Anti-proliferative and immunomodulatory properties of kaffir lime leaves and bioactive compounds on macrophages co-cultured with squamous cell carcinoma

PLOS ONE RESEARCH ARTICLE Anti-proliferative and immunomodulatory properties of kaffir lime leaves and bioactive compounds on macrophages co-cultured with squamous cell carcinoma Thitiya Luetragoon1,2, Yordhathai Thongsri2, Krai Daotak2, Pachuen Potup2, Kanchana Usuwanthim ID2* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Faculty of Allied Health Sciences, Department of Medical Technology, Nakhonratchasima College, Nakhonratchasima, Thailand, 2 Faculty of Allied Health Sciences, Cellular and Molecular Immunology Research Unit, Naresuan University, Phitsanulok, Thailand * Abstract OPEN ACCESS Citation: Luetragoon T, Thongsri Y, Daotak K, Potup P, Usuwanthim K (2023) Anti-proliferative and immunomodulatory properties of kaffir lime leaves and bioactive compounds on macrophages co-cultured with squamous cell carcinoma. PLoS ONE 18(2): e0281378. https://doi.org/10.1371/ journal.pone.0281378 Editor: Abdul Qadir Syed, Northwest University, UNITED STATES Received: February 22, 2022 Accepted: January 22, 2023 Published: February 21, 2023 Copyright: © 2023 Luetragoon et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was support by Thailand Science Research and Innovation, and Naresuan University (R2566B087). Competing interests: The authors have declared that no competing interests exist. Head and neck squamous cell carcinoma (HNSCC) is the seventh most common cancer worldwide. Late-stage patients have a significant chance of local recurrence and distant metastasis, as well as poor prognosis. Therapeutic goals for patients must be improved and personalized to reduce adverse effects. This study explored the anti-proliferative activity and immunomodulation potential of the constituents of crude kaffir lime leaf extract (lupeol, citronellal and citronellol) under co-culture. Results showed high cytotoxicity to human SCC15 cell line but not to human monocyte-derived macrophages. Treatment with crude extract and the contained compounds also suppressed cell migration and colony formation of SCC15 compared to the untreated control group, while high levels of intracellular ROS production were detected in the treatment group of SCC15. The MuseTM cell analyzer revealed cell cycle arrest at G2/M phase and apoptosis induction. Inhibition of Bcl-2 and activation of Bax, leading to induction of the downstream caspase-dependent death pathway were confirmed by Western blot analysis. Co-culture with activated macrophages, kaffir lime extract and its constituents enhanced the development of pro-inflammatory (M1) macrophages and boosted TNF-α production, resulting in SCC15 apoptosis. Findings revealed novel potential activities of kaffir lime leaf extracts and their constituents in inducing M1 polarization against SCC15, as well as direct anti-proliferative activity. Introduction Head and neck squamous cell carcinoma (HNSCC) was the seventh most common cancer worldwide in 2018 with 890,000 new cases and 450,000 deaths [1]. This cancer can arise from the mucosal epithelium in the oral cavity, oropharynx, hypopharynx and larynx [2]. HNSCC has been related to alcohol consumption, tobacco smoking, human papillomavirus (HPV) infection for cancers of the oropharyngeal region, the Epstein-Barr virus (EBV) associated with nasopharyngeal cancer and ultraviolet radiation from sunlight exposure for lip cancer [3]. PLOS ONE | https://doi.org/10.1371/journal.pone.0281378 February 21, 2023 1 / 23 PLOS ONE Anti-proliferative and immunomodulatory properties of kaffir lime leaves Cancer is a complicated disease involving tumor microenvironment (TME) that defines the nature of cancer, not by the genetics of the tumor cells alone but by the surrounding environment that the tumor cells need for survival, growth, proliferation, invasion, and metastasis. The production of numerous intercellular mediators such as cytokines, chemokines and vesicles attract non-transformed cells in the surrounding area. The consequences are TME formation and close interaction with cancer cells to support the development of cancer hallmarks [4–6]. The TME comprises cancer cells and multiple normal cell types like fibroblasts, myofibroblasts and immune cells as well as cytokines, vascular tissue, and the surrounding extracellular matrix. Hence, complex interaction of different cellular signaling pathways with immune cell components in TME influences cancer initiation, progression, and therapeutic response [7,8]. Macrophages play an important role in the innate immune system by maintaining immune homeostasis. They are involved in several processes of initiating and regulating the immune responses to foreign antigens [9]. Two major polarization states have been described for macrophages as classically activated macrophages (M1 macrophages) that exhibit pro-inflammatory responses in host defense systems against a variety of bacteria, protozoa and viruses and play a role in anti-tumor immunity, while alternatively activated macrophages or M2 macrophages have anti-inflammatory functions and regulate tissue repair and remodeling [10,11]. In cancer, tumor-derived factors drive macrophages toward an immunosuppressive M2 marker following their recruitment to the tumor. They are called tumor-associated macrophages (TAMs) and have been linked to immune suppression and growth, angiogenesis, metastasis, and invasion of cancers [12,13]. M2-like TAMs can inhibit T cell function directly by multiple mechanisms and are associated with poor prognosis outcomes in solid tumors [14]. Macrophages are involved in tumor development and suppression. Immunostimulation of macrophages contributes to tumor regulation as a promising therapeutic target. Numerous studies have revealed that many plants or natural compounds have an immunomodulatory effect on macrophages. Methanolic extract from Pouteria campechiana leaves stimulated the proliferation of murine macrophages, phagocytic activity, nitric oxide (NO), hydrogen peroxide (H2O2) and cytokines (interleukin (IL)-6 and tumor necrosis factor (TNF)-α) production [15]. Crude polysaccharide extract from Curcuma xanthorrhiza Roxb. significantly increased phagocytosis and release of NO, H2O2, TNF-α and PGE2 in a dose-dependent manner in macrophages by specific activation of nuclear factor-κB (Nf-κB) [16]. In Japan, Ando et al. (2002) revealed that polysaccharides from safflower petals (Carthamus tinctorius L.) activated macrophages by recognizing and binding to the specific Toll-like receptor 4 on their surfaces [17]. Moreover, Nordin et al. investigated the immunomodulatory effects of Clinacanthus nutans extracts on THP-1 macrophages in co-culture with triple-negative breast cance (...truncated)