Synthesis and Anticancer Activity of Ergosterol Peroxide Hybrids With Paclitaxel Side Chain Inducing Apoptosis in Human Hepatoma Carcinoma Cells

Abstract

The antitumor activities of natural paclitaxel (PTX), semisynthetic docetaxel, and cabazitaxel are highly dependent on their C-13 side chains. Therefore, using natural ergosterol peroxide (EP, 1) as the lead compound, two EP-PTX hybrids (EP-A2 and EP-B2) were prepared and their antitumor activities were evaluated against 4 kinds of human MCF-7, HepG2, HCT-116, and A549 cell lines in vitro. The results showed that both EP-A2 and EP-B2 inhibited the growth of all four kinds of tested tumor cell lines. For paclitaxel-resistant MCF-7 cells, both EP-A2 and EP-B2 showed significant inhibitory activity with relatively low IC₅₀ values (9.39 μM and 8.60 μM, respectively). In addition, EP-B2 inhibited the growth of the HepG2 cells (IC₅₀ = 7.82 μM) more successfully than EP. Preliminary studies of the mechanism suggest that EP-B2 could arrest the G1 phase transition in HepG2 cells. In addition, EP-B2 showed an obvious apoptosis-inducing effect in HepG2 cells, as detected by the Annexin V/PI binding assay and the Western blot assay. Hybrid EP-B2 has the potential to become a novel antitumor drug through further study of the mechanism of action and its structural modifications.

Keywords

structure modification paclitaxel ergosterol peroxide hybrids antitumor

Introduction

Natural products have a long history of medicinal use and are also the main sources of new drugs.^1–3 The structural modification of bioactive natural products is also a highly efficient and useful method for the development of novel antitumor drugs.^4–6 Ergosterol peroxide (EP, 1), is a natural endoperoxide steroid that our research group extracted from the traditional Chinese medicine Ganoderma lucidum (Figure 1), has extensive biological activities and potential medicinal value.^7,8 As it is a valuable lead compound, EP (1) has attracted attention over the last decade, with research focusing on its antitumor activities.^9–13 It is worth noting that the peroxide bridge of EP (1) is considered to be an important pharmacophore, and the cytotoxic effects of EP (1) on a variety of cancer cells are significantly enhanced compared to those of ergosterol, which lacks the peroxide bridge structure.¹⁴ Due to the amount of EP (1), isolated from fungi, was too little, which was not sufficient to be used clinically. Hence, we developed an approach to synthesize ergosterol peroxide from ergosterol.¹⁴ However, moderate biological activity and low bioavailability of ergosterol peroxide limit its further application in drug development. Therefore, we attempted to modify the structure of EP (1) to improve its antitumor activity.

Figure 1.

Structure of natural ergosterol peroxide (EP, 1).

Paclitaxel (PTX, 2) is a naturally occurring taxane diterpenoid antineoplastic agent that was first isolated from Taxus brevifolia in 1971 and then approved by the FDA in 1992.^15–17 Docetaxel (3) and cabazitaxel (4) (Figure 2), which are both semi-synthetic analogs of paclitaxel, are important chemotherapeutic drugs that are widely used for the treatment of various cancer.^18–20 Due to the important role of paclitaxel, docetaxel, and cabazitaxel, their structure–activity relationships have been studied at length. The results showed that their unique C-13 side chains play an indispensable role in their biological activity.²¹ Ojima and co-authors^22,23 reported several paclitaxel-mimics with a simplified structure, in which the taxane core was substituted by an indolizidine scaffold, and the C-13 side chain was retained. These compounds expressed modest cytotoxic activities. In addition, the structures and configurations of some natural compounds like dehydroepiandrosterone (DHEA), cholesterol, vitamin D2, talatisamine, and songorine are similar to the taxane, and these natural products are economical and easy to obtain. Guenard and co-authors²⁴ synthesized a series of novel compounds combining steroids with the side chain of docetaxel, in which the taxane skeleton was replaced by a steroid scaffold. These compounds exhibited weak cytotoxic activities. Zheng et al reported some taxane-like natural skeleton-C13 side chain hybrids and evaluated their cytotoxicity against several kinds of human tumor cell lines. For PTX-resistant human mammary glands (MCF-7), most compounds were superior to PTX in inhibiting cell line growth.^25–27 These benefits encourage us to further synthesize taxane-like natural skeleton-C13 side chain hybrids and we expect to extend the therapeutic use of PTX to PTX-resistant or insensitive tumors.

Figure 2.

Structures of paclitaxel, docetaxel, and cabazitaxel (2-4).

Previously, we prepared a series of C-17 modified derivatives of EP (1), and many of them exhibited strong inhibitory efficacy against different kinds of tested tumor cell lines.^28–31 In addition, esterification at C-3 of EP (1) as acrylate or propionate ester has strong efficacy against 3 kinds of human tumor cell lines (Figure 3).³² We also successfully introduced the carbamate side chain into the C-3 hydroxy group of EP (1), and most of these synthetic derivatives exhibited nanomole cytotoxic activity (Figure 3).³³ Based on the encouraging results mentioned above, our research group set out to further modify EP (1) by introducing richer types of heterocycle substituents into its hydroxy group.

Figure 3.

The design strategy of new ergosterol peroxide-paclitaxel (EP-PTX) ester derivatives.

In this work, 2 new EP (1) ester derivatives, which modify the C-3 hydroxy with a paclitaxel side chain, were synthesized (Figure 3). The cytotoxic activity of all of the new target hybrids and intermediates was evaluated using an in vitro MTT assay using 4 human cancer HepG2 (hepatoma carcinoma), MCF-7 (breast carcinoma), HCT-116 (colorectal carcinoma), and A549 (lung carcinoma) cell lines. In addition, the preliminary antitumor mechanism of the EP-PTX hybrid was explored through AO/EB staining, flow cytometric analysis, and Western blot tests.

Results and Discussion

The synthetic method of preparing EP-PTX ester derivatives is shown in Scheme 1. EP (1) was synthesized by a Diels-Alder reaction between ergosterol (EG) and pure oxygen (O₂) and photosensitizer phloxine B. Then, using EDCI and DMAP as condensation agents, EP (1) was esterified at C-3 hydroxy group with (4S,5R)-3-benzoyl-2-(4-methoxyphenyl)-4-phenyl-5-oxazolidinecarboxylic acid (5) or (4S,5R)-3-tert-butoxycarbony-2-(4-anisy)-4-phenyl-5-oxazolidinecarboxylic acid (6) to produce the intermediate EP-A1 or EP-B1, respectively. The acid-mediated oxazolidine cleavages of the esters EP-A1 and EP-B1 were then carried out using p-toluenesulfonic acid to produce the targeted hybrids EP-A2 and EP-B2 in 73.2% and 78.6% yields, respectively. The chemical structures of all new compounds were confirmed by HRMS, ¹H NMR, and ¹³C NMR spectra.

Scheme 1.

Synthetic pathway to EP-PTX ester derivatives. Reagents and conditions: (i) carboxylic acid substituent group 5 or 6, EDC, DMAP, dichloromethane, rt, 20 h, 84.0%, and 82.5%. (ii) p-Toluenesulfonic acid (PTSA), methanol, rt. 73.2% and 78.6%.

The in vitro cytotoxic activities of all intermediates and EP-PTX ester derivatives against MCF-7, HepG2, HCT-116, and A549 cell lines were systematically evaluated using an MTT assay. Paclitaxel (2) was selected as the positive drug. The IC₅₀ values of all compounds were calculated and are summarized in Table 1. The results showed that both EP-A2 and EP-B2 inhibited the growth of all 4 tested cell lines. For paclitaxel-resistant MCF-7 cells, both the EP-A2 and EP-B2 compounds showed significant inhibitory activity with IC₅₀ values < 10 μM. Significantly, EP-B2 inhibited the proliferation of the HepG2 cells (IC₅₀ = 7.82 μM) more strongly than EP (1).

Table 1.

In Vitro Cytotoxicity Activity of EP-PTX Derivatives in Human Cancer Cells.

Compound	IC₅₀ (μM)
Compound	MCF-7	A549	HepG2	HCT-116
EP-A1	> 50	> 50	> 50	> 50
EP-B1	> 50	> 50	> 50	> 50
EP-A2	9.39 ± 0.28	14.62 ± 0.95	11.47 ± 0.74	19.85 ± 1.32
EP-B2	8.60 ± 0.32	11.80 ± 0.54	7.82 ± 0.33	17.60 ± 1.11
EP (1)	18.62 ± 1.80	23.40 ± 1.13	20.43 ± 0.82	31.04 ± 1.35
Paclitaxel (2)	< 1.0	< 1.0	< 1.0	< 1.0

As far as we know, the action mechanism of paclitaxel and its analogs is mainly related to its ability to stabilize the tumor cell division cycle in the G2/M phase.³⁰ According to research findings, EP (1) inhibits the proliferation of the cells by blocking the tumor cell cycle's progression to the G1 phase. First, we explored whether EP-B2, as a new hybrid structure, exhibited a blocking effect on the cycle of tumor cells. To study the effect of EP-B2 on the progression of the tumor cell cycle, HepG2 cells were cultivated and treated with EP-B2 at different concentrations (0, 4, 8, and 16 μM) for 24 h, and then assessed using flow cytometry. As shown in Figure 4, a dose-dependent arrest of the G1 phase transition arrest was founded in HepG2 cells that were treated with EP-B2. Treatment of HepG2 cells with EP-B2 at the highest concentration of 16 μM could result in 66.54% of cells being arrested in the G1 phase, compared with 42.56% in the untreated (0 μM) group.

Figure 4.

Hybrid EP-B2 induced tumor cell cycle arrest at G1 phase in HepG2 cells. (A) HepG2 cells were treated with EP-B2 and then detected by flow cytometry. (B) Quantitative analysis of HepG2 cell cycle arrest. Data were represented as mean ± SD (n = 3). *P < .05, **P < .01 versus control group.

To assess whether EP-B2 could promote apoptosis in HepG2 cells, the cells were treated with EP-B2 at different concentrations (0, 4, 8, and 16 μM) for 24 h. Annexin V and the propidium iodide double staining methods were carried out using flow cytometry. As shown in Figure 5, after treatment with EP-B2, the total apoptosis levels of the HepG2 cells noticeably increased, from 1.14% (control) to 20.18%. This result indicates that EP-B2 may induce HepG2 cell apoptosis.

Figure 5.

Hybrid EP-B2 induced HepG2 cell apoptosis. (A) HepG2 cells were treated with EP-B2 and then detected apoptosis by flow cytometry. (B) Quantitative analysis of apoptotic cells. Data were represented as mean ± SD (n = 3). *P < .05, **P < .01 versus control group.

As EP-B2 showed positive anticancer activity and could induce HepG2 cell apoptosis, we further explored the expression status of related apoptotic markers that are affected by EP-B2 in HepG2 cells using Western blot analysis (Figure 6). Using glyceraldehyde 3-phosphate dehydrogenase as the control, we noted that treatment with hybrid EP-B2 (0, 4, 8, and 16 μM) for 24 h, resulted in an obvious increase in Bax, Cyt-c, and cleaved-caspase-3, and -9, and an obvious decrease in Bcl-2. The results also demonstrate that hybrid EP-B2 could promote the apoptosis of HepG2 cells.

Figure 6.

Levels of apoptosis-related proteins. (A) The expression of apoptosis-related proteins was analyzed by western blotting. (B) Quantitative analysis of the protein expression. Data were represented as mean ± SD (n = 3). *P < .05, **P < .01 ***P < .001 versus glyceraldehyde 3-phosphate dehydrogenase (GAPDH) group.

Experimental

General

All chemical materials and organic solvents were commercially available and were used without further purification. Both (4S,5R)-3-benzoyl-2-(4-methoxyphenyl)-4-phenyl-5-oxazolidinecarboxylic acid (5) and (4S,5R)-3-tert-butoxycarbony-2-(4-anisy)-4-phenyl-5-oxazolidinecarboxylic acid (6) are commercially available from Energy chemical (Zesheng Technology Co., LTD, Anhui, China). The crude products were purified by column chromatography with 200–400 mesh silica gel (Yinlong, China). High-resolution mass spectra (HRMS) data were detected by TripleTOF 4600 (AB SCIEX, USA) and UPLC G2-XS Q-TOF (Waters, USA). NMR spectra were recorded with a 600 MHz AVANCE NEO-600 spectrometer (Bruker, Berlin, Germany).

General Procedure for the Preparation of EP-A1 and EP-B1

A suspension of EP (1) (0.2 mmol), DMAP (0.2 mmol, 1.0 eq), EDC (0.24 mmol, 1.2 eq), and protected side chain 5 or 6 (0.24 mmol, 1.2 eq) in dichloromethane (20 mL) was steadily stirred for 20 h at room temperature. After completion of the reaction, the mixture was diluted with ice water (10 mL) and then extracted with DCM (20 mL) 3 times. The organic phases were combined and washed with brine solution, dried over MgSO₄, and then evaporated to get the crude product. The product was separated and purified by column chromatography to give ester EP-A1 or EP-B1.

5α,8α-Epidioxiergosta-6,22-dien-3β-yl-3-benzoyl-2-(4-methoxyphenyl)-4-phenyloxazolidine 5-carboxylate (EP-A1). White powder. Yield 84.0%. ¹H NMR (600 MHz, CDCl₃) δ 7.84 (2H, d, J = 8.7 Hz, Ar-H), 7.75 (2H, d, J = 6.9 Hz, Ar-H), 7.51 (1H, t, J = 7.4 Hz, Ar-H), 7.45 (4H, d, J = 7.9 Hz, Ar-H), 7.36 (2H, t, J = 7.6 Hz, Ar-H), 7.29 (2H, t, J = 7.3 Hz, Ar-H), 7.02 (1H, s, Ar-H), 7.01 (1H, s, H-3′), 6.52 (1H, d, J = 8.5 Hz, H-7), 6.23 (1H, d, J = 8.5 Hz, H-6), 5.77 (1H, d, J = 9.2 Hz, H-4′), 5.22 (1H, dd, J = 15.2, 7.6 Hz, H-2′), 5.15 (1H, m, H-23), 5.13 (1H, m, H-22), 4.60 (1H, m, H-3), 3.90 (3H, s, H-OCH₃), 2.14 (2H, d, J = 6.8 Hz), 2.03 (1H, d, J = 9.2 Hz), 1.94 (2H, d, J = 7.6 Hz), 1.86 (2H, d, J = 6.7 Hz), 1.75 (1H, d, J = 10.1 Hz), 1.67 (1H, s), 1.64 (1H, s), 1.61 (1H, s), 1.57 (1H, s), 1.48 (2H, d, J = 6.1 Hz), 1.44 (1H, d, J = 6.7 Hz), 1.38 (1H, d, J = 6.7 Hz), 1.34 (1H, d, J = 10.3 Hz), 1.25 (2H, s), 1.21 (1H, s), 0.99 (3H, H-18), 0.91 (6H, m, H-21, H-28), 0.82 (9H, m, H-19, H-26, H-27). ¹³C NMR (150 MHz, CDCl₃) δ 172.3 (C5′), 167.0 (C1′), 164.8 (Ar), 135.3 (Ar), 135.0 (Ar), 134.4 (C22), 132.5 (C6), 132.2 (C23), 131.9 (Ar), 131.2 (Ar), 130.1 (C7), 128.9 (Ar × 6), 128.0 (Ar), 127.2 (Ar × 2), 127.0 (Ar × 2), 114.5 (Ar × 6), 81.9 (C3′, C5), 79.7 (C2′, C8), 73.5 (C3), 72.7 (C4′), 56.3 (C17), 55.7 (OCH₃), 54.7 (C4′), 51.7 (C14), 51.2 (C9), 44.8 (C13), 42.9 (C24), 40.1 (C12), 39.5 (C20), 37.2 (C10), 34.4 (C4), 33.2 (C25), 28.9 (C2), 26.0 (C16), 23.7 (C15), 21.1 (C11), 20.7 (C21), 20.2 (C26), 19.7 (C27), 18.4 (C19), 17.8 (C28), 13.0 (C18). MS (ESI) m/z: [M + H]⁺ 814.5.

5α,8α-Epidioxiergosta-6,22-dien-3β-yl-3-(tert-butoxycarbonyl)-2-(4-methoxyphenyl)-4-phenyloxazolidine 5-carboxylate (EP-B1). White powder. Yield 82.5%. ¹H NMR (600 MHz, CDCl₃) δ 7.85 (1H, d, J = 8.7 Hz, H-Ar), 7.36 (3H, t, J = 8.6 Hz, H-Ar), 7.33 (2H, d, J = 10.9 Hz, H-Ar), 7.28 (1H, s, H-Ar), 7.01 (1H, d, J = 8.8 Hz, H-Ar), 6.87 (1H, d, J = 8.5 Hz, H-Ar), 6.52 (1H, d, J = 8.4, H-7), 6.45 (1H, d, J = 18.5 Hz, H-3′), 6.23 (1H, d, J = 8.5, H-6), 5.39 (1H, d, J = 10.4 Hz, H-4′), 5.24 (1H, t, J = 9.3 Hz, H-2′), 5.22 (1H, m, H-23), 5.15 (1H, m, H-22), 5.12 (1H, m, H-3), 3.86 (3H, d, J = 49.7 Hz, H-8′), 2.15 (1H, d, J = 5.1 Hz), 2.04 (2H, d, J = 6.1 Hz), 2.00 (2H, d, J = 8.1 Hz), 1.97 (1H, d, J = 11.2 Hz), 1.86 (1H, d, J = 6.8 Hz), 1.75 (1H, s), 1.66 (1H, s), 1.62 (1H, d, J = 3.3 Hz), 1.59 (1H, s), 1.56 (1H, d, J = 5.0 Hz), 1.52 (2H, d, J = 9.1 Hz), 1.48 (1H, d, J = 6.5 Hz), 1.46 (1H, d, J = 6.6 Hz), 1.43 (1H, s), 1.40 (6H, s, H-C7′), 1.32 (3H, s, H-7′), 1.26 (2H, s), 1.24 (1H, s), 1.01 (3H, H-18), 0.92 (6H, m, H-21, H-28), 0.84 (9H, m, H-19, H-26, H-27). ¹³C NMR (150 MHz, CDCl₃) δ 164.6 (C1′), 155.0 (Ar), 153.8 (C5′), 139.8 (Ar), 135.2 (C22), 134.9 (C6), 132.4 (C23), 132.0 (Ar), 131.0 (C7), 128.6 (Ar × 2), 127.7 (Ar), 127.1 (Ar × 2), 126.7 (Ar × 2), 114.3 (Ar × 2), 91.2 (C3′), 81.7 (C5, C2′), 79.5 (C6′, C8), 71.2 (C3), 56.2 (C17), 55.6 (C8′), 51.7 (C14), 51.1 (C9), 44.7 (C13), 42.9 (C24), 39.8 (C12), 39.4 (C20), 37.1 (C10), 34.2 (C4), 33.2 (C25), 29.7 (C1), 28.7 (C2), 28.2 (C7′), 26.2 (C16), 23.5 (C15), 20.8 (C11), 20.7 (C21), 20.2 (C26), 19.7 (C27), 18.2 (C19), 17.6 (C28), 13.0 (C18). MS (ESI) m/z: [M + H]⁺ 810.5.

General Procedure for the Preparation of EP-A2 and EP-B2

The ester EP-A1 or EP-B1 (0.02 mmol) was added to methanol (20 mL) and treated with p-toluenesulfonic acid (PTSA, 0.1 mmol). The mixture was steadily stirred for 5 h at room temperature and then diluted with ethyl acetate (50 mL). The organic phases were combined and washed with NaHCO₃ saturated aqueous solution and brine solution, dried over MgSO₄, and then evaporated in vacuo to get the crude product. The product was separated and purified by column chromatography to give the target compound EP-A2 or EP-B2.

5α,8α-Epidioxiergosta-6,22-dien-3β-yl-(2R,3S)-3-benzamido-2-hydroxy-3-phenylpropanoate (EP-A2). White powder. Yield 73.2%. ¹H NMR (600 MHz, CDCl₃) δ 7.75 (2H, d, J = 7.0 Hz, H-Ar), 7.52 (1H, m, H-Ar), 7.45 (4H, d, J = 7.6 Hz, H-Ar), 7.36 (2H, t, J = 7.7 Hz, H-Ar), 7.30 (1H, d, J = 7.4 Hz, H-Ar), 6.98 (1H, d, J = 9.4 Hz, NH-3′), 6.52 (1H, d, J = 8.5 Hz, H-7), 6.23 (1H, d, J = 8.4 Hz, H-6), 5.77 (1H, d, J = 9.3 Hz, H-3′), 5.22 (1H, m, H-23), 5.15 (1H, m, H-22), 5.13 (1H, m, H-3), 4.60 (1H, d, J = 2.1 Hz, H-2′), 4.31 (1H, t, J = 6.7 Hz, OH-2′), 2.22 (1H, t, J = 7.7 Hz), 2.13 (2H, d, J = 10.1 Hz), 2.01 (2H, d, J = 7.9 Hz), 1.95 (1H, d, J = 3.6 Hz), 1.86 (1H, d, J = 6.7 Hz), 1.74 (1H, d, J = 6.9 Hz), 1.64 (1H, s), 1.59 (1H, s), 1.57 (1H, s), 1.55 (1H, s), 1.48 (2H, d, J = 4.7 Hz), 1.45 (1H, s), 1.42 (1H, d, J = 1.9 Hz), 1.39 (1H, d, J = 5.1 Hz), 1.25 (2H, s), 1.21 (1H, s), 0.99 (3H, d, J = 6.6 Hz, H-18), 0.90 (6H, m, H-21, H-28), 0.82 (9H, m, H-19, H-26, H-27). ¹³C NMR (150 MHz, CDCl₃) δ 172.3 (C1′), 167.1 (C4′), 138.9 (Ar), 135.2 (C22), 135.0 (Ar), 134.3 (C6), 132.5 (C23), 131.9 (C23), 131.2 (Ar), 130.1 (C7), 128.9 (Ar × 4), 128.0 (Ar), 127.2 (Ar × 2), 127.0 (Ar × 2), 81.9 (C5), 79.7 (C8), 73.5 (C2′), 72.7 (C3), 56.3 (C17), 54.6 (C3′), 51.8 (C14), 51.1 (C9), 44.6 (C13), 43.1 (C24), 39.8 (C12), 39.4 (C20), 37.1 (C10), 34.3 (C4), 33.2 (C25, C1), 28.8 (C2), 26.1 (C16), 23.5 (C15), 21.1 (C11), 20.9 (C21), 20.2 (C26), 19.7 (C27), 18.4 (C19), 17.8 (C28), 13.0 (C18). MS (ESI) m/z: [M + H]⁺ 696.4.

5α,8α-Epidioxiergosta-6,22-dien-3β-yl-(2R,3S)-3-((tert-butoxycarbonyl)amino)-2-hydroxy-3-phenylpropanoate (EP-B2). White powder. Yield 78.6%. ¹H NMR (600 MHz, CDCl₃) δ 7.71 (1H, s, H-Ar), 7.53 (1H, s, H-Ar), 7.34 (2H, s, H-Ar), 7.28 (1H, d, J = 6.8 Hz, H-Ar), 6.53 (1H, d, J = 8.6 Hz, H-7), 6.24 (1H, d, J = 8.5 Hz, H-6), 5.42 (1H, d, J = 9.9 Hz, NH-3′), 5.21 (1H, s, H-3′), 5.15 (1H, m, H-23), 5.12 (1H, m, H-22), 4.43 (1H, m, H-3), 4.30 (1H, s, H-2′), 4.12 (1H, d, J = 7.2 Hz, OH-2′), 2.15 (1H, s), 2.04 (2H, s), 2.00 (2H, d, J = 12.2 Hz), 1.97 (1H, s), 1.85 (1H, d, J = 6.8 Hz, 1H), 1.71 (1H, s), 1.61 (1H, s), 1.60 (1H, s), 1.58 (1H, s), 1.56 (1H, d, J = 7.4 Hz), 1.51 (2H, d, J = 8.6 Hz), 1.47 (1H, s), 1.46 (1H, s), 1.44 (1H, s), 1.40 (9H, s, H-6′), 1.25 (2H, s), 1.22 (1H, s), 1.00 (3H, d, J = 6.6 Hz, H-18), 0.91 (6H, d, J = 6.0 Hz, H-28, H-21), 0.84 (3H, H-19), 0.81 (6H, d, J = 5.7 Hz, H-26, H-27). ¹³C NMR (150 MHz, CDCl₃) δ 167.8 (C1′), 155.0 (C4′), 139.4 (Ar), 135.3 (C22), 134.9 (C6), 132.4 (C23), 131.0 (C7), 128.6 (Ar × 2), 127.7 (Ar), 126.7 (Ar × 2), 81.8 (C5), 79.5 (C5′, C8), 73.6 (C2′), 72.3 (C3), 56.2 (C17), 55.7 (C3′), 51.5 (C14), 51.1 (C9), 44.6 (C13), 42.8 (C24), 39.9 (C12), 39.5 (C20), 37.2 (C10), 34.2 (C4), 33.1 (C25), 29.7 (C1), 28.7 (C2), 28.3 (C6′), 25.8 (C16), 23.4 (C15), 20.8 (C11), 20.5 (C21), 20.2 (C26), 19.9 (C27), 18.0 (C19), 17.5 (C28), 13.0 (C18). MS (ESI) m/z: [M + H]⁺ 692.5.

In Vitro Cytotoxic Activity

HepG2, MCF-7, HeLa, and A549 cells were cultured under fresh RPMI-1640 medium (Corning, USA) containing penicillin (100 kU/L), FBS (10%), and streptomycin (100 mg/L) in 5% CO₂ atmosphere. Cells were cultured into 96-well plates (100 μL per well) at a concentration of 1 × 10⁴/mL for 24 h. Then the cells were treated with gradient concentrations of tested compounds for 48 h, then 10 μL MTT solution was added into each well for 2 h. The MTT solution was poured off and then 100 μL DMSO was added. The OD value was recorded using a microplate. The IC₅₀ values were obtained by SPSS analysis. The MTT kit was obtained from Beyotime Biotechnology (Nanjing, China).

Cell Cycle Analysis

HepG2 cells were seeded into 6-well plates (1 × 10⁶ cells/well) and cultured overnight and then were treated with EP-B2 for 24 h. The tumor cells were washed with pre-cold PBS, and then cells were stained with 5 mL of PI solution (0.1 mg/ml) at 37 °C for 10 min. Then, the DNA content of cells was detected by flow cytometry for cell cycle distribution analysis. The cell cycle detection kit was obtained from Beyotime Biotechnology (Nanjing, China).

Cell Apoptosis Analysis

HepG2 cells were seeded into 6-well plates (1 × 10⁶ cells/well) and cultured overnight and then were treated with EP-B2 for 24 h. The tumor cells were washed with pre-cold PBS, and then cells were stained with 10 mL of Annexin VFITC (AV) solution and 5 mL of PI solution at 37 °C for 10 min. Then, the samples were detected by a flow cytometer. The total percentages of apoptotic tumor cells were calculated on the CellQuestTM software (BD Biosciences). The apoptosis detection kit was obtained from Beyotime Biotechnology (Nanjing, China).

Western Blot Assay

HepG2 cells were treated with EP-B2 for 24 h. Then the tumor cells were collected and lysed wholly with lysis buffer (Beyotime, Nanjing, China) under an ice-cold bath. The effects of EP-B2 on the related protein levels were detected by Western blot assay. After quantifying the protein concentration collected by the BCA method, the total protein was separated using 10% polyacrylamide gel electrophoresis (PAEG) and then transferred to a polyvinylidene fluoride (PVDF) membrane. The transferred membrane was incubated with 5% bovine serum albumin (BSA) solution for another 1.5 h and then with the appropriate antibody for 2 h. The blot bands were collected and detected carefully using an enhanced chemiluminescence reagent (Thermo Fish, USA).

Conclusions

In summary, 2 new EP-PTX ester derivatives were prepared and characterized. The cytotoxic activities of all the intermediates (EP-A1 and EP-B1) and EP-PTX hybrids (EP-A2 and EP-B2) against human breast cancer (MCF-7), human hepatoma carcinoma (HepG2), human colorectal (HCT-116), and human lung (A549) cell lines were evaluated. Among the compounds, EP-B2 inhibited the growth of the HepG2 cells (IC₅₀ = 7.82 μM) more efficiently than EP. The preliminary studies of the mechanisms suggest that EP-B2 can arrest the G1 phase transition in a dose-dependent manner in HepG2 cells. In addition, EP-B2 showed an obvious apoptosis-inducing effect in the HepG2 cells, according to an Annexin V/propidium iodide binding assay and a Western blot assay of the apoptosis-related proteins. The hybrid EP-B2 has the potential to become an effective anti-tumor agent through further study of its mechanisms of action and structural modifications. Moreover, this work elucidates an effective hybridization method by analyzing the structure–activity relationships of ergosterol peroxide and PTX derivatives.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X231166778 - Supplemental material for Synthesis and Anticancer Activity of Ergosterol Peroxide Hybrids With Paclitaxel Side Chain Inducing Apoptosis in Human Hepatoma Carcinoma Cells

Supplemental material, sj-docx-1-npx-10.1177_1934578X231166778 for Synthesis and Anticancer Activity of Ergosterol Peroxide Hybrids With Paclitaxel Side Chain Inducing Apoptosis in Human Hepatoma Carcinoma Cells by Xiaoshan Guo, Wenkang Ren, Zhen Lv, Gang Li, Hongling Li, Mingrui Sun, Xiaoming Li, Gang Chen, Zhiguo Zhang, Wenting Zhang and Ming Bu in Natural Product Communications

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by grants from the Fundamental Research Funds for Education Department of Heilongjiang Province (No. 2019-KYYWF-1217).

Ethical Approval

Ethical approval is not applicable to this article.

Statement of Human and Animal Rights

This article does not contain any studies with human or animal subjects.

Statement of Informed Consent

There are no human subjects in this article and informed consent is not applicable.

ORCID iDs

Zhen Lv

Ming Bu

Hongling Li

Supplemental Material

Supplemental material for this article is available online.

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