Fisetin, a dietary flavonoid, augments the anti-invasive and anti-metastatic potential of sorafenib in melanoma

Abstract

Melanoma is the most aggressive and deadly form of cutaneous neoplasm due to its propensity to metastasize. Oncogenic BRAF drives sustained activation of the BRAF/MEK/ERK (MAPK) pathway and cooperates with PI3K/AKT/mTOR (PI3K) signaling to induce epithelial to mesenchymal transition (EMT), leading to cell invasion and metastasis. Therefore, targeting these pathways is a promising preventive/therapeutic strategy. We have shown that fisetin, a flavonoid, reduces human melanoma cell invasion by inhibiting EMT. In addition, fisetin inhibited melanoma cell proliferation and tumor growth by downregulating the PI3K pathway. In this investigation, we aimed to determine whether fisetin can potentiate the anti-invasive and anti-metastatic effects of sorafenib in BRAF-mutated melanoma. We found that combination treatment (fisetin + sorafenib) more effectively reduced the migration and invasion of BRAF-mutated melanoma cells both in vitro and in raft cultures compared to individual agents. Combination treatment also effectively inhibited EMT as observed by a decrease in N-cadherin, vimentin and fibronectin and an increase in E-cadherin both in vitro and in xenograft tumors. Furthermore, combination therapy effectively inhibited Snail1, Twist1, Slug and ZEB1 protein expression compared to monotherapy. The expression of MMP-2 and MMP-9 in xenograft tumors was further reduced in combination treatment compared to individual agents. Bioluminescent imaging of athymic mice, intravenously injected with stably transfected CMV-luciferase-ires-puromycin.T2A.EGFP-tagged A375 melanoma cells, demonstrated fewer lung metastases following combination treatment versus monotherapy. Our findings demonstrate that fisetin potentiates the anti-invasive and anti-metastatic effects of sorafenib. Our data suggest that fisetin may be a worthy adjuvant chemotherapy for the management of melanoma.

Keywords: EMT; fisetin; invasion; melanoma; sorafenib.

Figure 1. Effect of fisetin, sorafenib and their combination on migration and invasion of BRAF-mutated melanoma cells

[A] After scratching, BRAF-mutated A375 and SK-MEL-28 cells were treated with 10 μM fisetin, 2 μM sorafenib and a combination of 10 μM fisetin and 2 μM sorafenib for 48 hrs. Images were acquired at 0 and 48 hrs under the microscope. The red lines define the areas lacking migratory cells. A representative picture from three independent experiments is shown here. Bar = 50 μm [B] BRAF-mutated A375 and SK-MEL-28 cells were incubated with 10 μM fisetin, 2 μM sorafenib and a combination of 10 μM fisetin and 2 μM sorafenib in a serum-reduced medium for 24 hrs in the upper chamber of a Boyden chamber while the lower chamber separated with matrigel coated polycarbonate membrane was filled with a medium supplemented with 10% FBS. After incubation, cells were fixed and stained with crystal violet and imaged under a microscope. A representative picture from three independent experiments is shown here. Bar = 5 μm. Average numbers of invaded cells were determined after counting four randomly selected microscopic fields on the membrane. *P < 0.05, **P < 0.01 significant difference versus control group. ^#P < 0.05, ^##P < 0.01 significant difference versus fisetin. ^$P < 0.05, ^$$P < 0.01 significant difference versus sorafenib. [C] The three-dimensional human melanoma skin equivalents containing A375 cells were treated with 20 μM fisetin, 5 μM sorafenib and a combination of 20 μM fisetin and 5 μM sorafenib for 7 days. After treatment, skin samples were collected and H&E was performed on 5 μm thin sections. A representative picture from three independent experiments is shown. Bar = 50 μm.

Figure 2. Effect of fisetin, sorafenib and their combination on protein expression of EMT-markers and EMT-inducers in BRAF-mutated melanoma cells

BRAF-mutated A375 and SK-MEL-28 melanoma cells were treated with fisetin (20 μM), Sorafenib (5 μM) and a combination these agents (fisetin 20 μM + sorafenib 5 μM) for 48 hrs. 40–50 μg protein from total cell lysates was resolved over 12% Tris-glycine gels and transferred onto a PVDF membrane followed by incubation with primary antibodies of [A] EMT marker proteins, [B] EMT-inducer proteins followed by chemiluminescence detection. After stripping, membranes were analyzed for β-actin to confirm equal loading of protein. The immunoblots shown here are from a representative experiment repeated three times with similar results.

Figure 3. Effect of fisetin, sorafenib and their combination on expression of EMT marker proteins in BRAF-mutated melanoma xenograft tumors

Athymic nude mice implanted with A375 or SK-MEL-28 cells were treated with fisetin 45 mg/kg p.o., sorafenib 45 mg/kg p.o., and their combination (fisetin 45 mg/kg + sorafenib 45 mg/kg) p.o. three times/week. All the mice were euthanized when tumor size reached 1200 mm³ in control group. [A] & [B] Images show representative tumors excised from the mice. [C] & [D] Tumor tissues were collected in 10% formalin and blocks were prepared in paraffin and immunostaining of EMT marker proteins (N-Cadherin, Vimentin and E-Cadherin) was performed. Images shown here are representative pictures taken from three independent tumor samples. Bar = 20 μm.

Figure 4. Effect of fisetin, sorafenib and their combination on expression of EMT-inducing protein in BRAF-mutated melanoma xenograft tumors

Tumor sections from the [A] A375 or [B] SK-MEL-28 xenografts of control and treated mice were stained with antibodies of EMT-inducing transcription factors (ZEB1, Snail1, Twist1 and Slug). Images shown here are representative pictures taken from three independent tumor samples. Bar = 20 μm.

Figure 5. Effect of fisetin, sorafenib and their combination on MMP-2 and MMP-9 expression in BRAF-mutated melanoma xenograft tumors

Tumor sections from the [A] A375 or [B] SK-MEL-28 xenografts of control and treated mice were stained with antibodies of MMP-2 and MMP-9. Images shown here are representative pictures taken from three independent tumor samples. Bar = 20 μm.

Figure 6. Effect of fisetin, sorafenib and their combination on lung metastasis (colonization) assay in nude mice intravenously injected with BRAF-mutated melanoma cells

Athymic nude mice were intravenously injected with 2×10⁶ A375 cells in 0.2 ml PBS in the tail vein. Mice were treated with fisetin 45 mg/kg p.o., sorafenib 45 mg/kg p.o., and their combination (fisetin 45 mg/kg + sorafenib 45 mg/kg) p.o. three times/week as described in “Methods” section. [A] Mice (n = 4) were imaged to record photon emission from tumors using a cooled CCD camera apparatus (IVIS, Xenogen) after i.p. injection of 100 μl of D-luciferin (30 mg/ml) (Xenogen, Alameda, CA, USA). [B] Living Image software (Xenogen) was used to analyze the data. [C] At the end of the experiment, all mice were euthanized, lungs were removed, and images were taken by digital camera. Images shown here are representative pictures taken from three mice of the same groups.