1,4-dihydroxy-2-naphthoic Acid Induces Apoptosis in Human Keratinocyte: Potential Application for Psoriasis Treatment

Abstract

Psoriasis, which affects approximately 1-3% of the population worldwide, is a chronic inflammatory skin disorder characterized by epidermal keratinocytes hyperproliferation, abnormal differentiation, and inflammatory infiltration. Decrease in keratinocyte apoptosis is a specific pathogenic phenomenon in psoriasis. Chinese herbs have been used for the treatment of psoriasis in China showing promising effect in clinical trials. A traditional Chinese medicine has relatively fewer side effects with longer remission time and lower recurrence rate. The extract of Rubia cordifolia L. (EA) was previously found by us to induce HaCaT keratinocytes apoptosis. In this study we identified one of the components in Rubia cordifolia L., the anthraquinone precursor 1,4-dihydroxy-2-naphthoic acid (DHNA), induces HaCaT keratinocytes apoptosis through G0/G1 cell cycle arrest. We have also demonstrated that DHNA acts through both caspase-dependent and caspase-independent pathways. Besides, cytotoxicity and IL-1 α release assays indicate that DHNA causes less irritation problems than dithranol, which is commonly employed to treat psoriasis in many countries. Since DHNA possesses similar apoptotic effects on keratinocytes as dithranol but causes less irritation, DHNA therefore constitutes a promising alternative agent for treating psoriasis. Our studies also provide an insight on the potential of using EA and DHNA, alternatively, as a safe and effective treatment modality for psoriasis.

Figure 1

The structure of 1,4-dihydroxy-2-naphthoic acid (DHNA).

Figure 2

Action of DHNA on HaCaT cells morphology. (a) Vehicle (0.24% DMSO) and (b) medium only treated HaCaT cells. (c to h) HaCaT cells treated with 30 μM (6.126 μg/mL; (c, d)), 60 μM (12.25 μg/mL; (e, f)) and 120 μM (24.5 μg/mL; (g, h)) DHNA for 72 h. After staining with Hoechst 33342, morphological examinations were carried out using fluorescent microscope. Cells with chromatin condensation (c, e) and nuclear fragmentation (d, f, h) are indicated by arrows. In (g), all cells undergo chromatin condensation. Three independent experiments were performed with similar results. Scale bar 50 μm.

Figure 3

Phosphatidylserine externalization is increased in DHNA-treated HaCaT cells. (a, b) Density plots and (c, d) bar chart showing effects of the DHNA on distribution of viable (lower left) and early/late apoptotic (lower/upper right) HaCaT cells. HaCaT cells were treated with vehicle (0.24% DMSO), medium only or various concentration of DHNA for 9 (a, c) and 24 h (b, d) and then analyzed by Annexin V/PI and flow cytometry. (e) DHNA-induced apoptosis in HaCaT cells was reduced by pan-caspase inhibitor. HaCaT cells were pretreated with 40 μM pan-caspase inhibitor Z-VAD-FMK for 1 h followed by DHNA treatment for 24 h. Three independent experiments with triplicates each time were performed with similar results. Data are expressed as mean ± SEM from one representative experiment and significant difference at *P < 0.05, ***P < 0.001 when versus vehicle control (c, d) or between DHNA treatment (e).

Figure 4

DHNA decreases MMP in HaCaT cells. HaCaT cells were treated with vehicle (0.24% DMSO), medium only, or various concentration of DHNA for 24 and 48 h. After staining with JC-1, HaCaT cells were analyzed by flow cytometry and the red/green fluorescence ratio in each sample was calculated. Three independent experiments with triplicates each time were performed with similar results. Data are expressed as mean ± SEM from one representative experiment and significant difference at **P < 0.01, ***P < 0.001 when versus vehicle control.

Figure 5

Effect of the DHNA on cell cycle distribution in HaCaT cells. (a) Bar chart and (b) graphical analysis showing the effect of the DHNA on cell cycle distribution of sub-G0/G1, G0/G1, S, and G2/M phase in HaCaT cells. HaCaT cells were treated with vehicle (0.24% DMSO), medium only or various concentration of DHNA for 24 h then stained with PI and cell cycle was analyzed by flow cytometry. The apoptotic proportion was recognized as the sub-G0/G1 population. Three independent experiments with triplicates each time were performed with similar results. Data are expressed as mean ± SEM from one representative experiment and significant difference at *P < 0.05, **P < 0.01, ***P < 0.001 when versus vehicle control.

Figure 6

DHNA induces DNA fragmentation in HaCaT cells. HaCaT cells were treated with vehicle (0.24% DMSO), medium only or various concentration of DHNA for 24 and 48 h. Cells were then assayed for DNA fragmentation by the Cell Death Detection ELISA^plus Kit. Three independent experiments with triplicates each time were performed with similar results. Data are expressed as mean ± SEM from one representative experiment and significant different at *P < 0.05, **P < 0.01, ***P < 0.001 when versus vehicle control.

Figure 7

Induction of apoptosis by DHNA in HaCaT cells measured by TUNEL staining. (a) Bar chart and (b) flow cytometric analysis of the effect of DHNA on TUNEL staining in HaCaT cells. HaCaT cells were treated with vehicle (0.24% DMSO), medium only or various concentration of DHNA for 24 h. The cell was analyzed for apoptosis by the In Situ Cell Death Detection kit (Fluorescein). In (b), HaCaT cells showing increased fluorescence (TUNEL staining) above that of control population (open graph) are considered apoptotic and their percentage populations are shown. Three independent experiments with triplicates each time were performed with similar results. Data are expressed as mean ± SEM from one representative experiment and significant difference at ***P < 0.001 when versus vehicle control.

Figure 8

Effects of DHNA on the expression of cell-cycle-related proteins in HaCaT cells. HaCaT cells were treated with vehicle (0.24% DMSO), medium only or various concentrations of DHNA for 12 h and then cells were harvested. Equal amount of cell extracts (10–20 μg) were loaded onto and separated by 12% to 15% SDS-PAGE and analyzed for the expression of cell cycle related proteins by Western blot. Equal protein loading for each sample was monitored by β-actin. Data are representative of three reproducible independent experiments.

Figure 9

Effects of DHNA on the expression of apoptosis related proteins in HaCaT cells. HaCaT cells were treated with vehicle (0.24% DMSO), medium only or various concentrations of DHNA for (a) 12 and (b) 24 h and then analyzed by Western blot. Equal protein loading was monitored by β-actin. Data are representative of three reproducible independent experiments.

Figure 10

DHNA causes nuclear translocation of AIF in HaCaT cells. HaCaT cells were treated with vehicle (0.24% DMSO; (a)), medium only (b), 120 μM (24.5 μg/mL; (c)) or 240 μM (49.01 μg/mL; (d)) DHNA for 24 h then analyzed by immunofluorescence staining. Cell nuclei were stained by DAPI. Data are representative of three reproducible independent experiments. Scale bar 25 μm.

Figure 11

DHNA causes nuclear translocation of endoG in HaCaT cells. HaCaT cells were treated with vehicle (0.24% DMSO; (a)), medium only (b), 120 μM (24.5 μg/mL; (c)) or 240 μM (49.01 μg/mL; (d)) DHNA for 24 h then analyzed by immunofluorescence staining. Cell nuclei were stained by DAPI. Data are representative of three reproducible independent experiments. Scale bar 25 μm.

Figure 12

Effect of DHNA and dithranol on IL-1α releasefrom NCTC 2544 cells. NCTC 2544 cells were treated with vehicle (0.2% DMSO), medium only or (a) 20 to 100 μM (4.08 to 20.42 μg/mL) DHNA or (b) 2 to 8 μM (0.45 to 1.81 μg/mL) dithranol for 72 h and then assayed for IL-1α release by the IL-1α ELISA Kit. Three independent experiments with duplicates each time were performed with similar results. Data are expressed as mean ± SEM from one representative experiment and significant difference at *P < 0.05, **P < 0.01, ***P < 0.001 when versus vehicle control.