Dehydroxyhispolon Methyl Ether, A Hispolon Derivative, Inhibits WNT/β-Catenin Signaling to Elicit Human Colorectal Carcinoma Cell Apoptosis

Abstract

Colorectal cancer (CRC) is the fourth leading cause of cancer mortality worldwide. Aberrant activation of WNT/β-catenin signaling present in the vast majority of CRC cases is indispensable for CRC initiation and progression, and thus is a promising target for CRC therapeutics. Hispolon is a fungal-derived polyphenol with a pronounced anticancer effect. Several hispolon derivatives, including dehydroxyhispolon methyl ether (DHME), have been chemically synthesized for developing lead molecules with stronger anticancer activity. Herein, a DHME-elicited anti-CRC effect with the underlying mechanism is reported for the first time. Specifically, DHME was found to be more cytotoxic than hispolon against a panel of human CRC cell lines, while exerting limited toxicity to normal human colon cell line CCD 841 CoN. Additionally, the cytotoxic effect of DHME appeared to rely on inducing apoptosis. This notion was evidenced by DHME-elicited upregulation of poly (ADP-ribose) polymerase (PARP) cleavage and a cell population positively stained by annexin V, alongside the downregulation of antiapoptotic B-cell lymphoma 2 (BCL-2), whereas the blockade of apoptosis by the pan-caspase inhibitor z-VAD-fmk attenuated DHME-induced cytotoxicity. Further mechanistic inquiry revealed the inhibitory action of DHME on β-catenin-mediated, T-cell factor (TCF)-dependent transcription activity, suggesting that DHME thwarted the aberrantly active WNT/β-catenin signaling in CRC cells. Notably, ectopic expression of a dominant-active β-catenin mutant (∆N90-β-catenin) abolished DHME-induced apoptosis while also restoring BCL-2 expression. Collectively, we identified DHME as a selective proapoptotic agent against CRC cells, exerting more potent cytotoxicity than hispolon, and provoking CRC cell apoptosis via suppression of the WNT/β-catenin signaling axis.

Keywords: Phellinus linteus; WNT/β-catenin; colorectal cancer; dehydroxyhispolon methyl ether; hispolon; hispolon derivatives.

Figure 1

Anti-colorectal cancer (CRC) effect of dehydroxyhispolon methyl ether (DHME). (A) Selective cytotoxicity of DHME against malignant but not normal human colorectal epithelial cell lines. A panel of human CRC cell lines, such as HCT 116, HCT-15, and LoVo, in addition to one normal human colorectal epithelial cell line, CCD 841 CoN, were treated with DHME for 48 h, followed by cell viability evaluation using MTS assay. (B) DHME suppresses CRC cells to form colonies. A total of 2 × 10² of human CRC cells, after 24 h of treatment with DHME, were allowed to grow in drug-free media for 10 days to form colonies, which were visualized by crystal violet staining. (C) Quantitative analysis of DHME-induced suppression of CRC clonogenicity. Colonies displayed in (B) were scored, and the results were subjected to statistical analysis. * p < 0.05; *** p < 0.001.

Figure 2

DHME is more potent than hispolon in inducing CRC cell death. HCT 116, HCT-15, and LoVo cells were treated with indicated concentration (0.00, 6.25, 12.50, and 50.00 μM) of hispolon or DHME for 48 h, and the viability of drug-treated cells was determined by MTS assay thereafter.

Figure 3

Apoptosis induction is essential for the anti-CRC action of DHME. (A) DHME induces poly (ADP-ribose) polymerase (PARP) cleavage. HCT 116, HCT-15, and LoVo cells were treated with DHME (0, 10, or 20 μM) for 24 h, followed by immunoblotting for the levels of cleaved PARP (c-PARP). The levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the loading control. (B) DHME enhances the levels of annexin V-positive cell population. Human CRC cell lines treated with DHME (0, 10, 20 μM) for 24 h were subjected to annexin V/Propidium iodide (PI) dual staining using flow cytometry analysis. Annexin V-positive cells were regarded as cells undergoing apoptosis. The levels of the cell population in each quadrant were expressed as the percentage of total cell population. The horizontal axis denotes the intensity of annexin V, and the vertical axis indicates PI levels. (C) Quantitative analysis of DHME-induced CRC cell apoptosis. The annexin V-positive (apoptotic) cell population shown in (B) were scored. (D–F) Apoptosis blockade by z-VAD-fmk (50 μM) attenuates DHME-induced apoptosis as well as clonogenicity in CRC cells. The levels of the protein-to-GAPDH ratio relative to DHME-untreated controls were quantitated by ImageJ algorithm and are indicated below each blot. * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 3

Apoptosis induction is essential for the anti-CRC action of DHME. (A) DHME induces poly (ADP-ribose) polymerase (PARP) cleavage. HCT 116, HCT-15, and LoVo cells were treated with DHME (0, 10, or 20 μM) for 24 h, followed by immunoblotting for the levels of cleaved PARP (c-PARP). The levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the loading control. (B) DHME enhances the levels of annexin V-positive cell population. Human CRC cell lines treated with DHME (0, 10, 20 μM) for 24 h were subjected to annexin V/Propidium iodide (PI) dual staining using flow cytometry analysis. Annexin V-positive cells were regarded as cells undergoing apoptosis. The levels of the cell population in each quadrant were expressed as the percentage of total cell population. The horizontal axis denotes the intensity of annexin V, and the vertical axis indicates PI levels. (C) Quantitative analysis of DHME-induced CRC cell apoptosis. The annexin V-positive (apoptotic) cell population shown in (B) were scored. (D–F) Apoptosis blockade by z-VAD-fmk (50 μM) attenuates DHME-induced apoptosis as well as clonogenicity in CRC cells. The levels of the protein-to-GAPDH ratio relative to DHME-untreated controls were quantitated by ImageJ algorithm and are indicated below each blot. * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 4

DHME inhibits WNT/β-catenin signaling in CRC cells. (A) Suppression of β-catenin–T-cell factor/lymphoid enhancer factor (TCF/LEF)-dependent transcription by DHME. HCT 116, HCT-15, and LoVo cells were transiently transfected with an M50 Super 8x TOPFlash plasmid (TOPFlash), a β-catenin luciferase reporter vector, followed by DHME treatment and then assessment of luciferase activity. The M51 Super 8x FOPFlash plasmid (FOPFlash) was used as a negative control for TOPFlash. * p < 0.05; ** p < 0.01. (B) DHME lowers the levels of c-MYC, cyclin D1, and survivin. Cell lysates of CRC cells, following 24 h treatment with DHME, were subjected to immunoblot analysis. GAPDH levels were used as the loading control. The levels of protein-to-GAPDH ratio relative to DHME-untreated controls were quantitated using ImageJ algorithm, and are indicated below each blot.

Figure 5

Blockade of WNT/β-catenin-mediated, pro-survival signaling is required for the anti-CRC action of DHME. (A) Persistent β-catenin activation abolishes DHME-induced PARP cleavage. HCT-15 and LoVo clones stably expressing HA–∆N90-β-catenin, an N-terminal, hemagglutinin (HA)-tagged, dominant-active β-catenin mutant (with N-terminal 90 amino acids deleted), were treated with DHME (0, 10, or 20 μM) for 24 h, followed by immunoblotting for the levels of HA (confirming ectopic expression of HA–∆N90-β-catenin), cleaved PARP (c-PARP), and BCL-2. GAPDH levels were used as the loading control. (B) Persistent β-catenin activation lowers the levels of the DHME-enhanced, annexin V-positive cell population. Stable vector or HA–∆N90-β-catenin clones of HCT-15 and LoVo cells were treated with DHME (0, 10, or 20 μM) for 24 h, followed by flow cytometry analysis for the levels of annexin V-positive (apoptotic) cell population. (C) Persistent β-catenin activation rescued DHME-mediated inhibition of clonogenicity. A total of 2 × 10² of stable vector or HA–∆N90-β-catenin clones of HCT-15 and LoVo cells after 24 h treatment of DHME (0, 10, or 20 μM) were assessed for their ability to form colonies. The levels of protein-to-GAPDH ratio relative to DHME-untreated vector controls were quantitated by ImageJ algorithm and are indicated below each blot. *** p < 0.001.

Figure 6

Schematic diagram depicting the anti-CRC mechanism of action of DHME elucidated in this study. In brief, DHME induces CRC cell apoptosis via the targeted inhibition of the pro-survival WNT/β-catenin–BCL-2 signaling axis. The chemical structure of DHME is adapted from Balaji et al. [18]. The dashed line denotes that our data implicated that the transcription of BCL-2 likely depends on the β-catenin–TCF/LEF transcription complex, but it still requires evidence to support the direct binding of TCF/LEF to the human BCL-2 promoter for driving BCL-2 transcription in the CRC cell lines used in this study.