IX in Lung Carcinoma
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Published OnlineFirst February 2, 2016; DOI: 10.1158/0008-5472.CAN-15-1484 Cancer Molecular and Cellular Pathobiology Research Phytoestrogen Suppresses Efflux of the Diagnostic Marker Protoporphyrin IX in Lung Carcinoma Hirofumi Fujita1, Keisuke Nagakawa1, Hirotsugu Kobuchi2, Tetsuya Ogino3, Yoichi Kondo1, Keiji Inoue4, Taro Shuin4, Toshihiko Utsumi5, Kozo Utsumi1,†, Junzo Sasaki1, and Hideyo Ohuchi1 Abstract One promising method to visualize cancer cells is based on the treatment, a significant increase in PpIX accumulation was detection of the fluorescent photosensitizer protoporphyrin IX observed in A549 cells (3.7-fold) and in other cell lines. Systemic (PpIX) synthesized from 5-aminolevulinic acid (ALA), but this preconditioning with genistein in a mouse xenograft model of method cannot be used in cancers that exhibit poor PpIX lung carcinoma resulted in a 1.8-fold increase in accumulated accumulation. PpIX appears to be pumped out of cancer cells by PpIX. Long-term genistein treatment stimulated the expression the ABC transporter G2 (ABCG2), which is associated with of genes encoding enzymes involved in PpIX synthesis, such as multidrug resistance. Genistein is a phytoestrogen that appears porphobilinogen deaminase, uroporphyrinogen decarboxyl- to competitively inhibit ABCG2 activity. Therefore, we investigat- ase, and protoporphyrinogen oxidase. Accordingly, the rate of ed whether genistein can promote PpIX accumulation in human PpIX synthesis was also accelerated by genistein pretreatment. lung carcinoma cells. Here we report that treatment of A549 lung Thus, our results suggest that genistein treatment effectively carcinoma cells with genistein or a specific ABCG2 inhibitor enhances ALA-induced PpIX accumulation by preventing promoted ALA-mediated accumulation of PpIX by approximately the ABCG2-mediated efflux of PpIX from lung cancer cells 2-fold. ABCG2 depletion and overexpression studies further and may represent a promising strategy to improve ALA-based revealed that genistein promoted PpIX accumulation via func- diagnostic approaches in a broader set of malignancies. tional repression of ABCG2. After an extended period of genistein Cancer Res; 76(7); 1–10. Ó2016 AACR. Introduction (3), and brain tumors (4), to identify precise tumor margins and prevent overlooking small lesions that are otherwise invisible. Protoporphyrin IX (PpIX) functions as a fluorescent photosen- However, ALA-based photodynamic diagnosis remains unsatis- sitizer, which is synthesized from 5-aminolevulinic acid (ALA). As factory in diagnosing some tumors that accumulate insufficient PpIX preferentially accumulates in malignant tissues (1), the amounts of PpIX (5–9). exogenous administration of ALA enables us to detect tumors Successful ALA-induced PpIX accumulation may rely on the exhibiting enhanced PpIX fluorescence. This technology, referred activity of enzymes that synthesize and metabolize PpIX and on to as photodynamic diagnosis, has been widely used clinically, the proteins that transport PpIX (10, 11). Exogenously added ALA especially during surgery for bladder cancer (2), prostate cancer is taken up by target cells and metabolized to coproporphyrino- gen III in the cytosol by several enzymes, which include porpho- 1Department of Cytology and Histology, Okayama University Gradu- bilinogen deaminase (PBGD), uroporphyrinogen III synthase ate School of Medicine, Dentistry and Pharmaceutical Sciences, (UROS), which is the rate-limiting enzyme of porphyrin metab- Okayama, Japan. 2Department of Cell Chemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, olism (12), and uroporphyrinogen decarboxylase (UROD). Okayama, Japan. 3Department of Nursing Science, Faculty of Health Coproporphyrinogen III is then translocated into mitochondria and Welfare Science, Okayama Prefectural University, Soja, Japan. through ATP-binding cassette (ABC) transporter B6 and metab- 4Department of Urology, Kochi University Medical School, Nankoku, Kochi, Japan. 5Department of Biological Chemistry, Faculty of Agri- olized to PpIX by coproporphyrinogen oxidase (CPOX) and culture, Yamaguchi University, Yamaguchi, Japan. protoporphyrinogen oxidase (PPOX; refs. 5, 7). PpIX is metab- Note: Supplementary data for this article are available at Cancer Research olized further to heme by ferrochelatase. Furthermore, accumu- Online (http://cancerres.aacrjournals.org/). lating evidence indicates that the elimination of PpIX from cells is H. Fujita and K. Nagakawa contributed equally to this article. carried out by ABC transporter G2 (ABCG2), which is a multidrug resistance-associated protein (11). Thus, heme synthesis enzymes †Deceased. and ABCG2 play important roles in regulating the cellular accu- Corresponding Author: Hirofumi Fujita, Okayama University Graduate School, mulation of PpIX in cancer. Our current research goal is to develop 2-5-1 shilatacho, Kitaku, Okayama 700-8558, Japan. Phone: 818-6235-7081; Fax: new combination regimens with compounds that enhance the 818-6235-7079; E-mail: [email protected] accumulation of PpIX to improve ALA-induced PpIX accumula- doi: 10.1158/0008-5472.CAN-15-1484 tion. A recent study reported that the systemic administration Ó2016 American Association for Cancer Research. of vitamin D3 for preconditioning significantly increased the www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on October 6, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst February 2, 2016; DOI: 10.1158/0008-5472.CAN-15-1484 Fujita et al. accumulation of PpIX in squamous tumor cell lines both in vitro cell lines were authenticated using short tandem repeat analysis and in vivo (13). The underlying mechanism involves increases in with the GenePrint 10 System (Promega) and Cell Line Authen- the expression of CPOX and decreases in that of ferrochelatase. We tication Database of JCRB and ATCC in 2015. ST-HEK cells were previously reported that the iron chelator deferoxamine promot- prepared by the stable transfection of ABCG2 and cells expressing ed the accumulation of PpIX in urothelial carcinoma in vitro and in high levels of the functional ABCG2 protein (18). These cells were vivo as well as in prostate cancer, oral squamous cell carcinoma, maintained in complete medium: RPMI1640 supplemented with and histiocytic lymphoma in vitro (14–17). Furthermore, the 10% heat-inactivated FBS, 100 U/mL penicillin, and 100 mg/mL inhibition of ABCG2 by specific inhibitors or knockdown using streptomycin in a humidified atmosphere with 5% CO2/air at RNAi facilitated the ALA-mediated accumulation of PpIX (8, 18, 37C. Typically, 1 Â 105 cells were seeded in 1.5 mL of complete 19). Therefore, the use of compounds that stimulate the synthesis medium on 3.5-cm dishes and cultured for 24 hours before of PpIX or block the efflux of PpIX may become a good strategy to each experiment. Unless otherwise indicated, chemicals were improve ALA-induced PpIX accumulation. added at the following final concentrations: 0.5 mmol/L of ALA, Estrogens are known to induce porphyria cutanea tarda, which 5to50mmol/L of genistein, 1 mmol/L of Ko143, and 300 mmol/L is characterized clinically by cutaneous photosensitivity and the of Noc18. excessive excretion of porphyrins (20). This effect of estrogen is fi supported by the ndings of another study using cancer-bearing Flow cytometry of cellular PpIX female rats in which estrogen depletion by ovariectomy caused a After being incubated, cells were rinsed three times with PBS fi signi cant reduction in ALA-induced PpIX levels and PBGD and harvested by trypsinization. After centrifugation at 800 Â g for activity in tumors (21). On the other hand, a phytoestrogen 5 minutes, the cells were resuspended in 0.4 mL of PBS. Cellular genistein known as a tyrosine kinase inhibitor was found to PpIX contents were measured using the flow cytometer FACScan exhibit estrogen-like activity by interacting with estrogen recep- (BD Biosciences) and quantified with CellQuest software (BD – tors (ER) in mammals (22 24). Furthermore, a previous study Biosciences). A total of 10,000 cells were analyzed in each sample reported that genistein reversed ABCG2-mediated multidrug (excitation 488 nm, emission 650 nm). resistance and genistein was likely to competitively inhibit the efflux of anticancer agents such as SN-38 and mitoxantrone by ABCG2 (25). Fluorescence microscopy fi After being treated with ALA, cells were stained with 1 mmol/L These ndings prompted us to hypothesize that genistein promotes the accumulation of PpIX by increasing the synthesis MitoTracker Green for 20 minutes at 37 C and then observed by fl of PpIX and/or reducing the efflux of PpIX. However, the effects of uorescence microscopy (Axiovert 200, Carl Zeiss Inc.). Mito- fl genistein on the accumulation of PpIX have not yet been Tracker Green is a uorescent dye compound that is used for the elucidated. detection of mitochondria. Fluorescence images were taken using Therefore, we herein determined whether genistein increased a highly light-sensitive thermoelectrically cooled charge-coupled fi the accumulation of PpIX in vitro and in a xenograft model using device camera (Axio-Cam CCD camera, Zeiss). The lter combi- fi the human lung carcinoma A549 cell line, which expresses high nations were a 450-nm excitation lter, 510-nm beam splitter, fi levels of the endogenous ABCG2 protein. and 515 to 565-nm emission lter for MitoTracker green; a 400-nm excitation filter, 580-nm beam splitter, and 590-nm long-pass emission filter for PpIX. Materials