Design, synthesis and biological evaluation of fluorescent derivatives of ursolic acid in living cells

Design, synthesis and biological evaluation of fluorescent derivatives of ursolic acid in living cells

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Wenyi Mei^a^,¹, Lijuan Xie^a^,¹, Xiaodong Zhang^a, Cunjian Shi^a, Fengzhi Wang^a, Qiqi Fu^a, Zhenjiang Zhao^a, Honglin Li^*^,^a^,^b^,^c, Yufang Xu^*^,^a, Zhuo Chen^*^,^a

Chinese Chemical Letters | 2024, 35(5) : 108825

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Chinese Chemical Letters | 2024, 35(5): 108825

• Communication •

Design, synthesis and biological evaluation of fluorescent derivatives of ursolic acid in living cells

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Wenyi Mei^a^,¹, Lijuan Xie^a^,¹, Xiaodong Zhang^a, Cunjian Shi^a, Fengzhi Wang^a, Qiqi Fu^a, Zhenjiang Zhao^a, Honglin Li^*^,^a^,^b^,^c, Yufang Xu^*^,^a, Zhuo Chen^*^,^a

Affiliations

^a Shanghai Key Laboratory of New Drug Design, State Key Laboratory of Bioreactor Engineering, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China

^b Innovation Center for AI and Drug Discovery, East China Normal University, Shanghai 200062, China

^c Lingang Laboratory, Shanghai 200031, China

Published: 2024-05-15 doi: 10.1016/j.cclet.2023.108825

Outline

Abstract

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Ursolic acid (UA) is a naturally occurring ursane triterpenoid, which exhibits a wide range of unique biological activities. To clarify its mechanism of action (MOA), a series of fluorescent derivatives of UA (5a–c) were designed and synthesized by conjugation with 7-nitrobenzo-2-oxa-1,3-diazole (NBD) fluorophore. Among them, 5c exhibited similar anti-proliferative activity with UA against HCT116 cells (half maximal inhibitory concentration (IC₅₀) = 9.21 ± 0.50 µmol/L). Cell imaging experiment indicated that 5c was rapidly taken up in HCT116 cells in a dose and time-dependent manner. Then, 5c was found to localize in endoplasmic reticulum (ER), lysosomes, and mitochondria, but not in nucleus of HCT116 cells by confocal microscopy studies. Preliminary MOA proved that UA induced autophagy with a unique intracellular distribution mechanism involving ER and lysosome. In all, our work provides new clues for revealing the molecular mechanism of UA as an antitumor agent.

Key words

Pentacyclic triterpenoid / Ursolic acid / Fluorescent derivatives / Autophagy / Apoptosis

Cite this Article

Wenyi Mei, Lijuan Xie, Xiaodong Zhang, Cunjian Shi, Fengzhi Wang, Qiqi Fu, Zhenjiang Zhao, Honglin Li, Yufang Xu, Zhuo Chen. Design, synthesis and biological evaluation of fluorescent derivatives of ursolic acid in living cells[J]. Chinese Chemical Letters, 2024 , 35 (5) : 108825 - . DOI: 10.1016/j.cclet.2023.108825

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Ursolic acid (UA, Fig. 1A) is widespread in fruits, vegetables, and medicinal herbs. It has attracted considerable attention due to its remarkable pharmacological activities, such as anti-inflammatory [1,2], antiviral [3–5], hepatoprotective [6,7]. It has also shown efficacy in treating solid tumors, including gallbladder carcinoma, breast cancer, and colorectal cancer [8–10]. UA is an interesting and important natural triterpenoid for cancer therapy, but it faces several challenges, including poor water solubility, poor pharmacokinetic properties, and unclear mechanism of action [11–13].

UA was reported to inhibit colorectal cancer (CRC) tumor growth and metastasis alone or in combination with other antitumor agents [14–16]. For example, UA synergized with oxaliplatin and inhibited CRC cell proliferation and tumor growth in vivo [14]. Given the significant potential of UA in cancer prevention and treatment, its detailed mechanism of action needs in-depth exploration. Recently, various technological and scientific developments have been employed to elucidate the actions of bioactive natural products, including biochip technology, drug affinity responsive target stability (DARTS), and activity-based protein profiling (ABPP). Among them, fluorescence imaging technology showed its advantages due to high sensitivity, fast response speed, in situ detection in living cells, practicality, and simplicity of operation [17]. Therefore, fluorescence imaging technology is widely used to investigate cellular uptake, cellular localization, and specific interactions associated phenotypes of bioactive natural products in living cells [18–26].

To date, the distribution of UA in cells has not been reported. Therefore, in this study, we designed and synthesized novel fluorescent derivatives based on UA to visualize their cellular uptake and subcellular localization.

The fluorescent derivatives were designed by UA combined with a fluorophore via a linker region. Firstly, UA should be derived from a position with minimal influence on its pharmacological profile. UA exhibited potent antiproliferative activity against HCT116 cells with half maximal inhibitory concentration (IC₅₀) value of 8.75 ± 0.13 µmol/L (Table 1). Replacement of 3β-OH with hydroxamic acid (UA-1) resulted in a slight loss of cytotoxicity (IC₅₀ = 26.49 µmol/L, Table S1 and Fig. S1 in Supporting information). Furthermore, structural modifications of 3β-OH with acetylation (UA-2) and dichloroacetate (UA-3), resulted in complete loss of against HCT116 cells activity (IC₅₀ > 50 µmol/L, Table S1 and Fig. S1). Therefore, 3β-OH was important for the antiproliferative activity against HCT116 cells. We selected C-28 carboxyl group for conjugating reactions. Secondly, 7-nitro-2,1,3-benzoxadiazole (NBD) was then selected as fluorophore due to its excellent fluorescence/pharmacological properties, such as water solubility, small size, cell permeability, and low toxicity [27]. Lastly, the conjugation strategies were important for development of fluorescent derivatives [28]. Thus, the carboxyl group of UA was extended by an alkyl linker and subsequently connected with NBD, affording three UA fluorescent derivatives (5a–c, Fig. 1B).

The antiproliferative activities of 5a–c (Fig. S2 in Supporting information) was evaluated in vitro in comparison with UA. As shown in Table 1, no obvious antiproliferative activities was detected for compounds 5a and 5b with two or four-carbon spacers (IC₅₀ > 50 µmol/L). The fluorescent derivatives with six-carbon spacers (5c) exhibits cytotoxicity equivalent to that of UA against HCT116 cells. NBD-C6-NH₂ with no UA moiety (Fig. S3 in Supporting information) has no antiproliferative activity on HCT116 cells (IC₅₀ > 50 µmol/L). Considering the favorable antiproliferative activity of 5c, it was selected for subcellular localization studies. As shown in Fig. S4 (Supporting information), 5c exhibits significant differences between maximum absorption (λabs = 480 nm) and emission (λem = 540 nm) wavelengths with a large stokes shift of 60 nm in an aqueous solution, which was suitable for the imaging studies.

To further investigate the uptake of 5c by HCT116 cells using fluorescence microscopy, the dose dependence and time dependence of 5c in HCT116 cells were tested. As shown in Fig. 2A, bright green fluorescence staining was observed inside the HCT116 cells when 5c was incubated at a concentration of 5, 10, and 20 µmol/L for 1 h, indicating that 5c could enter HCT116 cells in a concentration-dependent manner. Similarly, 5c began to be absorbed into HCT116 cells after 15 min of incubation (Fig. 2B), indicating that the uptake of 5c at this concentration is quick and in time-dependent manner.

To further investigate the subcellular localization of UA, we treated 5c in HCT116 cells with various organelle-specific dyes. As shown in Fig. 3, confocal microscopy of HCT116 cells contained with LysoTracker Red, ER Tracker, and Mito Tracker confirmed the localization of 5c in lysosomes, ER, and mitochondria with a Pearson's correlation coefficient (PCC) value of 0.88, 0.92, and 0.70, respectively. In contrast, 5c exhibited almost no colocalization in nucleus (Fig. S5 in Supporting information). Additionally, when we tested NBD-C6-NH₂, no obvious overlap was detected (Fig. S6 in Supporting information). Therefore, 5c provided visual evidence that UA primarily localized with mitochondria, lysosome, and ER in living cells.

The mitochondrion plays a critical role in apoptosis, while lysosomes and ER play critical roles in autophagy [29–32]. Previous reports have indicated that UA induced apoptosis and autophagy in various cancer cells, such as T47D, MCF-7, and PC-3 cells [33,34]. Our subcellular localization studies have shown that UA was distributed in the mitochondrion, ER, and lysosomes in HCT116 cells. To further clarify the subcellular distribution of UA and its regulation of cell function, we investigated the expression levels of apoptosis- and autophagy-related proteins by Western blot. UA could down-regulate the expression levels of caspase-3 and poly ADP-ribose polymerase (PARP) in a concentration-dependent manner. Additionally, the expression levels of cleaved caspase-3, cleaved PARP, and p53 were up-regulated in a concentration-dependent manner after incubation with UA for 24 h (Fig. S7A in Supporting information). Moreover, the expression levels of LC3-Ⅰ, LC3-Ⅱ, and p62 were up-regulated with increasing the concentrations of UA, and the ratio between LC3-Ⅱ and LC3-Ⅰ also increased (Figs. S7B and C in Supporting information). These results indicate that UA was widespread distributed in the ER, lysosome, and mitochondria and exert its effects through autophagy and apoptosis mechanisms.

In summary, we designed and synthesized three fluorescent UA derivatives with a large stokes shift. Luckily, 5c retained similar antitumor activity with UA against HCT116 cells using cell counting kit-8 (CCK-8) assays in vitro, with IC₅₀ value of 9.21 ± 0.50 µmol/L. Fluorescence microscopy studies confirmed that 5c could quickly transport into HCT116 cells in a concentration- and time-dependent manner. Confocal microscopy experiments revealed that 5c was distributed in the ER, lysosomes, and mitochondrial. Inspired by subcellular localization study, the involvement of autophagy and apoptosis mechanisms was demonstrated through Western blot analysis. Overall, our work provides deeper insights and visual tool for further studying the mechanisms in living cell.

Declaration of competing interest

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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.

Acknowledgments

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The research is supported in part by the National Key Research and Development Program of China (No. 2022YFC3400501), the National Natural Science Foundation of China (Nos. 81825020 and 82150208 to H.L.), the Shanghai Science and Technology Commission Biomedical Science and Technology Support Special Project (Nos. 21S11907900 and 20S11901000 to Z.Z.). Honglin Li is also sponsored by the National Program for Special Supports of Eminent Professionals and the National Program for Support of Top-Notch Young Professionals.

Supplementary materials

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Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2023.108825.

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Year 2024 volume 35 Issue 5

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Article Info

doi: 10.1016/j.cclet.2023.108825

Receive Date：2023-05-09
Online Date：2025-11-21
Published：2024-05-15

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Received：2023-05-09
Revised：2023-07-17
Accepted：2023-07-18

Affiliations

^a Shanghai Key Laboratory of New Drug Design, State Key Laboratory of Bioreactor Engineering, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China

^b Innovation Center for AI and Drug Discovery, East China Normal University, Shanghai 200062, China

^c Lingang Laboratory, Shanghai 200031, China

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表12种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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Fig. 1 Design fluorescent derivatives of UA. (A) Chemical structure of UA. (B) Fluorescent derivatives design strategy based on UA.

Fig. 2 The uptake of indicated 5c in HCT116 cells, images were collected using fluorescence microscopy. (A) The dose-course uptake of 20 µmol/L 5c in HCT116 cells, (a) 5 µmol/L 5c for 1 h, (b) 10 µmol/L 5c for 1 h, (c) 20 µmol/L 5c for 1 h. (B) The time-course uptake of 20 µmol/L 5c in HCT116 cells, (a) HCT116 cells were treated with 20 µmol/L 5c for 0.25 h, (b) 0.5 h, (c) 1 h, (d) 2 h, (e) 4 h. Enlarged view of representative cells, 10×.

Fig. 3 Subcellular localization of 5c. (A) Subcellular localization of 5c and ER Tracker (ER specific dye) in HCT116 cells. HCT116 cells incubated with 20 µmol/L 5c for 1 h. ER tracker at λex = 587 nm and λem = 615 nm. (B) Subcellular localization of 5c and LysoTracker (lysosomes specific dye) in HCT116 cells. HCT116 cells incubated with 20 µmol/L 5c for 1 h. LysoTracker Red at λex = 577 nm and λem = 590 nm. (C) Subcellular localization of 5c and Mito Tracker (mitochondria specific dye) in HCT116 cells. HCT116 cells incubated with 20 µmol/L 5c for 1 h. Mito Tracker at λex = 579 nm and λem = 599 nm. Scale bar: 20 µm. Images were collected by confocal microscopy (Nikon A1R).

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