Advanced malignant melanoma responds to Prunus mume Sieb. Et Zucc (Ume) extract: Case report and in vitro study

Abstract

Malignant melanoma (MM) is an aggressive chemoresistant skin cancer characterized by rapid metastasis and a poor prognosis. Therefore, the development of innovative effective therapies is critical. MK615 is an extract from the Japanese apricot Prunus mume Sieb. Et Zucc (Ume). At a neutral pH, it contains natural chemical substances such as triterpenoids that exert anti-neoplastic effects in several types of cancers. We found that in patients with advanced MM, MK615 dramatically suppressed the cutaneous in-transit metastasis of the disease. Pre- and post-treatment comparison of tumors showed that the apoptotic index was significantly increased by MK615. In vitro studies, MTT assay, flow cytometric cell cycle analysis and immunofluorescence microscopy revealed that MK615 inhibited the growth of SK-MEL28 cells in a dose-dependent manner, increased the proportion of cells in sub-G1 phase and induced apoptosis. We further examined the expression of the receptor for advanced glycation end products (RAGE). RAGE is a multi-ligand receptor that binds to a novel cytokine, high mobility group box protein 1 (HMGB1), as well as advanced glycation end products. There is evidence that RAGE/HMGB1 interactions enhance cell invasion in MM. Here, we present Western blotting and immunofluorescence microscopy data indicating that MK615 inhibited the expression of RAGE in SK-MEL28 cells, and suppressed the release of HMGB1 by SK-MEL28 cells. Our findings suggest that MK615 may be a valuable tool for treating MM and other malignant tumors.

Figure 1.

(a) A 67-year-old Japanese woman with MM on the sole of her left foot. (b) Multiple nodules on the left thigh indicating in-transit metastasis of MM. (c) Highly atypical cells and significant inflammatory infiltrates in the vicinity of the nodules in the dermis (hematoxylin and eosin; original magnification x40 and x100). (d) Positron emission tomography (PET) showed multiple glucose uptake on the left thigh.

Figure 2.

(a) Five months after the start of MK615 treatment, the number of in-transit metastases is clearly decreased. (b) PET showed improvement of the lesions on the left thigh after MK615 treatment.

Figure 3.

(a) The viability of SK-MEL28 cells treated with MK615 for 24 and 48 h was determined by MTT assay. Each time point represents the results of 6 experiments. The viability of SK-MEL28 cells treated with MK615 decreased significantly in a dose-dependent manner after a 24- and 48-h incubation. Statistical significance was determined by the Student’s t-test. *p<0.05 compared with MK615-untreated controls. (b) SK-MEL28 cells were treated with 0, 10 or 20 μl/ml MK615 for the indicated times. DNA components were analyzed by flow cytometry. MK615 treatment resulted in an increase in SK-MEL28 cells in the sub-G1 phase. (c) The effect of MK615 on phosphatidylserine externalization. Cells were treated with the indicated doses of MK615 for 2, 4 and 8 h. Phosphatidylserine externalization was determined by Annexin V-FITC binding assay. Nuclei were stained blue by DAPI and green by Annexin V.

Figure 4.

(a) RAGE expression in MK615-treated SK-MEL28 cells determined by Western blotting. SK-MEL28 cells were treated with 0, 2.5, 5 and 10 μl/ml MK615 for 16 h. RAGE expression of SK-MEL28 cells treated with MK615 decreased significantly in a dose-dependent manner. (b) SK-MEL28 cells were incubated with or without 5 μl/ml MK615 for 16 h. Non-treated and MK615-treated cells were incubated with anti-RAGE antibody and then with FITC-labeled anti-goat IgG as a secondary antibody. Nuclei were labeled with DAPI (original magnification, x400).

Figure 5.

SK-MEL28 cells were incubated with 0, 2.5, 5 and 10 μl/ml MK615 for 16 h. Supernatants were collected and analyzed by HMGB1 ELISA. MK615 significantly suppressed HMGB1 release in a dose-dependent manner. Values are expressed as the mean ± SE of triplicate experiments. *P<0.05.