Abstract
Background
Pancreatic neuroendocrine neoplasms (pNENs) represent a rare group of highly heterogeneous tumors derived from pancreatic epithelial cells exhibiting neuroendocrine differentiation properties. Everolimus, an oral inhibitor of mTOR, is the most promising drug for patients with unresectable, metastatic disease, particularly in progressive well-differentiated pNENs. Sorafenib is utilized in the treatment of hepatocellular carcinoma (HCC), renal cell carcinoma, and differentiated thyroid cancer. Furthermore, it plays an indispensable role in the management of multisystem malignancies. This study aims to investigate the effects and mechanisms of sorafenib, as well as its potential for combination use with everolimus in pNENs.
Methods
QGP-1and BON-1cells were collected from routine in vitro culture and treated with various concentrations of sorafenib, everolimus, and dual-drug combinations for 24 hours. The capacity of these medications to influence tumor activity was evaluated through the use of the Cell Counting Kit-8 (CCK-8) assay, cloning assay, 5-ethynyl-2'-deoxyuridine (EdU) assay, transwell assay, and analysis of the xenograft tumor model. Western blot analysis was conducted to detect the level of mTOR in QGP-1 and BON-1 cells.
Results
Compared to the control group, the proliferation and migration of sorafenib-treated cells were significantly inhibited. Furthermore, as drug concentration increased, the proliferation rates of both cell types decreased. Notably, the inhibition of cell proliferation was more pronounced in the sorafenib and everolimus combination group than in the single-drug group. Western blot results indicated that the expression level of mTOR was down-regulated in the experimental group after treatment with sorafenib, everolimus, and the dual-drug combination for 24 hours, compared to the control group. In experiments involving animals, tumors in the groups treated with both high and low doses of sorafenib were smaller than those observed in the control group, and liver metastasis was suppressed in the experimental groups when compared to the control.
Conclusions
Sorafenib can inhibit the proliferation and migration of pNENs by down-regulating the mTOR pathway. The combination of sorafenib and everolimus exhibits a stronger anti-tumor effect.
Keywords: Pancreatic neuroendocrine neoplasms (pNENs), sorafenib, everolimus, mTOR, proliferation
Highlight box.
Key findings
• Sorafenib and everolimus synergistically inhibits pancreatic neuroendocrine neoplasms (pNENs).
What is known and what is new?
• Everolimus inhibits the progression of pNENs.
• This study newly discovered that everolimus and sorafenib can combine to inhibit neoplasms and that they act synergistically.
What is the implication, and what should change now?
• The results of this study suggest that sorafenib can be used in the diagnosis and treatment of pNENs, and the next step is expected to start the clinical trial work.
Introduction
Neuroendocrine neoplasms (NENs) are rare, low-growing tumors originating from peptidergic neurons and neuroendocrine cells that possess the capability to produce and secrete biologically active peptides, neuramines, and hormones (1,2). The genesis of neuroendocrine tumors can involve multiple systems (3,4). Approximately two-thirds of NENs occur in the digestive system, with the highest incidence observed in the pancreas (5). Pancreatic neuroendocrine neoplasms (pNENs) represent a rare group of highly heterogeneous tumors derived from pancreatic epithelial cells exhibiting neuroendocrine differentiation properties. In recent years, the incidence of pNENs has increased rapidly, a trend that can be attributed, at least in part, to advancements in imaging and pathological diagnosis (6). Consequently, identifying effective treatment options for pNENs has become imperative. Sorafenib is a kinase inhibitor that targets a variety of intracellular kinases, including c-CRAF, BRAF, and mutant BRAF, as well as cell surface kinases such as KIT, FLT-3, RET, RET/PTC, VEGFR-1, VEGFR-2, VEGFR-3, and PDGFR-ß (7-9). This broad inhibition may lead to a reduction in tumor cell proliferation in vitro. Sorafenib is utilized in the treatment of hepatocellular carcinoma (HCC), renal cell carcinoma, and differentiated thyroid cancer. Furthermore, it plays an indispensable role in the management of multisystem malignancies (10,11). Recent studies suggest that sorafenib’s anti-tumor activity may involve the regulation of the mTOR/AKT/PI3K pathway. Recent research has shown that mTOR functions as a serine/threonine protein kinase that plays a role in the transmission of growth factor signaling dependent on the PI3K/AKT pathway, which is recognized to be disrupted in various human cancers, such as NENs. This pathway ultimately regulates cell metabolism, proliferation, apoptosis, and angiogenesis (12-14). Currently, everolimus, an oral inhibitor of mTOR, is the most promising drug for patients with unresectable, metastatic disease, particularly in progressive well-differentiated pNENs (15). Based on these findings, we aim to explore the effects and mechanisms of sorafenib, as well as the potential benefits of combining everolimus and sorafenib in pNENs. Our study indicates that sorafenib inhibits cell proliferation and migration in pNENs by decreasing mTOR activity, and that the combination of everolimus and sorafenib produces a synergistic effect. We present this article in accordance with the ARRIVE and MDAR reporting checklists (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2024-2424/rc).
Methods
Cell culture and reagents
QGP-1 and BON-1 cell lines were maintained in RPMI 1640 and DMEM/F12 media, respectively, both of which were enriched with 10% fetal bovine serum, 1% penicillin (100 U/mL), and 100 mg/L of streptomycin. These cells were incubated at 37 ℃ in a controlled environment with 5% CO2 and were subcultured every 2 to 3 days, with cells in the logarithmic growth phase being chosen for further experimentation. Sorafenib and everolimus were obtained from Medchem Express, while 0.25% trypsin, RPMI 1640, and DMEM/F12 medium were sourced from Gibco. Fetal bovine serum was procured from Hyclone (USA), and dimethylsulfoxide (DMSO) was purchased from Sigma (USA). Trizol was acquired from Invitrogen (USA), and the PrimeScript™ RT Reagent Kit, SYBR Premix Ex Taq™ Kit, Taq enzyme, dNTP Mix, RNAase inhibitor, and TUNEL Method Apoptosis Detection Kit were purchased from Yeasen (China). RIPA lysis solution and phenylmethanesulfonyl fluoride (PMSF) were obtained from Beyotime Company (Nantong, China). The low-temperature high-speed centrifuge and nucleic acid quantification instrument were sourced from Thermo Fisher (USA). The cell counter and plates were purchased from Reward, while the medical centrifuge and multifunctional enzyme labeling instrument were obtained from Thermo Fisher. Electrophoresis and membrane transfer instruments were purchased from Bio-Rad, and the FCM gel imaging system was sourced from Shanghai Tianneng Life Science Co. (Shanghai, China).
Drug’s half maximal inhibitory concentration (IC50)
Logarithmically grown cells were inoculated into 96-well plates at a density of 1×104 cells per well and cultured routinely for 24 hours. The experimental group was treated with various concentrations of sorafenib, everolimus, and dual-drug combination applications for 24 or 48 h, while the control group received an equivalent volume of the solvent DMSO. Subsequently, the cells were replaced with a complete culture medium containing 10% Cell Counting Kit-8 (CCK-8) reagent in a total volume of 100 µL for an additional 24 hours. Absorbance values at 450 nm for each well were measured using a fully automated enzyme marker after 3 hours of incubation in low light conditions. The mean and standard deviation for each experimental and control group were calculated, followed by the subtraction of the absorbance value of each group from that of the control group to obtain the net absorbance. This net absorbance was regressed against the drug concentration, and a curve of drug concentration versus percentage cell viability was plotted using GraphPad Prism 10. The IC50 value of the drug was determined at the point of 50% drug concentration versus percentage cell viability. The experiment was conducted in triplicate.
Colony formation assay
A total of 2×103 cells, which were growing logarithmically, were placed into each well of a 6-well plate. Following a standard incubation period of 24 hours, the cells were split into two groups: one experimental and one control. The experimental group received varying concentrations of sorafenib, everolimus, and combinations of both drugs. In contrast, the control group was treated with the same volume of complete medium. For a duration of 14 days, the cells were incubated, with the medium being refreshed every 3 days. After this 2-week period, colonies were fixed using 4% paraformaldehyde, then stained with crystal violet for observation and photography of colony formation. The experiment was performed in triplicate.
5-ethynyl-2'-deoxyuridine (EdU) assay
A EdU assay kit (Ribobio, Guangzhou, China) was utilized to assess cell proliferation ability. Cells were seeded into confocal plates at a density of 2×104 cells per well. Following drug treatment, the cells were incubated with 50 µM EdU buffer at 37 ℃ for 2 hours, fixed with 4% formaldehyde for 30 minutes, and permeabilized with 0.1% Triton X-100 for 20 minutes. The culture was then supplemented with the EdU solution, and nuclear staining was conducted using Hoechst. Visualization of the results was achieved with a fluorescence microscope (Zeiss, Oberkochen, Germany).
Transwell assays
The evaluation of cell invasion was conducted through a transwell assay. After treating the cells with sorafenib, everolimus, and a dual-drug combination for 24 hours, QGP-1 and BON-1 cells were resuspended in 0.2 mL of serum-free medium and placed in the upper chamber, which was either uncoated or featured a Matrigel (BD Biosciences) coated membrane. The lower chamber was supplied with medium containing 20% FBS. To guarantee the precision of the experimental findings, we quantified the number of viable cells. Following a 24-hour incubation period, the invaded cells were fixed using methanol, stained with crystal violet (Beyotime), and counted in three randomly selected fields for each sample.
Western blot
Following a 24-hour treatment with varying drug concentrations, QGP-1 and Bon-1 cells were harvested for total protein extraction. The proteins underwent denaturation by being placed in a metal bath at 100 ℃ for 5 minutes. Subsequently, the proteins were analyzed through electrophoresis, transferred onto a membrane, and incubated with 5% skimmed milk for 1 hour. After the blocking solution was removed, the membranes were treated with anti-GAPDH, AKT, PI3K, and mTOR antibodies (diluted 1:1,000) and incubated overnight at 4 ℃. The membranes were then washed three times with TBST for 15 minutes each. Following this, an IgG secondary antibody solution (diluted 1:1,000) was applied and incubated for 1 hour at room temperature. Visualization of the protein bands was achieved using an ECL system.
RNA sequence
Total RNA was extracted and purified with TRIzol reagent (Invitrogen, California, USA). From 1 µg of total RNA, poly(A) RNA was specifically captured using Dynabeads Oligo(dT)25-61005 (Thermo Fisher Scientific, Waltham, USA). The poly(A) RNA was subsequently fragmented into smaller pieces, and these cleaved RNA fragments underwent reverse transcription to generate complementary DNA (cDNA). The resulting cDNA was then amplified, producing fragments of roughly 300±50 bp. For the RNA sequencing analysis, the Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway database was employed to evaluate the enrichment of signaling pathways in differentially expressed genes from the drug-treated group in comparison to the control. A screening criterion of |log2FC| ≥1 and P<0.05 was set.
Combination index (CI)
Combination experiments that involved drugs were assessed utilizing SynergyFinder 3.0 along with the Zero Interaction Potency (ZIP) approach. A ZIP score lower than −10 signifies antagonistic interactions, whereas a score exceeding 10 indicates synergetic effects, and scores that fall within the range between the two suggest an additive effect.
Tumor xenograft model in nude mice
Five-week-old male BALB/c nude mice were sourced from the Animal Center at Nanjing Medical University. Bon-1 cells (5×106 cells per mouse) were suspended in 100 µL of PBS and injected subcutaneously into the right ventral axilla of each mouse. The mice received either normal saline (10 mg/kg) or a combination of sorafenib and everolimus (10 and 5 mg/kg for the low-dose group; 40 and 10 mg/kg for the high-dose group) via intraperitoneal injection every other day for four weeks after the cell injection, with each group consisting of five mice. Afterward, tumors were surgically removed and weighed. The tumor volumes were determined using the formula: 0.5 × length × width2. All animal procedures were approved by the Institutional Animal Care and Use Committee at Nanjing Medical University (No. IACUC-2408042), adhering to both national and institutional regulations regarding the care and use of animals. An experimental protocol, serving as the Master’s thesis proposal, is registered in the Graduate Management System of Nanjing Medical University.
Transferase dUTP nick-end labeling (TUNEL) assay
According to the instructions of the TUNEL Method Apoptosis Detection Kit, the sections were prepared and processed, labeled with TUNEL, and subsequently re-stained. The apoptosis of the sample cells was then observed and photographed under a fluorescence microscope, with the images analyzed using ImageJ version 1.8.0 software.
Statistical analysis
All data presented results from independent experiments conducted multiple times, expressed as mean ± standard deviation (SD) unless otherwise noted. GraphPad Prism (GraphPad Software) was utilized for all calculations. Data comparisons were performed using the appropriate statistical methods. A P value of <0.05 indicated statistical significance).
Results
Sorafenib inhibits pNENs proliferation and migration in vitro
The 24-hour IC50 cytotoxicity values of sorafenib are presented in Figure 1A,1B. We also treated normal liver cells (HPNE) with drugs and found that the results of colony formation assay in the drug-treated group were not statistically significant to the experimental group (Figure S1A-S1C). Compared with the results of 48 h treatment (Figure S1D,S1E), there was no statistically significant difference between the two, and in order to reduce the possibility of contamination during the operation, we chose 24 h as the time of drug treatment. Based on these results, an appropriate concentration was selected for subsequent experiments involving QGP-1 and BON-1 cells, with an incubation period of 24 hours. The colony formation assay (Figure 1C,1D), EdU assays (Figure 1E,1F), and transwell assay (Figure 1G,1H) demonstrated that sorafenib effectively reduced cell growth and metastasis in vitro.
Figure 1.
Sorafenib inhibits pNEN cells proliferation and migration in vitro. (A,B) IC50 values of BON-1 and QGP-1 cells treated with sorafenib for 24 hours. The 95% CI of sorafenib for BON-1 cells is 9.039–13.62, while for QGP-1 cells, the 95% CI is 26.01–30.16. (C,D) Both cell lines growth rates were determined with colony formation assay and stained with crystal violet. (E,F) The EdU assay results of BON-1, QGP-1 being treated with sorafenib and observed at 40×. (G,H) The Transwell assay results of BON-1, QGP-1 being treated with sorafenib, stained with crystal violet and observed at 40×. ***, P<0.001. CI, confidence interval; DAPI, 4'-6-diamidino-2-phenylindole; EdU, 5-ethynyl-2'-deoxyuridine; H-dose, twice the value of IC50; IC50, half maximal inhibitory concentration; L-dose, IC50 of drug; NC, negative control; pNEN, pancreatic neuroendocrine neoplasm; Sora, sorafenib.
Sorafenib suppresses pNENs via mTOR pathway
Next, RNA sequencing was conducted following sorafenib treatment. The results of the KEGG and Gene Ontology (GO) enrichment analyses indicated that the mTOR pathway was among the enriched signaling pathways (Figure 2A-2D).
Figure 2.
Sorafenib suppresses pNEN cells growth via inhibiting mTOR pathway. (A,B) Pathways enriched in KEGG; (C,D) results of GO enrichment. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; pNEN, pancreatic neuroendocrine neoplasm.
Sorafenib inhibits pNENs growth and metastasis in vivo
In the in vivo experiment, tumors in both the high-dose and low-dose drug-treated groups were smaller than those in the control group, with the difference being statistically significant (Figure 3A-3C). Additionally, the Ki67 index of the tumors decreased with increasing drug concentration, indicating that sorafenib significantly inhibited tumor progression (Figure 3D,3E). Furthermore, the TUNEL results support this conclusion (Figure 3F). H&E staining results indicated that liver metastasis was inhibited in the experimental group compared to the control group, with the inhibitory effect increasing significantly with higher drug concentrations (Figure 3G,3H).
Figure 3.
Sorafenib inhibits pNENs proliferation, migration and liver metastasis in vivo. (A-C) The tumors formed were inhibited significantly in experimental group in nude mice. (D,E) The results of Ki-67 of tumors formed in nude mice, positive cells are labeled by a chromogenic reaction (e.g., DAB staining) using an anti-Ki67 antibody that binds to the Ki67 protein in the tissue sections. (F) The results of TUNEL of tumors formed in nude mice and stained with fluorescein-labeled dUTP. (G,H) The liver metastasis in nude mice was suppressed after being treated with sorafenib and the H&E staining of liver specimens. **, P<0.01; ***, P<0.001. CON, control group; DAB, 3,3'-diaminobenzidine; H-dose, sorafenib (40 mg/kg); L-dose, sorafenib (10 mg/kg); pNEN, pancreatic neuroendocrine neoplasm.
Everolimus inhibits pNENs proliferation and migration in vitro
The 24-h IC50 cytotoxicity values of everolimus are presented in Figure 4A,4B. Based on these results, an appropriate concentration was selected for subsequent experiments involving QGP-1 and BON-1 cells, with a 24-h incubation period. The colony formation assay (Figure 4C,4D), EdU assays (Figure 4E,4F), and transwell assay (Figure 4G,4H) demonstrated that everolimus effectively reduced both cell growth and metastasis.
Figure 4.
Everolimus suppressed pNEN cells viability, proliferation and migration in vitro. (A,B) IC50 values of BON-1 and QGP-1 cells treated with everolimus for 24 hours. The 95% CI of Evo for BON-1 cells is 35.42–45.50, while for QGP-1 cells, the 95% CI is 46.40–52.05. (C,D) Both cell lines’ growth rates were determined with colony formation assay. (E,F) The EdU assay results of BON-1, QGP-1 being treated with everolimus, and observed at 40×. (G,H) The transwell assay results of BON-1, QGP-1 being treated with everolimus, stained with crystal violet, and observed at 40×. ***, P<0.001. CI, confidence interval; DAPI, 4'-6-diamidino-2-phenylindole; EdU, 5-ethynyl-2'-deoxyuridine; IC50, half maximal inhibitory concentration; NC, negative control; pNEN, pancreatic neuroendocrine neoplasm.
Combination of sorafenib and everolimus inhibits pNENs more significantly
The ZIP score revealed that sorafenib and everolimus were significantly synergistic over a range of drug concentrations (Figure 5A). Meanwhile, colony formation assay (Figure 5B), EdU assays (Figure 5C,5D), and transwell assay (Figure 5E,5F) revealed that combination of everolimus and sorafenib reduced cell growth and metastasis.
Figure 5.
Combination of sorafenib and everolimus suppressed pNEN cells more significantly in vitro. (A) ZIP score of combination of sorafenib and everolimus is 17.259.ZIP score below −10 indicates antagonism, above 10 synergy and in between an additive effect. (B-D) Both cell lines’ growth rates were determined with colony formation assay and EdU labelling analysis and observed at 40×. (E,F) The transwell assay results of BON-1, QGP-1 being treated with sorafenib and everolimus, stained with crystal violet and observed at 40×. ***, P<0.001. DAPI, 4'-6-diamidino-2-phenylindole; EdU, 5-ethynyl-2'-deoxyuridine; pNEN, pancreatic neuroendocrine neoplasm.
Everolimus and sorafenib inhibit mTOR of pNENs in vitro through PI3K/AKT/mTOR pathway
The results of western blot revealed that after treating with sorafenib, everolimus and dual-drugs treatment for 24 h, we observed that mTOR was significantly decreased after treatment with sorafenib, everolimus and dual-drugs treatment in these cells. This suggests that sorafenib, everolimus and dual-drugs treatment exert a suppressive effect on its activity (Figure 6A-6C), indicating that sorafenib, everolimus and dual-drugs treatment suppress pNENs and partially through mTOR pathway. Sorafenib inhibited mTOR of pNENs in vitro through PI3K/AKT/mTOR pathway (Figure 6D-6G).
Figure 6.
Everolimus and sorafenib inhibit mTOR of pNENs in vitro through PI3K/AKT/mTOR pathway. (A-C) Sorafenib, everolimus and dual-drugs treatment suppress pNENs and partially through mTOR pathway. (D-G) Sorafenib inhibited mTOR of pNENs in vitro through PI3K/AKT/mTOR pathway. ****, P<0.0001. C-H-dose, combine with everolimus and sorafenib at IC50; C-L-dose, combine with everolimus and sorafenib at IC25; CON, control group; NC, negative control; pNEN, pancreatic neuroendocrine neoplasm; Sora, sorafenib.
Discussion
NENs are a rare disease with an increasing incidence year by year (16,17). Based on this, pNENs is the most common type of NENs (18-20). Most pNENs is malignant, with up to 60% of patients having metastatic disease at diagnosis (21). Everolimus, approved for patients with advanced pNENs and non-functioning pulmonary and gastrointestinal NENs, inhibits mTOR, which is involved in cell growth, proliferation, and apoptosis (17,22). Despite progress in treatment strategies, finding effective therapies for pNENs continues to be challenging, underlining the necessity for novel options to manage tumor growth. Sorafenib acts as a kinase inhibitor targeting several protein kinases, such as vascular endothelial growth factor, platelet-derived growth factor receptor, and Raf kinase. It is currently authorized for use in treating advanced renal cell carcinoma, HCC, and thyroid cancer (23,24). Sorafenib exerts its oncogenic effect in renal cell carcinoma and thyroid cancer by inhibiting the Raf/MEK/ERK kinase pathway, while in HCC it exerts its oncogenic effect by inhibiting the Raf/MEK/ERK and EGFR/SRC/STAT3 kinase pathway (25,26). mTOR serves as a key regulator of protein synthesis by integrating mitogenic signals triggered by growth factors alongside the availability of resources. It also governs the initiation phase of mRNA translation and the biogenesis of ribosomes (27). Previous studies have demonstrated that activation of the PI3K/AKT/mTOR pathway promotes the development of pNENs (28). Furthermore, a recent investigation into the relationship between lipid metabolism and colorectal cancer development confirmed that inactivation of the mTOR pathway induces autophagy in cancer cells, thereby inhibiting colorectal cancer progression (29). In the context of pNENs, low expression of ALKBH5 acts synergistically with everolimus to inhibit mTOR activation and suppress cancer cell growth (30). In our research, we explored the potential of sorafenib as an anti-tumor agent against pNENs via decreasing the mTOR pathway, and the results of several experiments demonstrated that sorafenib significantly inhibited the proliferation, growth, and migration of pNENs cells both in vivo and vitro. Moreover, we further validated that the combination of sorafenib and everolimus is synergistic and significantly superior to single agent in a range of concentrations. Sorafenib and everolimus still have potential risks of antagonism in combination therapy, which needs to be further explored in future applications. In conclusion, our findings support the notion that sorafenib inhibits the mTOR pathway and the combination of everolimus and sorafenib regresses pNENs markedly, which provides a new option for the treatment of pNENs. In order to fully elucidate the potential of the combination of everolimus and sorafenib as a new therapeutic option for pNENs, it is necessary to further investigate the specific mechanisms by which drugs interact with and its broader implications for cancer progression and drug resistance. We still need to refine in vivo experiments to investigate the effect of the two-drug combination on pNENs.
Conclusions
In summary, our study reveals the anti-tumor effects of sorafenib, with an enhanced inhibition of pNENs growth and metastasis when combined with everolimus. These findings underscore the potential of the everolimus and sorafenib combination as a promising therapeutic strategy for the treatment of pNENs.
Supplementary
The article’s supplementary files as
Acknowledgments
None.
Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All animal procedures were approved by the Institutional Animal Care and Use Committee at Nanjing Medical University (No. IACUC-2408042), adhering to both national and institutional regulations regarding the care and use of animals.
Footnotes
Reporting Checklist: The authors have completed the ARRIVE and MDAR reporting checklists. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2024-2424/rc
Funding: This work was supported by Science Foundation Project of Ili & Jiangsu Joint Institute of Health (No. yl2023ms10) and Jiangsu Province Hospital (The Frist Affiliated Hospital with Nanjing Medical University) Clinical Capacity Enhancement Project (grant No. JSPH-MA-2021-4).
Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2024-2424/coif). The authors have no conflicts of interest to declare.
Data Sharing Statement
Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2024-2424/dss
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