Artemisinin Suppressed Melanoma Recurrence and Metastasis after Radical Surgery through the KIT/PI3K/AKT Pathway

Abstract

Cancer radical surgery is the primary treatment for melanoma, but almost all malignant melanoma patients get recurrence and metastasis after surgery and are eventually dead. This clinical dilemma appeals to better drugs for post-surgery therapy. Artemisinin is a safe and effective antimalarial drug used in the clinic for decades. However, no information is available regarding the effect of Artemisinins on melanoma recurrence and metastasis after tumor excision. In the present study, we established a post-surgery tumor model on balb/c nude mice, and we found that subclinical dosages of Artemisinin significantly blocked recurrence, metastasis, and extended survival time of mice after tumor excision. Similar results were obtained in the in vitro experiments with B16 and A375 cell lines. Further experiments confirmed that Artemisinin inhibits melanoma in vitro and in vivo after radical surgery by the c-KIT/PI3K/AKT signaling pathway. Our findings support the therapeutic potential of Artemisinin in malignant melanoma after surgery.

Keywords: AKT; artemisinin; c-KIT; melanoma; post-surgery model.

Figure 1

Artemisinin inhibited melanoma growth, recurrence and metastasis, and prolonged survival time. A. Mice before and after the post-surgery model. B. The size of recurrent melanoma three weeks after surgery in 16-induced tumor model. C. Survival analysis in B16-induced post-surgery model between different treatment groups. D. The typical pictures of lung metastasis in B16-induced post-surgery model. E. The ratio of lung metastasis in B16-induced post-surgery model. F. Mice in 3 weeks after surgery in A375-induced post-surgery model between artemisinin (Art) and negative control (NC) groups. G. The statistical result of recurrent tumor size in the two groups. H. Survival analysis in A375-induced post-surgery model. *P<0.05, **P<0.01.

Figure 2

Artemisinin inhibited proliferation, migration and invasion in B16 and A375 cells. A. MTT assay of B16 cells in different groups. B. Colony formation assay in B16 cells. C. Statistical result of colony formation assay in B16 cells. D. Wound healing assay in B16 cell line. E. Statistical result of wound healing assay in B16 cells. F. Transwell migration and invasion assay in B16 cells. G. Statistical result of transwell migration assay in B16 cells. H. Statistical result of transwell invasion assay in B16 cell line. I. Wound healing assay in A375 cells. J. Statistical result of wound healing assay in A375 cell line. K. MTT assay in A375 cells. *P<0.05, **P<0.01, ***P<0.001.

Figure 3

Pathways screening for artemisinin derived from RNA sequencing and high throughput analysis. A. Volcano Plot for differentially expressed genes of recurrent melanoma samples between recurrence-NC and recurrence-Art group. B. Volcano Plot for differentially expressed genes between primary tumor tissues and lung metastasis tissues. C. Volcano Plot for differentially expressed genes of B16 cell line between NC group and Art group. D. Top 20 pathways involving the differential genes between recurrence-NC group and recurrence-Art group. E. Top 20 pathways involving the differential genes between primary tumor and lung metastasis. F. Top 20 pathways involving the differential genes between B16-NC group and B16-Art group.

Figure 4

Targets screening for artemisinin with high throughput analysis. A. Venn diagram for Artemisinin-induced significant pathways between melanoma tissues and B16 cells. B. Bubble diagram for the 11 intersectant pathways in artemisinin-treated melanoma tissues and B16 cells, among which PI3K/AKT signaling pathway contained the most gene numbers. C. Heatmap of some differential genes between the recurrence-NC group and recurrence-Art group involved in the PI3K-AKT signaling pathway. D. Heatmap of some differential genes between the B16-NC group and B16-Art group involved in the PI3K-AKT signaling pathway. E. qRT-PCR confirmed c-KIT downregulation by artemisinin in B16 cells. F. The survival analysis of c-KIT based on 458 melanoma patients and related data in the TCGA database.

Figure 5

Artemisinin triggered c-KIT induced pathway. A. Western blot for c-KIT, phosphorylated PI3K, AKT and NFKB treated by artemisinin. B. Statistical result of the c-KIT relative expression. C. Statistical result of the relative expression of phosphorylated PI3K. D. Statistical result of the relative expression of phosphorylated AKT. E. Statistical result of the relative expression of phosphorylated NFKB. F. Western blot for Epithelial-Mesenchymal Transition (EMT) markers treated by artemisinin. G. Statistical result of the Snail relative expression. H. Statistical result of the E-cadherin relative expression. I. Statistical result of the N-cadherin relative expression. J. Statistical result of the Vimentin relative expression. K. Western blot for four groups in tissue from the post-surgery model. L-O. Statistical result of the relative expression of c-KIT, phosphorylated AKT, N-cadherin and vimentin in tissue samples. *P<0.05, **P<0.01, ***P<0.001.

Figure 6

Knockout of c-KIT with gRNA-Cas9 technique significantly inhibits melanoma. A. Colony formation assay between the B16-NC group and B16-KIT-gRNA group. B. Statistical result of the colony formation ratio. C. Statistical result of cell viability derived from MTT assay. D. Wound healing assay between the two groups to detect 24-hour migration. E. Statistical result of the wound distance by time. F. Western blot between B16-NC group and B16-KIT-gRNA group. G. Statistical result of the c-KIT relative expression. H. Statistical result of the relative expression of phosphorylated AKT (Thr308). I. Statistical result of the N-cadherin relative expression. J. Statistical result of the Vimentin relative expression. *P<0.05, **P<0.01, ***P<0.001.

Figure 7

Artemisinin suppressed melanoma by c-KIT related pathway, while rescued experiments with overexpression (OE) of KIT reversed the effects. A. Western blot in NC group, Art group and Art+KIT_OE group. B. Statistical result of the c-KIT relative expression. C. Statistical result of the relative expression of phosphorylated KIT (ser703). D. Statistical result of the relative expression of phosphorylated PI3K. E. Statistical result of the relative expression of phosphorylated AKT (Thr308). F. MTT assay of the three groups in B16 cells. G. Wound healing assay of the three groups in B16 cells. H. Statistical result of wound healing assay. I. Transwell migration and invasion assay for the three groups in B16 cells. J. Statistical result of transwell migration assay. K. Statistical result of transwell invasion assay. *P<0.05, **P<0.01, ***P<0.001.

Figure 8

PI3K/AKT inhibitor LY294002 decreased proliferation, migration and invasion in B16 and A375 melanoma cell lines. A. MTT assay among the NC group, LY294002-15μM group, and LY294002-30μM group in B16 cells. B. Wound healing assay of the above three groups in B16 cells. C. Statistical result of the wound healing assay in B16 cells. D. Transwell migration assay and transwell invasion assay among the NC group, LY294002-15μM group, and LY294002-30μM group. E. Statistical result of the transwell migration assay in B16 cells. F. Statistical result of the transwell invasion assay in B16 cells. G. MTT assay of A375 cells among NC group, LY294002-15μM group, and LY294002-30μM group. H. Wound healing assay of A375 cells among the three groups. I. Statistical result of the wound healing assay in A375 cells. J. Transwell migration assay and transwell invasion assay of A375 cells among the three groups in A375. E. Statistical result of the transwell migration assay in A375 cells. F. Statistical result of the transwell invasion assay in A375 cells. *P<0.05, **P<0.01, ***P<0.001.

Figure 9

AKT activator SC79 attenuated the anti-proliferative, anti-migratory and anti-invasive effect of Artemisinin in both B16 and A375 melanoma cells. A. MTT assay in B16 cells among the NC group, Art group, and Art+SC79 group. B. Wound healing assay in B16 cells among the three groups. C. Statistical result of the wound healing assay in B16 cells. D. Transwell migration assay and transwell invasion assay in B16 cells among the three groups. E. Statistical result of the transwell migration assay in B16 cells. F. Statistical result of the transwell invasion assay in B16 cells. G. MTT assay in A375 cells among the NC group, Art group, and Art+SC79 group. H. Wound healing assay in A375 cells. I. Statistical result of the wound healing assay in A375 cells. J. Transwell migration assay and transwell invasion assay in A375 cells. E. Statistical result of the transwell migration assay in A375 cells. F. Statistical result of the transwell invasion assay in A375 cells. *P<0.05, **P<0.01, ***P<0.001.

Figure 10

Dihydroartemisinin (DHA) and artesunate (AS) suppressed melanoma in vitro and in vivo after cancer radical surgery. A. Mice in 3 weeks after surgery in Negatvie control group (NC), dihydroartemisinin treating group (DHA), and artesunate treating group (AS). B. Statistical result of recurrent tumor size in the three groups. C. The typical pictures for lung metastasis in the three groups. D. Statistical result of the ratio of lung metastasis in the three groups. E. Survival analysis among the three groups in 60 days after surgery. F. MTT assay indicated artesunate (AS) inhibited B16 proliferation in vitro. *P<0.05, **P<0.01, ***P<0.001.

Figure 11

Artemisinin also suppressed breast and lung cancer in vitro and in post-surgery mice model. A. Typical pictures of mice in post-surgery model induced by breast cancer cell line MB231 in negative control (NC) group and artemisinin (Art) group. B. Statistical result of recurrent breast cancer after surgery. C. Typical pictures of mice in post-surgery model induced by lung cancer cell line A549 in NC group and Art treatment group. D. Statistical result of recurrent lung cancer after surgery. E. MTT assay of MB231 cells treated by a dose of artemisinin. F. MTT assay of A549 cells treated by a dose of artemisinin. *P<0.05, **P<0.01, ***P<0.001.

Figure 12

The schematic diagram of the mechanism that Artemisinin can inhibit KIT/PI3K/AKT and EMT signals through phosphorylation cascade to suppress growth, migration, invasion, recurrence and metastasis in melanoma.