Renoprotective Effects of Hypoxylonol C and F Isolated from Hypoxylon truncatum against Cisplatin-Induced Cytotoxicity in LLC-PK1 Cells

Abstract

Although cisplatin is the standard platinum-based anticancer drug used to treat various solid tumors, it can cause damage in normal kidney cells. Protective strategies against cisplatin-induced nephrotoxicity are, therefore, clinically important and urgently required. To address this challenge, we investigated the renoprotective effects of Hypoxylontruncatum, a ball-shaped wood-rotting fungus. Chemical investigation of the active fraction from the methanol extract of H.truncatum resulted in the isolation and identification of the renoprotective compounds, hypoxylonol C and F, which ameliorated cisplatin-induced nephrotoxicity to approximately 80% of the control value at 5 μM. The mechanism of this effect was further investigated using hypoxylonol F, which showed a protective effect at the lowest concentration. Upregulated phosphorylation of p38, extracellular signal-regulated kinases, and c-Jun N-terminal kinases following cisplatin treatment were markedly decreased after pre-treatment with hypoxylonol F. In addition, the protein expression level of cleaved caspase-3 was significantly reduced after co-treatment with hypoxylonol F. These results show that blocking the mitogen-activated protein kinase signaling cascade plays a critical role in mediating the renoprotective effect of hypoxylonol F isolated from H.truncatum fruiting bodies.

Keywords: Hypoxylon truncatum; hypoxylonol; renoprotective effect.

Figure 1

Comparison of protective effects of H. truncatum and its fractions in LLC-PK1 cells exposed to 25 μM of cisplatin for 24 h by MTT assay. Protective effect of (A) MeOH extract, (B) Hex fraction, (C) EA fraction, (D) BuOH fraction, and (E) water fraction in LLC-PK1 cells exposed to 25 μM of cisplatin for 24 h by MTT assay. Cell viability assays were done in triplicate for each assays and were repeated at least three times. * p < 0.05 compared with cisplatin-treated control.

Figure 2

Comparison of protective effects of compounds 1–3 isolated from H. truncatum in LLC-PK1 cells exposed to 25 μM of cisplatin for 24 h by MTT assay. (A) Chemical structures of isolated compounds 1–3. (B) Protective effects of hypoxylonol C (1) on cisplatin-induced cytotoxicity. (C) Protective effects of hypoxylonol F (2) on cisplatin-induced cytotoxicity. (D) Protective effects of BNT (3) on cisplatin-induced cytotoxicity. (E) Protective effects of N-acetylcysteine on cisplatin-induced cytotoxicity. Cell viability assays were done in triplicate for each assays and were repeated at least three times. * p < 0.05 compared with cisplatin-treated control.

Figure 3

Effect of hypoxylonol F (2) on apoptosis in LLC-PK1 cells exposed to 25 μM of cisplatin for 24 h by image-based cytometric assay. (A) Percentage of annexin V-positive-stained apoptotic cells. (B) Representative images for apoptosis detection. BF (bright field): cell morphology under light microscope. Image-based cytometric assays were done in triplicate and were repeated at least two times. * p < 0.05 compared with cisplatin-treated control.

Figure 4

Effect of hypoxylonol F (2) on the expression of apoptosis-related proteins in LLC-PK1 cells exposed to 25 μM of cisplatin for 24 h. (A) Expression of proteins in the MAPK-caspase-3 pathway. (B) Each bar graph shows the densitometric quantification of western blotting bands. Western blot assays were repeated at least three times. * p < 0.05 compared with cisplatin-treated control.