Synergistic therapy with tangeretin and 5-fluorouracil accelerates the ROS/JNK mediated apoptotic pathway in human colorectal cancer cell

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Highlights

  • Antioxidant level in colorectal cancer cells was decreased upon 5-FU treatment in combination with TAN.

  • TAN and 5-FU co-treatment suppressed the autophagy cell survival mechanism.

  • TAN synergistically accelerated 5-FU induced apoptotic event.

  • TAN and 5-FU induced the program cell death through the JNK mediated signaling pathway.

Abstract

Synergistic therapy is emerging as a promising strategy for improving the chemotherapeutic efficacy of anticancer drugs. Addition of adjuvants with standard anticancer drugs has shown successful reduction of adverse side effects. The synthetic drug 5-Fluorouracil (5-FU) shows several side effects upon prolonged chemotherapy, thereby restricting its long-term clinical application. Several studies have reported anticancer potential and anti-inflammatory activity of tangeretin (TAN) towards mammalian cells. Therefore, we investigate whether the combination of TAN with 5-FU increases their anticancer potential against colorectal cancer. In this study, we examined the synergistic activity of TAN and 5-FU on the viability of several human cancer and normal cells. Several possible mechanistic pathways were screened, and found that co-exposure of TAN and 5-FU accelerates oxidative-stress and increases endogenous-ROS generation, which sequentially triggers the DNA damage response and activates the apoptotic pathway, by down-regulating autophagy and DNA repair system in HCT-116 cells. TAN and 5-FU co-treatment also remarkably reduces the mitochondrial membrane potential, and sequentially decreases ATPase activity. Collectively, results indicate that combination of TAN and 5-FU significantly accelerates apoptosis via JNK mediated pathway. To our knowledge gained from literature, this study is the first to describe synergistic activity of TAN and 5-FU against colorectal cancer cells.

Graphical abstract

Tangeretin and 5-Fluorouracil synergistically promote the JNK mediated apoptotic cell death against human colorectal cancer cell.

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Introduction

Statistically, colorectal cancer is considered the second leading cause of death in women and third in men, among the most commonly diagnosed cancers in the western world (Bray et al., 2018). The mortality rate of people diagnosed with colorectal cancer is around 900,000 deaths annually (Dekker et al., 2019). In addition, reports state that metastatic colorectal cancer (mCRC) is more frequently diagnosed at an older age (greater than 65 years) (Yu and Cheung, 2018). In the early 1990s, 5-fluorouracil (5-FU) (Fig. 1A) was considered as the standard drug for colorectal cancer, which showed a significant survival advantage over chemotherapy (Goodwin and Asmis, 2009). 5-FU is an analog of uracil, having a fluorine atom at the C-5 position in place of hydrogen (Wohlhueter et al., 1980). Within the cell, 5-FU is converted into various active metabolites such as fluorodeoxyuridine monophosphate (FdUMP), fluorodeoxyuridine triphosphate (FdUTP) and fluorouridine triphosphate (FUTP) (Longley, 2003), which play crucial roles in biological activities such as disruption of RNA synthesis, and also exert anticancer effects through the inhibition of thymidylate synthase (TS). However, the acquired chemoresistance of cancer cell and low cell membrane permeability of 5-FU limits the therapy strategies (Farhat et al., 2014; Focaccetti et al., 2015). In addition, the major side effects of 5-FU including cognitive impairment upon prolonged administration (Wigmore et al., 2010).

Recently, synergistic adjuvant chemotherapy with other compounds, such as folinic acid, levamisole, rutin, and quercetin, has been indicated to potentiate the effect of 5-FU (Moertel et al., 1995; Masi et al., 2006; Dai et al., 2016; Satari et al., 2019). Substantial progress has been made towards finding more effective adjuvants that eliminate cancer cells efficiently in combination with the standard anticancer drugs, without being toxic to normal cells. This ongoing research has witnessed different approaches applied by researchers around the world, to uncover the best combinational therapeutics.

Tangeretin (TAN, Fig. 1B) is one such polymethoxylated flavonoid found in the peel of various citrus fruits (Rafiq et al., 2018). It exerts several biological activities against a broad spectrum of diseases such as anti-inflammatory properties (Xu et al., 2019), protection against renal tubular cell injury, efficiently blocks the cellular entry of arenaviruses that causes viral hemorrhagic fever, and also suppresses various cancers by modulating different pathways (Guo et al., 2015; Ting et al., 2015; Surichan et al., 2018; Wei et al., 2019). Therefore, in the current study, we evaluated the synergistic effect of TAN towards 5-FU, which could reduce the side effects associated with the prolonged usage of the latter. Through our study, we were able to determine that TAN, in combination with 5-FU, significantly accelerates the apoptotic events via JNK -dependent pathway against the human colorectal cancer cells. Based on these obtained results, we concluded that, tangeretin-5-FU combination may offer a new promising approach which can effectively control the growth and development of human colorectal cancer.

Section snippets

Chemicals and reagents

Roswell Park Memorial Institute −1640 (RPMI-1640), Hoechst 33342, Rhodamine 123, dichlorodihydrofluorescein diacetate (H2DCFDA), dimethylsulfoxide (DMSO) and, 3,-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich. Fetal bovine serum (FBS) was procured from Gibco. The details of antibodies used are provided (Supp. Table 1). Tangeretin (TAN) was purchased from Cayman Chemicals, and 5-FU was purchased from Sigma-Aldrich. All the solvents were of the

Combination of 5-FU with TAN synergistically enhances the rate of cell death in colorectal cancer cells

To enhance the anticancer effect and reduce side effects of 5-FU, the efficacy of 5-FU was evaluated in combination with a natural drug, against human colon carcinoma cells. Different concentrations of the drugs were determined based on the available literature (Morley et al., 2007), which was further validated by performing the formal synergism evaluation (Fig. 1A). In Table 1, we demonstrated the synergistic evaluation of TAN and 5-FU treatment against HCT-116, KM12C, HCT-15, and CCD 841 CoN

Discussion

5-FU is a chemotherapeutic drug used to suppress the growth of various cancer cells, including breast, colorectal, and pancreatic (Wigmore et al., 2010; Lee and Park, 2016). 5-FU acts in several ways, but primarily as a thymidylate synthase inhibitor. Briefly, 5-FU interrupts the action of thymidylate synthase. Thymidine is a nucleoside which are an essential component required for DNA replication, which ultimately affects DNA synthesis (Zhang et al., 2008). The IC50 value of 5-FU as a

CRediT authorship contribution statement

Debasish Kumar Dey: Conceptualization, Data curation, Formal analysis, Methodology, Project administration, Software, Supervision, Validation, Visualization, Writing - original draft. Sukkum Ngullie Chang: Writing - original draft, Data curation, Formal analysis, Methodology. Yellamandayya Vadlamudi: Funding acquisition, Writing - review & editing. Jae Gyu Park: Formal analysis, Investigation. Sun Chul Kang: Funding acquisition, Investigation, Project administration, Resources.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgment

This research was supported by the National Research Foundation of Korea grant, NRF2019R1A2B501070543 and 2019H1D3A1A01103012.

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