Hypoxia is the imbalance between oxygen consumption and supply[1]. Hypoxic regions within tumor is a common characteristic to a vast variety of solid tumors[2]. Hypoxia-inducible factor 1(HIF-1) is the main regulator of O2 homeostasis in mammalian cells[3]. HIF1 activity is dependent on stabilization of its alpha subunit(HIF-1α) which is under tight control of O2 availability in the cytosol. Even though, HIF-1α gene transcription is mainly regulated in an O2-independent manner by NF-kB and STAT3. NF-kB interacts with HIF-1α promoter at -197/-188 bp through its p50 and p65 subunits[4]. Moreover, it has been shown that HIF-1α expression is regulated by JAK/Stat signaling through a binding motif at -363/-355 bp of the HIF-1 α promoter[5]. Under normoxic conditions, HIF-1α is constantly synthesized and degraded. When oxygenation is sufficient, von Hippel–Lindau (VHL) binds HIF-1α and through interaction with Elongin C, recruits an E3 ubiquitin-protein ligase complex that ubiquitinates HIF-1 α and targets it for degradation by the 26S proteasome. In order to bind HIF-1α, VHL needs to be hydroxylated on proline residue 402 or 564 or both by prolyl hydroxylase PHD2. VHL uses O2 and α-ketoglutarate as substrates[6]. During hypoxia, this degradation mechanism is disturbed, resulting in accumulation of HIF-1α in cytoplasm. Upon stabilization of HIF-1α, it dimerizes with HIF-1β, HIF1 transcription factor forms in the nucleus and triggers the expression of more than 1500 genes[7].
HIF-1 is a major driver of tumor microenvironment(TME) formation. Elevated expression of glycolytic genes[8]–[11], and HIF1 induction of pyruvate dehydrogenase kinase 1(PDK1)[12] and lactate dehydrogenase A(LDHA)[10], drives tumor cell’s metabolism through anaerobic respiration, resulting in declined pH of solid tumor microenvironment[13], [14]. HIF1 upregulates carbonic anhydrase IX (CAIX)[15] which also has a role in decreased TME acidity[16], [17]. On the other hand, HIF1 upregulates proangiogenic genes Ang-1 and − 2, Tie-2 and VEGF[18], [19]. High permeability of newly formed vasculature and lack of lymphatics result in elevated interstitial fluid pressure (IFP) of tumors, another hallmark of TME[20]. This environment favors tumor cells with resistance to current chemotherapy and radiotherapy strategies[21].
Crucial involvement of HIF1 in several steps of carcinogenesis and tumor invasion and metastasis has made it a great target for cancer therapy. Though resistance to current therapeutic reagents has been a sustaining challenge. Melittin(MEL) is a short polypeptide of 26 aminoacids and constitutes about 50% of total dried weight of honey bee venom[22]. Several studies have reported anti-inflammatory, anti-arthritic, and anti-viral effects of MEL in various cell types. It also induces apoptosis, cell cycle arrest and growth inhibition in several cancer cell types[23]. it has been shown that MEL can inhibit angiogenesis through downregulation of VEGF in cervical cancer. MEL specifically suppressed EGF-induced VEGF secretion and new blood vessel formation by inhibiting HIF1 signaling pathway[24].
Nevertheless, there are no previous reports on how MEL can affect HIF-1α in breast cancer. in this study, we have investigated the effect of MEL on breast cancer cell line MDA-MB-231, and its inhibitory effect on HIF-1α mRNA expression and protein level. Also, the expression of genes involved in the formation of TME and downstream to HIF1 signaling has been investigated.