Tetrahydroanthraquinone Derivative (±)-4-Deoxyaustrocortilutein Induces Cell Cycle Arrest and Apoptosis in Melanoma Cells via Upregulation of p21 and p53 and Downregulation of NF-kappaB

Abstract

Background: Malignant melanoma is an aggressive type of skin cancer with high risk for metastasis and chemoresistance. Disruption of tightly regulated processes such as cell cycle, cell adhesion, cell differentiation and cell death are predominant in melanoma development. So far, conventional treatment options have been insufficient to treat metastatic melanoma and survival rates are poor. Anthraquinone compounds have been reported to have anti-tumorigenic potential by DNA-interaction, promotion of apoptosis and suppression of proliferation in various cancer cells.

Methods: In the current study, the racemic tetrahydroanthraquinone derivative (±)-4-deoxyaustrocortilutein (4-DACL) was synthesized and the cytotoxic activity against melanoma cells and melanoma spheroids determined by CellTiter-Blue viability Assay and phase contrast microscopy. Generation of reactive oxygen species (ROS) was determined with CellROX Green and Deep Red Reagent kit and microplate-based fluorometry. Luciferase reporter gene assays for nuclear factor kappa B (NF-κB) and p53 activities and western blotting analysis were carried out to detect the expression of anti-proliferative or pro-apoptotic (p53, p21, p27, MDM2, and GADD45M) and anti-apoptotic (p65, IκB-α, IKK) proteins. Cell cycle distribution and apoptosis rate were detected by flow cytometry, the morphological changes visualized by fluorescence microscopy and the activation of different caspase cascades distinguished by Caspase Glo 3/7, 8 and 9 Assays.

Results: We demonstrated that 4-DACL displayed high activity against different malignant melanoma cells and melanoma spheroids and only low toxicity to melanocytes and other primary cells. In particular, 4-DACL treatment induced mitochondrial ROS, reduced NF-κB signaling activity and increased up-regulation of the cell cycle inhibitors cyclin-dependent kinase inhibitor p21 (p21(WAF1/Cip1)) and the tumor suppressor protein p53 in a dose-dependent manner, which was accompanied by decreased cell proliferation and apoptosis via the intrinsic pathway.

Conclusion: According to these results, we suggest that 4-DACL may be a promising therapeutic agent for the treatment of malignant melanoma.

Keywords: 4-DACL; NF-κB; ROS; anthraquinone; apoptosis.; cell cycle arrest; melanoma; spheroids.

Figure 1

4-DACL (racemic 2,5-dihydroxy-7-methoxy-2-methyl-1,2,3,4-tetrahydroanthracene-9,10-dione).

Figure 2

4-DACL promotes cell toxicity in melanoma cells and melanoma spheroids in a dose dependent manner. (A) Cells were plated at 2×10⁵ cells/ml in 96 well plates for 6 h and stimulated with 200 µg/ml-0.04 µg/ml 4-DACL for 24 h before CellTiter blue was added (10%) and fluorescence intensity measured. (B) Primary cells (NHM_1, NHM_2, hKER) and tumor cells (melanoma, HaCaT, CaCo-2, MCF7) were treated as in (A) and the IC₅₀ determined. (C) The actin and nuclear of NHM, IGR37 and A375 were stained with Alexa Fluor 568 phalloidin and Hoechst 33342, respectively and viewed by fluorescence and phase contrast microscopy; bar represents 100 μm (D-F) NHM, IGR37 and A375 were cultured in matrigel for spheroid formation and spheroids treated with 5 µg/ml 4-DACL for 48 h or left untreated (n.c.). (D) Microscopic images. (E) Diameters of spheroids were determined before and after treatment and statistically evaluated. (F) Cell metabolism and viability of spheroids were analyzed using CellTiter blue assay.

Figure 3

4-DACL shows no DNA intercalation but a slight mitochondrial formation of reactive oxygen species (ROS). (A) Cells were fixed (3.5% PFA), permeabilized (0.5% TX100) and stained with Hoechst and Hoechst binding measured with a multiplate reader. Thereafter cells were treated with different concentrations of 4-DACL, DMSO (neg.contr.) and Ethidiumbromide (pos. contr.), washed, measured and Hoechst replacement calculated. (B) Supercoiled DNA was incubated with different concentrations of 4-DACL or EtBr 1 h later Topoisomerase I was added and relaxation of the supercoiled DNA allowed for 30 min at 37°C before DNA was loaded on an agarose gel. Control contain DNA and topoisomerase, but no compound. (C) Supercoild DNA was incubated with different concentrations of DMSO, mitoxantron and 4-DACL and assay performed as in (B). (D) As in (C) but with EtBr. (E) IGR37 cells were treated with TBHP (pos. ctrl), DMSO (neg. ctrl) or different concentrations of 4-DACL for 1.5 h and ROS was detected using CellRox green (mitochondrial) and deep-red (cytoplasm). (*, p<0.05; **, p<0.01, as compared with n.c.).

Figure 4

4-DACL reduces NF-κB activation in melanoma cells. A 5x NF-κB-luc reporter gene was transfected into (A) IGR37, (B) A375 and (C) MCM-1 cells and 24 h later treated with 10-0.62 µg/ml 4-DACL or left untreated. 2 h after treatment cells were non-stimulated or stimulated with TNF-α, LPS or 10% FCS. Luciferase levels were normalized for a co-transfected eGFP control. (D) IGR37 and A375 cells were analyzed by western blot with antibodies against p-IKK-α/β, p-65, p-IκB-α and tubulin. (*, p<0.05; **, p<0.01, as compared with n.c.).

Figure 5

4-DACL activates p53, p21 and GADD45G in melanoma cells in a dose dependent manner. (A) IGR37 and A375 cells were treated with 5 µg/ml 4-DACL for 0, 0.5, 1, 3, 6 and 24 hours or (B) with 0, 1 and 5 µg/ml 4-DACL for 24 hours. Total protein expression of p21^Waf1/Cip1, p53, and Gadd45G were detected by western blot analysis using specific antibodies. Tubulin was used as a loading control. p53-Luc reporter assays with (C) IGR37 and (D) A375 co-transfected with eGFP and p53 plasmid (p.c.). (*, p<0.05; **, p<0.01, as compared with cells incubated media alone).

Figure 6

4-DACL reduces cell proliferation in a dose- and time dependent manner in melanoma cells. IGR37 (A) and A375 (B) cells were treated with 0.5, 1 or 5µg/ml µg/ml 4-DACL or were left untreated (ctrl) and stained with cell tracker after 0, 24 and 72 h and fluorescence detected with a multiplate reader. (C) Melanoma cells were treated with 4-DACL and total protein expression of Ki67 detected 24 h later by western blot analysis. (D) Melanoma cells were immunostained with anti-Ki67 antibody, Alexa488-conjugated anti-rabbit as secondary reagent, and with Hoechst 33342 dye for nuclei staining and viewed by fluorescence and phase contrast microscopy; bar represents 100 μm (E) Melanoma cells were treated with 0, 1, 2.5 or 5 µg/ml 4-DACL for 24 hours, fixed, stained with propidium iodide solution and analyzed for cell cycle distribution using flow cytometry. (F) Flow cytometric analysis showing the cell cycle distribution of IGR37, A375 and NHM.

Figure 7

4-DACL enhances apoptosis in in a dose dependent manner. (A) IGR37 cells were treated with 1, 2.5, 5 and 10 µg/ml 4-DACL for 16 h and stained with Annexin V and PI and analyzed using flow cytometry. (B) IGR37 cells were treated with 100-0.4 µg/ml cisplatin or 4-DACL for 16 h and analyzed for caspase 3/7. NHM, IGR37 and A375 cells were treated with 0-10 µg/ml 4-DACL for 16 h and caspase 3 (C), caspase 9 (D) and caspase 8 (E) activity measured. (*, p<0.05; **p<0.01, as compared with NHM cells treated with the same concentrations).