Synergistic Anti-cancer Activity of MH-30 in a Murine Melanoma Model Treated With Cisplatin and its Alleviated Effects Against Cisplatin-induced Toxicity in Mice


Abstract

Background/aim: Although cisplatin is an effective anticancer drug, its toxic effects on normal tissues limit its use. We developed a herbal formula, MH-30, with increased fat-soluble polyphenols by improving the manufacturing method of HemoHIM. In this study, we examined whether the combination of MH-30 with cisplatin exerts synergistic antitumor effect while it reduces cisplatin-induced toxicities.

Materials and methods: MH-30 was produced by adding the ethanol-insoluble fraction to its extract after decocting herbs in 30% ethanol and water. We used a melanoma-bearing mice model to investigate synergistic anticancer effects. The NK cell activity and cytokine levels were measured by Cr51-release assay and ELISA. The AST, ALT, BUN, and creatinine levels were estimated in the serum.

Results: MH-30 effectively inhibited melanoma growth in vitro. Furthermore, MH-30 had a synergistic effect in combination with cisplatin on melanoma growth inhibition in vitro and in vivo. In melanoma-bearing mice, cisplatin alone decreased the activity of NK cells and the levels of IL-2 and IFN-γ, which were effectively restored by the combination of MH-30 with cisplatin. Combined treatment with MH-30 and cisplatin significantly inhibited the cisplatin-induced increase in the levels of AST, ALT, BUN, and creatinine.

Conclusion: Combination of MH-30 with cisplatin may be a beneficial anticancer treatment with reduced adverse effects.

Keywords: Chemotherapy; anticancer; cisplatin; cisplatin-induced toxicity; herbal; melanoma.

Conflict of interest statement

The Authors declare that they have no competing interests regarding this study

Figures

Figure 1
Figure 1. The scheme for producing the herbal formula MH-30
Figure 2
Figure 2. MH-30 inhibits melanoma cell growth and enhanced the effect of cisplatin in vitro. The melanoma cells were seeded at a density of 7×103 cells per well. (A) At 24 h after melanoma seeding, cells were treated with various concentration of MH-30 or HemoHIM for 48 h. (B) At 24 h after melanoma seeding, cells were treated with various concentrations of MH-30 and with or without various concentrations of cisplatin for 24 h. After incubation, the CCK-8 solution was added to each well and then the optical density was measured. Data are presented as the Mean±SD. *p<0.05, **p<0.01, ***p<0.005 and ****p<0.001
Figure 3
Figure 3. The experimental schedule used for assessing the synergistic anticancer efficacy of MH-30 and cisplatin in melanoma-bearing mice. B16F10 melanoma cells (2×105/mouse) were inoculated subcutaneously into the left femoral region of mice. MH-30 was given daily at 100 mg/kg B.W. until the end of the experiment. Cisplatin was intraperitoneally injected at 3 mg/kg B.W. on days 7, 8, 13, 14, 19 and 20 (total six injections)
Figure 4
Figure 4. MH-30 has synergistic anticancer effects in combination of cisplatin in melanoma-bearing mice. All mice treated as described in figure 3 were sacrificed on day 22 after B16F10 melanoma inoculation. (A) The tumor weight of each group was measured. (B) Photographs of melanoma solid tumors taken from all mice of each group. There were eighteen~twenty-two mice in each group. Data are presented as the Mean±SD. ?p<0.001 compared with control group; *p<0.005 compared with only cisplatin treated group
Figure 5
Figure 5. Effect of MH-30 on the cancer cell killing activity of NK cells and on the secretion levels of IL-2 and IFN-gamma in melanoma-bearing mice. On day 19 after B16F10 melanoma inoculation, spleen lymphocytes were isolated using a Ficoll-Hypaque density gradient centrifugation. (A) The cancer cell killing activity of NK cells was determined by 51Cr release assay as described in Materials and Methods. (B), (C) Spleen lymphocytes were cultured with ConA (1 mg/ml). After 1 or 2 days, IL-2 and IFN-gamma in the culture supernatants were measured by ELISA as described in Materials and Methods. There were six mice in each group. Data are presented as the Mean±SD. ?p<0.05 and ?p<0.01 compared with control group; *p<0.1 and **p<0.05 compared with only cisplatin treated group.
Figure 6
Figure 6. MH-30 administration prevents cisplatin-induced body, liver, and kidney weight loss. (A) The experimental schedule for assessing the cisplatin-induced damages in vivo. MH-30 was given daily at 100 mg/kg B.W. for 20 days. From day 3 after initial oral feeding of MH-30, cisplatin was intraperitoneally injected at 3 mg/kg B.W. twice a week for three weeks. (B) Body weight of all groups was measured once every three or four days. (C) On day 17 after initial injection of cisplatin, the liver and the kidneys of all groups were removed, and their weight was measured. There were twelve mice in each group. Data are presented as the Mean±SD. ?p<0.1 and ?p<0.01 compared with the control group; *p<0.05 and **p<0.01 compared with the only cisplatin treated group
Figure 7
Figure 7. MH-30 administration reduces cisplatin-induce hepatotoxicity. On day 17 after initial injection of cisplatin, serum was prepared through clotting of whole blood collected from the retro-orbital veins and the liver was removed and fixed in 10% buffered formalin for 2 days. (A) The levels of serum AST, and ALT were analyzed using the AU680 Chemistry System. (B) The paraffin-embedded sections (5 mm thick) were stained with hematoxylin and eosin (H&E) for histopathological examination and observed under light microscope at ×200 magnification. The result shown is representative from each group. There were twelve mice in each group. Data are presented as the Mean±SD. ?p<0.05 and ?p<0.01 compared with the control group; *p<0.1 and **p<0.05 compared with the only cisplatin treated group.
Figure 8
Figure 8. MH-30 administration reduces cisplatin-induce nephrotoxicity. On day 17 after initial injection of cisplatin, serum was prepared through clotting of whole blood collected from the retro-orbital veins and the kidneys were removed and fixed in 10% buffered formalin for 2 days. (A) The levels of serum BUN and creatinine were analyzed using the AU680 Chemistry System. (B) The paraffin-embedded sections (5 mm thick) were stained with hematoxylin and eosin (H&E) for histopathological examination and observed under light microscope at ′200 magnifications. The result shown is representative cortex from each group. There were twelve mice in each group. Data are presented as the Mean±SD. ?p<0.05 and ?p<0.01 compared with the control group; *p<0.01 compared with the only cisplatin treated group
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