Synergistic Anti-Cancer Activity of Melittin and Erlotinib in Non-Small Cell Lung Cancer

Abstract

Lung cancer remains a leading cause of cancer-related mortality worldwide. Despite advancements in current therapies, the development of drug resistance and the need for improved treatment outcomes necessitate the exploration of novel therapeutic approaches. This study aimed to investigate the synergistic anti-cancer effects of Melittin, a bee venom peptide, in combination with Erlotinib, an EGFR inhibitor, in non-small cell lung cancer (NSCLC). The study evaluated the combined effects of Melittin and Erlotinib on A549 NSCLC cells. Cell viability, proliferation, migration, and apoptosis were assessed using standard in vitro assays. Mechanistic studies investigated the impact of the combination treatment on key signaling pathways, including those involving JAK2 and JAK3. Molecular docking simulations were performed to predict the binding interactions between Melittin and these kinases. The combination of Melittin and Erlotinib significantly inhibited A549 cell proliferation and migration, with a marked reduction in cell viability and enhanced apoptosis compared to either agent alone. Mechanistically, Melittin demonstrated interactions with JAK2 and JAK3, key proteins involved in apoptotic signaling. Molecular docking simulations further supported these findings, predicting strong binding affinities between Melittin and both kinases. These findings demonstrate a synergistic anti-cancer effect of Melittin and Erlotinib in A549 NSCLC cells. The observed interactions with JAK2 and JAK3 suggest a potential mechanism for Melittin's activity. These results highlight the potential of Melittin as a promising adjuvant to Erlotinib for the treatment of NSCLC.

Keywords: Erlotinib; JAK2/JAK3 signaling; Melittin; lung cancer; synergistic anti-cancer effects.

Figure 1

Effect of Melittin and Erlotinib on the growth of A549 lung cancer cells and Beas-2B lung epithelial cell line. (A) Cell viability was determined by SRB assay. (B,C) Effect of Melittin and Erlotinib on the cell viability in A549 cells and Beas-2B. (D) Phase contrast images of treated A549 cells at 20× magnification. Data were expressed as the mean ± S.D. of three experiments. * p < 0.05 and ** p < 0.01 indicate statistically significant differences from control group.

Figure 2

Effect of Melittin and Erlotinib on the cell migration (wound healing activity) of A549 lung cancer cells. After 50% cell confluent in a 12-well plate, a scratch was made by using sterile tips in the middle of the well and treating the plate with Melittin 50 µM and Erlotinib 6 µM concentrations, respectively (A) Effect of Melittin and Erlotinib on the cell migration and wound healing activity for A549 cells and phase contrast images of treated cells at 20× magnification. (B) Cell migration was recorded in fold changes. Data were expressed as the mean ± S.D. of three experiments.

Figure 3

Effect of Melittin and synergistic effect on the expression of apoptosis regulatory markers. (A). Apoptotic activity of Melittin and Erlotinib on downstream apoptotic markers Caspase-3 and caspase-9 using the Elabscience colorimetric kit assay. (B). Expression of apoptosis regulatory proteins related intrinsic pathway was determined by quantitative PCR of JAK-2, Jak-3 and Stat-3 with GAPDH internal control. (C). Melting curve of qPCR expression values under expression fold indicate the cT values. Blue curve indicate Jak-2, Yellow curve indicate Jak-3, Red curve indicate State-3 and Violet curve indicate GAPDH. Data were expressed as the mean ± S.D. of three experiments. * p < 0.05 indicates statistically significant differences between groups and ** p < 0.01 represents significance between the groups.

Figure 4

Melittin interaction and virtual eray structure analysis. (A) Protein network analysis and its interaction with other protein was revealed using protein-protein interaction tool. (B) The 3D fold of mature Melittin protein. Illustration of the protein models folded using AlphaFold v2, including the monomeric conformation of Melittin. Additionally, a homo-tetrameric structure of Melittin is revealed through X-ray analysis.

Figure 5

Domain architecture and structural folding of Melittin, JAK2, JAK3, and STAT3. (A). Melittin: The full protein sequence 70 aa; the mature 26–amino acid peptide adopting a helix–hinge–helix motif; N–terminal region involves a helix–hinge–helix motif, where residues 1–11 form an α–helix, followed by a hinge region (residues 12–14), and another α–helix spanning residues 15–26, C–terminal region. (B). JAK2: Domain structure featuring B41 domain, SH2 (Src Homology 2) domain, pseudo–kinase domain (STyKc), and tyrosine kinase domain (TyrKc). (C). JAK3: Similar domain organization to JAK2, with approximately 50% sequence identity. Domains include FERM, SH2, STyKc, and TyrKc. (D) STAT3 exhibits the N–terminal domain (STAT_int), the coiled–coil domain (STAT_alpha), the DNA–binding domain (STAT_bind), and the Src Homology 2 (SH2) domain. Protein structures were predicted using SWISS–MODEL and AlphaFold. Domain boundaries are indicated by different colors.

Figure 6

Molecular docking of Melittin–human protein interactions. Representation of the 2D interaction graph depicting (A). Mel–JAK2 complex, (B). Mel–JAK3 complex; (C). Mel–STAT3 complex. In the graph, red color denotes human proteins, while blue represents Melittin. In the graph, red colour denotes human programmed cell death proteins, while blue represents Melittin in a monomer stat.