۱- Introduction

۲- Materials and methods

۳- Results

۴- Discussion

References

Abstract

Diosmetin is a natural flavonoid obtained from citrus fruits and some medicinal herbs. Previous studies have reported the anti-cancer activity of diosmetin in some types of tumors. However, it is still unclear whether diosmetin exerts anti-cancer effects, particularly anti-angiogenic effects, in skin cancer. In this study, we used B16F10 melanoma cells and human umbilical vein endothelial cells to investigate the inhibitory effect of diosmetin on cell proliferation, migration and tube formation in vitro. Rat aorta ring assays were performed to determine the effect of diosmetin on ECs sprouting ex vivo. Furthermore, a B16F10 mouse melanoma model was used to observe the effect of diosmetin on tumor growth, angiogenesis, and metastasis in vivo. Our results showed that diosmetin not only suppressed tumor cell proliferation and migration but also induced cell apoptosis via the caspase pathway in B16F10 cells, and potently inhibited tube formation and cell migration in HUVECs. Rat aorta ring assays showed that diosmetin attenuated the ECs sprouting. Moreover, the mouse melanoma model showed that diosmetin significantly delayed tumor growth by inhibiting tumor vessels sprouting and expansion during tumor progression. Notably, diosmetin induced the normalization of tumor vasculature through the downregulation of angiopoietin-2 and the improvement of pericyte coverage, leading to suppression of metastasis formation in lungs and lymph nodes. In conclusion, our results demonstrate that diosmetin suppresses tumor progression and metastasis by inducing tumor cell death and inhibiting tumor angiogenesis as well as normalizing the defective tumor vasculature, suggesting that diosmetin is a potential adjuvant chemotherapy agent.

Introduction

Malignant melanoma is the most lethal form of skin cancer, accounting for approximately 90% of the deaths caused by this disease [1,2]. In recent decades, the incidence of melanoma has steadily risen. Worldwide, there were about 55,000 deaths in 2012 [3] and according to the International Agency for Research on Cancer, in 2030, an estimated 351,396 new cases and 87,725 deaths are expected to occur [4]. The aggressiveness of melanoma is associated with its high metastatic potential [5]. If recognized during the early stages, melanoma can be removed by surgical excision. However, once it metastasizes, it is difficult to treat and has poor prognosis. Chemotherapy is the most commonly chosen treatment option for the management of melanoma that has disseminated. In general, however, the current chemotherapies to treat melanoma have low efficacy and cause severe adverse effects [6]. Therefore, the development of anti-cancer drug that are both effective and safe is urgently required. Diosmetin, the aglycone part of the flavonoid glycosides diosmin, is abundant in citrus fruits, oregano and is also obtained from some medicinal herbs such as Rosa agrestis Savi (Rosaceae) and Anastatica hierochuntica L. (Brassicaceae) [7–۱۰]. Several therapeutic activities of diosmetin, such as antibacterial, anti-inflammatory, and antioxidant properties, have been reported [11–۱۳]. It has been also demonstrated that diosmetin has a potent anti-proliferative effect in some tumor cells, including MDA-MB 468 breast cancer cells [14], uterine cervical carcinoma cells injected in mice [15], and HepG2 hepatocellular carcinoma cells [16,17]. However, the therapeutic activity of diosmetin in malignant melanoma is still unknown. Angiogenesis, the formation of new vasculature through the sprouting and extension from pre-existing vessels, occurs in several pathological conditions including various malignant solid tumors [18,19]. In tumor progression, tumor-induced angiogenesis supplies nutrients and oxygen and removes metabolites, resulting in further tumor growth [20]. Moreover, tumor vasculature is one of the routes to the spreading of tumor cells to distant organs [21]. Therefore, inhibition of tumor-induced angiogenesis has become a promising strategy for cancer therapy. On the other hand, tumor blood vessels have impaired structure and functionality [21–۲۴].