World J Oncol
World Journal of Oncology, ISSN 1920-4531 print, 1920-454X online, Open Access
Article copyright, the authors; Journal compilation copyright, World J Oncol and Elmer Press Inc
Journal website https://www.wjon.org

Original Article

Volume 15, Number 3, June 2024, pages 492-505

Synergistic Effects of Neratinib in Combination With Palbociclib or Miransertib in Brain Cancer Cells

Figures

Figure 1.
Figure 1. The expression levels of various growth factor receptors in human brain cancer cell lines determined by flow cytometry and represented as histograms. EGFR: epidermal growth factor receptor; HER: human epidermal growth factor receptor; c-MET: mesenchymal-epithelial transition factor; ALK7: anaplastic lymphoma kinase 7: CD44: cluster differentiation 44.
Figure 2.
Figure 2. Effect of doubling dilutions of various agents targeting HER family members and other cell signaling molecules on the growth of brain cancer cells when cultured in medium containing 2% FBS. Tumor cells that were examined when grown in control wells (i.e., only medium) were confluent. Each point represents the mean ± SD of the triplicate sample. FBS fetal bovine serum; HER: human epidermal growth factor receptor; SD: standard deviation.
Figure 3.
Figure 3. Effects of tyrosine kinase inhibitors (TKIs) on cell signaling on brain tumor cell line A172 in the presence or absence of ligands. The cells were cultured in 10% FBS DMEM medium to near confluency. Cells were washed once with 0.5% FBS DMEM medium and incubated with selected agents (400 nM) for 1 h and then stimulated with 30 nM ligands (EGF or HB-EGF) for 15 min. Cells were then lysed, separated using SDS-PAGE, transferred onto PVDF membranes, probed with the antibodies of interest, and visualized using LI-COR software. DMEM: Dulbecco’s modified Eagles medium; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis; PVDF: polyvinylidene fluoride; EGF: epidermal growth factor; HB-EGF: heparin-binding EGF-like growth factor; EGFR: epidermal growth factor receptor; MAPK: mitogen-activated protein kinase; AKT: serine/threonine protein kinase; STAT3: signal transducer and activator of transcription 3; SRC: proto-oncogene tyrosine kinase SRC.
Figure 4.
Figure 4. The effect of various agents on migration of brain cancer cell line A172. Migration is determined using the IncuCyte Clear View 96 well IncuCyte Chemotaxis system. Cells were seeded into the top layer of a 96-well cell migration assay plate in 0.5% FBS DMEM together with TKIs at IC50 concentrations, while 10% FBS DMEM (chemoattractant) was added to the bottom layer. Cells were incubated at 37 °C for 48 h, with images taken from chamber wells and were analyzed every 3 h for 48 h using the IncuCyte chemotaxis software. Most TKIs inhibited migration of A172 cell line, to varying degrees. Each point is a representative of the mean ± SD of triplicate samples. NS: not significant; IC50: 50% inhibitory concentration; TKIs: tyrosine kinase inhibitors; SD: standard deviation; FBS fetal bovine serum; DMEM: Dulbecco’s modified Eagles medium.

Tables

Table 1. Surface Expression of Various Growth Factor Receptors in Human Brain Cancer Cell Line (Determined by Flow Cytometry)
 
Cell lineMean fluorescence intensity (MFI)
ControlEGFRHER2HER3HER4C-METALK-7CD44
The data are presented as the mean fluorescence intensity (MFI) ± standard deviation (SD) of gated events. N/A: not available. ALK anaplastic lymphoma kinase; c-MET: hepatocyte growth factor receptor; CD: cluster differentiation; EGFR: epidermal growth factor receptor; HER: human epidermal growth factor receptor.
LN-183.732.98.84.23.95.84.43.9
U118MG3.528.55.83.94.05.43.91,354.6
A1723.043.69.43.53.47.73.81,439.0
T-98G3.233.88.93.53.65.93.63,259.8
HN52.91071.7N/AN/AN/AN/AN/AN/A
SKOV33.8N/A233.3N/AN/AN/AN/AN/A
CaCo22.4N/AN/AN/AN/AN/AN/A49.51

 

Table 2. IC50 Values of Various Agents on HBCCLs as Assessed by SRB Colorimetric Assay: (A) HER-Family Targeting TKIs and Other Downstream Signaling Molecules and (B) Other TKIs and Chemotherapeutic Agents
 
IC50 value (µM)% FBSLN-18A172U118MGT-98G
Each value is the mean of triplicate samples. IC50: 50% inhibitory concentration; SRB: sulforhodamine B; TKIs: tyrosine kinase inhibitors; STAT: signal transducer and activator of transcription; Abl: Abelson murine leukemia viral oncogene homolog; AKT: serine/threonine protein kinase; ALK anaplastic lymphoma kinase; c-MET: hepatocyte growth factor receptor; CDK: cyclin dependent kinase; EGFR: epidermal growth factor receptor; EPGN: epithelial mitogen; FBS fetal bovine serum; FGFR: fibroblast growth factor receptor; HBCCLs: human brain cancer cell lines; HER: human epidermal growth factor receptor; PDGFR: platelet-derived growth factor receptor; VEGFR: vascular endothelial growth factor.
A
  Erlotinib (EGFR inhibitor)2%6.4210.007.456.84
10%> 10.00> 10.00> 10.00> 10.00
  Lapatinib (EGFR/HER2 inhibitor)2%1.0810.005.603.25
10%8.5510.006.3910.00
  Neratinib (EGFR/HER2/HER4 inhibitor)2%0.300.350.330.69
10%1.551.120.441.94
  Afatinib (EGFR/HER2/HER4 inhibitor)2%1.111.241.491.85
10%2.972.501.424.04
  Palbociclib (CDK4/CDK6 inhibitor)2%1.941.160.450.56
10%2.074.692.464.78
  Dinaciclib (CDK1/CDK2/ CDK5/CDK9)2%0.0060.0040.0140.013
10%0.0080.0030.01250.010
  Ribociclib (CDK4/CDK6 inhibitor)2%1.625.761.435.50
10%5.804.131.00> 10.00
  Capmatinib (C-MET inhibitor)2%> 10.00> 10.004.22> 10.00
10%> 10.00> 10.00> 10.00> 10.00
  Dasatinib (Abl/Src/c-Kit)2%0.0410.040.010.01
10%1.802.960.100.06
  Stattic (STAT3 inhibitor)2%1.210.611.139.68
10%3.760.721.00> 10.00
B
  Ponatinib (Abl/ PDGFRα/VEGFR2/FGFR1 inhibitor)2%0.100.030.400.19
10%0.410.190.880.42
  Entrectinib (TrkA/B/C/ROS/ALK inhibitor)2%0.110.781.190.95
10%2.952.852.933.47
  AZD4547 (FGFR 1/2/3 inhibitor)2%1.610.014.900.39
10%6.622.684.103.83
  Trametinib (MEK 1/2 inhibitor)2%0.020.010.170.12
10%6.290.1050.04>10.00
  Selumetinib (MEK/ERK1/ERK2 inhibitor)2%> 10.006.72> 10.00>10.00
10%8.404.394.20>10.00
  Miransertib (AKT1/2/3 inhibitor)2%0.603.035.252.56
10%3.258.171.669.90
  Lorlatinib (ALK/Ros1 inhibitor)2%>10.009.50> 10.08.76
10%8.918.797.20> 10.00
  Docetaxel (depolymerisation of microtubules)2%1.970.6920.111.04
10%0.0021.7971.441.25
  Paclitaxel (microtubule polymer stabiliser)2%0.120.0150.0180.01
10%0.030.0390.0020.03

 

Table 3. Effect on Cell Cycle Distribution on LN-18, U118MG, A172 and T-98G Cells Following Treatment With HER-Family Inhibitor Neratinib in Combination With Various Agents
 
Cell linesCell cycle phase (% of gated cells)
TreatmentSub G1G0/G1SG2/M
Each value is expressed as mean ± standard deviation (SD). HER: human epidermal growth factor receptor.
LN-18Control0.8 ± 0.0273.7 ± 3.99.6 ± 0.915.07 ± 4.7
Neratinib11.9 ± 1.171.59 ± 2.86.2 ± 0.98.8 ± 2.3
Dinaciclib51.3 ± 7.533.5 ± 0.24.4 ± 1.19.7 ± 4.8
Dasatinib3.9 ± 1.182.8 ± 8.29.0 ± 0.810.5 ± 0.4
Stattic98.8 ± 0.30.81 ± 0.140.5 ± 0.010.15 ± 0.05
Paclitaxel11.63 ± 3.959.6 ± 10.715.9 ± 1.913.3 ± 1.5
U118MGControl6.9 ± 1.176.9 ± 6.912.2 ± 7.93.6 ± 0.7
Neratinib5.6 ± 4.484.4 ± 0.66.05 ± 3.63.5 ± 1.0
Dinaciclib42.7 ± 18.350.1 ± 13.35.4 ± 4.91.4 ± 0.3
Dasatinib8.2 ± 4.488.3 ± 3.12.1 ± 0.71.0 ± 0.7
Stattic37.5 ± 29.354.1 ± 19.88.1 ± 9.61.5 ± 1.7
Paclitaxel22.1 ± 9.870.3 ± 13.210.4 ± 7.52.9 ± 0.05
A172Control2.8 ± 0.990.4 ± 1.84.1 ± 0.32.5 ± 0.01
Neratinib45.1 ± 34.136.3 ± 13.318.5 ± 21.72.2 ± 2.2
Dinaciclib67.7 ± 7.325.8 ± 1.087.3 ± 7.70.2 ± 0.3
Dasatinib12.6 ± 5.672.3 ± 10.616.5 ± 20.51.3 ± 0.1
Stattic9.1 ± 9.682.2 ± 4.56.8 ± 5.52.7 ± 1.4
Paclitaxel55.6 ± 13.625.2 ± 6.412.4 ± 4.96.0- ± 3.2
T-98GControl1.9 ± 1.0488.9 ± 2.36.7 ± 1.52.6 ± 0.4
Neratinib1.67 ± 0.783.1 ± 6.812.9 ± 8.23.2 ± 0.5
Dinaciclib28.8 ± 0.53.2 ± 0.075.07 ± 0.0259.7 ± 0.6
Dasatinib14.1 ± 3.370.0 ± 2.013.4 ± 5.63.5 ± 0.9
Stattic48.3 ± 65.741.5 ± 52.78.9 ± 12.21.5 ± 1.9
Paclitaxel64.1 ± 18.825.8 ± 13.97.6 ± 2.91.9 ± 1.3

 

Table 4. The Effect of Treatment With the Pan-HER Family Inhibitor Neratinib When Used In Combination With Other Drugs on Cell Lines LN-18, A172 and T-98G
 
Drug combinationCombination index mean (range)
LN-18A172T-98G
10%2%10%2%10%2%
Combination Index < 0.9 = synergistic effect, 0.9 - 1.1 = additive effect, > 1.1 = antagonistic. HER: human epidermal growth factor receptor.
NeratinibPalbociclib0.430.180.680.450.340.88
Dinaciclib3.781.141.121.060.840.61
Capmatinib0.280.351.471.150.600.78
Dasatinib0.751.781.820.710.750.88
Stattic0.830.710.721.29.170.78
Ponatinib0.630.973.10.970.990.85
AZD45471.010.251.10.080.580.18
Trametinib0.890.771.021.150.910.26
Miransertib0.240.520.590.700.910.21
Paclitaxel0.341.441.270.871.370.85

 

Table 5. Linear Regression Analysis of the Expression of Various Receptors Against the Sensitivity of Human Brain Cancer Cell Lines to Treatment With Various TKIs, CDK Inhibitors, STAT3 Inhibitor and Cytotoxic Agents
 
Drugs/cell surface markers2%10%
EGFRHER2CD44EGFRHER2CD44
R2 (P value)R2 (P value)R2 (P value)R2 (P value)R2 (P value)R2 (P value)
N/A: not available; TKIs: tyrosine kinase inhibitors; STAT: signal transducer and activator of transcription; CDK: cyclin dependent kinase; EGFR: epidermal growth factor receptor; HER: human epidermal growth factor receptor; CD44: cluster differentiation 44.
Erlotinib0.784 (0.114)0.717 (0.153)0.221 (0.530)N/AN/AN/A
Lapatinib0.901 (0.051)0.634 (0.204)0.070 (0.735)0.019 (0.863)0.528 (0.273)0.125 (0.647)
Neratinib0.164 (0.595)0.069 (0.737)0.372 (0.390)0.531 (0.271)0.023 (0.850)0.004 (0.941)
Afatinib0.042 (0.796)0.011 (0.897)0.775 (0.120)0.461 (0.321)0.047 (0.783)0.004 (0.939)
Palbociclib0.107 (0.673)0.031 (0.824)0.694 (0.167)0.065 (0.745)0.796 (0.108)0.004 (0.939)
Dinaciclib0.023 (0.849)0.811 (0.099)0.064 (0.746)0.543 (0.263)0.003 (0.947)0.102 (0.681)
Ribociclib0.049 (0.778)0.837 (0.085)0.007 (0.916)0.476 (0.310)0.036 (0.810)0.021 (0.855)
Dasatinib0.226 (0.525)0.460 (0.322)0.814 (0.098)0.152 (0.610)0.275 (0.475)0.904 (0.032)
Stattic0.297 (0.455)0.012 (0.891)0.405 (0.364)0.560 (0.251)0.002 (0.961)0.251 (0.499)
Ponatinib0.002 (0.958)0.328 (0.428)0.656 (0.190)0.000 (0.985)0.479 (0.308)0.475 (0.311)
Entrectinib0.272 (0.479)0.066 (0.743)0.507 (0.288)0.128 (0.643)0.126 (0.644)0.288 (0.464)
AZD45750.023 (0.850)0.481 (0.307)0.212 (0.539)0.557 (0.254)0.692 (0.168)0.024 (0.846)
Trametinib0.000 (0.992)0.145 (0.619)0.904 (0.049)0.813 (0.098)0.039 (0.803)0.090 (0.700)
SelumetinibN/AN/AN/A0.856 (0.075)0.051 (0.773)0.052 (0.771)
Miransertib0.455 (0.325)0.008 (0.909)0.308 (0.445)0.001 (0.963)0.610 (0.219)0.000 (0.994)
Lorlatinib0.047 (0.784)0.303 (0.450)0.139 (0.627)0.310 (0.443)0.136 (0.631)0.005 (0.927)
Docetaxel0.222 (0.529)0.194 (0.560)0.086 (0.707)0.009 (0.953)0.634 (0.204)0.064 (0.747)
Paclitaxel0.872 (0.066)0.218 (0.533)0.024 (0.846)0.352 (0.407)0.308 (0.445)0.291 (0.461)

 

　

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