Author Manuscript Published OnlineFirst on February 2, 2016; DOI: 10.1158/0008-5472.CAN-15-1484 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Phytoestrogen suppresses efflux of the diagnostic marker protoporphyrin IX (PpIX) in lung carcinoma Running title: Genistein promotes ALA-mediated PpIX accumulation HIROFUMI FUJITAa,#,*, KEISUKE NAGAKAWAa,#, HIROTSUGU KOBUCHIb, TETSUYA OGINOc, YOICHI KONDOa, KEIJI INOUEd, TARO SHUINd, TOSHIHIKO UTSUMIe, KOZO UTSUMIa†, JUNZO SASAKIa, HIDEYO OHUCHIa Departments of aCytology and Histology, bCell Chemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8558, Japan, cDepartment of Nursing Science, Faculty of Health and Welfare Science, Okayama Prefectural University, Soja, Japan, dDepartment of Urology, Kochi University Medical School, Nankoku, Kochi 1 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2016 American Association for Cancer Research. Author Manuscript Published OnlineFirst on February 2, 2016; DOI: 10.1158/0008-5472.CAN-15-1484 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 783-8505, Japan, eDepartment of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan # Authors made an equal contribution. *Corresponding author. E-mail: [email protected] The authors disclose no potential conflicts of interest. 2 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2016 American Association for Cancer Research. Author Manuscript Published OnlineFirst on February 2, 2016; DOI: 10.1158/0008-5472.CAN-15-1484 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. ABSTRACT One promising method to visualize cancer cells is based on detection of the fluorescent photosensitizer protoporphyrin IX (PpIX) synthesized from 5-aminolevulinic acid (ALA), but this method can not be used in cancers which exhibit poor PpIX accumulation. PpIX appears to pumped out of cancer cells by the ABC transporter G2 (ABCG2), which is associated with multidrug resistance. Genistein is a phytoestrogen that appears to competitively inhibit ABCG2 activity. Therefore, we investigated whether genistein can promote PpIX accumulation in human lung carcinoma cells. Here we report that treatment of A549 lung carcinoma cells with genistein or a specific ABCG2 inhibitor promoted ALA-mediated accumulation of PpIX by ~2-fold. ABCG2 depletion and overexpression studies further revealed that genistein promoted PpIX accumulation via functional repression of ABCG2. After an extended period of genistein treatment, a significant increase in PpIX accumulation was observed in A549 cells (3.7-fold) and in other cell lines. Systemic preconditioning with genistein in a mouse xenograft model of 3 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2016 American Association for Cancer Research. Author Manuscript Published OnlineFirst on February 2, 2016; DOI: 10.1158/0008-5472.CAN-15-1484 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. lung carcinoma resulted in a 1.8-fold increase in accumulated PpIX. Long-term genistein treatment stimulated the expression of genes encoding enzymes involved in PpIX synthesis, such as porphobilinogen deaminase, uroporphyrinogen decarboxylase, and protoporphyrinogen oxidase. Accordingly, the rate of PpIX synthesis was also accelerated by genistein pre-treatment. Thus, our results suggest that genistein treatment effectively enhances ALA-induced PpIX accumulation by preventing the ABCG2-mediated efflux of PpIX from lung cancer cells, and may represent a promising strategy to improve ALA-based diagnostic approaches in a broader set of malignancies. 4 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2016 American Association for Cancer Research. Author Manuscript Published OnlineFirst on February 2, 2016; DOI: 10.1158/0008-5472.CAN-15-1484 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. INTRODUCTION Protoporphyrin IX (PpIX) functions as a fluorescent photosensitizer, which is synthesized from 5-aminolevulinic acid (ALA). Since PpIX preferentially accumulates in malignant tissues (1), the exogenous administration of ALA enables us to detect tumors exhibiting enhanced PpIX fluorescence. This technology, referred to as photodynamic diagnosis, has been widely used clinically, especially during surgery for bladder cancer (2), prostate cancer (3), and brain tumors (4), in order to identify precise tumor margins and prevent overlooking small lesions that are otherwise invisible. However, ALA-based photodynamic diagnosis remains unsatisfactory in diagnosing some tumors that accumulate insufficient amounts of PpIX (5-9). Successful ALA-induced PpIX accumulation may rely on the activity of enzymes that synthesize and metabolize PpIX and on the proteins that transport PpIX (10, 11). Exogenously added ALA is taken up by target cells and metabolized to coproporphyrinogen III in the cytosol by several enzymes, which include porphobilinogen deaminase (PBGD), uroporphyrinogen III 5 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2016 American Association for Cancer Research. Author Manuscript Published OnlineFirst on February 2, 2016; DOI: 10.1158/0008-5472.CAN-15-1484 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. synthase (UROS), which is the rate-limiting enzyme of porphyrin metabolism (12), and uroporphyrinogen decarboxylase (UROD). Coproporphyrinogen III is then translocated into mitochondria through ATP-binding cassette (ABC) transporter B6 and metabolized to PpIX by coproporphyrinogen oxidase (CPOX) and protoporphyrinogen oxidase (PPOX) (5, 7). PpIX is metabolized further to heme by ferrochelatase (FECH). Furthermore, accumulating evidence indicates that the elimination of PpIX from cells is carried out by ABC transporter G2 (ABCG2), which is a multi-drug resistance-associated protein (11). Thus, heme synthesis enzymes and ABCG2 play important roles in regulating the cellular accumulation of PpIX in cancer. Our current research goal is to develop new combination regimens with compounds that enhance the accumulation of PpIX in order to improve ALA-induced PpIX accumulation. A recent study reported that the systemic administration of vitamin D3 for preconditioning significantly increased the accumulation of PpIX in squamous tumor cell lines both in vitro and in vivo (13). The underlying 6 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2016 American Association for Cancer Research. Author Manuscript Published OnlineFirst on February 2, 2016; DOI: 10.1158/0008-5472.CAN-15-1484 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. mechanism involves increases in the expression of CPOX and decreases in that of FECH. We previously reported that the iron chelator deferoxamine (DFX) promoted the accumulation of PpIX in urothelial carcinoma in vitro and in vivo as well as in prostate cancer, oral squamous cell carcinoma, and histiocytic lymphoma in vitro (14-17). Furthermore, the inhibition of ABCG2 by specific inhibitors or knockdown using RNAi facilitated the ALA-mediated accumulation of PpIX (8, 18, 19). Therefore, the use of compounds that stimulate the synthesis of PpIX or block the efflux of PpIX may become a good strategy to improve ALA-induced PpIX accumulation. Estrogens are known to induce porphyria cutanea tarda, which is characterized clinically by cutaneous photosensitivity and the excessive excretion of porphyrins (20). This effect of estrogen is supported by the findings of another study using cancer-bearing female rats in which estrogen depletion by ovariectomy caused a significant reduction in ALA-induced PpIX levels and PBGD activity in tumors (21). On the other hand, a phytoestrogen genistein known as a tyrosine kinase inhibitor was found to 7 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2016 American Association for Cancer Research. Author Manuscript Published OnlineFirst on February 2, 2016; DOI: 10.1158/0008-5472.CAN-15-1484 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. exhibit estrogen-like activity by interacting with estrogen receptors in mammals (22-24). Furthermore, a previous study reported that genistein reversed ABCG2-mediated multidrug resistance and genistein was likely to competitively inhibit the efflux of anticancer agents such as SN-38 and mitoxantrone by ABCG2 (25). These findings prompted us to hypothesize that genistein promotes the accumulation of PpIX by increasing the synthesis of PpIX and/or reducing the efflux of PpIX. However, the effects of genistein on the accumulation of PpIX have not yet been elucidated. Therefore, we herein determined whether genistein increased the accumulation of PpIX in vitro and in a xenograft model using the human lung carcinoma A549 cell line, which expresses high levels of the endogenous ABCG2 protein. MATERIALS AND METHODS 8 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2016 American Association for Cancer Research. Author Manuscript Published OnlineFirst on February 2, 2016; DOI: 10.1158/0008-5472.CAN-15-1484 Author manuscripts have been peer reviewed and accepted for publication but