A novel chalcone derivative has antitumor activity in melanoma by inducing DNA damage through the upregulation of ROS products

Abstract

Background: Melanoma is one of the most aggressive tumors with the remarkable characteristic of resistance to traditional chemotherapy and radiotherapy. Although targeted therapy and immunotherapy benefit advanced melanoma patient treatment, BRAFi (BRAF inhibitor) resistance and the lower response rates or severe side effects of immunotherapy have been observed, therefore, it is necessary to develop novel inhibitors for melanoma treatment.

Methods: We detected the cell proliferation of lj-1-59 in different melanoma cells by CCK 8 and colony formation assay. To further explore the mechanisms of lj-1-59 in melanoma, we performed RNA sequencing to discover the pathway of differential gene enrichment. Western blot and Q-RT-PCR were confirmed to study the function of lj-1-59 in melanoma.

Results: We found that lj-1-59 inhibits melanoma cell proliferation in vitro and in vivo, induces cell cycle arrest at the G2/M phase and promotes apoptosis in melanoma cell lines. Furthermore, RNA-Seq was performed to study alterations in gene expression profiles after treatment with lj-1-59 in melanoma cells, revealing that this compound regulates various pathways, such as DNA replication, P53, apoptosis and the cell cycle. Additionally, we validated the effect of lj-1-59 on key gene expression alterations by Q-RT-PCR. Our findings showed that lj-1-59 significantly increases ROS (reactive oxygen species) products, leading to DNA toxicity in melanoma cell lines. Moreover, lj-1-59 increases ROS levels in BRAFi -resistant melanoma cells, leading to DNA damage, which caused G2/M phase arrest and apoptosis.

Conclusions: Taken together, we found that lj-1-59 treatment inhibits melanoma cell growth by inducing apoptosis and DNA damage through increased ROS levels, suggesting that this compound is a potential therapeutic drug for melanoma treatment.

Keywords: Chalcone; DNA damage; Melanoma; P53; ROS (reactive oxygen species).

Fig. 1

lj-1-59 inhibits the proliferation of human melanoma cells. a Structure of lj-1-59. b SK-Mel-28 (upper panel) and SK-Mel-5 (lower panel) were prepared in 96-well plates. The cells were treated with increasing dose lj-1-59 for 0-72 h. Cell viability was determined by CCK-8 assay. The results represent the means (n = 6) ± SD. Significant differences were evaluated using Student’s t-test, and an asterisk (*) indicates a significant difference (p < 0.05). c The IC₅₀ values of lj-1-59 in SK-Mel-28 (left panel) and SK-Mel-5 (right panel) cells were automatically calculated for 48 h by GraphPad Prism software. d SK-Mel-28 (left panel) and SK-Mel-5 (right panel) cells were prepared in 6-well plates. The cells were treated with increasing dose lj-1-59 for 24 h. After 2 weeks, the number of colonies was assessed and quantified as described in “Methods”. The data represent the mean (n = 4) ± SD, and an asterisk (*) indicates a significant difference (p < 0.05, Student’s t-test)

Fig. 2

lj-1-59 suppresses xenograft tumor growth in vivo. a The tumor volume of nude mice. b The body weight of nude mice. The results in a and b are shown as the mean (n = 5) ± SD, and asterisk (*) indicates a significant difference (p < 0.05 one way ANOVA). c Representative images of IHC staining of Ki67 in tumor tissues. d Quantification of the Ki67 staining. Five images fields were analyzed per tumor slice. The results represent the means (n = 5) ± SD, and asterisk (*) indicates a significant difference (p < 0.05, Student’s t-test)

Fig. 3

lj-1-59 arrest the cell cycle at G2/M phase and induce apoptosis in melanoma cells. a Cell cycle analysis of SK-Mel-28 (left panel) and SK-Mel-5 (right panel) cells with increasing dose lj-1-59 for the 48 h. The cell cycle distribution was detected by flow cytometry as described in “Methods”. The results represent the means (n = 4) ± SD, and asterisk (*) indicates a significant difference (p < 0.05, Chi-square test). b Apoptosis analysis of SK-Mel-28 (left panel) and SK-Mel-5 (right panel) cells with increasing dose lj-1-59 for 48 h. The results represent the means (n = 4) ± SD, and asterisk (*) indicates a significant difference (p < 0.05, Student’s t-test). c Western Blot analysis of apoptosis-associated proteins in SK-Mel-28 (left panel) and SK-Mel-5 (right panel) cells with lj-1-59 treatment for 48 h

Fig. 4

RNA-seq analyses of the effect of lj-1-59 on the gene expression profile. a The heatmap of SK-Mel-28 after lj-1-59 treatment. b Top 20 enriched KEGG pathways after lj-1-59 treated. c GSEA enrichment plots after lj-1-59 treated, and Normalized enrichment score (NES) and Normalized p-value (P) are shown in each plot. d SK-Mel-28 cells were treated with 5 µM lj-1-59 for 48 h. Then extract total RNA to Q-RT-PCR analysis as described in “Methods”. The results are expressed as the mean (n = 6) ± SD. Significant differences were evaluated using Student’s t-test, and an asterisk (*) indicates a significant difference (p < 0.05)

Fig. 5

lj-1-59 treatment induces DNA damage by increasing ROS. a The level of ROS of SK-Mel-5 (upper panel) and SK-Mel-28 (lower panel) cells were treated with 5 µM lj-1-59 for 0–6 h. b Western Blot analysis of cell cycle-associated proteins and DNA damage-associated proteins in SK-Mel-5 (left panel) and SK-Mel-28 (right panel) cells with increasing does lj-1-59 treatment for 48 h. c, d γH2AX of SK-Mel-28 (left panel) and SK-Mel-5 (right panel) cells were stained by immunofluorescence after 5 µM lj-1-59 treated and calculated. The results in d was represent as the mean (n = 6) ± SD, and asterisk (*) indicates a significant difference using Student’s t-test (p < 0.05)

Fig. 6

Effect of lj-1-59 on BRAFi-resistant melanoma cells. a BRAFi-resistant melanoma cells (RA) were generated as described in “Methods”. RA (left panel) and parental A375 (right panel) cells were prepared in 96-well plates. The cells were treated with PLX4032. Cell viability was determined by CCK-8 assay. The results represent the means (n = 6) ± SD, and asterisk (*) indicates a significant difference (p < 0.05, Student’s t-test). b RA cells were treated with increasing dose lj-1-59 for 0-72 h (left panel). Cell viability was determined by CCK-8 assay. The results represent the means (n = 6) ± SD, and asterisk (*) indicates a significant difference (p < 0.05, Student’s t-test). The IC₅₀ values of lj-1-59 in RA cells were automatically calculated by GraphPad Prism software (right panel). c RA cells were prepared in 6-well plates. The cells were treated with increasing dose lj-1-59 for 24 h. After 2 weeks, the number of colonies was assessed and quantified as described in “Methods”. The results represent the means (n = 5) ± SD, and asterisk (*) indicates a significant difference (p < 0.05, Student’s t-test). d Cell cycle analysis of RA cells with increasing dose lj-1-59 for the 48 h. The cell cycle distribution was detected by flow cytometry as described in “Methods”. The results are expressed as the means (n = 4) ± SD, and asterisk (*) indicates a significant difference (p < 0.05, Chi-square). e RA cells were treated with increasing dose lj-1-59 for the 48 h. Apoptosis was detected by flow cytometry as described in “Methods”. The results are expressed as the means (n = 4) ± SD, and asterisk (*) indicates a significant difference (p < 0.05, Student’s t-test). f Western Blot analysis of apoptosis-associated proteins in RA cells with lj-1-59 treatment for 48 h. GAPDH was used as a loading control

Fig. 7

lj-1-59 induces DNA damage by increasing ROS in RA cells. a RA cells were treated with 5 μM lj-1-59 for 0-6 h, the level of ROS was measured by flow cytometry. The results are expressed as the means (n = 4) ± SD, and asterisk (*) indicates a significant difference (p < 0.05, Student’s t-test). b Western Blot analysis of cell cycle-associated proteins and DNA damage-associated proteins in RA cells with increasing does lj-1-59 treatment for 48 h. α-tubulin was used as a loading control. c RA cells were treated with 5 μM for 0–48 h, and γH2AX was stained by immunofluorescence (left panel) and calculated (right panel). The results are expressed as the mean (n = 5) ± SD, and asterisk (*) indicates a significant difference (p < 0.05, Student’s t-test). d RA cells were treated with 5 μM lj-1-59 for 48 h. Then extract total RNA to Q-RT-PCR analysis as described in “Methods”. The results are expressed as the mean (n = 5) ± SD. Significant differences were evaluated using Student’s t-test, and an asterisk (*) indicates a significant difference (p < 0.05)

Fig. 8

Schematic diagram of the mechanism of lj-1-59. lj-1-59 induces DNA damage by increasing intracellular ROS levels. ATM and ATR are activated after DNA damage, then regulating downstream target protein P53, leading to cell cycle arrest and apoptosis