Halorotetin A: A Novel Terpenoid Compound Isolated from Ascidian Halocynthia rotetzi Exhibits the Inhibition Activity on Tumor Cell Proliferation

Abstract

Halocynthia roretzi, the edible ascidian, has been demonstrated to be an important source of bioactive natural metabolites. Here, we reported a novel terpenoid compound named Halorotetin A that was isolated from tunic ethanol extract of H. roretzi by silica gel column chromatography, preparative layer chromatography (PLC), and semipreparative-HPLC. ¹H and ¹³C NMRs, ¹H-¹H COSY, HSQC, HMBC, NOESY, and HRESIMS profiles revealed that Halorotetin A was a novel terpenoid compound with antitumor potentials. We therefore treated the culture cells with Halorotetin A and found that it significantly inhibited the proliferation of a series of tumor cells by exerting cytotoxicity, especially for the liver carcinoma cell line (HepG-2 cells). Further studies revealed that Halorotetin A affected the expression of several genes associated with the development of hepatocellular carcinoma (HCC), including oncogenes (c-myc and c-met) and HCC suppressor genes (TP53 and KEAP1). In addition, we compared the cytotoxicities of Halorotetin A and doxorubicin on HepG-2 cells. To our surprise, the cytotoxicities of Halorotetin A and doxorubicin on HepG-2 cells were similar at the same concentration and Halorotetin A did not significantly reduce the viability of the normal cells. Thus, our study identified a novel compound that significantly inhibited the proliferation of tumor cells, which provided the basis for the discovery of leading compounds for antitumor drugs.

Keywords: Halocynthia roretzi; antitumor; ascidian; hepatocellular carcinoma; terpenoid.

Figure 1

Isolation and activity verification of Halorotetin A. (A) Halocynthia roretzi and its three components. Bar = 1 cm (B) Relative cell viability of HepG-2 Cells after treatment with TE (200 μL/mL). Data represent the mean ± s.e.m.; n = 5 samples. Significance was determined by the t-test after Welch’s correction. (C–E), Relative cell viability of HepG-2 Cells after treatment with SGC-I~II (600 μg/mL) (C) and PLC-I~VI (50 μg/mL) (D) and H-I~VI (25 μg/mL) (E). Significance was determined by the one-way ANOVA test after Brown–Forsyths and Welch test. For boxplot: The vertical line represents 1.5 IQR, the upper and lower hinges present the 75% quantiles and 25% quantiles, respectively, and the center line means the median value. Each point represents a sample; *** p < 0.001, **** p < 0.00001.

Figure 2

Structural identification of Halorotetin A. (A) The purity detection of Halorotetin A for subsequent structural identification. (B) The key COSY, HMBC, and NOESY correlations and chemical structure of Halorotetin A.

Figure 3

The inhibitory effects of Halorotetin A and the IC₅₀ values on different tumor cells. (A) Relative cell viability of different tumor cells after treatment with different Halorotetin A concentrations. (B) IC₅₀ values of Halorotetin A on tumor cells. All curves were generated by the nonlinear regression. (C) Relative cell viability of HepG-2 cells and L929 after treatment with Halorotetin A at the concentration of 60 μM for 24, 48, and 72 h, respectively. Data represent the mean ± s.e.m.; n = 5 samples. Significance was determined by the one-way ANOVA test after Brown–Forsyths and Welch test. *** p < 0.001, **** p < 0.00001.

Figure 4

The cytotoxicity of Halorotetin A on HepG-2 cells and L929 cells. (A) Relative cell viability of HepG-2 and L929 cells treated with 16 μg/mL Halorotetin A and 16 μg/mL dox for 48 h, respectively. The vertical line represents 1.5 IQR, the upper and lower hinges present the 75% quantiles and 25% quantiles, respectively, and the center line means the median value. Each point represents a sample. Significance was determined by the one-way ANOVA test after Brown–Forsyths and Welch test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.00001 (B) Morphological features of L929 and HepG-2 cells treated with 16 μg/mL Halorotetin A and 16 μg/mL dox for 48 h, respectively, as observed under light microscopy. Bar = 50 μm.

Figure 5

Gene expression profiles of HepG-2 cells after Halorotetin A treatment. qRT-PCR showed the expression levels of multiple oncogenes and suppressor genes in HepG-2 cells with or without 40 μM Halorotetin A treatment for 16 h. The same volume of methanol was used as the experimental group. Data represent the mean ± s.e.m.; n = 4 samples. Significance was determined by the t-test after Welch’s correction. * p < 0.05, ** p < 0.01, *** p < 0.001.