KM-H2

Melittin is Toxic for KM-H2 HL Cells

The high toxicity of melittin (MEL) for HL cell lines KM-H2. IC₅₀ values were found to be 0.75 +/− 0.073 µM for KM-H2 cells. The toxicity of MEL for these HL cells was higher than the toxicity for normal blood cells. KM-H2 cells with normal peripheral blood mononuclear cells (PBMC) were co-cultured in the presence of different concentrations of MEL. HL cells and PBMC were discriminated against using their different cell size. As shown in Fig. 1, the percentage of living tumor cells decreased with increasing MEL concentrations. Interestingly, a shift in the composition of the surviving normal PBMCs. As shown in Fig. 2, a strong increase in the monocyte population. This increase was also visible in cultures without HL cells. After staining with specific antibodies, a strong increase in the percentage of CD14-positive monocytes and a less pronounced but significant increase in the percentage of CD56-positive NK cells (Fig. 3). Percentages of CD19-positive B cells and CD8-positive cytotoxic T cells decreased whereas CD4-positive T helper cells remained unchanged.

Fig. 1 Toxicity of MEL for HL cell lines KM-H2. (Kreinest T, et al., 2020)

Fig. 2 MEL increases monocyte numbers. (Kreinest T, et al., 2020)

Fig. 3 Changes in blood cell populations after treatment with MEL. (Kreinest T, et al., 2020)

mir148a Overexpression Decreases Cell Proliferation in KM-H2

To put the observation of mir-148a downregulation in classical HL (cHL) in a functional context, three cHL cell lines stably overexpressed mir-148a (KM-H2, L-540, L-1236) were established.

By analyzing these cell lines using the CCK8 assay, a significant decrease (p < 0.05) in cell proliferation after miR-148a overexpression in the KM-H2 cell line. On day 8 of the experiment, a 32% reduction in cell proliferation was observed in the mir-148a-expressing cell line as compared to cells with the empty vector. miR-148a overexpression did not affect the proliferation of L-1236 and L-540 cell lines (Fig. 4).

To analyze if the decrease in cell proliferation observed using the CCK8 test is related to differences in DNA replication, the flow cytometry assay was conducted based on the measurement of newly synthesized DNA in cells in S-phase. In line with the CCK8 test results, a decrease in DNA synthesis was observed for the KM-H2 cell line. On day 3 of culture, KM-H2 cells transduced with miR-148a expression vector showed a significantly lower percentage of cells with newly synthesized DNA as compared to cells transduced with the empty vector (35% vs. 49%, p = 0.03).

Fig. 4 cHL cell lines (KM-H2, L-540, L-1236) viability after mir-148a overexpression (CCK-8 test). (Paczkowska J, et al., 2020)