The proto-oncogene c-Src is a non-receptor tyrosine kinase that plays a key role in multiple signalling pathways to regulate cell growth and metastasis in several cancer types [23]. The deregulation of its activity or expression is also related to drug resistance in cancer patients and is associated with poor prognoses [24]. ELISA is widely used as a diagnostic and analytical tool in clinical practice and basic research to detect and quantify specific antigens or antibodies in a given sample [25]. Moreover, ELISA has several advantages over other screening techniques because of its simplicity, selectivity, and sensitivity. In this study, using Src pY419 ELISA as the drug-screening platform to screen a compound library with more than 400 plant active ingredients, we found that peruvoside is a potential Src inhibitor that significantly inhibits NSCLC cell functions in vitro and tumorigenesis in vivo. Moreover, we determined that peruvoside has a synergistic effect when used in combination with gefitinib.
Peruvoside, a cardiac glycoside (CG), is a natural ingredient extracted from oleander seeds. CGs are a class of organic compounds comprising a sugar (glycoside) and an aglycone (steroid) moiety. They are used to treat heart ailments, such as congestive heart failure, ischaemia and cardiac arrhythmia. Interestingly, in recent years, several studies have revealed that some CGs possess potent anticancer effects in various cancers [26]. Peruvoside has also been shown to have anti-proliferative and anticancer effects by regulating the expression of various key proteins involved in cell cycle arrest, caspase activation and autophagic cell death in several cancers, including myeloid leukaemia, breast cancer and lung cancer cells [27–29]. Furthermore, previous studies have indicated that peruvoside inhibits AKT phosphorylation and β-catenin expression in H460 lung cancer cells (EGFR wild-type) [29], as well as induces autophagy and apoptosis through MAPK, Wnt/β-catenin and mTOR signalling in A549 lung cancer cells (EGFR wild-type) [30]. However, these studies did not investigate the effect of peruvoside on NSCLC with activating and acquired resistance EGFR mutations, the Src-EGFR-related signalling pathways involved, or its effect on tumorigenesis in vivo. Moreover, the functional role of peruvoside in cancer and mechanism underlying its antitumour activity are still largely unknown. Here, we elucidated the multi-faceted role of peruvoside in NSCLC and signalling pathways in which it may be involved. To our knowledge, this is the first study to indicate that peruvoside can inhibit NSCLC progression by regulating multiple Src-related signalling pathways.
EGFR is overexpressed in approximately 40%-80% of NSCLC tumours; therefore, EGFR activity and mutations that can trigger downstream signalling pathways are important factors in lung cancer treatment that must be considered by clinicians attempting to manage this disease [31]. Similar to EGFR, c-Src is also overexpressed in many types of cancer and is co-overexpressed with EGFR in several types of tumours, including carcinomas of the colon, breast, and lung [14, 32]. A previous study showed that Src inhibitors not only suppress Src activity but also inhibit EGFR tyrosine kinase activation and downstream signalling pathways. Moreover, depending on the EGFR/Ras mutational profile, different Src inhibitors may exhibit divergent anticancer effects in NSCLCs [33]. Therefore, Src can serve as a therapeutic target to improve NSCLC treatment [13]. The Src inhibitor dasatinib has been approved for clinical use in patients with chronic myeloid leukaemia (CML) [34] and can improve the efficacy of cetuximab and cisplatin in tumour-negative breast cancer (TNBC) when used in combination [35]. Moreover, dasatinib was recently shown to be a multi-kinase inhibitor that affects the STAT5, c-kit, and PDGFR pathways [36]. However, similar to gefitinib, dasatinib cannot inhibit the growth of NSCLC cells with wild-type EGFR (A549) or a T790M mutation (H1975) [37]. Compared to dasatinib, peruvoside had a cytotoxic effect on all NSCLC cell lines tested, namely A549, PC9, PC9/gef, H3255 and H1975 cells, regardless of their EGFR mutation status. Furthermore, peruvoside was relatively less toxic to BEAS-2B cells than to cancer cells in this study.
c-Src has been reported to bind to EGFR and phosphorylate tyrosine residues on Y845, resulting in the activation of various downstream pathways [38]. Therefore, c-Src and activated EGFR cooperate to induce cell transformation, and cancer development is critical for EGFR-mediated oncogenesis [39]. Because of the crosstalk between Src and EGFR, inhibiting the activity of both proteins may facilitate the successful treatment of NSCLC patients without EGFR-activating mutations or with acquired resistance mutations. Previous studies have shown that the inhibition of c-Src kinase activity sensitises EGFR-TKI-resistant cells and significantly decreases AKT activation, cell survival and migration, indicating that Src inhibitors might overcome resistance to EGFR inhibitors in lung cancer cells [40]. Thus, the effects of combination therapy with dasatinib and EGFR TKIs (erlotinib, gefitinib and afatinib) on NSCLC were investigated in several clinical trials in recent years [41, 42]. However, these phase I/II clinical trials did not obtain ideal results because of few or no clinical responses in NSCLC patients with acquired EGFR-TKI-resistant mutations or with wild-type EGFR [41, 43]. Our data indicated that peruvoside significantly sensitised gefitinib-resistant lung adenocarcinoma cells (A549, PC9/gef, and H1975) to gefitinib treatment in vitro, suggesting that this compound may reduce the gefitinib dose, enhance gefitinib efficacy, and decrease targeted therapy costs and patient loads. These findings indicate that peruvoside may be a new candidate compound that can be used instead of dasatinib in combination therapy regimens comprising one of the two kinase inhibitors.
Src has been identified as an important oncogenic driver in many signalling pathways to enhance cancer cell motility, tumorigenesis, angiogenesis, and metastasis [22]. Among these pathways, some pivotal pathways have been demonstrated to modulate cancer progression, including the PI3K/AKT, STAT3, MEK/ERK, JNK, FAK, Paxillin, and p130cas pathways [23]. The PI3K/AKT pathway can be activated by EGFR and Src, leading to aberrant cell survival and cell cycle progression [44]. Our data showed that peruvoside inhibits the activity of Src and EGFR as well as the phosphorylation and expression of PI3K in EGFR mutant (H3255 and H1975) and wild-type (A549) cells. FAK-Src signalling through Paxillin, ERK, and p130cas regulates actin cytoskeletal reorganisation to promote cell migration [45]. Furthermore, JNK is the transcriptional regulator of matrix metalloproteinase (MMP)-2 and MMP-9; thus, JNK activation can lead to proteolysis and increased cell invasion [46]. Our data revealed that peruvoside significantly represses FAK, JNK, Paxillin, and p130cas phosphorylation or protein expression in H3255, H1975 and A549 cells, leading to the inhibition of cancer cell invasion and migration. A previous study showed that MEK and ERK might be involved in the Src-related signalling pathway, resulting in increased cell proliferation [47]. In this study, peruvoside decreased MEK and/or ERK phosphorylation levels in the indicated cell lines but promoted their phosphorylation in A549 cells. Similar results were reported in a previous study [18] and suggested that peruvoside may also affect other signalling pathways, growth factors or protein kinases to inhibit cell growth in a certain cell line. Additionally, we found that the reductions in certain proteins caused by peruvoside may be due to its effect on transcriptional regulation rather than increased ubiquitination, which is one of the effects that is often observed with antitumour drugs such as palbociclib (a CDK inhibitor) and fludarabine (premature transcription chain terminator) [48].
In summary, our findings indicate that peruvoside may directly or indirectly affect the expression of Src and downstream or related proteins, thereby inhibiting cancer progression. However, we cannot rule out the possibility that peruvoside may affect multiple targets. Recently, based on the multiple target effects of drugs inducing different anticancer responses, the concept of polypharmacology has been developed [49]. Multi-target drugs can treat diseases more effectively than single-target drugs, regardless of whether these multi-target drugs are used alone or in combination with other agents, and multi-target agents are expected to provide more efficacious and safer therapeutic solutions that are less prone to drug resistance phenomena [50]. For example, sorafenib, a VEGFR, PDGFR, KIT, FLT3, and RAF inhibitor, was recently confirmed in clinical trials for its effectiveness in advanced gastrointestinal stromal tumour patients [51]. Moreover, the safety and efficacy of anlotinib, a novel multi-target TKI that inhibits VEGFR2/3, FGFR1-4, PDGFR α/β, c-Kit, and Ret [52], in patients with refractory advanced NSCLC have been verified in a randomised phase II trial.