Oleanolic acid (OA) and its derivatives show potent anticancer function. Pancreatic cancer (PC) is the fourth core motive of cancer-related deaths worldwide. Epidermal growth factor receptor (EGFR) has been implicated in PC and has been validated as a therapeutic target. Our study demonstrated that K73-03, an OA derivative, was identified as a potent inhibitor of EGFR by using reverse pharmacophore screening and molecular dynamics simulation assays. Moreover, Western blot analysis showed that K73-03 markedly suppressed the levels of phosphorylated-EGFR (p-EGFR) and phosphorylated-Akt (p-Akt). The inhibitory effect of K73-03 on PC cells was assessed in vitro and in vivo. Mechanistically, K73-03 effectively inhibited the cell proliferation of PC cells, and induced apoptosis and autophagy of ASPC-1 cells in a dose-dependent manner. Additionally, pretreatment with chloroquine, an autophagy inhibitor, significantly inhibited K73-03-induced autophagy and enhanced K73-03-induced apoptotic cell death. K73-03 also strongly repressed ASPC-1 cells xenograft growth in vivo. Thus, all these findings provided new clues about OA analog K73-03 as an effective anticancer agent targeted EGFR against ASPC-1 cells, it is worth further evaluation in the future.

1 INTRODUCTION

Pancreatic cancer (PC), one of the most aggressive and lethal solid malignancies, has become the fourth leading cause of cancer deaths worldwide (Cykowiak et al., 2021; Siegel et al., 2019). It is associated with lack of symptomatology and extremely dreary prognosis, less than 10% of patients would be alive at 5 years after diagnosis. Currently, chemotherapy is of vital importance therapy for sufferers with PC (Balsano et al., 2019). However, serious adverse side effects and the problem of multidrug resistance limit their clinical applicability (Cykowiak et al., 2021; Tuan Anh et al., 2018). Therefore, developing novel drugs with high efficiency and minimal adverse effects is urgently required.

Natural products and their derivatives have been shown considerable structural diversity and better drug-like properties compared to the synthetic compounds in the aspects of preventive and anticancer effects, and remain a major resource of new drugs discovery (Li et al., 2015; Y. Wang, Lia, et al., 2018). Oleanolic acid (OA) is a pentacyclic triterpenoid that is widely present in plant species. OA and its natural derivatives exerted a variety of biological activities, including hepatoprotective, anti-inflammatory, and especially anticancer effect in many cancer types (Baer-Dubowska et al., 2021; Tang et al., 2022). Therefore, OA derivatives remain essential fields regarding their synthesis and potential anticancer efficacy to PC.

The epidermal growth factor receptor (EGFR) family comprises four closely related receptors (EGFR/HER1/ErbB1, HER2/ErbB2, HER3/ErbB3, and HER4/ErbB4) and plays a major regulatory role in cell proliferation, differentiation, survival, and angiogenesis (Grapa et al., 2019; Mirgany et al., 2021). Structurally, EGFR is composed of an extracellular ligand-binding domain, a transmembrane region, and an intracellular tyrosine kinase domain. EGFR activation is involved in both the PC initiation and its progression to metastatic cancer (Navas et al., 2012). The inhibition of EGFR activity has been considered to be a prospective strategy for clinically treating pancreatic tumors. It has been reported that OA inhibited proliferation possibly through modulating EGFR activity in A375 cells which would qualify it as a potent anticancer agent (Ghosh et al., 2014). We previously synthesized several OA derivatives, named SZC009, 013, 014, 015, 017. Among them, SZC015 exhibited antitumor activity against PC cells (Chu et al., 2017; Song et al., 2018). Recently, we synthesized a novel OA derivative called K73-03. Our previous study has elucidated that K73-03 induced mitochondrial damage that led to apoptosis of PC through epigenetic SPINK1 suppression by miR-421 upregulation (Shopit et al., 2020). In the current study, we investigated the antitumor ability of K73-03 as a potent inhibitor of EGFR on PC, which induced apoptosis and autophagy through blocking EGFR/(Akt) pathway. In this respect, our data suggest that K73-03 may be a potential candidate for treating PC.

2 MATERIALS AND METHODS

2.1 Materials

K73-03 are provided in our previous study (Shopit et al., 2020). The primary antibodies against β-actin, EGFR, p-EGFR, Akt, p-Akt, LC3B, Beclin1 Caspase-3, Cleaved-Caspase-3, Caspase-9, Bax, and Bcl-2 and all the secondary antibodies were purchased from Proteintech. All other chemicals were purchased from Sigma Chemical Co. Human pancreatic ductal epithelial cells (HPDE6-C7), human PC cell lines ASPC-1, and PANC-1 were purchased from Shanghai Institute of Biochemistry and Cell Biology.

2.2 Targets predicted by PharmMapper

PharmMapper is an open-source online platform, which aims identify potential target candidates for the given probe small molecules via a pharmacophore mapping approach (Liu et al., 2010; X. Wang, Shen, et al., 2017). Molecular file of K73-03 was uploaded to the PharmMapper servers for in silico screening. The number of reserved matched targets was set to 300, whereas other parameters were considered as default.

2.3 Molecular docking

The high-resolution crystal structure of EGFR was obtained from the PDB bank (PDB code: 3W2S), Molecular docking analysis was performed using AutoDock 4.2 with MGL tools 1.5.6. The protein structures were cleaned by removing crystallographic water and prepared by the addition of polar hydrogen atoms, followed by the addition of Gasteiger charges. The residual interactions at the protein drug interface were evaluated using LigPlot (Laskowski & Swindells, 2011).

2.4 Molecular dynamics (MD) simulation

The conformation of the complexes formed between ligands and EGFR target protein were predicted using AutoDock 4.2 program, and all of the MD simulations were carried out by Gromacs 5.1.5 with Amber99sb force field (Y. Wang et al., 2019). After MD simulation, the molecular mechanics energies combined with the Molecular Mechanics/Poisson Boltzmann Surface Area (MM/PBSA) methods were employed to calculate the binding free energy of OA and K73-03 with EGFR target protein.

2.5 Cell culture and cell viability assay

HPDE6-C7, ASPC-1, and PANC-1 cells were cultured in high-glucose DMEM supplemented with 10% fetal bovine serum, 100 units/ml penicillin and 100 µg/ml streptomycin and maintained in 5% CO₂ at 37°C in saturated humidity. Cell viability was measured by Cell Counting Kit-8 (CCK-8; Dojindo) assay. Cells were seeded into 96-well plates with 5 × 10³ cells/well and incubated for the cell attachment, and were treated with K73-03 with different concentrations for 24 h. Then, each well was treated with 10 µl CCK-8 solution. Two hours later, the absorption values were measured at 450 nm using a microplate reader (Varioskan™ LUX; Thermo Co).

2.6 Transient transfection of EGFR small interfering RNA (siRNA) in ASPC-1

Transfection was performed with Lipofectamine 3000 (Invitrogen) according to the manufacturer's protocol. Briefly, Cells were seeded in 6-well or 96-well plates at approximately 50% confluency for 24 h before siRNA treatment. Then, siRNA transfection was performed 24 h before K73-03 treatment. The siRNAs were synthesized by GenePharma (Shanghai, China) with following sequences: siNC: 5′-UUCUCCGAACGUGUCACGUdTdT-3′, siEGFR:5′-CCUAUGCCUUAGCAGUC UUTT -3′.

2.7 Western blot analysis

Equal amounts of cell lysate proteins were loaded onto a 8%−12% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE), separated by gel electrophoresis and then electrically transferred onto a polyvinylidene difluoride membrane. The membrane was sealed with 5% skim milk for 2 h at room temperature and incubated with specific antibody. The blots were then detected using enhanced chemiluminescence reagent and photographed by Bio-Spectrum Gel Imaging System (Bio-Rad).

2.8 In vitro migration assay

Cell migration was observed by Scratch assay (wound healing assay). The cells were inoculated into six-well plates, incubated at 37°C and 5% CO₂ overnight, and then wounded with a 200 µl sterile pipette tip after 6 h of starvation in incomplete medium. Cells were treated with indicated doses of K73-03 in full medium. After incubation at 37°C for 24 h, cells were washed three times with phosphate buffered saline (PBS), and images of the cells were obtained using microscope.

2.9 Cell cycle analysis

To evaluate the cell cycle distribution after exposure to K73-03, ASPC-1 cells were incubated with K73-03 for 24 h. After the treatment, the cells were collected following centrifugation and fixed in ice-cold 70% ethanol overnight at 4°C. The samples were then suspended with staining reagent (50 µg/ml of PI and 100 µg/ml of RNase A in PBS) in the dark at 37°C for 30 min. Finally, the DNA content of cells was detected using FACScan flow cytometry (BD FACSAria II; BD Co).

2.10 Apoptosis assay

To determine whether K73-03 can trigger apoptosis in ASPC-1 cell line. Cells (5 × 10⁵) cultured in six-well plates were treated with different concentrations of K73-03 for 24 h. The cells were collected and stained with annexin-V–FITC and propidium iodide (PI) for 30 min in the dark at 37°C according to the instructions of the manufacturer. The samples were immediately analyzed using flow cytometry (BD FACSAria II; BD Co). Flow cytometry data were finally analyzed using FlowJo version 7.6.1 (FlowJo).

2.11 Transmission electron microscopy

ASPC-1 cells were plated in six-well plates, incubated at 37°C and treated with K73-03 for 24 h. Cells were collected and fixed with 2% glutaraldehyde in 0.1 M PBS overnight at 4°C. The samples were subsequently dehydrated, embedded, sectioned, and double stained with uranyl acetate and lead citrate. Electron micrographs were taken on a Transmission Electron Microscope (TEM) (JEM-2000EX; JEOL Co).

2.12 In vivo studies

The laboratory animal center of Dalian Medical University (SCXK: 2013-0006) provided BALB/c nude mice with a body weight of 15−20 g for 4−6 weeks. All animals were maintained under sterile conditions, and animal experiments are conducted in accordance with the guidelines of the Animal Care and Use Committee. To generate tumor xenografts, 5 × 10⁵ ASPC-1 cells in 0.2 ml of PBS were injected into the subcutaneous area of the right flank of mice. When the tumor diameter reached 6−9 mm, these mice were randomly divided into three groups (n = 5 per group): Control group, low-dose (25 mg/kg) of K73-03-treated group, and high-dose (50 mg/kg) of K73-03-treated group. Mice from the experimental groups were administered (intraperitoneal injection, [ip]) with either 25 or 50 mg/kg K73-03 once a day for 3 weeks. Tumor volume was measured every 3 days with Vernier calipers and calculated using the following formula: V = A × B²/2, where A and B represent the maximum and minimum diameters (Peirce et al., 2011). Finally, mice were Euthanized, and tumors were weighed, measured and photographed.

2.13 Hematoxylin and eosin (H&E) staining

Before paraffin embedding, primary tumors were fixed in 4% paraformaldehyde overnight. 5-µm serial slicing, sections were dyed with H&E as previously described. For H&E, histopathological changes were observed using a light microscope (Olympus).

2.14 Statistical analysis

The results were expressed as mean ± SD. Statistical differences between groups were assessed using a One-way analysis of variance. Two groups were compared using two-tailed Student's t-test using Graph Pad Prism 8.0 (Graph Pad Software, Inc.). Bonferroni test was used to correct multiple comparison and the p < 0.05 were considered statistically significant.

3 RESULTS

3.1 Potential target prediction and screening using PharmMapper and molecular docking

The chemical structure of OA and K7303 are shown in Supporting Information: Figure S1A. Potential protein target for K73-03 was identified by virtual screening procedures with PharmMapper. Significantly, EGFR ranked 29 among 2241 human protein targets (Figure 1a). Moreover, total 22 kinases from top 100 ranked potential protein targets were subjected to further molecular docking computation using Autodock 4.2. The results of this analysis are shown in Table 1, where the docking score in the EGFR complex (−11.34 Kcal/mol) was best among the top 100 of potential protein targets.

Details are in the caption following the image — **Figure 1**
Open in figure viewer PowerPoint

K73-03 inhibits EGFR/AKT signaling pathways and knocking down EGFR attenuates the inhibitory effect of ASPC-1 cell growth by K73-03. (a) Putative targets of K73-03 were identified by PharmMapper and Pharmacophoric features of EGFR protein aligned over K73-03. (b, c) K73-03 suppress EGFR/AKT signaling pathways. ASPC-1 cells were treated with K73-03 at the indicated doses. After 24 h of treatment, the p-EGFR, EGFR, p-AKT and AKT protein levels were analyzed by Western blots. Histogram data are presented as mean ± SD, n = 3. *p < .05; **p < .01 versus the control group. (d) ASPC-1 cell lines were transfected with EGFR siRNA. The levels of EGFR in the lysates were checked using Western blot analysis. (e) Inverted contrast-phase microscopy revealed the morphological changes of ASPC-1 cells treated with K73-03 and EGFR siRNA for 24 h. Scale bar = 100 μm (f) K73-03 has less effect on cell growth of EGFR siRNA transfected cells than that of NC siRNA cells. EGFR, epidermal growth factor receptor; SD,standard deviation; siRNA, Small interfering RNA.

Table 1. Prediction of potential targets of K73-03 by PharmMapper and molecular docking

PharmMapper rank	PDB ID	Name	Target gene	Docking score (kcal/mol)
8	1UNH	Cyclin-dependent kinase 5 activator 1	CDK5R1	−9.39
13	3BGQ	Proto-oncogene serine/threonine-protein kinase Pim-1	PIM1	−6.15
14	2G01	Mitogen-activated protein kinase 8	MAPK8	−8.97
19	2RKU	Serine/threonine-protein kinase PLK1	PLK1	−8.54
29	2ITZ	Epidermal growth factor receptor	EGFR	−11.34
41	1QPD	Proto-oncogene tyrosine-protein kinase LCK	LCK	−9.13
42	1SM2	Tyrosine-protein kinase ITK/TSK	ITK	−3.14
44	2BU5	Pyruvate dehydrogenase kinase 2	PDK2	−7.68
47	2UW9	RAC-beta serine/threonine-protein kinase	AKT2	−9.69
61	2VX0	Ephrin type-B receptor 4	EPHB4	−5.73
63	2I6B	Adenosine kinase	ADK	−9.75
65	1RW8	TGF-beta receptor type-1	TGFBR1	−9.46
69	1PMN	Mitogen-activated protein kinase 10	MAPK10	−9.15
72	2F57	Serine/threonine-protein kinase PAK 7	PAK5	−9.55
82	1S9J	Dual specificity mitogen-activated protein kinase kinase 1	MAP2K1	−8.77
87	1NXK	MAP kinase-activated protein kinase 2	MAPKAPK2	−10.36
89	2E9V	Serine/threonine-protein kinase Chk1	CHEK1	−9.15
91	1OKY	3-phosphoinositide-dependent protein kinase 1	PDPK1	−5.92
92	1P2A	Cell division protein kinase 2	CDK2	−9.51
94	1Q3D	Glycogen synthase kinase-3 beta	GSK3B	−7.19
96	1M51	Phosphoenolpyruvate carboxy kinase	PCK1	−5.10
97	1XJD	Protein kinase C theta type	PRKCQ	−9.66

Note: The K73-03 potential targets with High Fit Score were screened by PharmMapper. Total 22 kinases from top 100 ranked potential protein targets were subjected to further molecular docking computation using Autodock4.2.

3.2 K73-03 decreases the levels of p-EGFR and downstream targets of EGFR signaling pathway in ASPC-1 cells

CCK8 assay were performed to examine the effect of OA and K73-03 on cell viability in ASPC-1, PANC-1, and HPDE6-C7 cell lines. The results showed that K73-03 decreased the cell viability of the ASPC-1 and PANC-1 cell lines in a concentration and time-dependent manner. However, the toxicity of k73-03 to HPDE6-C7 cells was less, suggesting that K73-03 possesses a promising anti-PC effect (Supporting Information: Figure S1B, C). Interestingly, as an OA derivative, K7303 exhibited a more significant reduction in the cell viability of ASPC-1 cells when compared with OA (Supporting Information: Figure S1D). The 50% inhibitory concentration (IC₅₀) after 24 h of K73-03 treatment was 3.3 µM in ASPC-1 cells and 4.5 µM for PANC-1 cells respectively. These results demonstrated that the K73-03 exhibited potent antitumor activity and ASPC-1 cell line showed more sensitive to the agent. Therefore, ASPC-1 cell line was selected to make a further exploration due to its low IC₅₀ value.

Western blots were used to detect the protein level of p-EGFR and its downstream PI3K/AKT signaling transduction. As shown in Figure 1b,c, K73-03 significantly suppressed the protein expression levels of both p-EGFR and p-AKT in a concentration dependent manner. Moreover, although both K73-03 and OA were able to decrease the expression of p-EGFR, 2.5 µM K73-03 treatment could almost completely inhibit the expression of p-EGFR in ASPC-1 cells (Supporting Information: Figure S2).

To better understand whether the effects of K73-03 were mediated directly through EGFR, we compared the effects of ASPC-1 cells transfected with NC siRNA or EGFR siRNA. Western blot analysis confirmed the downregulation of EGFR in ASPC-1 cells (Figure 1d). Transfected and NC ASPC-1 cells were cultured, and then cell viability was determined using CCK-8 assay. The results showed that the proliferation of NC siRNA cells was decreased with K73-03 treatment compared to knocked down cells (Figure 1f). These results suggested that EGFR could be a direct target for K73-03 to suppress cancer cell growth. Interestingly, treatment EGFR siRNA markedly caused cellular morphological changes, the vacuolation was observed as they were consistent with treatment with 2.5 µM K73-03 (Figure 1e).

3.3 Binding mechanism of K73-03 to kinase domain of EGFR

A large number of EGFR crystal structures have been reported in the literature. The availability of EGFR crystal structure allowed us to utilize molecular docking to identify the novel EGFR inhibitors (Ahmed et al., 2018; Haddad et al., 2021). In this study, high resolution crystal structures (PDB ID:3W2S) was further considered for the molecular docking and MD simulations studies. As shown in Figure 2a,b, the interactions showed by LigPlot are mediated by hydrogen bonds and by hydrophobic interactions. Specifically, In the adenosine-triphosphate (ATP) binding site of the EGFR kinase domain, K73-03 interacted with the active amino acid residues (Asp837 and Cys797) to formed two hydrogen bonds. Furthermore, K73-03 maintains hydrophobic interactions with the catalytic residue Asp837 and its surrounding residues Gly796, Leu844, Leu718, Val726, Lys745, Asp855, Phc723, Asn842, Ser720, Ala722, Gly721, Arg841, Gly719, and Ala743 in the active site. Collectively, these results suggested K73-03 inhibits EGFR activity by targeting the ATP binding site.

3.4 MD simulations

The K73-03 is subjected to MD simulation of 100 ns to make sure the ligand stays inside the active site pocket and to observe the interaction properties to ensures the ligand functionality. The RMSD trajectory of K73-03 was stable, indicating that the small molecule K73-03 was stable in combination with EGFR protein receptor (Figure 2c). The interaction between the K73-03 and each residue was analyzed by the MM/PBSA methods. Binding free energy calculation listed in Table 2 where the binding energy in the EGFR-K73-03 complex (−242.78 kJ/mol) was better than in the EGFR-OA complex (−164.95 kJ/mol). The results also showed Top 10 hot residues, which made a favorable contribution to the binding of the inhibitor and protein (Figure 2d).

Table 2. The PBSA binding free energies of OA and K73-03 relative to EGFR protein targets were calculated according to molecular dynamics simulation and MM/PBSA methods

Complex	ΔE_vdW	ΔE_ele	ΔG_PB	ΔG_SA	ΔG_binding
EGFR-OA	−191.78	−109.38	158.35	−21.89	−164.95
EGFR-K73-03	−226.41	−232.14	240.35	−24.71	−242.78

Note: Snapshots extracted from the last 0.5 ns MD simulation were submitted to MmPbSaStat.py for the free energy calculation. All the energies are in kJ/mol.
Abbreviations: MM/PBSA, Molecular Mechanics/Poisson Boltzmann Surface Area; OA, Oleanolic acid.

Further, hydrogen bond analysis showed that the hydrogen bond interaction between Cys797-K73-03 was stable during the last 5 ns simulation in the binding system, and the hydrogen bond occupancy was 100%. The hydrogen bond occupancies between Lys745-K73-03, Asp800-K73-03, and Arg841-K73-03 in the system were 47.32%, 4.86%, and 0.90%, respectively. Figure 2e summarized hydrogen-bond interactions and hydrogen bond occupancy between ligand atoms and residues during simulations.

3.5 Suppression migration of ASPC-1 cells by K73-03

As shown in Figure 3a, in the control group of the migrating ASPC-1 cells, the part of gap or wounding space between cell layers after making a scratch was occupied almost 24 h after making a scratch, while in the cells treated with K73-03, due to the damage of migration ability, the migration failed to occupy the scratched space.

3.6 Effect of K73-03 on cell cycle distribution of ASPC-1 cells

Cell cycle arrest is an important mechanism to inhibits cancer cell growth (F. Wang, Tian, et al., 2018; Yao et al., 2019). We next study the effect of K73-03 on cell cycle progression in ASPC-1 cells. Our data showed that K73-03 treatment caused an accumulation of ASPC-1 cell lines in the G2/M-phase. As seen in Figure 3b, the percentage of ASPC-1 cells in the G2/M-phase was increased from 9.87% in the control group to 22.83% in the 5 µM K73-03 group. Our findings suggest that K73-03 induces G2/M-phase arrest of the ASPC-1 cells.

3.7 K73-03 induces apoptosis in the PC cells

To detect whether the antitumor activity of K73-03 was associated with apoptosis in ASPC-1 cells, flow cytometry analysis using Annexin V-FITC and PI double staining was performed. The cells apoptotic rates were noticeably increased from 5.79% (control group) to 7.43%, 14.49%, and 23.89% in ASPC-1 cells treated with 1.25, 2.5 and 5 µM of K73-03 for 24 h, respectively (Figure 3c). The ratio of Bax/Bcl-2 which determines the susceptibility to apoptosis by regulating mitochondrial functions was increased by K73-03. The levels of procaspase-9 and procaspase-3 were suppressed, while the level of cleaved caspase-3 was increased by K73-03 (Figure 3d). The results confirmed that K73-03 induced apoptosis in ASPC-1 cells in a concentration-dependent manner.

3.8 K73-03 induces autophagy in ASPC-1 cells

The micro-morphological change of K73-03 treated ASPC-1 cells were also observed by TEM, which is the most convincing standard method to verify autophagy (Gao et al., 2015; Song et al., 2017; F. Wang, Xu, et al., 2017). The intact cell and nuclear membrane, rough endoplasmic reticulum, distinct nucleus, and homogeneous cytoplasm maintained normal micromorphology in the untreated cells, while the results showed that 2.5 µM K73-03 treatment resulted in numerous vacuoles in the cytoplasm changes of autophagosomes in the ASPC-1 (Figure 4a). Moreover, microtubule-associated protein 1, light chain 3 (LC3), the specific marker of autophagy was detected by Western Blots. As shown in Figure 4b, compared with the control group, the ratio of LC3-II/LC3-I was increased after treatment with K73-03. Our data showed that K73-03 induced autophagy in ASPC-1 cells in a concentration-dependent manner. Furthermore, to determine the relationship between autophagy and apoptosis induced by K73-03, we evaluated level of cleaved caspase-3 after pretreatment with Chloroquine, a late-stage inhibitor of autophagy. Our study showed pretreatment with 10 µM Chloroquine dramatically increased expression of cleaved caspase-3, as compared with only K73-03 (2.5 µM) group (Figure 5). These data suggest that K73-03-induced autophagy not only prevent cells from K73-03-induced cell death, but also inhibit apoptosis triggered by K73-03 in ASPC-1 cells.

3.9 K73-03 suppresses ASPC-1 cell xenograft growth

To investigate the effect of K73-03 on tumor formation of PC in vivo, ASPC-1 cell xenograft mouse models (BALB/c nude mice) were used to evaluate antitumor activity of K73-03. As illustrated in Figure 6a−c, K73-03 significantly decreased the growth of ASPC-1 cell xenograft tumors compared with those of control tumors. Moreover, 50 mg/kg K73-03 treatment exhibited a more significant inhibition of tumor volumes and weights compared with 25 mg/kg K73-03, whereas the body weight was comparable between K73-03-treated and control groups (Figure 6d). H&E staining further showed that K73-03 administration could lead to several morphological changes such as the decrease of tumor cell number, tumor tissue cavitation, tumor cell density and necrosis area in tumor tissue (Figure 6e). Meanwhile, results from immunoblotting analyses confirmed that p-EGFR expression was markedly reduced in xenograft tissues receiving K73-03 administration (Figure 6f). Therefore, our results indicate that K73-03 exerts potent antitumor effect tumors against PC in vivo mediated by EGFR.

4 DISCUSSION

In recent years, the technologies of target fishing fulfill an important role in investigating the mechanism of bioactive small molecules (Alvarez et al., 2006; Giordano et al., 2013). It has become a hot spot in drug research for using computer tools to identify novel interactors or novel targets for drugs. OA is a pentacyclic triterpenoid that is widely present in plant species. OA and its natural derivatives exerted a variety of biological activities, including hepatoprotective, anti-inflammatory, and especially anticancer effect in many cancer types. However, the molecular mechanism of action still be obscure. Recently, we synthesized a novel OA derivative called K73-03. In this study, the potential protein targets of K73-03 were screened via reverse pharmacophore screening and molecular docking study. Our primary investigation showed that EGFR was the best potential kinase protein target among 2241 human protein targets. Moreover, EGFR overexpression was one of the indicators for clinical outcome implicated in pancreatic carcinomas (Guo et al., 2016). Furthermore, our result using Western blot also confirmed that K73-03 suppressed the protein level of p-EGFR and its downstream PI3K/AKT signaling transduction in ASPC-1 cells.

To better understand whether the effects of K73-03 were mediated directly through EGFR, we compared the effects of ASPC-1 cells transfected with NC siRNA or EGFR siRNA. The proliferation of NC siRNA cells was decreased by the treatment with K73-03 comparing to knocked down cells (Figure 1f), suggesting that EGFR could be a direct target for K73-03 to suppress cancer cell growth. Interestingly, the treatment with EGFR siRNA markedly caused cellular morphological changes, the vacuolation was observed as they were consistent with treatment with 2.5 µM K73-03 (Figure 1e).

Molecular docking calculation was performed to gain insight into the binding mode of K73-03-EGFR complex and OA-EGFR complex. Subsequently, 100 ns MD simulation was subjected to further observe the interactions between K73-03 and EGFR. As shown in Table 2, the binding free energy in the EGFR-K73-03 complex (−242.78 kJ/mol) was better than in the EGFR-OA complex (−164.95 kcal/mol). Further, the hydrogen bond interaction between Cys797-K73-03 was stable during the last 5 ns simulation in the binding system, and the hydrogen bond occupancy was 100%. The results indicated the carboxy of K73-03 is a reactive group and Cys797 could be a vital residue instead of Thr790. Therefore, K73-03 has the potential to become an EGFR tyrosine kinase inhibitor to overcoming the mutation of T790M resistance.

PC is a highly lethal malignancy, and the treatment outcomes including chemotherapy at present are rather ineffective (Rabi & Catapano, 2016; Rossi et al., 2014). EGFR is a rational target for PC treatment because it is usually expressed at high levels in PC. Similarly, the present study demonstrated that K73-03 markedly inhibited cell viability in ASPC-1 and PANC-1 cells. Interestingly, as an OA derivative, K73-03 exhibited a more significant reduction in the cell viability of ASPC-1 cells when compared with OA, and exerted a less pronounced antiproliferative effect on HPDE6-C7 normal pancreatic cells. Moreover, K73-03 exerts potent antitumor effect tumors against PC in vivo mediated by EGFR, suggesting that K73-03 could be used as a protective agent in therapies targeted against PC.

In multicellular organisms, there exists a delicate balance between the cell-generating effects of mitosis and apoptosis-induced cell death (Cotter, 2009; Huang et al., 2016; Prakobwong et al., 2011). The induction of apoptosis and cell cycle arrest are currently two important processes in the development of anticancer drugs (Curtin, 2013; Gao et al., 2016). Consequently, flow cytometric analysis was performed to determine whether K73-03 could induce PC cells death, which was associated with apoptosis. As shown in Figure 3 and 6, K73-03 markedly increased in the Bax/Bcl-2 ratio and caspase 3 activity both in vitro and in vivo, suggesting that the action of K73-03 likely has been linked with intrinsic apoptosis, which is mediated via targeting of the mitochondria (Y. Wang et al., 2019; Yao et al., 2019). The cell cycle, featuring its various components and stages, and cell growth and division, forms a complex biological process (Martín-Renedo et al., 2008; Tao et al., 2014). In the present study, K73-03 induced antiproliferative effects on pancreatic cells by restricting cell cycle progression, which was verified by blocking ASPC-1 cells in the G2/M phase of cell cycle.

Several natural products have been demonstrated to possess anticancer activity, mainly through autophagy-dependent mechanisms (Bredholt et al., 2009; Nie et al., 2016; Zhang et al., 2013). Autophagy, as an evolutionarily conserved lysosomal degradation process, has an important role in regulating prosurvival and prodeath signaling pathways in numerous diseases, especially in cancer (Mizushima et al., 2010). Our result using TEM revealed that the autophagosomes of the ASPC-1 cells upon treatment with K73-03 exhibited the typical appearance of containing cytoplasmic organelles and an engulfed bulk cytoplasm. Furthermore, a significant transformation from LC3I to LC3II, which is a typical sign of autophagy, was also detected (Baehrecke, 2005; Mizushima et al., 2010). Therefore, to determine the association between autophagy and apoptosis induced by K73-03, the extent of apoptosis in the cells, and the level of cleaved caspase-3 following pretreatment with chloroquine, were evaluated. The findings supported that apoptosis triggered by K73-03 was significantly enhanced by blocking autophagy (Figure 5), suggesting that autophagy induced by K73-03 may serve as an inhibitor role in the process of apoptosis in ASPC-1 cells.

5 CONCLUSIONS

In summary, we report the OA derivative K73-03 as a novel type inhibitor of EGFR. Moreover, K73-03 inhibited the cell proliferation and cell migration of ASPC-1 cells, and subsequently induced autophagy and apoptosis of ASPC-1 cells, which were associated with the EGFR/AKT signaling pathway. Furthermore, K73-03 also strongly inhibited ASPC-1 xenograft growth in vivo. These findings provide a strong theoretical basis that K73-03 possesses a potential to be further developed in the treatment of PC.

ACKNOWLEDGMENTS

This study was supported by the Natural Science Foundation of China (no. 82073768) and the Dalian High-level Talent Innovation Support Program (no. 2019RD03).

CONFLICT OF INTEREST

The authors declare no conflict of interest.

ETHICS STATEMENT

The animal study was reviewed and approved by the Animal Experimental Ethics Committee of Dalian Medical University.

Open Research

DATA AVAILABILITY STATEMENT

The data or materials generated or used during the study are available from the corresponding author upon reasonable request.

Supporting Information

Filename

Description

cbin11866-sup-0001-Fig_S1.tif8.8 MB

FIGURE S1 The Structure of K73-03 and OA and cell viability. (A) The Structure of K73-03 and OA. (B) Inhibitory effect of K73-03 on the ASPC-1, PANC-1 and HPDE6-C7 cell lines. Cells were treated with K73-03 at concentrations ranging from 1.25-10 μM, and cell viability was measured by the CCK-8 assay after treatment for 24 h. (C) Inhibitory effect of K73-03 on the ASPC-1 cell lines. Cells were treated with K73-03 at concentrations ranging from 1.25-10 μM, and cell viability was measured by the CCK-8 assay after treatment for 24, 48 and 72 h. (D) The inhibition effect of OA and K73-03 on cell viability in ASPC-1 cells. Cells were treated with OA and K73-03 at concentrations ranging from 1.25-10 μM, and cell viability was assessed by the CCK-8 assay after treatment for 24 h. Values are expressed as the mean ± SD of 3 independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001 Vs. the control group.

cbin11866-sup-0002-Fig_S2.tif484.4 KB

FIGURE S2 Western blot analysis of p-EGFR was evaluated in ASPC-1 cells. ASPC-1 cells were treated with K73-03 (2.5 μM) and OA (10 μM). After 24 h of treatment, the p-EGFR and EGFR protein levels were analyzed by western blots. The values are expressed as mean ± SD of three replicates. *P < 0.05; **P < 0.01 Vs. the control group.

Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

REFERENCES

Ahmed, H., Ihmaid, S. K., Omar, A. M., Shehata, A. M., & Elaasser, M. M. (2018). Design, synthesis, molecular docking of new lipophilic acetamide derivatives affording potential anticancer and antimicrobial agents. Bioorganic Chemistry, 76, 332–342. https://doi.org/10.1016/j.bioorg.2017.11.019
10.1016/j.bioorg.2017.11.019
CAS PubMed Web of Science® Google Scholar
Alvarez, J. V., Greulich, H., Sellers, W. R., Meyerson, M., & Frank, D. A. (2006). Signal transducer and activator of transcription 3 is required for the oncogenic effects of non-small-cell lung cancer-associated mutations of the epidermal growth factor receptor. Cancer Research, 66(6), 3162–3168. https://doi.org/10.1158/0008-5472.CAN-05-3757
10.1158/0008-5472.CAN-05-3757
CAS PubMed Web of Science® Google Scholar
Baehrecke, E. H. (2005). Autophagy: Dual roles in life and death? Nature Reviews Molecular Cell Biology, 6(6), 505–510. https://doi.org/10.1038/nrm1666
10.1038/nrm1666
CAS PubMed Web of Science® Google Scholar
Baer-Dubowska, W., Narożna, M., & Krajka-Kuźniak, V. (2021). Anti-cancer potential of synthetic oleanolic acid derivatives and their conjugates with NSAIDs. Molecules (Basel, Switzerland), 26(16), 4957. https://doi.org/10.3390/molecules26164957
10.3390/molecules26164957
CAS PubMed Web of Science® Google Scholar
Balsano, R., Tommasi, C., & Garajova, I. (2019). State of the art for metastatic pancreatic cancer treatment: Where are we now. Anticancer Research, 39(7), 3405–3412. https://doi.org/10.21873/anticanres.13484
10.21873/anticanres.13484
CAS PubMed Web of Science® Google Scholar
Bredholt, T., Dimba, E. A., Hagland, H. R., Wergeland, L., Skavland, J., Fossan, K. O., & Gjertsen, B. T. (2009). Camptothecin and khat (Catha edulis forsk.) induced distinct cell death phenotypes involving modulation of c-FLIPL, Mcl-1, procaspase-8 and mitochondrial function in acute myeloid leukemia cell lines. Molecular Cancer, 8, 101. https://doi.org/10.1186/1476-4598-8-101
10.1186/1476-4598-8-101
CAS PubMed Web of Science® Google Scholar
Chu, P., Li, H., Luo, R., Ahsan, A., Qaed, E., Shopit, A., & Tang, Z. (2017). Oleanolic acid derivative SZC014 inhibit cell proliferation and induce apoptosis of human breast cancer cells in a ROS-dependent way. Neoplasma, 64(5), 681–692. https://doi.org/10.4149/neo_2017_505
10.4149/neo_2017_505
CAS PubMed Web of Science® Google Scholar
Cotter, T. G. (2009). Apoptosis and cancer: The genesis of a research field. Nature Reviews Cancer, 9(7), 501–507. https://doi.org/10.1038/nrc2663
10.1038/nrc2663
CAS PubMed Web of Science® Google Scholar
Curtin, N. J. (2013). Inhibiting the DNA damage response as a therapeutic manoeuvre in cancer. British Journal of Pharmacology, 169(8), 1745–1765. https://doi.org/10.1111/bph.12244
10.1111/bph.12244
CAS PubMed Web of Science® Google Scholar
Cykowiak, M., Kleszcz, R., Kucińska, M., Paluszczak, J., Szaefer, H., Plewiński, A., & Krajka-Kuźniak, V. (2021). Attenuation of pancreatic cancer in vitro and in vivo via modulation of Nrf2 and NF-κB signaling pathways by natural compounds. Cells, 10(12), 3556. https://doi.org/10.3390/cells10123556
10.3390/cells10123556
CAS PubMed Web of Science® Google Scholar
Gao, L., Wang, Y., Xu, Z., Li, X., Wu, J., Liu, S., & Tang, Z. (2015). SZC017, a novel oleanolic acid derivative, induces apoptosis and autophagy in human breast cancer cells. Apoptosis: An International Journal on Programmed Cell Death, 20(12), 1636–1650. https://doi.org/10.1007/s10495-015-1179-0
10.1007/s10495-015-1179-0
CAS PubMed Web of Science® Google Scholar
Gao, L., Xu, Z., Wang, Y., Sun, B., Song, Z., Yang, B., & Tang, Z. (2016). Anticancer effect of SZC017, a novel derivative of oleanolic acid, on human gastric cancer cells. Oncology Reports, 35(2), 1101–1108. https://doi.org/10.3892/or.2015.4447
10.3892/or.2015.4447
CAS PubMed Web of Science® Google Scholar
Ghosh, S., Bishayee, K., & Khuda-Bukhsh, A. R. (2014). Oleanolic acid isolated from ethanolic extract of Phytolacca decandra induces apoptosis in A375 skin melanoma cells: drug-DNA interaction and signaling cascade. Journal of Integrative Medicine, 12(2), 102–114. https://doi.org/10.1016/S2095-4964(14)60015-7
10.1016/S2095-4964(14)60015-7
PubMed Google Scholar
Giordano, C., Vizza, D., Panza, S., Barone, I., Bonofiglio, D., Lanzino, M., & Andò, S. (2013). Leptin increases HER2 protein levels through a STAT3-mediated up-regulation of Hsp90 in breast cancer cells. Molecular Oncology, 7(3), 379–391. https://doi.org/10.1016/j.molonc.2012.11.002
10.1016/j.molonc.2012.11.002
CAS PubMed Web of Science® Google Scholar
Grapa, C. M., Mocan, T., Gonciar, D., Zdrehus, C., Mosteanu, O., Pop, T., & Mocan, L. (2019). Epidermal growth factor receptor and its role in pancreatic cancer treatment mediated by nanoparticles. International Journal of Nanomedicine, 14, 9693–9706. https://doi.org/10.2147/IJN.S226628
10.2147/IJN.S226628
CAS PubMed Web of Science® Google Scholar
Guo, M., Luo, G., Liu, C., Cheng, H., Lu, Y., Jin, K., & Yu, X. (2016). The prognostic and predictive role of epidermal growth factor receptor in surgical resected pancreatic cancer. International Journal of Molecular Sciences, 17(7), 1090. https://doi.org/10.3390/ijms17071090
10.3390/ijms17071090
PubMed Web of Science® Google Scholar
Haddad, Y., Remes, M., Adam, V., & Heger, Z. (2021). Toward structure-based drug design against the epidermal growth factor receptor EGFR. Drug Discovery Today, 26(2), 289–295. https://doi.org/10.1016/j.drudis.2020.10.007
10.1016/j.drudis.2020.10.007
CAS PubMed Web of Science® Google Scholar
Huang, L., Hu, B., Ni, J., Wu, J., Jiang, W., Chen, C., & Wang, X. (2016). Transcriptional repression of SOCS3 mediated by IL-6/STAT3 signaling via DNMT1 promotes pancreatic cancer growth and metastasis. Journal of Experimental & Clinical Cancer Research: CR, 35, 27. https://doi.org/10.1186/s13046-016-0301-7
10.1186/s13046-016-0301-7
PubMed Web of Science® Google Scholar
Laskowski, R. A., & Swindells, M. B. (2011). LigPlot+: multiple ligand-protein interaction diagrams for drug discovery. Journal of Chemical Information and Modeling, 51(10), 2778–2786. https://doi.org/10.1021/ci200227u
10.1021/ci200227u
CAS PubMed Web of Science® Google Scholar
Li, F., Zhang, Y., Qiu, D., & Kang, J. (2015). Screening of epidermal growth factor receptor inhibitors in natural products by capillary electrophoresis combined with high performance liquid chromatography-tandem mass spectrometry. Journal of Chromatography A, 1400, 117–123. https://doi.org/10.1016/j.chroma.2015.04.055
10.1016/j.chroma.2015.04.055
CAS PubMed Web of Science® Google Scholar
Liu, X., Ouyang, S., Yu, B., Liu, Y., Huang, K., Gong, J., & Jiang, H. (2010). PharmMapper server: A web server for potential drug target identification using pharmacophore mapping approach. Nucleic Acids Research, 38(Web Server issue), W609–W614. https://doi.org/10.1093/nar/gkq300
10.1093/nar/gkq300
Google Scholar
Martín-Renedo, J., Mauriz, J. L., Jorquera, F., Ruiz-Andrés, O., González, P., & González-Gallego, J. (2008). Melatonin induces cell cycle arrest and apoptosis in hepatocarcinoma HepG2 cell line. Journal of Pineal Research, 45(4), 532–540. https://doi.org/10.1111/j.1600-079X.2008.00641.x
10.1111/j.1600-079X.2008.00641.x
CAS PubMed Web of Science® Google Scholar
Mirgany, T. O., Abdalla, A. N., Arifuzzaman, M., Motiur Rahman, A., & Al-Salem, H. S. (2021). Quinazolin-4(3H)-one based potential multiple tyrosine kinase inhibitors with excellent cytotoxicity. Journal of Enzyme Inhibition and Medicinal Chemistry, 36(1), 2055–2067. https://doi.org/10.1080/14756366.2021.1972992
10.1080/14756366.2021.1972992
CAS PubMed Web of Science® Google Scholar
Mizushima, N., Yoshimori, T., & Levine, B. (2010). Methods in mammalian autophagy research. Cell, 140(3), 313–326. https://doi.org/10.1016/j.cell.2010.01.028
10.1016/j.cell.2010.01.028
CAS PubMed Web of Science® Google Scholar
Navas, C., Hernández-Porras, I., Schuhmacher, A. J., Sibilia, M., & Barbacid, M. (2012). EGF receptor signaling is essential for k-ras oncogene-driven pancreatic ductal adenocarcinoma. Cancer Cell, 22(3), 318–330. https://doi.org/10.1016/j.ccr.2012.08.001
10.1016/j.ccr.2012.08.001
CAS PubMed Web of Science® Google Scholar
Nie, H., Wang, Y., Qin, Y., & Gong, X. G. (2016). Oleanolic acid induces autophagic death in human gastric cancer cells in vitro and in vivo. Cell Biology International, 40(7), 770–778. https://doi.org/10.1002/cbin.10612
10.1002/cbin.10612
CAS PubMed Web of Science® Google Scholar
Peirce, S. K., Findley, H. W., Prince, C., Dasgupta, A., Cooper, T., & Durden, D. L. (2011). The PI-3 kinase-Akt-MDM2-survivin signaling axis in high-risk neuroblastoma: A target for PI-3 kinase inhibitor intervention. Cancer Chemotherapy and Pharmacology, 68(2), 325–335. https://doi.org/10.1007/s00280-010-1486-7
10.1007/s00280-010-1486-7
CAS PubMed Web of Science® Google Scholar
Prakobwong, S., Gupta, S. C., Kim, J. H., Sung, B., Pinlaor, P., Hiraku, Y., & Aggarwal, B. B. (2011). Curcumin suppresses proliferation and induces apoptosis in human biliary cancer cells through modulation of multiple cell signaling pathways. Carcinogenesis, 32(9), 1372–1380. https://doi.org/10.1093/carcin/bgr032
10.1093/carcin/bgr032
CAS PubMed Web of Science® Google Scholar
Rabi, T., & Catapano, C. V. (2016). Aphanin, a triterpenoid from Amoora rohituka inhibits K-Ras mutant activity and STAT3 in pancreatic carcinoma cells. Tumour Biology: The Journal of the International Society for Oncodevelopmental Biology and Medicine, 37(9), 12455–12464. https://doi.org/10.1007/s13277-016-5102-2
10.1007/s13277-016-5102-2
CAS PubMed Google Scholar
Rossi, M. L., Rehman, A. A., & Gondi, C. S. (2014). Therapeutic options for the management of pancreatic cancer. World Journal of Gastroenterology, 20(32), 11142–11159. https://doi.org/10.3748/wjg.v20.i32.11142
10.3748/wjg.v20.i32.11142
PubMed Web of Science® Google Scholar
Shopit, A., Li, X., Tang, Z., Awsh, M., Shobet, L., Niu, M., & Tang, Z. (2020). miR-421 up-regulation by the oleanolic acid derivative K73-03 regulates epigenetically SPINK1 transcription in pancreatic cancer cells leading to metabolic changes and enhanced apoptosis. Pharmacological Research, 161, 105130. https://doi.org/10.1016/j.phrs.2020.105130
10.1016/j.phrs.2020.105130
CAS PubMed Web of Science® Google Scholar
Siegel, R. L., Miller, K. D., & Jemal, A. (2019). Cancer statistics, 2019. CA: A Cancer Journal for Clinicians, 69(1), 7–34. https://doi.org/10.3322/caac.21551
10.3322/caac.21551
PubMed Web of Science® Google Scholar
Song, Y., Gao, L., Tang, Z., Li, H., Sun, B., Chu, P., & Tang, Z. (2018). Anticancer effect of SZC015 on pancreatic cancer via mitochondria-dependent apoptosis and the constitutive suppression of activated nuclear factor κB and STAT3 in vitro and in vivo. Journal of Cellular Physiology, 234(1), 777–788. https://doi.org/10.1002/jcp.26892
10.1002/jcp.26892
PubMed Web of Science® Google Scholar
Song, Y., Kong, L., Sun, B., Gao, L., Chu, P., Ahsan, A., & Tang, Z. (2017). Induction of autophagy by an oleanolic acid derivative, SZC017, promotes ROS-dependent apoptosis through Akt and JAK2/STAT3 signaling pathway in human lung cancer cells. Cell Biology International, 41(12), 1367–1378. https://doi.org/10.1002/cbin.10868
10.1002/cbin.10868
CAS PubMed Web of Science® Google Scholar
Tang, Z. Y., Li, Y., Tang, Y. T., Ma, X. D., & Tang, Z. Y. (2022). Anticancer activity of oleanolic acid and its derivatives: Recent advances in evidence, target profiling and mechanisms of action. Biomedicine & Pharmacotherapy, 145, 112397. https://doi.org/10.1016/j.biopha.2021.112397
10.1016/j.biopha.2021.112397
CAS PubMed Web of Science® Google Scholar
Tao, L., Fu, R., Wang, X., Yao, J., Zhou, Y., Dai, Q., & Wang, W. (2014). LL-202, a newly synthesized flavonoid, inhibits tumor growth via inducing G(2)/M phase arrest and cell apoptosis in MCF-7 human breast cancer cells in vitro and in vivo. Toxicology Letters, 228(1), 1–12. https://doi.org/10.1016/j.toxlet.2014.04.002
10.1016/j.toxlet.2014.04.002
CAS PubMed Web of Science® Google Scholar
Tuan Anh, H. L., Tran, P. T., Thao, D. T., Trang, D. T., Dang, N. H., Van Cuong, P., Kiem, P. V., Minh, C. V., & Lee, J. H. (2018). Degalactotigonin, a steroidal glycoside from Solanum nigrum, induces apoptosis and cell cycle arrest via inhibiting the EGFR signaling pathways in pancreatic cancer cells. BioMed Research International, 2018, 3120972. https://doi.org/10.1155/2018/3120972
10.1155/2018/3120972
PubMed Web of Science® Google Scholar
Wang, F., Tian, X., Zhang, Z., Ma, Y., Xie, X., Liang, J., & Yang, Y. (2018). Demethylzeylasteral (ZST93) inhibits cell growth and enhances cell chemosensitivity to gemcitabine in human pancreatic cancer cells via apoptotic and autophagic pathways. International Journal of Cancer, 142(9), 1938–1951. https://doi.org/10.1002/ijc.31211
10.1002/ijc.31211
CAS PubMed Web of Science® Google Scholar
Wang, F., Xu, C., Reece, E. A., Li, X., Wu, Y., Harman, C., & Yang, P. (2017). Protein kinase C-alpha suppresses autophagy and induces neural tube defects via miR-129-2 in diabetic pregnancy. Nature Communications, 8, 15182. https://doi.org/10.1038/ncomms15182
10.1038/ncomms15182
CAS PubMed Web of Science® Google Scholar
Wang, X., Shen, Y., Wang, S., Li, S., Zhang, W., Liu, X., Lai, L., Pei, J., & Li, H. (2017). PharmMapper 2017 update: A web server for potential drug target identification with a comprehensive target pharmacophore database. Nucleic Acids Research, 45(W1), W356–W360. https://doi.org/10.1093/nar/gkx374
10.1093/nar/gkx374
CAS PubMed Web of Science® Google Scholar
Wang, Y., Lai, H., Fan, X., Luo, L., Duan, F., Jiang, Z., & Yao, X. (2018). Gossypol inhibits non-small cell lung cancer cells proliferation by targeting EGFRL858R/T790M. Frontiers in Pharmacology, 9, 728. https://doi.org/10.3389/fphar.2018.00728
10.3389/fphar.2018.00728
CAS PubMed Web of Science® Google Scholar
Wang, Y., Peng, C., Wang, G., Xu, Z., Luo, Y., Wang, J., & Zhu, W. (2019). Exploring binding mechanisms of VEGFR2 with three drugs lenvatinib, sorafenib, and sunitinib by molecular dynamics simulation and free energy calculation. Chemical Biology & Drug Design, 93(5), 934–948. https://doi.org/10.1111/cbdd.13493
10.1111/cbdd.13493
CAS PubMed Web of Science® Google Scholar
Yao, Y., Zhou, L., Liao, W., Chen, H., Du, Z., Shao, C., & Ding, K. (2019). HH1-1, a novel Galectin-3 inhibitor, exerts anti-pancreatic cancer activity by blocking Galectin-3/EGFR/AKT/FOXO3 signaling pathway. Carbohydrate Polymers, 204, 111–123. https://doi.org/10.1016/j.carbpol.2018.10.008
10.1016/j.carbpol.2018.10.008
CAS PubMed Web of Science® Google Scholar
Zhang, D. M., Liu, J. S., Deng, L. J., Chen, M. F., Yiu, A., Cao, H. H., & Ye, W. C. (2013). Arenobufagin, a natural bufadienolide from toad venom, induces apoptosis and autophagy in human hepatocellular carcinoma cells through inhibition of PI3K/Akt/mTOR pathway. Carcinogenesis, 34(6), 1331–1342. https://doi.org/10.1093/carcin/bgt060
10.1093/carcin/bgt060
CAS PubMed Web of Science® Google Scholar

Citing Literature

Volume46, Issue11

November 2022

Pages 1801-1813

An oleanolic acid derivative, K73-03, inhibits pancreatic cancer cells proliferation in vitro and in vivo via blocking EGFR/Akt pathway

Abstract