Evaluation of Riboflavin Transporters as Targets for Drug Delivery and Theranostics

The retention and cellular internalization of drug delivery systems and theranostics for cancer therapy can be improved by targeting molecules. Since an increased uptake of riboflavin was reported for various cancers, riboflavin and its derivatives may be promising binding moieties to trigger internalization via the riboflavin transporters (RFVT) 1, 2, and 3. Riboflavin is a vitamin with pivotal role in energy metabolism and indispensable for cellular growth. In previous preclinical studies on mice, we showed the target-specific accumulation of riboflavin-functionalized nanocarriers in cancer cells. Although the uptake mechanism of riboflavin has been studied for over a decade, little is known about the riboflavin transporters and their expression on cancer cells, tumor stroma, and healthy tissues. Furthermore, evidence is lacking concerning the representativeness of the preclinical findings to the situation in humans. In this study, we investigated the expression pattern of riboflavin transporters in human squamous cell carcinoma (SCC), melanoma and luminal A breast cancer samples, as well as in healthy skin, breast, aorta, and kidney tissues. Low constitutive expression levels of RFVT1–3 were found on all healthy tissues, while RFVT2 and 3 were significantly overexpressed in melanoma, RFVT1 and 3 in luminal A breast cancer and RFVT1–3 in SCC. Correspondingly, the SCC cell line A431 was highly positive for all RFVTs, thus qualifying as suitable in vitro model. In contrast, activated endothelial cells (HUVEC) only presented with a strong expression of RFVT2, and HK2 kidney cells only with a low constitutive expression of RFVT1–3. Functional in vitro studies on A431 and HK2 cells using confocal microscopy showed that riboflavin uptake is mostly ATP dependent and primarily driven by endocytosis. Furthermore, riboflavin is partially trafficked to the mitochondria. Riboflavin uptake and trafficking was significantly higher in A431 than in healthy kidney cells. Thus, this manuscript supports the hypothesis that addressing the riboflavin internalization pathway may be highly valuable for tumor targeted drug delivery.

[1]  H. Said,et al.  Mechanism and regulation of riboflavin uptake by human renal proximal tubule epithelial cell line HK-2. , 1998, American journal of physiology. Renal physiology.

[2]  P. Swaan,et al.  Riboflavin-Targeted Polymer Conjugates for Breast Tumor Delivery , 2013, Pharmaceutical Research.

[3]  N. K. Jain,et al.  Development and Characterization of the Paclitaxel loaded Riboflavin and Thiamine Conjugated Carbon Nanotubes for Cancer Treatment , 2016, Pharmaceutical Research.

[4]  Olivia Freeman,et al.  Talking points personal outcomes approach: practical guide. , 2012 .

[5]  Joseph W. Nichols,et al.  Odyssey of a cancer nanoparticle: from injection site to site of action. , 2012, Nano today.

[6]  S. Arora,et al.  Riboflavin and health: A review of recent human research , 2017, Critical reviews in food science and nutrition.

[7]  I. Goldman,et al.  Mechanisms of membrane transport of folates into cells and across epithelia. , 2011, Annual review of nutrition.

[8]  Y. Moriyama Riboflavin transporter is finally identified. , 2011, Journal of biochemistry.

[9]  Kenneth W Dunn,et al.  A practical guide to evaluating colocalization in biological microscopy. , 2011, American journal of physiology. Cell physiology.

[10]  B. Grant,et al.  Intracellular trafficking. , 2006, WormBook : the online review of C. elegans biology.

[11]  Gideon Blumenthal,et al.  Assessment of benefits and risks in development of targeted therapies for cancer — The view of regulatory authorities , 2015, Molecular oncology.

[12]  W. Wang,et al.  SLC52A3 expression is activated by NF-κB p65/Rel-B and serves as a prognostic biomarker in esophageal cancer , 2018, Cellular and Molecular Life Sciences.

[13]  Satohiro Masuda,et al.  Identification and comparative functional characterization of a new human riboflavin transporter hRFT3 expressed in the brain. , 2010, The Journal of nutrition.

[14]  L. Matherly,et al.  The human proton-coupled folate transporter , 2012, Cancer biology & therapy.

[15]  Katsuhisa Inoue,et al.  Identification and functional characterization of rat riboflavin transporter 2. , 2009, Journal of biochemistry.

[16]  D. Kamei,et al.  The intracellular trafficking pathway of transferrin. , 2012, Biochimica et biophysica acta.

[17]  K. Thorisdottir,et al.  Neutrophilic eccrine hidradenitis. , 1993, Journal of the American Academy of Dermatology.

[18]  L. Matherly,et al.  Membrane transporters and folate homeostasis: intestinal absorption and transport into systemic compartments and tissues , 2009, Expert Reviews in Molecular Medicine.

[19]  G. Mansoori,et al.  Utilizing the folate receptor for active targeting of cancer nanotherapeutics , 2012, Nano reviews.

[20]  J. Laissue,et al.  Distribution of somatostatin receptors in normal and neoplastic human tissues: recent advances and potential relevance. , 1997, The Yale journal of biology and medicine.

[21]  W. Wilson,et al.  Targeting hypoxia in cancer therapy , 2011, Nature Reviews Cancer.

[22]  S. Bohlega,et al.  Riboflavin Has Neuroprotective Potential: Focus on Parkinson’s Disease and Migraine , 2017, Front. Neurol..

[23]  S. Nakagawa,et al.  Riboflavin Transporters RFVT/SLC52A Mediate Translocation of Riboflavin, Rather than FMN or FAD, across Plasma Membrane. , 2017, Biological & pharmaceutical bulletin.

[24]  S. Weinberg,et al.  Targeting mitochondria metabolism for cancer therapy. , 2015, Nature chemical biology.

[25]  O. Jo,et al.  Riboflavin transport by isolated perfused rabbit renal proximal tubules. , 2000, American journal of physiology. Cell physiology.

[26]  Satohiro Masuda,et al.  Identification and functional characterization of a novel human and rat riboflavin transporter, RFT1. , 2008, American journal of physiology. Cell physiology.

[27]  Jin-Hu Fan,et al.  RFT2 is overexpressed in esophageal squamous cell carcinoma and promotes tumorigenesis by sustaining cell proliferation and protecting against cell death. , 2014, Cancer letters.

[28]  Shreya Singh,et al.  Tumor markers: A diagnostic tool , 2016, National journal of maxillofacial surgery.

[29]  Mohan G. Hebsur,et al.  Development and Characterization , 1998 .

[30]  F. Kiessling,et al.  Balancing Passive and Active Targeting to Different Tumor Compartments Using Riboflavin-Functionalized Polymeric Nanocarriers. , 2017, Nano letters.

[31]  P. Varalakshmi,et al.  Riboflavin Responsive Mitochondrial Dysfunction in Neurodegenerative Diseases , 2017, Journal of clinical medicine.

[32]  F. Kiessling,et al.  Photoacoustic imaging of tumor targeting with riboflavin-functionalized theranostic nanocarriers , 2017, International journal of nanomedicine.

[33]  Sandeep Arora,et al.  Breast cancers: MR imaging of folate-receptor expression with the folate-specific nanoparticle P1133. , 2010, Radiology.

[34]  H. Birn The kidney in vitamin B12 and folate homeostasis: characterization of receptors for tubular uptake of vitamins and carrier proteins. , 2006, American journal of physiology. Renal physiology.

[35]  R. Finnell,et al.  Renal tubular reabsorption of folate mediated by folate binding protein 1. , 2005, Journal of the American Society of Nephrology : JASN.

[36]  C. Dann,et al.  Structures of human folate receptors reveal biological trafficking states and diversity in folate and antifolate recognition , 2013, Proceedings of the National Academy of Sciences.

[37]  F. Sotgia,et al.  Targeting flavin-containing enzymes eliminates cancer stem cells (CSCs), by inhibiting mitochondrial respiration: Vitamin B2 (Riboflavin) in cancer therapy , 2017, Aging.

[38]  Gerd Ulrich Nienhaus,et al.  New views on cellular uptake and trafficking of manufactured nanoparticles , 2013, Journal of The Royal Society Interface.

[39]  H. McMahon,et al.  Mechanisms of endocytosis. , 2009, Annual review of biochemistry.

[40]  I. Vergote,et al.  Vintafolide: a novel targeted therapy for the treatment of folate receptor expressing tumors , 2015, Therapeutic advances in medical oncology.

[41]  D. Hanahan,et al.  Hallmarks of Cancer: The Next Generation , 2011, Cell.

[42]  F. Kiessling,et al.  Amphiphilic Phospholipid-Based Riboflavin Derivatives for Tumor Targeting Nanomedicines. , 2016, Bioconjugate chemistry.

[43]  P. Choyke,et al.  Super enhanced permeability and retention (SUPR) effects in tumors following near infrared photoimmunotherapy. , 2016, Nanoscale.

[44]  M. Raj,et al.  Elevation of serum riboflavin carrier protein in breast cancer. , 1999, Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology.

[45]  A. Mitra,et al.  Molecular and functional characterization of riboflavin specific transport system in rat brain capillary endothelial cells , 2012, Brain Research.

[46]  P. Low,et al.  Intraoperative tumor-specific fluorescence imaging in ovarian cancer by folate receptor-α targeting: first in-human results , 2011, Nature Medicine.

[47]  E. Li,et al.  Plasma Riboflavin Level is Associated with Risk, Relapse, and Survival of Esophageal Squamous Cell Carcinoma , 2017, Nutrition and cancer.

[48]  Thomas D. Schmittgen,et al.  Dynamin 2 Regulates Riboflavin Endocytosis in Human Placental Trophoblasts , 2007, Molecular Pharmacology.

[49]  Y. Sunami Targeting Metabolism , 2020, Translational Pancreatic Cancer Research.

[50]  P. Low,et al.  Folate receptor expression in carcinomas and normal tissues determined by a quantitative radioligand binding assay. , 2005, Analytical biochemistry.

[51]  M. V. Vander Heiden,et al.  Targeting Metabolism for Cancer Therapy. , 2017, Cell chemical biology.

[52]  Hui‐Ting Yang,et al.  Riboflavin at high doses enhances lung cancer cell proliferation, invasion, and migration. , 2013, Journal of food science.

[53]  A. Yonezawa,et al.  Novel riboflavin transporter family RFVT/SLC52: identification, nomenclature, functional characterization and genetic diseases of RFVT/SLC52. , 2013, Molecular aspects of medicine.

[54]  P. Choyke,et al.  Improving Conventional Enhanced Permeability and Retention (EPR) Effects; What Is the Appropriate Target? , 2013, Theranostics.

[55]  K. Vaidyanathan,et al.  Organ Specific Tumor Markers: What’s New? , 2011, Indian Journal of Clinical Biochemistry.

[56]  Kristina M. Ilieva,et al.  Targeting folate receptor alpha for cancer treatment , 2016, Oncotarget.

[57]  Qiong Wang,et al.  Overexpression of riboflavin transporter 2 contributes toward progression and invasion of glioma , 2016, Neuroreport.

[58]  P. Swaan,et al.  Intracellular processing of riboflavin in human breast cancer cells. , 2008, Molecular pharmaceutics.

[59]  Mark A Feitelson,et al.  Sustained proliferation in cancer: Mechanisms and novel therapeutic targets. , 2015, Seminars in cancer biology.

[60]  J. Ledermann,et al.  Targeting the folate receptor: diagnostic and therapeutic approaches to personalize cancer treatments. , 2015, Annals of oncology : official journal of the European Society for Medical Oncology.

[61]  A. Adjei,et al.  Targeting Angiogenesis in Cancer Therapy: Moving Beyond Vascular Endothelial Growth Factor. , 2015, The oncologist.

[62]  F. Kiessling,et al.  Refinement of adsorptive coatings for fluorescent riboflavin-receptor-targeted iron oxide nanoparticles. , 2016, Contrast media & molecular imaging.

[63]  N. Jana,et al.  Supramolecular Host–Guest Chemistry-Based Folate/Riboflavin Functionalization and Cancer Cell Labeling of Nanoparticles , 2017, ACS omega.

[64]  P. Swaan,et al.  Involvement of Endocytic Organelles in the Subcellular Trafficking and Localization of Riboflavin , 2003, Journal of Pharmacology and Experimental Therapeutics.

[65]  M. Cifuentes,et al.  Differential distribution of the Sodium-vitamin C cotransporter-1 along the proximal tubule of the mouse and human kidney. , 2008, Kidney international.

[66]  Gaurav Sahay,et al.  Endocytosis of nanomedicines. , 2010, Journal of controlled release : official journal of the Controlled Release Society.

[67]  Carolyn J. Anderson,et al.  Folate Receptor-Targeted Multimodality Imaging of Ovarian Cancer in a Novel Syngeneic Mouse Model , 2014, Molecular pharmaceutics.