Vitexin inhibited the invasion, metastasis, and progression of human melanoma cells by targeting STAT3 signaling pathway

In human melanoma cells, resistance to conventional chemotherapy and radiotherapy and rapid metastasis give melanoma a remarkable feature of the most aggressive and lethal. The low response rate of melanoma to existing treatment modalities is a substantial threat to patients and researchers. It is crucial to identify new therapeutic agents for the fatal malignancy melanoma. Vitexin is a flavonoid compound in many traditional Chinese medicines that exhibits antioxidant, anti-inflammatory and anti-tumour activities in many cancer cells. In our study, we elucidated the inhibitory effects of vitexin on invasion and metastasis in human melanoma A375 and C8161 cells in vitro. After vitexin treatment for 24 h or 48 h, the invasive ability and migration of melanoma cells were decreased in a dose- and time-dependent manners. In western blot analysis, we verified that vitexin inhibited the expression levels of MMP-2, MMP-9, vimentin, Slug and Twist which are known as the regulators of protein degradation and promote various cell behaviours such as migration and invasion. To further investigate the target signal that may be influenced by vitexin, immunofluorescence assay was performed to observe STAT3 localization and western blot results showed that vitexin decreased the expression of the phosphorylation of kinases that inducing STAT3 activation. Accordingly, we provide inspiring insight into the basic inhibition mechanism of vitexin, which will soon be an issue due to its scientific potential for further development as a novel anti-tumour agent for the clinical therapy of human melanoma.


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With a poor prognosis and a progressively increasing incidence, cutaneous melanoma is attracting 30 considerable interest as the most aggressive and deadly type of skin cancer (1). As a malignant tumour, 31 melanoma originates from pigmentary cells originally derived from the neuroectoderm and can occur 32 throughout the skin, iris, and rectum(2). Additionally, it has been reported that the third most common 33 cause of brain metastasis is melanoma, following in lung and breast cancer, and more than 75% of 34 melanoma patients suffer brain metastases, which cause death in 95% of all cases(3). The survival rate 35 in patients with metastatic melanoma for 5 years, who receive traditional therapy is consistently <10% 36 (4). However, chemotherapy, a strong anti-tumour strategy, yet fails to alleviate melanoma that shows 37 drug resistance. Chemical drugs kill normal cells and cause toxic side effects that are unbearable for 38 patients. In addition to fatal side effects, no obvious benefit in survival time is achieved with 39 immunotherapy and targeted inhibitors(5). Consequently, developing novel therapeutic drugs is an 40 urgent need, and such drugs should exert better anticancer activity, less toxicity, and the ability to 41 coordinate current chemotherapeutic drugs to enhance their antitumor efficacy against human melanoma.

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As described above, cells can acquire invasive and migratory properties; these transformed cells 43 progress to become life-threatening cancers(6). Degradation of stromal extracellular matrix (ECM) and 44 basement membranes is one crucial step for malignant cells to invade and metastasize. As the family 3 45 members of zinc-dependent matrix-degrading enzymes, matrix metallopeptidases (MMPs)(7, 8), MMP-2 46 (Gelatinase A, 72 kDa) and MMP-9 (Gelatinase B, 92 kDa) are two well-known gelatinases, and MMP-2 47 contributes to tumour cell migration after being directly activated by binding to αvβ3 integrin(9, 10). It 48 has been reported that the expression of MMP-9 is vitally related to the invasiveness of melanoma cancer; 49 thus, blockade of MMP-9 inhibits melanoma cells invasion(11). Epithelial-mesenchymal transition 50 (EMT) is another metastasis pathway activated in cancer cells that also contributes to cell invasion and 51 migration in various cancers (12)(13)(14). Some related proteins, such as vimentin, Slug and Twist, have been 52 found to work synergistically in the regulation of EMT(15-17); thus, in this study, we sought to address 53 whether vitexin could suppress the expression of EMT-related proteins and MMPs to further determine 54 the underlying mechanism of vitexin inhibition.

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Meanwhile signal transducer and activator of transcription 3 (STAT3), as an oncogenic transcription 56 factor, plays a major role in malignant transformation (18,19). During the progression of oncogenesis 57 STAT3 is responsible for the transmitting of signals, from plasma membrane to nucleus, which results

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in the alternated expression of target genes related to cell proliferation, inflammatory response, 59 metastasis, and immune response(18). In the STAT3 signaling pathway, the receptor-associated Janus 60 kinase (JAK) that regulating the phosphorylation of STAT3 initially modulates the activation of STAT3 early-phase clinical trials are giving promising results. In current study, two human melanoma cell lines 68 A375 and A2058 were used to verify the involvement of STAT3 in the anti-tumor effects of vitexin.

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The aromatic shrub Vitex negundo L (Verbenaceae) is abundant in Asian countries due to its anti-70 inflammatory effect, has been widely used as a Chinese folk medicine for the treatment of asthma, cough, 71 and arthritis. Recently the effective constituent of vitex negundo L was found to be vitexin (apigenin-8-

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Vitexin is an active compound of vitex negundo L, but it can also be concentrated from various medicinal

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In summary, the primary aim of this paper is to provide empirical and theoretical evidence for the 82 claim that vitexin can inhibit the invasion and metastasis of melanoma cells in vitro through the 83 suppression of the related proteins MMP-2 and MMP-9, and briefly discuss the involvement of STAT3 84 signaling pathway in these effects.

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Chemicals and reagents.

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Vitexin (purity >98%), which is purified from the seeds of the Chinese herb Vitex negundo, was

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To observe the migration of A375 and C8161 cells, an in vitro wound-healing assay was performed, 118 following vitexin treatment. Cells (1x10 6 cells/ml) were seeded into a 6-well plate and incubated for 24 119 h until they reached full confluence. The centre of the cell monolayer was scraped with a sterile 100-μL 120 pipette tip to create a straight gap of constant width as a "wound". Then, the cells were exposed to various 121 concentrations of vitexin (0, 5, 10 and 20 µM) or DMSO (10 µM). After 24 h, 4% paraformaldehyde was 122 used to fixed the cells, wound closure was imaged using a fluorescence microscope (Olympus Optical

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To assess the motility of A375 and C8161 melanoma cells treated with vitexin, this invasion assay was 127 performed using a modified Boyden chamber coated with 50 µl of Matrigel. The cells were then 128 suspended at a density of 5 × 10 4 cells/chamber and treated with vitexin (10 µM) or DMSO (10 µM).

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Medium containing 2% FBS was added to the upper wells while medium containing 10% FBS was added 130 to the lower chambers. Then, the plates were incubated for 24 h at 37˚C. Next, 4% paraformaldehyde 131 was used to fix the invaded cells on the bottom surface of the insert, and the cells were stained with 0.1% 132 crystal violet at room temperature. Images were obtained by using an inverted microscope (Olympus).

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The inhibitory effect of vitexin on human melanoma cell viability was determined. A375 and C8161

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A wound-healing assay was performed to investigate whether vitexin inhibits the migration of melanoma 185 cells in vitro. A375 and C8161 cells were exposed to various concentrations of vitexin (0, 5, 10 and 20 186 µM) and the positive control (DMSO, 10 µM). At 24 h, the results revealed that vitexin reduced the 187 migration of both A375 and C8161 cells in a dose-dependent manner ( Fig. 2A, B and C).

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In the Transwell assay, the invasion of A375 and C8161 cells treated with vitexin was observed.

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Following a 24 h treatment with vitexin (10 µM), there was a marked decrease in A375 and C8161 cells 192 invasion, which indicated that vitexin can inhibit cell invasion at a low concentration in the Matrigel 193 invasion assay (Fig. 3A). The number of invaded cells also demonstrated that when vitexin was added at 194 a concentration of 10 µM, the inhibitory effect on cell invasion was almost the same in A375 and C8161 195 cells (Fig. 3B).

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Vitexin suppresses the expression of migration-related proteins.

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To determine the proteins that vitexin may affect to inhibit cell migration, we performed western blotting

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The cellular-distribution of activated STAT3 dimer plays a vital role for its transcription. Thus, STAT3 206 was determined by immunofluorescence assay to elucidate whether vitexin exposure can affect its 207 nuclear translocation. Immunocytochemistry data indicated that treatment with vitexin downregulated 208 the level of STAT3 in the nucleus in melanoma cells (Fig. 5A). To further explore the mechanism of 209 vitexin on the inactivation of STAT3 signaling pathway, we performed western blot assay to evaluated 210 the expression level of the phosphorylation of kinases that regulating the activation of STAT3. The 211 regulation of STAT3 activation is closely associated with tyrosine kinases of the Src kinase and JAK 212 families. As is shown in Fig. 5B, the expression of Scr and both JAK1 and JAK2 were persistently active.

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However, after vitexin treatment, the expression of phosphorylated Scr, JAK1 and JAK2 were 214 significantly suppressed in a time-dependent manner (Fig. 5B).

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An epidemiological study has demonstrated that the major risk factors for developing melanoma are

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Pharmacologists revealed the quantity of stable radical (a total of 7) and the order of the hydroxyls in

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Inspired by other studies, which have demonstrated that STAT3 could promote tumorigenesis in many 262 malignant tumors by regulating several genes (52-54), the relationship between the anti-melanoma 263 effects of vitexin and the involvement of STAT3 was elucidated in our study. As mentioned above,

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TWIST gene expression is responsible for cancer cell EMT which is known to be directly mediated by 13 265 STAT3(55), and MMP-2 is also a STAT3-target gene that is involved in cell metastasis (56), indicating 266 that inhibition of STAT3 is the possible mechanism of anti-migration in the anti-melanoma effects of 267 vitexin. Through a reciprocal interaction with SH2 domain (a phosphotyrosine recognition domain),

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STAT3 is activated to be phosphorylated and transform to STAT3 dimers(57). Then, to transcribe various 269 target genes, STAT3 dimers needs to translocate to the nucleus which was found to be inhibited after 270 vitexin treatment in current study. Furthermore, it has been verified that STAT3 phosphorylation is

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In conclusion, our present study revealed that vitexin could function as a novel inhibitor that decreased