The In Vitro and In Vivo Anticancer Properties of Chalcone Flavokawain B through Induction of ROS-Mediated Apoptotic and Autophagic Cell Death in Human Melanoma Cells

Abstract

Melanoma is the most prevalent type of skin cancer with high mortality rates. This study demonstrates the in vitro and in vivo anticancer properties of chalcone flavokawain B (FKB) induced ROS-mediated apoptosis and autophagy in human melanoma (human epithelial melanoma cell line A375 and/or human skin lymph node derived melanoma cell line A2058) cells. Cell viability was calculated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the expression patterns of various apoptosis, autophagy-associated proteins were determined by Western blot methods. Annexin V was detected by flow cytometry, whereas acidic vesicular organelles (AVOs) and intracellular ROS levels were measured by fluorescence microscopy. The in vivo anticancer properties of FKB were evaluated by xenografting the A375 cells into nude mice. The results convey that FKB inhibited cell viability, B-Raf proto-oncogene, serine/threonine kinase (BRAF)/extracellular signal-regulated kinase (ERK) expression in human melanoma cells. Caspase-3 activation, poly (ADP-ribose) polymerase (PARP) cleavage pathway, and Bcl2 associated X (Bax)/B-cell lymphoma 2 (Bcl-2) dysregulation were involved in the execution of apoptosis. Moreover, FKB-induced autophagy was observed through increased microtubule-associated protein 1A/1B-light chain 3B (LC3-II) accumulation and AVOs formation, which was also associated with an increase in sequestosome 1 (SQSTM1/p62), decreased protein kinase B (AKT)/mammalian target of rapamycin (mTOR) expressions, and dysregulated Beclin-1/Bcl-2 levels. Autophagy inhibitors [3-methyladenine (3-MA)/chloroquine (CQ)] and LC3 silencing suppressed FKB-induced apoptosis by decreasing caspase-3 in melanoma cells. The antioxidant N-acetylcysteine (NAC) diminished FKB-induced apoptotic and autophagic cell death. However, the inhibition of apoptosis decreased FKB-induced autophagy (LC3-I/II). The in vivo study confirmed that FKB inhibited melanoma growth in A375-xenografted nude mice. This study concluded that FKB is critically associated with the execution and generation of ROS-modulated apoptotic and autophagic cell death of melanoma cells. FKB also repressed tumor growth in xenografted nude mice. Therefore, flavokawain B might be a potential anti-tumor agent in human melanoma treatment.

Keywords: ROS; apoptosis; autophagy; flavokawain B; melanoma cells.

Figure 1

Flavokawain B (FKB) inhibited the growth of human melanoma cells. (A–D) human epithelial melanoma cell line A375, human skin lymph node derived melanoma cell line A2058, human primary epidermal melanocyte cell line (HEMn), and human skin keratinocyte cell line (HaCaT) cells were exposed to 0–20 µg/mL concentrations of FKB for 24 h. Then, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was carried out to determine cell viability. The IC₅₀ value for each cell type was determined as described in the methods section (inset: IC₅₀ values of respective cell lines). (E) FKB mediated morphological changes in A375 and A2058 cells were examined using the phase-contrast microscope. (F) Expression patterns of BRAF and p-ERK proteins were determined by Western blot to discover the effect of FKB concentrations (0–10 μg/mL) for 24 h. β-actin was used as an internal control. Each value was expressed as mean ± standard deviation of three experiments. Statistical significance was assigned as ** p < 0.01 and *** p < 0.001 as compared to the untreated control cells.

Figure 2

FKB induced apoptosis in human melanoma cells. Different concentrations of FKB were treated to A375 (0–10 μg/mL) and A2058 (0–15 μg/mL) cells for 24 h. (A,B) The expression levels of FKB-induced activation of caspase-3 and PARP cleavage were measured by Western blot in both A375 and A2058 cells. (C,D) The expression levels of apoptosis activator; Bax and inhibitor proteins; Bcl-2 were measured by Western blot in both A375 and A2058 cells. Data were expressed as fold-over control of the Bax/Bcl-2 ratio. (E) 10 μg/mL of FKB was treated to A375 for 0–24 h. The effect of time on the expressions of Bax and Bcl-2 proteins was measured by Western blot. β-actin was used as an internal control. The Bax/Bcl-2 ratio was represented as fold-over control whose value was arbitrarily assigned as one. Each value was expressed as mean ± standard deviation (SD) of three experiments and the statistical significance was assigned as * p < 0.05, ** p < 0.01, and *** p < 0.001 as compared to the untreated control cells.

Figure 3

FKB induced apoptotic cell death of A375 and A2058 cells. Cells were pretreated with pan-caspase inhibitor z-Val-Ala-Asp fluoromethyl ketone (Z-VAD-FMK) (20 μM) for 1 h followed by treatment with various concentrations of FKB (0–10 μg/mL for A375 and 0–15 μg/mL for A2058) for 24 h. (A,B) MTT assay was performed to measure the viability of A375 and A2058 cells. (C,D) Changes in A375 and A2058 cell membrane morphology were examined under a phase-contrast microscope. (E) Flow cytometry analysis of FKB-mediated early and late apoptotic cell death in A375 cells using annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining. A375 cells were exposed to 0, 2.5, 5, and 10 μg/mL of FKB for 24 h. The results in each quadrant were interpreted as described in the methods section. Each value was expressed as mean ± standard deviation (SD) of three experiments. Statistical significance was assigned as ** p < 0.01 and *** p < 0.001 as compared to untreated control cells. ^# p < 0.05, ^## p < 0.01, and ^### p < 0.001 as compared to the treated cells.

Figure 4

FKB induced autophagy LC3-II and sequestosome 1 (p62) expression in human melanoma cells. (A) A375 and (B) A2058 cells were treated with various concentrations of FKB (0–10 or 0–15 μg/mL) for 24 h. These cells were subjected to Western blot analysis to determine the conversion of LC3-I to LC3-II and expression of p62 proteins. (C,D) FKB (0–10 μg/mL) was treated to A375 cells for 24 h. Immunofluorescence staining (100× magnification) was used to measure the accumulation of LC3. The data were expressed as fold-over untreated control cells. Each value was expressed as mean ± standard deviation (SD) of three experiments. Statistical significance was assigned as ** p < 0.01, and *** p < 0.001 as compared to untreated control cells.

Figure 5

FKB triggered acidic vesicular organelle (AVO) formation in A375 and A2058 cells. A375 and A2058 cells were exposed to different concentrations of FKB (0–10 or 0–15 μg/mL) for 24 h. Acridine orange stain was used to detect the AVOs formation that can be observed through a red filter fluorescence microscope (100× magnification). (A,B) Compared to the untreated control cells, FKB treatment dose-dependently significantly upregulated the formation of AVOs in A375 and (C,D) A2058 cells. Histogram depicting the number of acridine orange (AO) stained cells. The data were expressed as fold-over untreated control cells in which control was assigned as one. Each value was expressed as mean ± standard deviation (SD) of three experiments. Statistical significance was assigned as * p < 0.05, and *** p < 0.001 as compared to untreated control cells.

Figure 6

FKB dysregulated Beclin-1/Bcl-2 ratio and suppressed p-AKT/p-mTOR expressions in melanoma cells. Cells were treated with increasing concentrations of FKB (0–10 and 0–15 μg/mL) for 24 h. After the incubation period, cells were harvested and subjected to the Western blot method as described in the methods section. (A,B) FKB dose-dependently significantly upregulated the ratio between Beclin-1 and Bcl-2 in both A375 and A2058 cells. (C) FKB dose-dependently suppressed the phosphorylation of AKT and mTOR proteins. Both these effects shifted the cellular homeostasis towards autophagy. Each value was expressed as mean ± standard deviation (SD) of three experiments. Statistical significance was assigned as * p < 0.05 compared to untreated control.

Figure 7

Effect of autophagy inhibitors on the FKB-mediated AVOs formation in A375 cells. (A,B) A375 cells were pretreated with 3-methyladenine (3-MA) (1 mM) or chloroquine (CQ) (10 μM) for 1 h followed by FKB treatment (10 μg/mL) for 24 h and then stained with acridine orange (AO) to observe the AVOs formed in the cells. These AVOs were visualized using the fluorescence microscope under a red filter (100× magnification). The fluorescence intensity was proportionate to the number of AVOs formed in the cells. Histogram representing the fold change of AVOs formation in different experimental conditions, in which the control value was arbitrarily assigned as one. Each value was expressed as mean ± standard deviation (SD) of three experiments. Statistical significance was assigned as * p < 0.05, ** p < 0.01, and *** p < 0.001 as compared to the untreated control cells.

Figure 8

FKB induced autophagy signaling cascades mediated the cell death mechanisms in human melanoma cells. (A,B) A375 and (C,D) A2058 cells were pretreated with early and late autophagy inhibitors (1 mM 3-MA, and 10 μM CQ) for 1 h followed by FKB (0–10 or 0–15 μg/mL) treatment, respectively, for 24 h. MTT assay was performed to determine cell viability. Each value was expressed as mean ± standard deviation (SD) of three experiments. Statistical significance was assigned as ** p < 0.01 and *** p < 0.001 as compared to the untreated control cells. ^# p < 0.05 and ^## p < 0.01 as compared to the FKB alone treated cells.

Figure 9

Intracellular ROS was induced by FKB in A375 cells. (A) A375 cells were treated with FKB (10 μg/mL) for 0–90 min. (B) Cells were pretreated with or without 5 mM N-acetylcysteine (NAC) (a ROS inhibitor) for 1 h followed by FKB (10 μg/mL) for 30 min. The non-fluorescent probe 2′-7′dichlorofluorescin diacetate (DCFH₂-DA) was used to measure the intracellular ROS generation. DCFH₂-DA reacted with cellular ROS and metabolized into fluorescent dichlorofluorescein (DCF), which was proportionate to the generation of ROS. The data were represented as fold-over change in the ROS levels compared to the control, which was arbitrarily assigned as one. Each value was expressed as mean ± standard deviation (SD) of three experiments. Statistical significance was assigned as * p < 0.05 and *** p < 0.001 compared to untreated control cells. ^### p < 0.001 compared to FKB alone treated cells.

Figure 10

ROS-mediated apoptotic and autophagy cell death in A375 cells was induced due to FKB. A375 and A2058 cells were pretreated with 5 mM NAC for 1 h then followed by treatment with 0–10 and 0–15 μg/mL of FKB, respectively, for 24 h. (A,B) Cells were subjected to MTT assay to measure the percentage of cell viability. (C) Western blot was used to measure the expression levels of LC3-I/II, caspase-3, and BRAF proteins both in the absence or presence of NAC pretreatment conditions. β-actin acts as an internal control. Each value was expressed as mean ± standard deviation (SD) of three experiments. Statistical significance was assigned as ** p < 0.01 and *** p < 0.001 compared to untreated control cells. ^# p < 0.05, ^## p < 0.01 and ^### p < 0.001 compared to FKB alone treated cells.

Figure 11

Inhibition of either apoptosis or autophagy compromised occurrence of FKB-induced cell death in A375 cells. (A) Western blot showing the effect of time on the expression patterns of LC3-I/II and caspase-3 with response to FKB (10 μg/mL) for 0–24 h. (B–D) Western blot showing the expression of LC3-I/II and caspase-3 levels in different conditions. (B) Cells were pretreated with caspase inhibitor (Z-VAD-FMK, 20 μM) and (C,D) early or late autophagy inhibitors (3-MA, 1 mM and CQ, 10 μM) for 1 h followed by FKB (10 μg/mL) for 24 h. (E) Control small interfering RNA (siRNA) and LC3 knockdown cells (siLC3) were treated with FKB (10 μg/mL) for 24 h, followed by the measurement of the expressions of LC3-I/II and caspase-3 proteins through the Western blot. β-actin was used as an internal control. Results are expressed as the mean ± standard deviation (SD) of three experiments.

Figure 12

FKB-mediated in vivo inhibition of tumor growth in A375 xenograft athymic nude mice. Athymic nude mice were subcutaneously xenografted with A375 cells as described in the methods section. Mice were subjected to the vehicle (control) or FKB (5 mg/kg, intraperitoneal) for 26 days of therapy. (A) The body weight (in grams) and (B) tumor volumes (in mm³) were measured every 2 days. (C–E) On the twenty sixth day, animals were photographed, sacrificed and their tumor tissues were removed and weighed. Results are expressed as the mean ± standard deviation (SD) of three experiments. Statistical significance was assigned as * p < 0.05, ** p < 0.01, and *** p < 0.001 as compared to vehicle-treated control group.