Notoamide-type alkaloid induced apoptosis and autophagy via a P38/JNK signaling pathway in hepatocellular carcinoma cells

Abstract

Bioassay-guided fractionation of a coral-associated fungus Aspergillus ochraceus LZDX-32-15 resulted in the isolation of eleven notoamide-type alkaloids, including four new congeners, namely notoamides W-Z (1-4). The structures of the new alkaloids were determined by extensive analyses of spectroscopic data (1D and 2D NMR, HRESIMS), while ECD data were used for the configurational assignment. Three alkaloids (6, 10, 11) exerted potent inhibition against a panel of hepatocellular carcinoma (HCC) cell lines with IC₅₀ values ranging from 0.42 to 3.39 μM, that are comparable to the data for paclitaxel. Notoamide G (6) inhibited the viability of HepG2 and Huh-7 cells via both apoptosis and autophagy pathways. Notoamide G activated the expression of caspase-3, caspase-8, and caspase-9, in association with the degradation of the downstream gene PARP in a dose-dependent manner, suggesting that notoamide G induced apoptosis via a mitochondrial pathway and a dead receptor-mediated pathway. In addition, notoamide G increased the autophagic vacuole in both HepG2 and Huh-7 cells in a dose-dependent manner after 24 h through the significant upregulation of the key proteins Beclin1 and LC3B. Further investigation revealed that notoamide G promoted P38 and JNK phosphorylation, whereas the total protein of P-38 and JNK was slightly influenced. Accordingly, the antitumor proliferation of notoamide G in HCC cells was mechanistically mediated by apoptosis and autophagy through a P38/JNK signaling pathway, while notoamide G was considered as a potent lead for further development as an antitumor agent.

Fig. 1. Structures of notoamide-type alkaloids 1–11.

Fig. 2. Key NOE correlations of 1–4.

Fig. 3. Experimental ECD spectra of 1–3 in MeOH.

Fig. 4. Experimental ECD spectra (200–500 nm) of 4 in MeOH and the calculated ECD spectra of 4 at the B3LYP/6-311++G(2d,2p) level.

Fig. 5. Spontaneous conversion of 11 to 10 in DMSO after 24 h. (a) ¹H NMR spectrum of 11, (b) ¹H NMR spectrum of 11 in DMSO-d₆ after 24 h, (c) ¹H NMR spectrum of 10. ¹H NMR spectra were measured in DMSO-d₆.

Scheme 1. Biogenetic speculation of the relationship among 1–11.

Fig. 6. Cytotoxic effects of alkaloids on human HCC cells. (A) Cytotoxic effects of 11 alkaloid compounds on HCT-8, MCF-7, SMMC7721 and HUVEC were detected by MTT assays. Cells were treated with 10 μM of each compound for 48 h. MTT solution (1 mg mL⁻¹ MTT, 100 μL) was added into each well and incubated for 2 h at 37 °C. Then, the medium was changed with the same volume of DMSO. Cell viability was measured at absorbance of 570 nm. Paclitaxel (PTX) at concentration of 10 μM was used as positive control. (B) Seven liver cancer cell lines were seeded into 96-well plates at a concentration of 1 × 10⁴ per well and treated with indicated concentrations of compounds 6, 10 and 11, respectively. Cell viability was assessed by CCK8 assays after 48 h of exposure to compounds. Data is represented as mean ± S.D.

Fig. 7. Compounds 6, 10 and 11 induced apoptosis in HCC cells. (A) Morphological changes of HepG2 and Huh7 treated with increasing concentrations of (0 μM, 1 μM, 2 μM, 4 μM) of compounds 6, 10 and 11 were shown. (B) Functions of compounds 6, 10 and 11 in cell apoptosis in HepG2 and Huh7 cells. Cells were treated with 4 μM compound for 48 h. Annexin V/PI double staining with flow cytometry analysis was executed for cell apoptosis. DMSO was used as negative control. ***p < 0.001.

Fig. 8. Functions of compound 6 on expression of apoptosis-related proteins. HepG2 and Huh7 cells were treated with increasing concentrations of (0 μM, 1 μM, 2 μM, 4 μM) of compound 6. (A) Expression of caspase-3 and PARP fragment (B) and caspase-8 fragment, caspase-9 fragment, cytochrome c, Bcl-2 and Bax were detected by Western blot assays. DMSO was used as negative control. Tubulin was used as a loading control for all whole cell extract samples.

Fig. 9. Compound 6 induced autophagy in HCC cells. (A and B) HepG2 and Huh7 cells were incubated at 37 °C for 30 minutes with 0.1 μM DAPGreen working solution, and then treated with increasing concentrations of (0, 1, 2, 4 μM) of compound 6. Fluorescence was observed by fluorescence microscope (A) or flow cytometry (B). (C) Expression level of Beclin1 and LC3B (I and II) were detected by Western blot analysis. DMSO was used as negative control. Tubulin was used as a loading control for all whole cell extract samples.

Fig. 10. Compound 6 induced p38/JNK phosphorylation in HCC cells. HepG2 and Huh7 cells were treated with indicated increasing doses of compound 6 and immunoblotted with indicated antibodies. DMSO was used as negative control. Tubulin was used as a loading control for all whole cell extract samples.