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Mar Drugs
2014 Jul 07;127:4069-85. doi: 10.3390/md12074069.
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Ovothiol isolated from sea urchin oocytes induces autophagy in the Hep-G2 cell line.
Russo GL
,
Russo M
,
Castellano I
,
Napolitano A
,
Palumbo A
.
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Ovothiols are histidine-derived thiols isolated from sea urchin eggs, where they play a key role in the protection of cells toward the oxidative burst associated with fertilization by controlling the cellular redox balance and recycling oxidized glutathione. In this study, we show that treatment of a human liver carcinoma cell line, Hep-G2, with ovothiol A, isolated from Paracentrotus lividus oocytes, results in a decrease of cell proliferation in a dose-dependent manner. The activation of an autophagic process is revealed by phase contrast and fluorescence microscopy, together with the expression of the specific autophagic molecular markers, LC3 II and Beclin-1. The effect of ovothiol is not due to its antioxidant capacity or to hydrogen peroxide generation. The concentration of ovothiol A in the culture media, as monitored by HPLC analysis, decreased by about 24% within 30 min from treatment. The proliferation of normal human embryonic lung cells is not affected by ovothiol A. These results hint at ovothiol as a promising bioactive molecule from marine organisms able to inhibit cell proliferation in cancer cells.
Figure 2. Analysis of ovothiol A purified from sea urchins. (A) Elutographic profile of ovothiol A obtained by ion exchange chromatography purification of the sea urchin extracts. Detection at 254 (black trace) and 280 (red trace) nm. Inset: UV-Vis absorption spectrum of ovothiol A; (B) Mass spectrum of ovothiol A.
Figure 4. Ovothiol A induces autophagy in Hep-G2 cells. (A) After stimulation for 24 h with increasing doses of ovothiol (50–200 μM) or positive controls (quercetin, sorafenib and rapamycin, at 25, 20 and 1 μM, respectively), autophagy was detected and measured using a specific kit, as described in the Materials and Methods Section. At least 100 cells in two independent fields were counted using a phase contrast microscope, and the presence of positive autophagic cells was evaluated by fluorescence in the same fields. The figure shows representative images of cells treated with ovothiol, quercetin, sorafenib and rapamycin at the indicated concentrations (optical microscope Axiovert 200 M Zeiss; 400×, phase contrast in a–g and fluorescence FITC in a’–g’). Numbers on the bottom of panels a’–g’ indicate percentage (means ± SD compared to untreated cells of two separate experiments) of positive autophagic cells; (B) Western blot analysis of LC3 I, LC3 II and Beclin-1 expression in Hep-G2 cells treated for 24 h with indicated doses of ovothiol, quercetin and sorafenib. Bands are representative of one out of three separate experiments performed; (C) Densitometric analysis of blots shown in Panel B (optical density of LC3 II/α-tubulin, left and Beclin-1/α-tubulin, right). Values are presented as the mean ± SD Symbols indicate significance: p < 0.05 (*), p < 0.01 (**), p < 0.001 (***) with respect to untreated cells.
Figure 6. Ovothiol A levels decrease in the incubation medium. The HPLC trace of the Hep-G2 culture medium following treatment with 200 μM ovothiol; (A) T = 0 min; (B) T = 30 min. Detection at 254 (black trace) and 280 (red trace) nm. Highlighted is the peak corresponding to ovothiol A.
Figure 8. Ovothiol does not induce cytotoxicity in WI-38 cells. Cells were treated for 24–48 h with increasing doses of ovothiol A (10–200 μM), and cell viability was measured by the crystal violet assay. Values are presented as the mean ± SD compared to untreated cells.
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