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 2, April 2024, pages 192-208

The Combination of Afatinib With Dasatinib or Miransertib Results in Synergistic Growth Inhibition of Stomach Cancer Cells

Figure 1. The membrane bound expression level of various growth factor receptors determined by flow cytometry in human stomach cancer cell lines and represented as histograms. EGFR: epidermal growth factor receptor; HER: human epidermal growth factor receptor; c-MET: mesenchymal-epithelial transition factor; ALK: anaplastic lymphoma kinase; CD44: cluster differentiation 44; CD133: cluster differentiation 133; FITC: fluorescein isothiocyanate.

Figure 2. Effect of doubling dilutions of TKIs on the growth inhibition of stomach cancer cell lines. (a) Effect of doubling dilutions of TKI targeting HER family members and CDK inhibitors. (b) Effect of doubling dilutions of TKI targeting downstream signaling pathway inhibitors and cytotoxic agent. Cells were grown in 2% FBS growth medium with or without drugs until control cells (only medium) were confluent. Each point represents the mean ± SD of the triplicate sample. FBS: fetal bovine serum; TKI: tyrosine kinase inhibitor; CDK: cyclin dependent kinase; SD: standard deviation.

Figure 3. Effect of afatinib, dinaciclib, dasatinib, stattic and miransertib with or without ligands (EGF, HB-EGF) on the phosphorylation of EGFR and downstream cell signaling molecules including MAPK, AKT, STAT3, SRC in MKN1 (a) and NCI-N87 (b) cells. The cells were cultured in 10% FBS RPMI-1640 medium to near confluency. Cells were washed once with 0.5% FBS RPMI-1640 medium and incubated with selected agents (400 nM) for 1 h and then stimulated with 30 nM ligands (EGF, HB-EGF and IGF-II) for 15 min. Cells were then lysed, separated using SDS-PAGE, transferred onto PDVF membranes, probed with the antibody of interest and visualized using LI-COR software. FBS: FBS: fetal bovine serum; RPMI-1640: Roswell Park Memorial Institute-1640 medium; EGF: epidermal growth factor; HB-EGF: heparin-binding EGF-like growth factor; IGF: insulin-like growth factor; EGFR: epidermal growth factor receptor; MAPK: mitogen-activated protein kinase; AKT: protein kinase B or PKB; STAT3: signal transducer and activator of transcription 3; SRC: proto-oncogene tyrosine kinase SRC; SDS-PAGE: sodium dodecyl sulphate-polyacrylamide gel electrophoresis.

**Table 1.** The Cell Surface Expression of HER Family Members, c-MET, ALK7 and Cancer Stem Cell Markers Measured by FACS Analysis in Human Stomach Cancer Cell Lines
	Mean fluorescence intensity (MFI)
The data are presented as MFI. HN5, SKBR3 and CaCo2 were used as positive controls. c-MET: hepatocyte growth factor receptor; EGFR: epidermal growth factor receptor; HER: human epidermal growth factor receptor; ALK: anaplastic lymphoma kinase; N/A: not available.
Cell line
HGC-27 (lymph node metastasis)	3.1	12.5	14.3	3.3	3.4	3.7	3.2	67.1	2.2
AGS (primary tumor)	3.3	72.3	14.2	5.6	3.8	6.8	5.8	15.8	3.2
FU97 (primary tumor	3.8	5.2	7.6	6.6	4.3	4.3	4.2	3.2	3.1
MKN1 (lymph node metastasis)	2.9	80.5	9.1	3.7	3.0	4.5	8.0	2281.8	4.8
MKN74 (liver metastasis)	3.6	104.7	15.3	9.5	4.2	5.6	6.5	5.1	3.8
NCI-N87 (liver metastasis)	3.2	46.6	595.9	7.9	3.1	13.2	4.8	18.5	2.6
Controls
HN5	3.1	641.3	N/A	N/A	N/A	N/A	N/A	N/A	N/A
SKBR3	4.0	N/A	226	N/A	N/A	N/A	N/A	N/A	N/A
Caco-2	2.2	N/A	N/A	N/A	N/A	N/A	N/A	47.3	88.2

Table 1. The Cell Surface Expression of HER Family Members, c-MET, ALK7 and Cancer Stem Cell Markers Measured by FACS Analysis in Human Stomach Cancer Cell Lines

Mean fluorescence intensity (MFI)

Control

EGFR

HER-2

HER-3

HER-4

c-Met

ALK7

CD44

CD133

The data are presented as MFI. HN5, SKBR3 and CaCo2 were used as positive controls. c-MET: hepatocyte growth factor receptor; EGFR: epidermal growth factor receptor; HER: human epidermal growth factor receptor; ALK: anaplastic lymphoma kinase; N/A: not available.

Cell line

HGC-27 (lymph node metastasis)

3.1

12.5

14.3

3.3

3.4

3.7

3.2

67.1

2.2

AGS (primary tumor)

3.3

72.3

14.2

5.6

3.8

6.8

5.8

15.8

3.2

FU97 (primary tumor

3.8

5.2

7.6

6.6

4.3

4.2

3.2

3.1

MKN1 (lymph node metastasis)

2.9

80.5

9.1

3.7

3.0

4.5

8.0

2281.8

4.8

MKN74 (liver metastasis)

3.6

104.7

15.3

9.5

4.2

5.6

6.5

5.1

3.8

NCI-N87 (liver metastasis)

3.2

46.6

595.9

7.9

3.1

13.2

4.8

18.5

2.6

Controls

HN5

3.1

641.3

N/A

SKBR3

4.0

N/A

226

N/A

Caco-2

2.2

N/A

47.3

88.2

**Table 2.** IC₅₀ Values for Various Types of HER TKIs, CDK, C-MET, SRC and STAT3 Inhibitors
	IC50 value (µM)
HER: human epidermal growth factor receptor family; CDK: cyclin dependent kinase; TKI: tyrosine kinase inhibitor; FBS: fetal bovine serum; IC₅₀: 50% inhibitory concentration; EGFR: epidermal growth factor receptor; c-MET: hepatocyte growth factor receptor; STAT3: signal transducer and activator of transcription 3.
HGC-27	> 10.00	> 10.00	3.35 ± 0.15	> 10.00	2.4 ± 0.53	2.71 ± 0.69	3.05 ± 0.34	3.26 ± 0.15	2.21 ± 1.55	3.73 ± 1.18	0.009 ± 0.001	0.01 ± 0	> 10.00	> 10.00	> 10.00	> 10.00	0.04 ± 0.001	4.89 ± 1.72	1.67 ± 0.17	1.69 ± 0.18
AGS	7.22 ± 0.88	> 10.00	> 10.00	> 10.00	0.17 ± 0.08	2.43 ± 1.03	0.96 ± 0.08	3.97 ± 0.75	4.52 ± 1.12	> 10.00	0.009 ± 0.000	0.009 ± 0.001	> 10.00	> 10.00	> 10.00	> 10.00	0.03 ± 0.001	0.24 ± 0.31	1.23 ± 0.01	2.29 ± 0.18
FU97	1.48 ± 0.60	2.95 ± 0.84	0.48 ± 0.27	2.48 ± 0.76	0.46 ± 0.12	0.04 ± 0.02	0.26 ± 0.07	0.66 ± 0.13	0.007 ± 0.01	0.05 ± 0.03	0.004 ± 0.00	0.005 ± 0.00	> 10.00	> 10.00	> 10.00	> 10.00	0.002 ± 0.00	0.12 ± 0.008	0.38 ± 0.09	0.83 ± 0.28
MKN1	0.06 ± 0.02	4.19 ± 2.72	0.15 ± 0.06	3.98 ± 0.67	0.03 ± 0.008	0.13 ± 0.06	0.015 ± 0.001	0.86 ± 0.10	5.36 ± 0.81	> 10.00	0.023 ± 0.001	0.003 ± 0.001	> 10.00	> 10.00	> 10.00	> 10.00	0.004 ± 0.001	0.015 ± 0.004	0.77 ± 0.11	1.67 ± 0.22
MKN74	8.87 ± 1.08	> 10.00	1.22 ± 0.22	8.39 ± 0.04	2.06 ± 0.21	2.57 ± 0.18	3.33 ± 0.10	4.32 ± 0.20	4.03 ± 0.13	3.94 ± 0.22	0.011 ± 0.001	0.014 ± 0.001	> 10.00	> 10.00	> 10.00	> 10.00	8.34 ± 0.77	> 10.00	2.21 ± 0.04	2.42 ± 0.46
NCI-N87	0.89 ± 0.17	0.23 ± 0.17	0.004 ± 0.001	0.013 ± 0.001	0.001 ± 0.17	0.001 ± 0.001	0.002 ± 0.19	0.003 ± 0.00	2.90 ± 2.01	7.64 ± 0.17	0.008 ± 0.001	0.001 ± 0.001	> 10.00	> 10.00	> 10.00	> 10.00	0.12 ± 0.001	0.19 ± 0.07	0.87 ± 0.11	1.39 ± 0.05

Table 2. IC₅₀ Values for Various Types of HER TKIs, CDK, C-MET, SRC and STAT3 Inhibitors

IC50 value (µM)

Erlotinib (reversible EGFR inhibitor)

Lapatinib (reversible EGFR/HER2 inhibitor)

Neratinib (irreversible EGFR/HER2 inhibitor)

Afatinib (irreversible EGFR/HER2/HER4 inhibitor)

Palbociclib (CDK4/CDK6 inhibitor)

Dinaciclib (CDK1/CDK2/ CDK5/CDK9 inhibitor)

Ribociclib (CDK4/CDK6 inhibitor)

Capmatinib (c-MET inhibitor)

Dasatinib (Abl/Src/c-Kit inhibitor)

Stattic (STAT3 inhibitor)

FBS 2%

FBS 10%

FBS 2%

FBS 10%

FBS 2%

FBS 10%

FBS 2%

FBS 10%

FBS 2%

FBS 10%

FBS 2%

FBS 10%

FBS 2%

FBS 10%

FBS 2%

FBS 10%

FBS 2%

FBS 10%

FBS 2%

FBS 10%

HER: human epidermal growth factor receptor family; CDK: cyclin dependent kinase; TKI: tyrosine kinase inhibitor; FBS: fetal bovine serum; IC₅₀: 50% inhibitory concentration; EGFR: epidermal growth factor receptor; c-MET: hepatocyte growth factor receptor; STAT3: signal transducer and activator of transcription 3.

HGC-27

> 10.00

3.35 ± 0.15

> 10.00

2.4 ± 0.53

2.71 ± 0.69

3.05 ± 0.34

3.26 ± 0.15

2.21 ± 1.55

3.73 ± 1.18

0.009 ± 0.001

0.01 ± 0

> 10.00

0.04 ± 0.001

4.89 ± 1.72

1.67 ± 0.17

1.69 ± 0.18

AGS

7.22 ± 0.88

> 10.00

0.17 ± 0.08

2.43 ± 1.03

0.96 ± 0.08

3.97 ± 0.75

4.52 ± 1.12

> 10.00

0.009 ± 0.000

0.009 ± 0.001

> 10.00

0.03 ± 0.001

0.24 ± 0.31

1.23 ± 0.01

2.29 ± 0.18

FU97

1.48 ± 0.60

2.95 ± 0.84

0.48 ± 0.27

2.48 ± 0.76

0.46 ± 0.12

0.04 ± 0.02

0.26 ± 0.07

0.66 ± 0.13

0.007 ± 0.01

0.05 ± 0.03

0.004 ± 0.00

0.005 ± 0.00

> 10.00

0.002 ± 0.00

0.12 ± 0.008

0.38 ± 0.09

0.83 ± 0.28

MKN1

0.06 ± 0.02

4.19 ± 2.72

0.15 ± 0.06

3.98 ± 0.67

0.03 ± 0.008

0.13 ± 0.06

0.015 ± 0.001

0.86 ± 0.10

5.36 ± 0.81

> 10.00

0.023 ± 0.001

0.003 ± 0.001

> 10.00

0.004 ± 0.001

0.015 ± 0.004

0.77 ± 0.11

1.67 ± 0.22

MKN74

8.87 ± 1.08

> 10.00

1.22 ± 0.22

8.39 ± 0.04

2.06 ± 0.21

2.57 ± 0.18

3.33 ± 0.10

4.32 ± 0.20

4.03 ± 0.13

3.94 ± 0.22

0.011 ± 0.001

0.014 ± 0.001

> 10.00

8.34 ± 0.77

> 10.00

2.21 ± 0.04

2.42 ± 0.46

NCI-N87

0.89 ± 0.17

0.23 ± 0.17

0.004 ± 0.001

0.013 ± 0.001

0.001 ± 0.17

0.001 ± 0.001

0.002 ± 0.19

0.003 ± 0.00

2.90 ± 2.01

7.64 ± 0.17

0.008 ± 0.001

0.001 ± 0.001

> 10.00

0.12 ± 0.001

0.19 ± 0.07

0.87 ± 0.11

1.39 ± 0.05

**Table 3.** IC₅₀ Values for Growth Factor Receptor Inhibitors, Downstream Signaling Molecules Inhibitors and Cytotoxic Agents
	IC50 value (µM)
TKI: tyrosine kinase inhibitor; FBS: fetal bovine serum; IC₅₀: 50% inhibitory concentration; EGFR: epidermal growth factor receptor; ALK anaplastic lymphoma kinase; CDK: cyclin dependent kinase; PDGFR: platelet-derived growth factor receptor; VEGFR: vascular endothelial growth factor receptor; FGFR: fibroblast growth factor receptor.
	FBS 2%	FBS 10%	FBS 2%	FBS 10%	FBS 2%	FBS 10%	FBS 2%	FBS 10%	FBS 2%	FBS 10%	FBS 2%	FBS 10%	FBS 2%	FBS 10%	FBS 2%	FBS 10%	FBS 2%	FBS 10%
HGC-27	0.37 ± 0.03	1.02 ± 0.18	0.71 ± 0.03	2.46 ± 0.54	2.90 ± 0.45	> 10.00	8.13 ± 1.63	> 10.00	> 10.00	> 10.00	0.08 ± 0.001	0.81 ± 0.55	> 10.00	> 10.00	0.0001 ± 0.05	0.003 ± 0.25	0.07 ± 0.006	0.08 ± 0.004
AGS	0.39 ± 0.03	0.85 ± 0.01	1.05 ± 0.17	2.73 ± 0.16	8.53 ± 2.57	> 10.00	0.005 ± 0.001	0.002 ± 0.00	0.05 ± 0.01	1.53 ± 2.47	0.60 ± 0.06	0.39 ± 0.12	8.34 ± 4.79	> 10.00	0.0001 ± 0.01	0.003 ± 0.06	0.15 ± 0.001	0.12 ± 0.02
FU97	0.015 ± 0.002	0.05 ± 0.003	0.06 ± 0.02	0.26 ± 0.09	0.04 ± 0.007	0.08 ± 0.002	0.03 ± 0.006	0.005 ± 0.001	0.16 ± 0.03	0.30 ± 0.03	0.017 ± 0.004	0.07 ± 0.005	4.56 ± 2.30	> 10.00	0.009 ± 0.36	0.002 ± 0.11	0.04 ± 0.004	0.06 ± 0.003
MKN1	0.09 ± 0.04	0.74 ± 0.07	0.62 ± 0.11	4.18 ± 0.47	4.20 ± 2.91	> 10.00	0.006 ± 0.001	0.014 ± 0.001	0.07 ± 0.08	1.99 ± 0.52	0.15 ± 0.03	0.75 ± 0.75	> 10.00	> 10.00	0.001 ± 0.001	0.002 ± 0.48	0.004 ± 0.12	0.007 ± 0.001
MKN74	1.02 ± 0.20	2.12 ± 0.03	1.24 ± 0.06	2.55 ± 0.31	6.07 ± 0.64	> 10.00	0.008 ± 0.008	1.35 ± 1.06	6.80 ± 0.16	> 10.00	3.51 ± 0.73	7.27 ± 0.96	> 10.00	> 10.00	0.0001 ± 0.001	0.0004 ± 0.001	0.06 ± 0.001	0.07 ± 0.001
NCI-N87	0.34 ± 0.06	1.03 ± 0.02	0.39 ± 0.18	2.11 ± 0.20	3.40 ± 1.22	7.27 ± 1.84	0.003 ± 0.001	0.03 ± 0.003	0.52 ± 0.03	0.47 ± 0.20	0.30 ± 0.05	1.69 ± 0.35	> 10.00	> 10.00	0.008 ± 0.19	0.001 ± 0.001	0.005 ± 0.26	0.001 ± 0.001

Table 3. IC₅₀ Values for Growth Factor Receptor Inhibitors, Downstream Signaling Molecules Inhibitors and Cytotoxic Agents

IC50 value (µM)

Ponatinib (Abl/ PDGFRa/VEGFR2/FGFR1 inhibitor)

Entrectinib (TrkA/B/C/ROS/ALK inhibitor)

AZD4547 (FGFR 1/2/3 inhibitor)

Trametinib (MEK 1/2 inhibitor)

Selumetinib (MEK/ERK1/ERK2 inhibitor)

Miransertib (AKT1/2/3 inhibitor)

Lorlatinib (ALK/Ros1 inhibitor)

Docetaxel (inhibits of depolymerization of microtubules)

Paclitaxel (microtubule polymer stabilizer)

TKI: tyrosine kinase inhibitor; FBS: fetal bovine serum; IC₅₀: 50% inhibitory concentration; EGFR: epidermal growth factor receptor; ALK anaplastic lymphoma kinase; CDK: cyclin dependent kinase; PDGFR: platelet-derived growth factor receptor; VEGFR: vascular endothelial growth factor receptor; FGFR: fibroblast growth factor receptor.

FBS 2%

FBS 10%

FBS 2%

FBS 10%

FBS 2%

FBS 10%

FBS 2%

FBS 10%

FBS 2%

FBS 10%

FBS 2%

FBS 10%

FBS 2%

FBS 10%

FBS 2%

FBS 10%

FBS 2%

FBS 10%

HGC-27

0.37 ± 0.03

1.02 ± 0.18

0.71 ± 0.03

2.46 ± 0.54

2.90 ± 0.45

> 10.00

8.13 ± 1.63

> 10.00

0.08 ± 0.001

0.81 ± 0.55

> 10.00

0.0001 ± 0.05

0.003 ± 0.25

0.07 ± 0.006

0.08 ± 0.004

AGS

0.39 ± 0.03

0.85 ± 0.01

1.05 ± 0.17

2.73 ± 0.16

8.53 ± 2.57

> 10.00

0.005 ± 0.001

0.002 ± 0.00

0.05 ± 0.01

1.53 ± 2.47

0.60 ± 0.06

0.39 ± 0.12

8.34 ± 4.79

> 10.00

0.0001 ± 0.01

0.003 ± 0.06

0.15 ± 0.001

0.12 ± 0.02

FU97

0.015 ± 0.002

0.05 ± 0.003

0.06 ± 0.02

0.26 ± 0.09

0.04 ± 0.007

0.08 ± 0.002

0.03 ± 0.006

0.005 ± 0.001

0.16 ± 0.03

0.30 ± 0.03

0.017 ± 0.004

0.07 ± 0.005

4.56 ± 2.30

> 10.00

0.009 ± 0.36

0.002 ± 0.11

0.04 ± 0.004

0.06 ± 0.003

MKN1

0.09 ± 0.04

0.74 ± 0.07

0.62 ± 0.11

4.18 ± 0.47

4.20 ± 2.91

> 10.00

0.006 ± 0.001

0.014 ± 0.001

0.07 ± 0.08

1.99 ± 0.52

0.15 ± 0.03

0.75 ± 0.75

> 10.00

0.001 ± 0.001

0.002 ± 0.48

0.004 ± 0.12

0.007 ± 0.001

MKN74

1.02 ± 0.20

2.12 ± 0.03

1.24 ± 0.06

2.55 ± 0.31

6.07 ± 0.64

> 10.00

0.008 ± 0.008

1.35 ± 1.06

6.80 ± 0.16

> 10.00

3.51 ± 0.73

7.27 ± 0.96

> 10.00

0.0001 ± 0.001

0.0004 ± 0.001

0.06 ± 0.001

0.07 ± 0.001

NCI-N87

0.34 ± 0.06

1.03 ± 0.02

0.39 ± 0.18

2.11 ± 0.20

3.40 ± 1.22

7.27 ± 1.84

0.003 ± 0.001

0.03 ± 0.003

0.52 ± 0.03

0.47 ± 0.20

0.30 ± 0.05

1.69 ± 0.35

> 10.00

0.008 ± 0.19

0.001 ± 0.001

0.005 ± 0.26

0.001 ± 0.001

**Table 4.** Effect of Various Agents on the Cell Cycle Distribution of Human Stomach Cancer Cell Lines
Cell line/treatment	Sub G1	G0/G1	S	G2/M
Each value is expressed as mean ± SD. SD: standard deviation.
HGC-27
Control	0.17 ± 0.05	65.49 ± 2.24	10.50 ± 4.31	18.87 ± 6.49
Afatinib	1.71 ± 1.84	45.86 ± 12.56	14.32 ± 2.84	35.53 ± 16.45
Dinaciclib	34.36 ± 9.44	25.12 ± 2.98	30.05 ± 10.77	5.33 ± 0.15
Dasatinib	0.37 ± 0.20	58.19 ± 8.75	10.13 ± 0.88	29.02 ± 8.82
Stattic	0.70 ± 0.70	46.01 ± 0.98	12.77 ± 0.78	36.58 ± 1.99
Paclitaxel	31.20 ± 15.22	24.62 ± 1.21	16.47 ± 3.59	23.18 ± 10.97
AGS
Control	3.77 ± 0.28	49.68 ± 1.33	16.90 ± 0.42	23.89 ± 0.25
Afatinib	18.65 ± 6.12	51.16 ± 5.66	10.22 ± 2.73	16.97 ± 3.42
Dinaciclib	36.70 ± 1.02	28.18 ± 0.50	16.21 ± 0.01	16.47 ± 0.93
Dasatinib	33.98 ± 6.68	30.27 ± 3.59	11.87 ± 1.59	14.79 ± 2.02
Stattic	31.62 ± 1.41	35.20 ± 4.85	15.27 ± 2.27	15.09 ± 0.68
Paclitaxel	53.82 ± 3.24	20.95 ± 9.72	12.94 ± 4.62	17.49 ± 0.45
FU97
Control	1.79 ± 0.67	80.85 ± 4.69	6.64 ± 2.09	9.38 ± 2.90
Afatinib	3.31 ± 2.43	82.15 ± 4.90	7.30 ± 3.27	5.87 ± 3.68
Dinaciclib	53.01 ± 2.88	35.57 ± 3.56	6.03 ± 0.11	3.27 ± 0.14
Dasatinib	6.29 ± 4.44	84.68 ± 3.34	2.07 ± 0.51	5.03 ± 0.06
Stattic	5.62 ± 4.13	81.35 ± 1.13	4.79 ± 2.78	6.43 ± 1.50
Paclitaxel	55.28 ± 0.69	17.79 ± 0.99	4.38 ± 0.81	21.13 ± 1.25
MKN1
Control	1.51 ± 0.68	72.33 ± 6.87	8.41 ± 1.22	14.65 ± 3.56
Afatinib	1.82 ± 0.49	78.58 ± 1.47	7.76 ± 1.53	8.63 ± 0.52
Dinaciclib	18.12 ±2.73	5.19 ± 2.57	71.36 ± 4.97	4.11 ± 0.06
Dasatinib	2.33 ± 0.37	75.78 ± 0.75	8.01 ± 1.28	10.23 ± 1.43
Stattic	2.29 ± 0.04	83.23 ± 1.26	6.03 ± 0.24	6.40 ± 0.18
Paclitaxel	10.32 ± 0.34	52.10 ± 2.15	13.67 ± 0.20	21.73 ± 1.59
MKN74
Control	4.99 ± 0.69	53.79 ± 2.41	13.88 ± 3.04	25.20 ± 0.54
Afatinib	8.54 ± 1.78	63.30 ± 1.29	11.16 ± 1.75	15.88 ± 0.86
Dinaciclib	26.75 ± 4.00	47.79 ± 3.20	7.85 ± 2.74	15.11 ± 2.60
Dasatinib	4.52 ± 0.04	64.39 ± 4.00	13.56 ± 0.47	15.43 ± 3.83
Stattic	16.56 ± 1.61	52.37 ± 1.30	11.94 ± 0.53	16.83 ± 2.67
Paclitaxel	50.35 ± 1.63	13.35 ± 0.05	13.31 ± 1.87	18.07 ± 1.24
NCI-N87
Control	2.09 ± 0.16	56.23 ± 2.75	15.71 ± 3.40	22.41 ± 1.63
Afatinib	8.15 ± 1.97	56.54 ± 3.95	10.66 ± 3.81	18.63 ± 0.11
Dinaciclib	52.70 ± 4.74	37.20 ± 2.65	2.72 ± 0.13	4.35 ± 0.70
Dasatinib	8.10 ± 3.78	62.53 ± 4.48	7.28 ± 0.86	14.91 ± 1.27
Stattic	11.95 ± 5.08	55.87 ± 7.33	10.33 ± 5.08	17.04 ± 8.41
Paclitaxel	39.35 ± 7.01	21.15 ± 7.60	13.28 ± 3.00	17.64 ± 9.47

Table 4. Effect of Various Agents on the Cell Cycle Distribution of Human Stomach Cancer Cell Lines

Cell line/treatment

Sub G1

G0/G1

G2/M

Each value is expressed as mean ± SD. SD: standard deviation.

HGC-27

Control

0.17 ± 0.05

65.49 ± 2.24

10.50 ± 4.31

18.87 ± 6.49

Afatinib

1.71 ± 1.84

45.86 ± 12.56

14.32 ± 2.84

35.53 ± 16.45

Dinaciclib

34.36 ± 9.44

25.12 ± 2.98

30.05 ± 10.77

5.33 ± 0.15

Dasatinib

0.37 ± 0.20

58.19 ± 8.75

10.13 ± 0.88

29.02 ± 8.82

Stattic

0.70 ± 0.70

46.01 ± 0.98

12.77 ± 0.78

36.58 ± 1.99

Paclitaxel

31.20 ± 15.22

24.62 ± 1.21

16.47 ± 3.59

23.18 ± 10.97

AGS

Control

3.77 ± 0.28

49.68 ± 1.33

16.90 ± 0.42

23.89 ± 0.25

Afatinib

18.65 ± 6.12

51.16 ± 5.66

10.22 ± 2.73

16.97 ± 3.42

Dinaciclib

36.70 ± 1.02

28.18 ± 0.50

16.21 ± 0.01

16.47 ± 0.93

Dasatinib

33.98 ± 6.68

30.27 ± 3.59

11.87 ± 1.59

14.79 ± 2.02

Stattic

31.62 ± 1.41

35.20 ± 4.85

15.27 ± 2.27

15.09 ± 0.68

Paclitaxel

53.82 ± 3.24

20.95 ± 9.72

12.94 ± 4.62

17.49 ± 0.45

FU97

Control

1.79 ± 0.67

80.85 ± 4.69

6.64 ± 2.09

9.38 ± 2.90

Afatinib

3.31 ± 2.43

82.15 ± 4.90

7.30 ± 3.27

5.87 ± 3.68

Dinaciclib

53.01 ± 2.88

35.57 ± 3.56

6.03 ± 0.11

3.27 ± 0.14

Dasatinib

6.29 ± 4.44

84.68 ± 3.34

2.07 ± 0.51

5.03 ± 0.06

Stattic

5.62 ± 4.13

81.35 ± 1.13

4.79 ± 2.78

6.43 ± 1.50

Paclitaxel

55.28 ± 0.69

17.79 ± 0.99

4.38 ± 0.81

21.13 ± 1.25

MKN1

Control

1.51 ± 0.68

72.33 ± 6.87

8.41 ± 1.22

14.65 ± 3.56

Afatinib

1.82 ± 0.49

78.58 ± 1.47

7.76 ± 1.53

8.63 ± 0.52

Dinaciclib

18.12 ±2.73

5.19 ± 2.57

71.36 ± 4.97

4.11 ± 0.06

Dasatinib

2.33 ± 0.37

75.78 ± 0.75

8.01 ± 1.28

10.23 ± 1.43

Stattic

2.29 ± 0.04

83.23 ± 1.26

6.03 ± 0.24

6.40 ± 0.18

Paclitaxel

10.32 ± 0.34

52.10 ± 2.15

13.67 ± 0.20

21.73 ± 1.59

MKN74

Control

4.99 ± 0.69

53.79 ± 2.41

13.88 ± 3.04

25.20 ± 0.54

Afatinib

8.54 ± 1.78

63.30 ± 1.29

11.16 ± 1.75

15.88 ± 0.86

Dinaciclib

26.75 ± 4.00

47.79 ± 3.20

7.85 ± 2.74

15.11 ± 2.60

Dasatinib

4.52 ± 0.04

64.39 ± 4.00

13.56 ± 0.47

15.43 ± 3.83

Stattic

16.56 ± 1.61

52.37 ± 1.30

11.94 ± 0.53

16.83 ± 2.67

Paclitaxel

50.35 ± 1.63

13.35 ± 0.05

13.31 ± 1.87

18.07 ± 1.24

NCI-N87

Control

2.09 ± 0.16

56.23 ± 2.75

15.71 ± 3.40

22.41 ± 1.63

Afatinib

8.15 ± 1.97

56.54 ± 3.95

10.66 ± 3.81

18.63 ± 0.11

Dinaciclib

52.70 ± 4.74

37.20 ± 2.65

2.72 ± 0.13

4.35 ± 0.70

Dasatinib

8.10 ± 3.78

62.53 ± 4.48

7.28 ± 0.86

14.91 ± 1.27

Stattic

11.95 ± 5.08

55.87 ± 7.33

10.33 ± 5.08

17.04 ± 8.41

Paclitaxel

39.35 ± 7.01

21.15 ± 7.60

13.28 ± 3.00

17.64 ± 9.47

**Table 5.** Combination Index Values of Afatinib When Combined With Palbociclib, Dinaciclib, Capmatinib, Dasatinib, Stattic, Ponatinib, AZD4547, Trametinib, Miransertib, Paclitaxel in Human Stomach Cancer Cell Lines
	Combination index (CI)
TKI: tyrosine kinase inhibitor; FBS: fetal bovine serum.
TKI combination	FBS 2%	FBS 10%	FBS 2%	FBS 10%	FBS 2%	FBS 10%
Afatinib + palbociclib	0.36	1.02	0.38	1.89	1.07	1.31
Afatinib + dinaciclib	0.95	1.08	0.42	0.53	1.58	2.23
Afatinib + capmatinib	0.62	0.85	0.24	0.48	1.55	1.28
Afatinib+ dasatinib	0.48	0.46	0.37	0.23	1.02	0.90
Afatinib + stattic	0.87	0.86	0.87	0.68	1.06	2.60
Afatinib+ ponatinib	2.51	1.23	0.45	0.43	2.63	1.19
Afatinib + AZD4547	1.32	1.60	0.20	1.14	1.69	1.14
Afatinib+ trametinib	0.37	1.11	0.40	1.16	1.22	0.8
Afatinib + miransertib	0.60	0.82	0.26	0.16	0.76	0.71
Afatinib + paclitaxel	0.07	1.20	1.33	0.41	1.64	2.04

Table 5. Combination Index Values of Afatinib When Combined With Palbociclib, Dinaciclib, Capmatinib, Dasatinib, Stattic, Ponatinib, AZD4547, Trametinib, Miransertib, Paclitaxel in Human Stomach Cancer Cell Lines

Combination index (CI)

AGS

MKN1

NCI-N87

TKI: tyrosine kinase inhibitor; FBS: fetal bovine serum.

TKI combination

FBS 2%

FBS 10%

FBS 2%

FBS 10%

FBS 2%

FBS 10%

Afatinib + palbociclib

0.36

1.02

0.38

1.89

1.07

1.31

Afatinib + dinaciclib

0.95

1.08

0.42

0.53

1.58

2.23

Afatinib + capmatinib

0.62

0.85

0.24

0.48

1.55

1.28

Afatinib+ dasatinib

0.48

0.46

0.37

0.23

1.02

0.90

Afatinib + stattic

0.87

0.86

0.87

0.68

1.06

2.60

Afatinib+ ponatinib

2.51

1.23

0.45

0.43

2.63

1.19

Afatinib + AZD4547

1.32

1.60

0.20

1.14

1.69

1.14

Afatinib+ trametinib

0.37

1.11

0.40

1.16

1.22

0.8

Afatinib + miransertib

0.60

0.82

0.26

0.16

0.76

0.71

Afatinib + paclitaxel

0.07

1.20

1.33

0.41

1.64

2.04

**Table 6.** Linear Regression Analysis of the Expression of Various Receptors Against the Sensitivity of Human Stomach Cancer Cell Lines to Treatment With Various TKIs, CDK Inhibitors, STAT3 Inhibitor and a Cytotoxic Agent
Drugs/cell surface markers	2%	10%
TKI: tyrosine kinase inhibitor; EGFR: epidermal growth factor receptor; CDK: cyclin dependent kinase; HER: human epidermal growth factor receptor; STAT3: signal transducer and activator of transcription 3.
Erlotinib	0.013 (0.830)	0.173 (0.412)	0.258 (0.303)	0.089 (0.566)	0.448 (0.146)	0.050 (0.671)
Lapatinib	0.015 (0.929)	0.099 (0.544)	0.90 (0.564)	0.052 (0.663)	0.436 (0.153)	0.041 (0.701)
Neratinib	0.003 (0.916)	0.142 (0.461)	0.130 (0.483)	0.074 (0.602)	0.206 (0.366)	0.170 (0.417)
Afatinib	0.028 (0.753)	0.157 (0.537)	0.154 (0.442)	0.166 (0.422)	0.308 (0.253)	0.115 (0.511)
Palbociclib	0.737 (0.029)	0.004 (0.905)	0.311 (0.250)	0.330 (0.233)	0.048 (0.678)	0.255 (0.307)
Dinaciclib	0.347 (0.218)	0.038 (0.710)	0.877 (0.006)	0.098 (0.545)	0.355 (0.212)	0.161 (0.431)
Dasatinib	0.399 (0.179)	0.019 (0.797)	0.051 (0.667)	0.143 (0.460)	0.076 (0.596)	0.092 (0.558)
Stattic	0.234 (0.331)	0.050 (0.671)	0.092 (0.560)	0.631 (0.059)	0.068 (0.618)	0.001 (0.943)
Ponatinib	0.384 (0.190)	0.001 (0.952)	0.153 (0.443)	0.490 (0.121)	0.003 (0.918)	0.028 (0.752)
Entrectinib	0.585 (0.076)	0.101 (0.540)	0.004 (0.900)	0.448 (0.146)	0.010 (0.850)	0.495 (0.119)
AZD4547	0.661 (0.066)	0.057 (0.649)	0.307 (0.254)	0.413 (0.169)	0.004 (0.901)	0.072 (0.607)
Trametinib	0.259 (0.302)	0.038 (0.710)	0.031 (0.737)	0.185 (0.394)	0.050 (0.671)	0.043 (0.693)
Selumetinib	0.006 (0.883)	0.093 (0.557)	0.86 (0.574)	0.032 (0.734)	0.135 (0.473)	0.042 (0.697)
Miransertib	0.490 (0.121)	0.027 (0.754)	0.056 (0.651)	0.001 (0.966)	0.722 (0.032)	0.231 (0.335)
Docetaxel	0.008 (0.965)	0.980 (< 0.001)	0.16 (0.812)	0.182 (0.399)	0.549 (0.092)	0.007 (0.915)
Paclitaxel	0.014 (0.821)	0.182 (0.399)	0.219 (0.349)	0.000 (0.995)	0.333 (0.230)	0.266 (0.295)

Table 6. Linear Regression Analysis of the Expression of Various Receptors Against the Sensitivity of Human Stomach Cancer Cell Lines to Treatment With Various TKIs, CDK Inhibitors, STAT3 Inhibitor and a Cytotoxic Agent

Drugs/cell surface markers

10%

EGFR

HER2

CD44

EGFR

HER2

CD44

R² (P value)

TKI: tyrosine kinase inhibitor; EGFR: epidermal growth factor receptor; CDK: cyclin dependent kinase; HER: human epidermal growth factor receptor; STAT3: signal transducer and activator of transcription 3.

Erlotinib

0.013 (0.830)

0.173 (0.412)

0.258 (0.303)

0.089 (0.566)

0.448 (0.146)

0.050 (0.671)

Lapatinib

0.015 (0.929)

0.099 (0.544)

0.90 (0.564)

0.052 (0.663)

0.436 (0.153)

0.041 (0.701)

Neratinib

0.003 (0.916)

0.142 (0.461)

0.130 (0.483)

0.074 (0.602)

0.206 (0.366)

0.170 (0.417)

Afatinib

0.028 (0.753)

0.157 (0.537)

0.154 (0.442)

0.166 (0.422)

0.308 (0.253)

0.115 (0.511)

Palbociclib

0.737 (0.029)

0.004 (0.905)

0.311 (0.250)

0.330 (0.233)

0.048 (0.678)

0.255 (0.307)

Dinaciclib

0.347 (0.218)

0.038 (0.710)

0.877 (0.006)

0.098 (0.545)

0.355 (0.212)

0.161 (0.431)

Dasatinib

0.399 (0.179)

0.019 (0.797)

0.051 (0.667)

0.143 (0.460)

0.076 (0.596)

0.092 (0.558)

Stattic

0.234 (0.331)

0.050 (0.671)

0.092 (0.560)

0.631 (0.059)

0.068 (0.618)

0.001 (0.943)

Ponatinib

0.384 (0.190)

0.001 (0.952)

0.153 (0.443)

0.490 (0.121)

0.003 (0.918)

0.028 (0.752)

Entrectinib

0.585 (0.076)

0.101 (0.540)

0.004 (0.900)

0.448 (0.146)

0.010 (0.850)

0.495 (0.119)

AZD4547

0.661 (0.066)

0.057 (0.649)

0.307 (0.254)

0.413 (0.169)

0.004 (0.901)

0.072 (0.607)

Trametinib

0.259 (0.302)

0.038 (0.710)

0.031 (0.737)

0.185 (0.394)

0.050 (0.671)

0.043 (0.693)

Selumetinib

0.006 (0.883)

0.093 (0.557)

0.86 (0.574)

0.032 (0.734)

0.135 (0.473)

0.042 (0.697)

Miransertib

0.490 (0.121)

0.027 (0.754)

0.056 (0.651)

0.001 (0.966)

0.722 (0.032)

0.231 (0.335)

Docetaxel

0.008 (0.965)

0.980 (< 0.001)

0.16 (0.812)

0.182 (0.399)

0.549 (0.092)

0.007 (0.915)

Paclitaxel

0.014 (0.821)

0.182 (0.399)

0.219 (0.349)

0.000 (0.995)

0.333 (0.230)

0.266 (0.295)