[논문]Synergistic Effects of Bee Venom and Natural Killer Cells on B16F10 Melanoma Cell Growth Inhibition through IL-4-mediated Apoptosis

Sin, Dae Chul; Kang, Mi Suk; Song, Ho Sueb

doi:10.13045/acupunct.2017069

Synergistic Effects of Bee Venom and Natural Killer Cells on B16F10 Melanoma Cell Growth Inhibition through IL-4-mediated Apoptosis 원문보기

The acupuncture = 대한침구의학회지, v.34 no.1, 2017년, pp.1 - 9

Sin, Dae Chul (Department of Acupuncture & Moxibustion Medicine, College of Oriental Medicine, Gachon University) , Kang, Mi Suk (Department of Acupuncture & Moxibustion Medicine, College of Oriental Medicine, Gachon University) , Song, Ho Sueb (Department of Acupuncture & Moxibustion Medicine, College of Oriental Medicine, Gachon University)

Abstract ▼ AI-Helper

Objectives : We investigated the synergistic effects of bee venom (BV) and natural killer (NK) cells on B16F10 melanoma cell apoptosis mediated by IL-4. Methods : We performed a cell viability assay to determine whether BV can enhance the inhibitory effect of NK-92MI cells on the growth of B16F10 melanoma cells, and western blot analysis to detect changes in the expression of IL-4, $IL-4R{\alpha}$, and other apoptosis-related proteins. EMSA was performed to observe the activity of STAT6. To confirm that the inhibitory effect of BV and NK cells was mediated by IL-4, the above tests were repeated after IL-4 silencing by siRNA (50 nM). Results : B16F10 melanoma cells co-cultured with NK-92MI cells and simultaneously treated by BV ($5{\mu}g/ml$) showed a higher degree of proliferation inhibition than when treated by BV ($5{\mu}g/ml$) alone or co-cultured with NK-92MI cells alone. Expression of IL-4, $IL-4R{\alpha}$, and that of other pro-apoptotic proteins was also enhanced after co-culture with NK-92MI cells and simultaneous treatment with BV ($5{\mu}g/ml$). Furthermore, the expression of anti-apoptotic bcl-2 decreased, and the activity of STAT6, as well as the expression of STAT6 and p-STAT6 were enhanced. IL-4 silencing siRNA (50 nM) in B16F10 cells, the effects of BV treatment and NK-92MI co-culture were reversed. Conclusion : These results suggest that BV could be an effective alternative therapy for malignant melanoma by enhancing the cytotoxic and apoptotic effect of NK cells through an IL-4-mediated pathway.

주제어

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문제 정의

3), further confirming the synergistic action of BV and NK-92MI cells in the regulation of IL-4-mediated biological responses. This study shows that such an action can inhibit proliferation and differentiation of B16F10 melanoma cells through IL-4-mediated apoptosis. Indeed, siRNA-mediated IL-4 silencing in B16F10 followed by BV treatment reversed the effects and cell viability increased (Fig.

제안 방법

It is reported that IL-4 induces STAT6 activation, and that snake venom toxin also influencesSTATs. To investigate whether BV activates STAT6 in B16F10 cells, the DNA binding activity of STAT6 was assessed by EMSA. I found that B16F10 cells not treated with BV or co-cultured with NK-92MI cells showed low constituted activation of STAT6.
To investigate whether BV treatment and NK-92MI cells synergistically influence IL-4-mediated STAT6 activation in B16F10 cells, I transfected B16F10 cells with50nM siRNA against IL-4 for 24 h, followed by treatment with BV (5 μg/ml) for an additional 24 h, and assessed the DNA binding activity of STAT6 by EMSA.

대상 데이터

T4 polynucleotide kinase was obtained from Promega(Madison, WI). Poly (dI-dC), a horseradish peroxidase-labeled donkey anti-rabbit secondary antibody, and ECL detection reagent were obtained from Amersham Pharmacia Biotech (Piscataway, NJ). Reagents for sodium dodecyl sulfate (SDS)–polyacrylamidegel electrophoresis were purchased from Bio-Rad (Hercules, CA).

데이터처리

Data are presented as mean ± standard deviation (SD) values. The differences between different data groups were assessed by one-way analysis of variance (ANOVA). When the pvalue in the ANOVA test indicated statistical significance, the differences were assessed by the Dunnett’s test.
When the pvalue in the ANOVA test indicated statistical significance, the differences were assessed by the Dunnett’s test.

성능/효과

(Fig. 6) Based on the above results, it can be concluded that the synergistic cytotoxic and apoptotic effects of BV and NK-92MI cells on B16F10 cells is mediated by IL-4.
The results of the present study show that B16F10 cell viability decreased when the cells were co-cultured with NK-92MI cells and then treated with BV (5 μg/ml) to a greater extent than it did after NK-92MI cell co-culture or BV treatment alone, although, compared to the control, cell viability decreased also in the latter two conditions (Fig. 1).
The results show that siRNA-mediated IL-4 silencing reversed the effect of simultaneous BV treatment and co-culture with NK-92MI cells on the expression of IL-4andIL-4Rα,and of other pro-apoptotic proteins, such as Bax, caspase-3, cleaved caspase-3, caspase-9 and cleaved caspase-9, leading to decreased protein levels.
Cell growth was then assessed by direct cell counting after trypan blue staining. The results show that siRNA-mediatedIL-4 silencing reversed the effect of simultaneous BV treatment and co-culture with NK-92MI cells, resulting in increased B16F10 cell viability (Fig. 4).

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