Peruvoside is a new Src inhibitor that suppresses NSCLC cell growth and motility by downregulating multiple Src-EGFR-related pathways

The tyrosine kinase Src plays an essential role in the progression of many cancers and is involved in several signalling pathways regulated by EGFR. To improve the ecacy of lung cancer treatments, this study aimed to identify novel compounds that can disrupt the Src-EGFR interaction to inhibit lung cancer progression and are less dependent on the EGFR mutation status than other compounds. We used Src pY419 ELISA as the drug-screening platform to screen a compound library of more than 400 plant active ingredients and identied peruvoside as a candidate Src-EGFR crosstalk inhibitor. Human Non-small cell lung cancer cell lines (A549, PC9, PC9/gef, H3255 and H1975) with different EGFR statuses were used to perform cell cytotoxicity and proliferation assays after peruvoside or dasatinib treatment. Src and Src-related protein expression was evaluated by western blotting in peruvoside-treated A549, H3255 and H1975 cells. The effects of peruvoside on cancer cell function were assessed in A549 cells. The synergistic effects of getinib and peruvoside were assessed by CI-isobologram analysis in getinib-resistant cell lines. The ecacy of peruvoside in vivo was determined using nude mice subjected to compound or vehicle treatments.

Introduction Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-related mortality worldwide.
The ve-year survival rate of lung cancer patients after diagnosis has plateaued at only 12%-15% [1].
Mutations in oncogenic driver genes are usually the cause of dysfunction of normal cells. Most of these mutations occur in kinases related to signal transduction, such as HER2, KRAS, AKT1, MEK, and EGFR, causing them to be constitutively activated and overexpressed, thereby inducing abnormal cancer cell growth and metastasis, which leads to poor prognoses and poor treatment responses [2,3]. Therefore, targeting these molecules to inhibit tumour growth is essential to improve the prognosis of NSCLC patients and increase the treatment e ciency for this disease [4].
In recent years, targeted therapies have been developed to treat NSCLC patients with certain driver mutations. EGFR tyrosine kinase inhibitors (TKIs), such as ge tinib and erlotinib, function by competing for the ATP-binding site and have been used in clinical treatment [5]. The sensitivity of these drugs is related to the position of the mutation on the tyrosine kinase domain of EGFR. For example, the lack of exon 19 and L858R mutation in exon 21 can increase sensitivity to the drug [6]. Conversely, the EGFR T790M mutation can lead to TKI resistance and is positively associated with lung cancer recurrence [7]. Drug resistance is a crucial issue in cancer treatment. Therefore, the development of new therapeutic strategies or targeted drugs, including the second-generation TKI afatinib [8] and third-generation TKIs osimertinib [9] and olmutinib [6], has become the main objective of current research focusing on cancer treatment. Nevertheless, approximately 10% of NSCLC patients show primary TKI resistance, and the underlying mechanism remains unclear.
The expression of the proto-oncogene Src is related to tumour development and a poor prognosis because Src regulates signalling pathways that affect its downstream protein expression [10]. Src is a member of Src family kinases (SFKs), which are non-receptor kinases. Under normal conditions, approximately 90%-95% of Src is phosphorylated at the Tyr530 position, which indicates a closed and inactive structure. In its activated form, Src is dephosphorylated at Tyr530 and auto-phosphorylated at Tyr416 in its kinase domain [11]. Mutations in EGFR and EGFR family-related proteins enhance Src expression and activate Src downstream signals through the ERK, Akt and STAT pathways [12]. Src and EGFR show synergistic effects through mutual phosphorylation and activation [13], and their coexpression induces cell transformation [14].
Because of the crosstalk between Src and EGFR, inhibiting Src may improve NSCLC treatment [15].
Several known Src inhibitors, including dasatinib (BMS-354825), saracatinib (AZD0530), and ponatinib (AP24534), have been developed as therapeutic agents, and their effectiveness against solid tumours has been evaluated in clinical trials [16]. Src inhibitors induce apoptosis in various NSCLC cells and inhibit cell survival and oncogenic malignant transformation regulated by EGFR [13]. Thus, inhibition of the Src pathway alone can induce cell apoptosis, and combinations with ge tinib can further enhance the effects of Src inhibitors on EGFR and HER2.
To improve the e cacy of lung cancer treatments, we aimed to identify compounds that disrupt the Src-EGFR interaction and are less dependent on the EGFR mutation status than other compounds. In this study, using Src pY419 enzyme-linked immunosorbent assay (ELISA) as the drug-screening platform to screen a compound library of more than 400 plant active ingredients, we identi ed peruvoside (PubChem CID: 12314120) as a candidate Src-EGFR crosstalk inhibitor. We also investigated the functional mechanism underlying the ability of peruvoside to suppress lung cancer cell progression using in vitro and in vivo approaches. Our ndings may promote the development of new anticancer drugs and therapeutic strategies useful to treat lung cancer in the future.

Cell culture
The human bronchial epithelial cell line BEAS2B (ATCC CRL-9609) and human lung adenocarcinoma cell lines A549 (ATCC CCL-185) and H1975 (ATCC CRL-5908) were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). The human lung adenocarcinoma cell lines PC9, PC9/gef and H3255 were a kind gift from Dr. Chih-Hsin Yang (National Taiwan University Hospital, Taiwan). The cell lines were grown in RPMI (Gibco, Breda, The Netherlands) supplemented with 10% foetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin at 37 °C in a humidi ed atmosphere of 5% CO 2 .

Drug treatment and herbal compound library
The herbal compound library, representing a collection of 415 pure products and their derivatives, was purchased from Sigma-Aldrich (St. Louis, MO) and ChromaDex (Irvine, CA) and contained a range of alkaloids, diterpenes, pentacyclic triterpenes, sterols, and many other diverse representatives. Peruvoside was purchased from Sigma-Aldrich, and a stock solution of peruvoside was prepared in dimethyl sulfoxide (DMSO) and stored at -20 °C. The compound was diluted in fresh medium before each experiment, and the nal DMSO concentration was lower than 0.1%. To determine whether peruvoside promotes protein degradation through the ubiquitination pathway, A549 cells were treated with 10 µM MG132, a proteasome inhibitor (Sigma-Aldrich) for 2 h and then with peruvoside for an additional 24 h.
The cell lysates were extracted and then subjected to western blot analysis of the speci c proteins.

Enzyme-linked immunosorbent assay (ELISA)
To accelerate the screening of the compound library, ELISA (Human Phospho-Src (Y419) DuoSet IC ELISA; R&D Systems, Minneapolis, MN, USA) was performed in the early stage. The details of the procedures, including plate preparation and signal detection, are described in the manufacturer's instructions. Brie y, A549 cells were seeded at 1.5 × 10 5 cells per well and then were treated with various compounds for 24 h. After extraction and quanti cation, the cell lysates or standards were added to 96well microplates coated with the diluted capture antibody for 2 h. Next, the detection antibody and streptavidin-HRP (1:2000) were consecutively added to each well. After incubating the samples with the substrate solution and stop solution, the absorbance was measured at 450 nm (570 nm as the reference) using a multilabel plate reader (Vector3; Perkin-Elmer, USA). The absorbance at 570 nm was subtracted from the absorbance at 450 nm to calculate the amount of Src phosphorylation.

Western blot analysis
Western blotting was used to examine the protein activity and expression levels of Src and related proteins after peruvoside treatment. The detailed procedures were described previously [17]. Monoclonal mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:5000; Upstate Biotechnology, Lake Placid, NY, USA) was used as a loading control. All the experiments were performed at least three times in duplicate.

Cell viability and proliferation assay
The PrestoBlue (Invitrogen) cell viability reagent was used to evaluate the cytotoxic and proliferative effects of peruvoside used for various durations as described previously [17]. Brie y, the tested cell lines were seeded in 96-well plates at a density of 5 × 10 3 cells/well and incubated for 24 h. Next, the cells were treated with different concentrations of peruvoside for 24, 48, 72 and 96 h. Subsequently, PrestoBlue solution was added to the culture medium in the wells. After a further 1.5 h of incubation, the colour intensity was measured at 570 nm (600 nm as the reference) using a multi-label plate reader (Vector3; Perkin-Elmer, USA).

Colony formation assay
For the anchorage-dependent growth assay, 500 cells were seeded in six-well plates and treated with peruvoside. After one week, the cells were washed with PBS and xed with 4% paraformaldehyde. The xed cells were stained with 0.05% crystal violet. For the anchorage-independent growth assay, CL1-5 cells were seeded at 1 × 10 3 cells per well in soft agar. After solidi cation, the cells were treated with different concentrations of peruvoside. The cells were incubated for 2 weeks and then stained with 0.5 mg/ml of p-iodonitrotetrazolium violet. Colonies with a diameter greater than 1 mm were counted under an inverted microscope. The assay was performed as described previously [18].

Cell migration and invasion assays
The motility of cancer cells was measured using a Transwell device, according to our previous study [19].
Brie y, Transwell membranes (8-µm pore size, 6.5-mm diameter; Corning CoStar Corporation) coated with or without Matrigel (2.5 mg/ml; BD Biosciences Discovery Labware) were used for the invasion and migration assays, respectively. Medium with 10% FBS was added to the lower wells of the chambers, and the upper wells were lled with serum-free medium containing 1 × 10 4 or 2 × 10 4 A549 cells per well. The medium contained different concentrations of peruvoside (10 and 50 nM), and 0 µM represented 0.1% DMSO as the solvent control. After 12 (migration) or 24 h (invasion) of incubation, the cells on the upper wells were removed, and the cells that migrated onto the lower surface of the membranes were xed with methanol and stained with 20% Giemsa solution (Sigma-Aldrich). The cells were counted under a light microscope. The experiments were performed in triplicate.

In vivo animal studies
The mouse experiments were approved by the Institutional Animal Care and Use Committee of the National Chung Hsing University, and tumorigenesis experiments were performed in the mice according to previously described protocols [17]. Six-week-old nude mice (obtained from the National Laboratory Animal Center, Taipei, Taiwan) were housed at six mice per cage. Brie y, 1 × 10 6 A549 cells in 100 µl of PBS were injected subcutaneously into the nude mice. When the tumour size reached 50-100 mm 3 , the mice were randomly grouped into peruvoside-treated or untreated groups. Peruvoside was suspended in DMSO and dosed i.p. once daily at 0.1 mg/kg for 4 weeks of treatment. After intraperitoneal injection, the mice were measured every 7 days for tumour appearance. After 4 weeks, the mice were killed, and their tumour sizes were analysed. The tumour volumes were estimated from the calliper-measured lengths (a) and widths (b) using the formula V = 0.4 × ab 2 [20].

Real-time reverse transcription-PCR (RT-PCR) and immunohistochemical staining
The expression levels of Src and related genes were detected by real-time RT-PCR using an ABI prism 7300 sequence detection system (Applied Biosystems, Foster, CA). TATA-box-binding protein (TBP) was used as the internal control (GenBank X54993). The detailed procedures and calculations have been described previously [18]. Immunohistochemical staining was used to investigate the phosphorylation level of Src in tumour tissue from nude mice. Brie y, the para n-embedded tumour tissue sections were reacted with the rabbit anti-human Src pY418 polyclonal primary antibody (Invitrogen), and then the DAKO EnVision System, containing the horseradish peroxidase-conjugated anti-rabbit secondary antibody and enzyme substrate 3,3'-diaminobenzidine, was used to detect Src activity.

Drug synergy analysis
To elucidate the synergistic effects of peruvoside and ge tinib on ge tinib-resistant lung cancer cells in vitro, A549, PC9/gef and H1975 cells were subjected to combination treatment and then cell viability assays. The above data were further analysed using CalcuSyn software (Biosoft, Cambridge, UK) and the combination index (CI)-isobologram equation, as described previously [21]. CI<1, CI=1 and CI>1 represent the synergistic, additive and antagonistic effects of the indicated compounds, respectively.

Statistical analysis
All the in vitro experiments were performed at least in triplicate, and the results are presented as the mean ± standard deviation where appropriate. Statistical analyses were performed using two-tailed Student's t test, and signi cant differences were de ned as P values of less than 0.05.

Results
Drug bank screening via the Src pY419 ELISA platform ELISA was performed to determine the inhibitory effect of the 415 pure compounds in the library on Src activity. The screening criteria and procedures of the drug bank are illustrated in Additional le 1: Fig. S1.
After two runs of ELISA screening, only 20 compounds showed a signi cant inhibitory effect compared with the control (Additional le 1: Fig. S2). Among these compounds, ve had more than 50% inhibitory activity on Src phosphorylation and were identi ed as candidates. To focus our research, peruvoside (P7897), a cardiac glycoside with inotropic and chronotropic effects, was selected for further in vitro and in vivo studies.
Effect of peruvoside treatment on cell viability and Srcrelated protein phosphorylation Lung cancer A549 cells were treated with various concentrations of peruvoside and then were subjected to the cell cytotoxic assay at 24, 48, 72, and 96 h. The results are presented as the percentage relative to the DMSO-treated control. The corresponding IC 50 values of each time point are shown in Table S1. The cytotoxicity results at 24 h indicated that cell viability was less in uenced by peruvoside at 10 nM, a dose that is approximately equivalent to IC 20 . Therefore, to determine the effects on cellular functions in subsequent experiments, we used concentrations no higher than 50 nM (IC 50 ), which are more appropriate concentrations for the following investigations (Fig. 1a).
To determine the inhibitory effects of peruvoside on Src, EGFR and STAT3 phosphorylation, western blotting was performed. Peruvoside inhibited the activation and expression of Src, EGFR and STAT3 in A549 cells at 24 h in a dose-and time-dependent manner (Fig. 1b). Similar results were obtained in two other lung cancer cell lines, H3255 (Fig. 1c, left panel) and H1975 (Fig. 1c, right panel).

Inhibition of cell viability and proliferation ability in EGFR mutant lung cancer cell lines by peruvoside
To identify the cytotoxic effect of peruvoside on EGFR mutant cell lines, PC9 and PC9/gef cells with exon 19 deletion, H3255 cells harbouring the L858R mutation and H1975 cells harbouring the L858R and T790M mutations were used. The viabilities of both TKI-sensitive or TKI-resistant cell lines were signi cantly inhibited by 50 nM peruvoside at all tested time points (Fig. 2a-d). Furthermore, the immortalised BEAS-2B cell line was used to evaluate the cytotoxicity of peruvoside in relatively normal cells. Peruvoside showed lower cytotoxicity in normal cells than in cancer cell lines (Additional le 1: Fig.   S3). Additionally, the results from the cytotoxicity assays were analysed using CalcuSyn software (Biosoft, Cambridge, UK) to determine the IC 50 Table S1).
Dasatinib is a well-known Src inhibitor whose effective dose is usually 100 nM. The same concentration of peruvoside and dasatinib was used to treat the lung cancer cell lines with different EGFR mutation statuses and to further compare the inhibitory effect on proliferation ability. Both peruvoside and dasatinib suppressed A549, PC9, PC9/gef, H3255 and H1975 cell proliferation and peruvoside; in particular, had a marked inhibitory effect. Therefore, whether the cell line was EGFR wild-type or mutant, peruvoside effectively inhibited cell proliferation-even better than dasatinib (Fig. 3a-e).
Anticancer effect of peruvoside on cancer cell functions The colony formation, migration and invasion assays were performed to investigate the anticancer effects of peruvoside. We demonstrated that peruvoside inhibited the anchorage-dependent growth of A549 cells (Fig. 4a) and anchorage-independent growth of CL1-5 cells (Fig. 4b) in a concentrationdependent manner. Notably, peruvoside at a concentration as low as 5 nM, inhibited colony formation. To investigate the effects of peruvoside on cell motility, A549 cells were pre-treated with peruvoside for 24 h and then were subjected to Transwell migration and invasion assays for 12 h and 24 h, respectively.
Peruvoside signi cantly inhibited the cell migratory and invasive abilities, even at the low concentration of 10 nM (Fig. 4c and d).

Suppression of in vivo tumour growth by peruvoside
To further examine the effect of peruvoside in vivo, A549 cells were injected subcutaneously into nude mice. The mice were then treated with or without peruvoside (i.p., 0.1 mg/kg/day) and were observed every 7 days for tumour growth. Twenty-eight days after treatment, the peruvoside-treated group had an average tumour size of 129 mm 3 (95% CI = 86-178 mm 3 ; P = 0.0063), which was signi cantly reduced compared with the average tumour size of 348 mm 3 (95% CI = 195-501 mm 3 ) in the control group (Fig. 5a). After 4 weeks, the mice were killed, and their tumour sizes were analysed. The tumour weights were also signi cantly decreased from 0.55 g to 0.26 g (P = 0.0126; Fig. 5b). To determine the change in Src activity in tumours, the tissues were sectioned and immunostained with human anti-phospho-Src antibody. The immunohistochemistry data indicated that peruvoside signi cantly decreased Src Y418 phosphorylation in tumour tissues compared with control tissues (Fig. 5c). Additionally, the lung adenocarcinoma cell lines A549, PC9/gef, and H1975 were treated with different combinations of peruvoside and ge tinib for 72 h, and then the combined effects of the two compounds on the cells were  Tables S2-S4). Speci cally, the combination of 0.005, 0.075, or 0.01 µM peruvoside and low-dose ge tinib (0.01 or 0.05 µM) had synergistic effects on A549 cells (Fig. 5d, upper  panels). Furthermore, the combination of 0.025 or 0.05 µM peruvoside and ge tinib increased the sensitivity of the PC9/gef and H1975 cell lines to ge tinib, even when peruvoside was administered at concentrations as low as 0.01 µM (Fig. 5d, middle and lower panels). In summary, 0.025 µM and 0.05 µM peruvoside could render A549, PC9/gef, and H1975 ge tinib-resistant lung cancer cells sensitive to lower ge tinib concentrations.

Effect of peruvoside on Src downstream pathways
To determine the mechanisms by which peruvoside inhibits cancer cell functions, western blotting of previously reported Src-related signalling pathways was performed. These proteins included PI3K and AKT for the survival pathway; MEK and ERK for the proliferation pathway; and FAK, paxillin, p130 CAS and JNK for the migration and invasion pathways [22]. A549 cells were treated with the designated concentrations of peruvoside for 24 h, and peruvoside inhibited PI3K, AKT, FAK, SAPK/JNK, Paxillin and p130 CAS phosphorylation in these cells. However, the phosphorylation of MEK and ERK was not reduced by peruvoside treatment (Fig. 6a). In addition to using the EGFR wild-type A549 cell line, we also used the EGFR mutant H3255 and H1975 cell lines to evaluate whether peruvoside can effectively inhibit the expression of these proteins in NSCLC cell lines with TKI-sensitive or TKI-resistant EGFR mutations. These two cell lines were treated with sublethal doses of peruvoside (50 nM and 100 nM) for 24 h and then subjected to western blotting. In the H3255 cell line, the proteins and pathways affected by peruvoside were similar to those in the A549 cell line (Fig. 6b), except for p-MEK, p-ERK and Paxillin. Furthermore, in the H1975 cell line, peruvoside suppressed the phosphorylation of PI3K, AKT, MEK, ERK FAK, SAPK/JNK, Paxillin and p130 CAS (Fig. 6c). Overall, the results in the three cell lines also showed that the expression of Src and some Src-related proteins, such as EGFR, STAT3 and FAK, was downregulated by peruvoside ( Fig. 1 and Fig. 6).

Reduced mRNA expression of Src and related pathways by peruvoside
The expression levels of Src and some of its related proteins were decreased compared with those in the control ( Fig. 1 and Fig. 6). The reduced protein levels could be explained by transcription and protein stability alterations. Therefore, real-time PCR was performed to investigate the effect of peruvoside on the transcriptional regulation of the genes tested in this study. The mRNA expression levels of EGFR, Src, STAT3 and FAK were signi cantly downregulated by peruvoside, even when the drug was administered at a relatively low concentration (25 nM) (Fig. 7a). However, to explore whether the ubiquitin-proteasome system plays a role in peruvoside-induced protein reduction, the proteasome inhibitor MG132 was used in cell culture. Peruvoside decreased the expression of the tested proteins as described previously in this study, but co-treatment with MG132 could not restore their expression compared with that in the vehicle control (Fig. 7b). Therefore, we speculate that the protein reduction caused by peruvoside might occur via the regulation of RNA levels. However, further investigations are needed to clarify the detailed regulatory mechanisms.

Discussion
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 speci c 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 signi cantly inhibits NSCLC cell functions in vitro and tumorigenesis in vivo. Moreover, we determined that peruvoside has a synergistic effect when used in combination with ge tinib.
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][28][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 rst 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 pro le, 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 e cacy 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 ge tinib, 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-TKIresistant cells and signi cantly 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, ge tinib 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-TKIresistant mutations or with wild-type EGFR [41,43]. Our data indicated that peruvoside signi cantly sensitised ge tinib-resistant lung adenocarcinoma cells (A549, PC9/gef, and H1975) to ge tinib treatment in vitro, suggesting that this compound may reduce the ge tinib dose, enhance ge tinib e cacy, and decrease targeted therapy costs and patient loads. These ndings 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 identi ed 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 signi cantly 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 udarabine (premature transcription chain terminator) [48].
In summary, our ndings 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 e cacious 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 con rmed in clinical trials for its effectiveness in advanced gastrointestinal stromal tumour patients [51]. Moreover, the safety and e cacy 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 veri ed in a randomised phase II trial.

Conclusions
We found that peruvoside may have multi-target inhibitory effects on NSCLC and may have a synergistic effect when combined with ge tinib. These results indicate that peruvoside has potential as a treatment method for cancer. Therefore, whether it is used alone or in combination with other drugs, peruvoside may be the basis for future therapies and should be evaluated in future drug development studies.    Table S1. Each experiment was performed independently and in triplicate. *P<0.05 compared with the vehicle control.  Suppressive effects of peruvoside on anchorage-dependent/independent cell growth and cell motility. The colony formation assay was used to determine the inhibitory effect of peruvoside on clonogenicity.   were measured by immunoblot analysis using the corresponding antibodies. GAPDH was used as an internal control. Ctrl: 0.1% DMSO. Each experiment was performed independently and in triplicate.