br Conclusion These results suggest that combined treatment with

Conclusion: These results suggest that combined treatment with naringenin and hesperetin might be a promising anti-cancer strategy for pancreatic cancers without eliciting toxicity on normal cells.
Introduction
Pancreatic cancer has a serious prognosis and is one of the most dangerous cancers (Onishi and Katano, 2014; Ryan et al., 2014). De-spite continuous endeavors over the past decades, the 5-year survival rate is only ∼7% (Siegel et al., 2016). More seriously, the prognosis of pancreatic cancer patients remains at a standstill, in spite of significant improvements in overall survival rates of other malignant cancers (Koay et al., 2014). Thus, development of effective therapies for pan-creatic cancer is urgently needed.
Many chemotherapeutic agents are widely used for pancreatic cancer patients as various types of treatment such as single, in
combination, and in conjunction with surgery or radiotherapy (Remesh, 2013). However, some agents can be toxic to normal ME2906 and organs, and they can cause immediate adverse reactions that are a re-sult of their biochemical nature rather than their expected action (Lazarou et al., 1998). Gemcitabine has been implicated as a potential agent for pancreatic cancer. However, its clinical benefit is weak or non-existent. Treatment of gemcitabine with 5-fluorouracil (5-FU) ex-tended overall survival by only 1 month compared with single treat-ment of 5-FU (Burris et al., 1997; Heinemann et al., 2008). Develop-ment of anti-cancer compounds with an optimal strategy is urgently required for pancreatic cancer patients.
Citrus unshiu (Citrus unshiu Markov.), which is known as kam-gyul in
Abbreviations: εCUP, Enzymatic hydrolysis of Citrus unshiu peel; ƒCUP, Fermented extraction of Citrus unshiu peel; FAK, Focal adhesion kinase; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; HUVEC, Human umbilical vein endothelial cell; LOD, Limit of detection; LOQ, Limit of quantification
Corresponding authors at: Faculty of Biotechnology, College of Applied Life Science, SARI, Jeju National University, 102 Jejudaehak-ro, Jeju-si, Jeju-do 690-756, Republic of Korea. E-mail addresses: [email protected] (J. Lee), [email protected] (J.H. Kim).
South Korea, is mainly cultivated on Jeju Island. Compared with its flesh, Citrus unshiu peel has many different biologically active com-pounds such as vitamin C, phenolic acids, and flavonoids. Dried peel of the young fruit has been used as an ingredient of traditional medicine for various diseases (Choi et al., 2007). Recently, it has been reported that Citrus unshiu peel can suppress allergic reactions, inflammation, and oxidative stress through the action of various bioactive compounds including hesperidin, naringin, and nobiletin (Higashi-Okai et al., 2002; Jeong et al., 2004; Kim et al., 1999; Murakami et al., 2000). However, little or limited research has been performed to elucidate the possible anti-cancer effects of Citrus unshiu peel extract (Jin et al., 2013; Manthey et al., 2001). We have previously reported that the products of enzymatic hydrolysis of Citrus unshiu peel (εCUP) and fermented ex-traction of Citrus unshiu peel (ƒCUP) have significant anti-cancer effects on human pancreatic cancer both in vitro and in vivo (Lee et al., 2017a; Lee et al., 2018). Despite their different characteristics and effects, the underlying mechanism remains elusive.
In this study, we determined that the combined administration of naringenin and hesperetin at an optimal ratio maximizes the anti-cancer effects in human pancreatic cancer in vitro and in vivo. Our re-sults indicate that a proper combination of naringenin and hesperetin might be a safe and effective treatment strategy for pancreatic cancer.
Materials and methods
Cell culture and reagents