Synergistic effects of combination treatment using EGCG and suramin against the chikungunya virus

https://doi.org/10.1016/j.bbrc.2017.07.157Get rights and content

Highlights

  • The green tea compound EGCG inhibits CHIKV entry, replication and release in U2OS cells.

  • EGCG and suramin possess the synergistic anti-CHIKV activity.

  • The synergistic anti-CHIKV effect of EGCG and suramin is also proved by CHIKV two clinical isolates.

Abstract

Chikungunya is a severe disease that results from infection with the chikungunya virus (CHIKV), an arbovirus. Thus, we (1) explored a new approach to combining previously researched drugs that have shown the potential to inhibit CHIKV infection; and (2) demonstrated the antiviral effects of (−)-Epigallocatechin-3-gallate (EGCG) and the underlying mechanisms. Specifically, we used U2OS cells infected with CHIVK to assess the synergistic antiviral activities of EGCG and suramin. EGCG presented the ability to inhibit the viral RNA, progeny yield, and cytopathic effect (CPE) of CHIKV and also demonstrated the ability to protect against virus entry, replication, and release. Moreover, the results confirmed that EGCG and suramin can have synergistic effects against CHIKV strain S27 infection and two other clinical isolates of CHIKV. Our findings suggest that treatment with a combination of EGCG and suramin could provide a basis for the development of novel stretages against CHIKV infection.

Introduction

The Chikungunya virus (CHIKV) is an enveloped, positive-stranded alphavirus that replicates within mosquito and human hosts and can cause severe disease. Specifically, CHIKV is transmitted to humans via mosquitoes of the Aedes genus. The first case of CHIKV was reported in Tanzania in 1952 [1], and the term “chikungunya” comes from the Makonde language. The English translation of this word means that the pain associated with the disease causes patients to become contorted. CHIKV can lead to fever, headache, joint pain, muscle pain, joint swelling, and rash [2]. Unfortunately, there is currently a lack of FDA approved antiviral drugs capable of treating CHIKV, and the development of new antiviral drugs requires significant investment for the design and validation of compounds. Nonetheless, the persistent threat of viruses such as CHIKV necessitates the implementation of measures capable of addressing future outbreaks. As an alternative to developing new antiviral drugs, natural compounds can also be used or previously approved drugs can be repurposed [3].

Green tea is believed to confer multiple physiological and pharmacological benefits. (−)-Epigallocatechin-3-gallate (EGCG) is the main ingredient in extracts of dry Camellia sinensis (tea plant) leaves [4] and, in green tea it is the most abundant bioactive catechin. EGCG has been shown to have formidable preventive effects against viral infection, many cancers, cardiovascular diseases, and neurodegenerative diseases [5], [6]. EGCG also has been shown to possess anti-inflammatory as well as anti-atherosclerotic effects, which make this compound a potent antioxidant [7], [8], [9]. Furthermore, EGCG can be safely administered to healthy individuals [10], and a previous study reported that EGCG has health benefits for human skin and does not lead to negative side effects when consumed orally or applied topically [11].

EGCG has shown effective antiviral activity against multiple virus families, including CHIKV (Weber et al., 2015), coxsackievirus B3 (CVB3) [12], ebola virus (EBOV) [13], hepatitis B virus (HBV) [14], hepatitis C virus (HCV) [15], enterovirus 71 (EV71) [16], human immunodeficiency virus (HIV) [6], herpes simplex virus (HSV) [17], influenza virus (FLU) [18], and zika virus (ZIKV) [19]. In hepatoma cell lines or primary human hepatocytes, green tea extract and EGCG can both reduce the infectivity of HBV [20] and has also been found to inhibit the mechanism of HBV virion entry [21] by directly binding to the virion, thereby causing a bulge to form on the viral envelope. EGCG inhibit HCV entry by interrupting viral attachment to host cells at concentrations as low as 1–10 μg/ml [22]. Furthermore, EGCG is able to reduce EBOV infectivity in human cells by inhibiting host proteins in a dose-dependent manner [13].

In the treatment of human HIV, studies have shown that EGCG has strong binding affinity to the CD4 protein [23], which reduces the ability of HIV vgp120 to bind to human CD4+ cells [24]. EGCG can also inhibit the infectivity of HIV-1 in human CD4+ cells, and the inhibitory effects of EGCG are similar among multiple subtypes of HIV strains (The half-maximal inhibitory concentration (IC50): 4.5 μM–12 μM). Moreover, the effectiveness of EGCG in treating HIV is comparable to that of the anti-HIV drugs ritonavir (1.4 μM) and zidovudine (10 μM) [6]. EGCG has also been shown to inhibit HIV infectivity through its galloyl moiety, which binds to viral integrase [25], and the mechanisms underlying the inhibition of HIV infectivity could involve multiple interactions between EGCG and cell surface molecules of host CD4 and viral integrase [4]. Another study found that higher concentrations of EGCG (>100 μM) were capable of inhibiting ZIKV entry by at least 1-log (>90%); however, pre-treating cells with EGCG did not have any effects on virus attachment at Vero E6 cells [19]. Taken together, the results from previous studies suggest that a variety of mechanisms underlie the ability of EGCG to inhibit different viral infections, including viral entry, viral replication, viral transmission, or interactions with host proteins.

Weber et al. (2015) demonstrated that EGCG is able to inhibit CHIKV infection via the CHIKV-mCherry-490 pseudovirus. Those researchers also showed that EGCG can (1) inhibit CHIKV entry by blocking the entry of CHIKV Env-pseudotyped lentiviral vectors and (2) inhibit CHIKV attachment to target cells [26]. However, their results would have been strengthened if the experiments had been conducted on CHIKV S27 strains and clinical isolates of CHIKV.

Our own previous studies revealed that, suramin is able to inhibit CHIKV entry and transmission [27] and as well as reduce CHIKV pathogenesis [28]. Therefore, in the current study, we sought to determine whether synergistic effects exist between EGCG and suramin. Specifically, we used human U2OS cells infected with CHIKV S27 to investigate the anti-CHIKV activities of EGCG as well as the underlying mechanisms. We also assessed the synergistic antiviral activities of EGCG and suramin against CHIKV. Finally, we found that a combination treatment of EGCG with suramin enhanced the inhibition of CHIKV infection.

Section snippets

Cell and virus cultures

U2OS cells (ATCC: HTB-96) and BHK-21 cells (ATCC: CCL-10) were cultured in DMEM (Biological Industries, catalog# 01-052-1) with 12.5 mM HEPES (Biological Industries, catalog# 03-025-1B), L-Alanyl-l-Glutamine (Biological Industries, catalog# 03-022-1B), antibiotics (Biological Industries, catalog# 03-033-1B), and 5% fetal bovine serum (Biological Industries, catalog# 04-001-1) at a temperature of 37 °C under 5% CO2. CHIKV strain S27 (ATCC-VR-64, African prototype) as well as two clinical strains

Anti-CHIKV activities of EGCG

RT-qPCR, TCID50, and microneutralization assays were used to determine the inhibitory effects of EGCG on CHIKV infection. U2OS cells, commonly been used to investigate anti-CHIKV infection [27], [31], [32], were infected with CHIKV strain S27 at an MOI of 0.01 for 1 h in the presence of indicated concentrations of EGCG. Cells were then provided with fresh medium that contained the same concentration of EGCG and were incubated at 37 °C for 16 h. Following this, the cell lysate and culture

Discussion

CHIKV was identified in 1952 [1] due to its possible association with cases of severe disease [2]. Scientists are currently trying to reduce the harmful risks of this virus through the development of vaccines or effective drugs. Results of previous investigations have suggested that EGCG exerts antiviral effects through a diverse array of mechanisms [13], [22], [33].

In this study, we evaluated the inhibitory activities of EGCG against CHIKV, in particular its ability to interfere with the

Acknowledgements

We would like to thank the members of BSL-3 lab in the Institute of Preventive Medicine, National Defense Medical Center. The two clinical strains of CHIKV (0611aTw [Singapore/0611aTw/2006/FJ807896] and 0810bTw [Malaysia/0810bTw/2008/FJ807899]) were kindly provided by Centers for Disease Control, R.O.C. (Taiwan).

Funding

This study was supported by grants (104-9-B19, 105-G6-1(3), 106-P7) from the Institute of Preventive Medicine, National Defense Medical Center.

Conflict of interest

The authors declare that they have no conflicts of interest.

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