Background: Bee venom is a promising agent for cancer treatment due to its selective cytotoxic potential for cancer cells through apoptotic pathways. However, there is no evidence for changes in the epigenome and mitochondrial DNA copy numbers after bee venom application. The purpose of this study was to determine the impact of bee venom on cytosine modifications and mitochondrial DNA copy number variation. Methods: A broad range of methods was applied to elucidate the impact of bee venom on neoplastic cells. These included MTT assay for detection of cytotoxicity, immunostaining of cytosine modifications and mitochondria, assessment of cellular morphology by flow cytometry, and quantification of mitochondrial DNA copy numbers using QPCR. Results: Bee venom-induced cell death was selective for cancer cells, where it triggered a response characterized by alteration of cytosine modification. In contrast, normal cells were more resistant to DNA modifications. Furthermore, application of the venom resulted in variation of mitochondrial membrane permeability and mitochondrial DNA copy numbers, together with alterations in cell morphology, manifesting as reduced affected cell size. Conclusion: The study findings suggest that bee venom can be used as a selective DNA (de)methylating agent in cancer. Various agents (such as decitabine and 5-azacytidine) have been synthesized and developed for cancer treatment, and a range of syntheses and preparation and application methods have been described for these patented drugs. However, to the best of our knowledge, no previous research has investigated the use of bee venom or any component thereof for epigenetic therapy in cancer cells.

Cell viability detection is important in cell culture applications including measurement of cell proliferation i.e for understanding cytotoxic effects of compounds on cells. There are some cell viability methods based on fluorescence or non-fluorescence detection. More simplified evaluation for cell viability, such as trypan blue staining, can be preferred before performing fluorescence assays. This appears advantageous when to have a large number of cell samples in ELISA plates after treatments with different concentrations of drug candidates. Thus, further fluorescence assays can include less concentrations rather than experiencing all used along 96-well plates. For this, trypan blue exclusion method is an option. Traditionally, treated cells are harvested by centrifugation and incubated with trypan blue within tubes followed by transferring the mixture into a hemacytometer with two chambers and assessed under the microscope. Nevertheless, using a hemacytometer limits practicability of this method when analyzing various cell samples into 96-well plates at the same time. This study was aimed to adapt trypan blue method to in situ staining of adherent cells cultured on ELISA plates. For this, cells were fixed with different fixatives after trypan blue incubation to maintain cells in impenetrable meshwork, and paraformaldehyde was the most effective fixative. This modified protocol was validated by testing the effect of dimethylsulfoxide-a cytotoxic agent-on cells, and expectedly found that cell viability reduced with higher concentrations of dimethylsulfoxide suggesting that in situ detection of cell viability by trypan blue can be a useful tool for preliminary detection of cells cultured on ELISA plates before performing automatized experiments with such flow cytometer and/or microplate reader.

Death‑associated protein kinase‑1 (DAPK1) is a pro‑apoptotic gene that induces cellular apoptosis in response to internal and external apoptotic stimulants. The silencing of DAPK1 can result in uncontrolled cell proliferation, indicating that it may have a role in tumor suppression. DAPK1 activity can be inhibited by the cytosine methylation that occurs in its promoter region. These methylation changes in the promoter region of DAPK1 have been reported in a range of solid and hematological malignancies. In the present study, DAPK1 methylation was investigated in chronic myeloid leukemia patients (n=43) using bisulfite conversion followed by methylation‑specific polymerase chain reaction. The present study included a number of patients who were identified to be resistant to the common chemotherapeutic agent imatinib (STI571, Gleevec®, Glivec®), exhibiting at least one mutation in the breakpoint cluster region‑Abelson murine leukemia (BCR‑ABL) gene. Thus, the patients in the present study were divided into two groups according to their response to imatinib therapy: Non‑resistant (n=26) and resistant (n=17) to imatinib. Resistant patients were characterized by the presence of single or multiple mutations of the BCR‑ABL gene: i) T315I, ii) M351T, iii) E255K, iv) T315I and M351T or v) T315I, M351T and E255K. The present study identified that: i) The incidence of DAPK1 methylation was significantly higher in the resistant patients compared with the non‑resistant patients; ii) the extent of resistance varied between mutation types; and iii) there was no DAPK1 methylation in any of the healthy controls. These findings indicate that DAPK1 methylation may be associated with a signaling pathway for imatinib resistance in chronic myeloid leukemia.