Today, large numbers of studies are being conducted to explore the antitumor action of BV towards different types of cancers and the underlying mechanisms. The anticancer effect is mainly accredited to a basic polypeptide, MEL, that makes up about 50% of the dry BV. Havas ^[1][112] was one of the first who recorded the impact of BV on cancer cells. Afterward, Mufson and colleagues ^[2][113] reported that MEL can pass through a phospholipid bilayer, and thus display its ability. The relation between MEL and cell membranes caused impairment of the phospholipid’s acyl groups, higher sensitivity to phospholipid hydrolysis by phospholipase, and increased synthesis of prostaglandins from arachidonic acid released from phospholipids. Furthermore, McDonald et al. ^[3][114] examined BV’s anticancer property in a mortality study which involved 580 beekeepers. Beekeepers were identified through the obituaries published in three different US beekeeping industry journals between 1949 and 1978. Based on the obituaries, they established the cause of death of the beekeepers and made a comparison with the general population. Results showed a slightly lower incidence of death from cancer in beekeepers professionally exposed to BV during their working life compared to the rest of the population and a significantly lower death rate from lung cancer, while the mortality from other diseases was equal to the rest of the general population. The obtained results were among the first to suggest the possible anticancer potential of BV. After that, numerous studies showed anticancer properties of BV and its major component MEL ^{[4][5][6][7][8][9][10][11][12][13][14]}[3,12,15,16,17,21,27,47,115,116,117].

Hait et al. ^[15][118] were the first to demonstrate an inhibiting potential of MEL in vitro. They showed that MEL, as an inhibitor of calmodulin, inhibits the growth and clonogenic capacity of human leukemia cells. Lee and Hait ^[16][119] have also observed an inhibitory impact of MEL on astrocytoma cell growth. Lazo et al. ^[17][120] noted a comparable mechanism of action of MEL as an inhibitor of calmodulin in leukemia cells. They also noted that MEL enhances bleomycin toxicity in human granulocyte macrophages and erythroid stem cell colonies ^[18][121]. Hait and Lee ^[19][122] noted that the cytotoxicity of MEL is proportional to the antagonistic effect of calmodulin. The aforementioned studies support the pharmacological role of calmodulin as a potential intracellular target of MEL antiproliferative activity.

Additionally, Killion and Dunn ^[20][123] showed that leukemia cells are more sensitive to MEL action compared to normal mouse spleen cells and bone marrow cells, reasoning that bone marrow cells have several binding sites on the membrane for carbohydrates, and these places disappear in the adult spleen cells, while they are almost absent after neoplastic changes that could make cancer cells more sensitive to the peptide. Zhu et al. ^[21][124] have reported that MEL does not prevent the growth of normal cells at a concentration that prevents the proliferation of cancer cells such as lung cancer cells. The observed cell response differences indicated an unalike activation of signaling pathways between normal and cancer cells. MEL has proven particularly effective in cultured cells that express high levels of the ras oncogene ^[22][23][125,126]. MEL also enhances the PLA₂ activation in the ras oncogene-transformed cells resulting in its selective destruction. The results suggest that the enhanced activation of PLA₂ by MEL could be the target of MEL’s cytotoxicity against cancer cells ^[5][12].

MEL causes increased activation of PLA₂ activity and calcium intake in ras-transformed cells, which could be the basis for the antitumor activity of this compound ^[23][126]. Following these findings, a large number of studies made a connection between PLA₂ activity and MEL’s cytotoxic effect on a variety of tumor cells ^{[24][25][26][27]}[127,128,129,130]. Activation of PLA₂ could play a role in the cytotoxicity of tumor cells through several different cell changes such as a synergistic effect of PLA₂ and phosphatidylinositol (3,4)-bisphosphate in the induction of cell death ^[28][131]. Death caused by PLA₂ and phosphatidylinositol (3,4)-bisphosphate is associated with the disruption of cell membrane integrity, abolition of signal transduction, and creation of a cytotoxic lyso-phosphatidylinositol (3,4)-bisphosphate. It was also found that their combined effect results in the formation of a tumor lysate that enhances the maturation of human monocyte-derived immunostimulatory dendritic cells. Such a tumor lysate, which is a complex mixture of tumor antigens with potential activity, has everything needed for a potential tumor vaccine ^[29][132].

One of the main issues in anticancer therapy is related to the concentration of the substance used, as it may cause serious side effects. Therefore, drug intake should be adequate and specific. A large number of insect lithic peptides, including those isolated from BV, have an amphipathic structure that binds and incorporates into negatively charged cell membranes. Compared to normal cells, which have a low membrane potential, the membrane of cancer cells has a high membrane potential ^{[5][6][30][31]}[12,15,133,134] and that is why numerous lytic peptides selectively disrupt the membrane structure of cancer cells rather than the normal cell membrane. MEL should thus have a suitable role in anticancer therapy ^[5][12]. Gawronska et al. ^[32][135] have thus found that MEL is toxic to ovarian cancer cells and that the toxicity is dose-dependent.

Bee venom-induced apoptosis has been observed both in vitro and in vivo. Liu et al. ^[33][136] observed that BV inhibits the proliferation of melanoma cancer cells both in vitro and in vivo. The apoptosis observed in those cells was regarded as one of the possible mechanisms of action by which BV inhibits proliferation and induces differentiation of those same cells in vitro. Apoptosis was also observed in lung cancer cells by inhibition of cyclooxygenase 2 (COX-2) ^[34][137] and in osteosarcoma cells by increased Fas expression after BV treatment ^[35][138]. Holle et al. ^[31][134] observed that the MEL avidin conjugate has strong cytolytic activity in cells with a high metalloproteinase activity, such as prostate and ovary cancer cells. In contrast, the same activity was much lower in normal cells with limited metalloproteinase activity in vitro. In vivo, a significant reduction in tumor size was observed after treatment with the MEL avidin conjugate compared to untreated tumors. These studies also suggest the possible application of MEL avidin conjugate for therapeutic purposes. Moon et al. ^[30][133] suggested a molecular mechanism by which BV causes apoptosis in leukemia. Apoptosis was induced by reduced regulation of ERK and Akt signaling pathway. Furthermore, apoptosis induced by BV was associated with the downregulation of Bcl-2, caspase-3 activation, and cleavage of poly (ADP-ribose) polymerase (PARP). Moreover, induction of apoptosis was accompanied by a reduced regulation of inhibitory apoptosis protein (IAP proteins). BV also activated p38, MAPK, and JNK and decreased regulation of ERK and Akt ^[5][12].

These results indicate that the induction of apoptosis might have a role in the anticancer activity of BV and MEL, although the mechanisms behind this induction have still not been fully elucidated. Moreover, the apoptosis induction in cancer cells is also shown throughout gene therapy with MEL ^[36][139]. As the possibility of using the peptides from BV in anticancer therapy has been attracting increasing attention in recent years, Hu et al. ^[37][140] also found that these peptides could successfully kill liver cancer cells both in vitro and in vivo. A major mechanism of cancer growth inhibition by these peptides is again cell death induced by apoptosis. Oršolić et al. ^[38][141] have found that intravenous application of BV significantly reduces the number of lung metastases in mice. However, subcutaneous BV intake failed to show such a good effect on metastases, indicating route dependence as well as the proximity effects of BV when used for anticancer purposes.

Previous studies indicated that BV and MEL can induce strong toxic effects in various cancer cells such as lung, liver, kidney, breast, prostate, bladder, and leukemic cells, with a less pronounced effect in normal cells ^[5][6][7][12,15,16]. The proposed mechanisms of action are mainly related to the activation of PLA₂, caspase, and matrix metalloproteinases that destroy cancer cells ^[30][31][133,134]. Conjugation of MEL with hormone receptors and MEL gene therapy could be useful in the future treatment of breast and prostate cancer ^{[36][39][40][41]}[139,142,143,144]. Accordingly, MEL as an amphipathic protein may have a desirable role in therapeutic purposes. MEL is particularly active against cultured cells that express high levels of the ras oncogene ^[22][23][125,126]. Additionally, MEL enhances PLA₂ activity in the ras oncogene-transformed cells, which results in their selective destruction, suggesting that such hyperactivation of PLA₂ by MEL could be one of the major pathways of MEL’s cytotoxic activity against cancer cells ^[5][12].

In the past few decades, numerous studies showed quite potent anticancer effects of BV and MEL towards various cancer cells such as hepatocellular cells, prostate cells, lung cells, bladder cells, ovarian cells, mammary cells, and melanocyte cells, as well as in leukemia through different mechanisms of action ^{[5][7][9][42]}[12,16,21,145].

The numerous cellular effects of BV and MEL summarized above need explanation on a molecular level, and the main issue here has to do with the trigger of the apoptotic cascade. Apoptosis could be either a consequence of the plasmatic membrane fenestration or the result of the direct interaction of BV components with pro-apoptotic and anti-apoptotic factors. Interaction of BV peptides and enzymes with the plasma membrane is a crucial step in the whole process.

Application of biophysical methods showed that MEL brought a small decrease in local membrane fluidity in homogeneous lipid membranes, as the lipids appear to be more closely packed in the proximity of the MEL pore. On the contrary, in heterogeneous lipid membranes in cells, the local order of lipids is diminished by the peptide ^[43][146]. The selective affinity of MEL to cancer cells is determined mostly by acidic phosphatidylserine exposure to the outer layer of the cell membrane in cancer cells ^[44][147]. The binding of MEL to the membranes causes the formation of non-bilayer lipid phases in the membranes ^[45][148]. According to data from computer modeling, after penetration, the lipid bilayer MEL can adopt either a transmembrane or a U-shaped conformation. Several peptides of different conformations aggregate to form a pore. In the pores, peptides are organized in a manner such that polar residues face inward and hydrophobic residues face outward, which stabilizes the pores and forms water channels ^[46][149]. Depending on the local concentration of MEL, it can induce toroidal pores owing to the collective insertion of multiple MEL peptides from the N-termini. The pore formation is initiated by a local increase in membrane curvature in the vicinity of the peptide aggregate. Pore formation can be also achieved by a detergent-like mechanism when lipids are extracted or bursting, causing rapid formation of a large pore in a strongly curved membrane ^[47][150]. Membrane cholesterol impedes pore formation by MEL ^[48][151]. Membrane deformities induced by MEL enhance the activity of PLA₂, and the synergistic action of the two BV components enhances the lytic effect of the venom ^[49][152].

Besides membrane lipids, MEL can directly interact with plasma membrane proteins, Na/K ATPase, for example. Binding causes inhibition of the enzyme ^[50][153]. MEL stimulates TRPM2 Ca²⁺ channels in glioblastoma cells, decreasing their resistance to chemotherapy ^[51][154]. BV and MEL suppress the activation of EGFR and HER2 in triple-negative and HER2-enriched breast cancer cells by interfering with the phosphorylation of these receptors in the plasma membrane ^[52][155]. Reports about the suppression of the Wnt/β-catenin pathway by MEL suggest the destruction of the Wnt receptors by the peptide ^[53][156].

What happens after the formation of the pore? Sure, it enables an influx of free radicals which can damage the cell; however, it seems that MEL action is better targeted. It was shown that MEL directly affected the mitochondrial membrane of the human lung adenocarcinoma cells A549. MEL caused changes in mitochondrial membrane potential, triggered mitochondrial ROS burst, and activated the mitochondria-related apoptosis pathway Bax/Bcl-2 ^[54][157]. Interaction with the mitochondrial membrane is localized to the cardiolipin-rich sites, where non-bilayer structures are formed ^[55][158]. Indeed, this effect is already sufficient to induce the terminal stage of apoptosis—leakage of cytochrome C, formation of apoptosomes, activation of executioner caspases, and fragmentation of chromatin.

MEL can also interact with proteins involved in different regulation pathways. MEL and calmodulin complexes can even be crystallized ^[56][159] and used as a model system of protein–protein complexes. Multiple binding modes exist. Whereas the helical structure of MEL remains, the swapping of its salt bridges and partial unfolding of its C-terminal segment can occur. Different sets of residues can anchor at the hydrophobic pockets of calmodulin, which are considered the main recognition sites ^[57][160]. A block of calmodulin can cause disruptions of the PI3K/Akt and other pathways caused by BV; numerous data have been summarized in recent exhaustive reports ^[42][58][22,145].

Being a positively charged peptide, MEL can directly interact with DNA and RNA ^[59][60][161,162]. Data about these interactions are few and might indicate direct damage of DNA by MEL or interference in the transcription mechanism. Treatment with BV triggers the intensive accumulation of the γ-H2AX histone, a marker of the DNA double-strand breaks, in cancer cell nuclei, but the effect is not observed in normal fibroblasts ^[61][163]. MEL binds centrin, an enzyme involved in nucleotide-excision repair. Binding is stabilized by the hydrophobic triads—leucine–leucine–tryptophan ^[59][161]. BV causes changes in the mitochondrial genome by modification of the methylation pattern and mitochondrial DNA copy number ^[62][164].