Targeted inhibition of mammalian target of rapamycin (mTOR) enhances radiosensitivity in pancreatic carcinoma cells

Figures & data

Table 1 Primer sequences and annealing temperature of the genes in RT-PCR

Gene	Gene sequences	Product size (bp)	Annealing temperature (°C)
DDB1	Forward primer 5′-ATCATCCGGAATGGAATTGGAA-3′ Forward primer 5′-TCAGACCGCAGTGGCCATAA-3′	83	60
RAD51	Forward primer 5′-TGGGAACTGCAACTCATCTGG-3′ Forward primer 5′-GCGCTCCTCTCTCCAGCAG-3′	157	61
XRCC5	Forward primer 5′-GCCATATCAGTGAACCTTTAGAGAC-3′ Forward primer 5′-GGAACTGGAACTCAAGGCAAG-3′	268	59
PCNA	Forward primer 5′-CTGTAGCGGCGTTGT-3′ Reverse primer 5′-ACTTTCTCCTGGTTTGG-3′	362	52
ABCC4	Forward primer 5′-GCTCACTGGATTGTCTTCATTTTC-3′ Reverse primer 5′-CTCGGTTACATTTCCTCCTCCAT-3′	147	59
β-actin	Forward primer 5′-CCTGAGGCTCTTTTCCAGCC-3′ Reverse primer 5′-TAGAGGTCTTTACGGATGTCAACGT-3′	110	60

Notes: Five genes, including DDB1, RAD51, XRCC5, PCNA, and ABCC4 were confirmed to their expression, in microarray analysis using RT- PCR, and were significantly expressed after RPM treatment in PC-2 cells. The primer pairs and the predicted sizes of the amplified PCR products and the annealing temperatures of PCR were listed. β-actin is the housekeeping gene for RT- PCR analysis. The primer sequences were designed with Primer Premier 5.0 software (Premier Biosoft, Palo Alto, CA, USA).

Abbreviations: PCR, polymerase chain reaction; RPM, rapamycin; RT, reverse transcription.

Figure 1 Growth inhibiting effects of RPM on pancreatic cancer cell lines PC-2 and PANC-1.

Notes: Pancreatic cancer cells were treated with RPM (0, 10, 20, 30, 40, 50, 60, and 70 nmol/L) for 48 hours. Cell viability was determined by the MTT method. This assay was performed in triplicate. A dose-dependent inhibition of cell growth could be observed (P < 0.05, ANOVA analysis).
Abbreviations: ANOVA, analysis of variance; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; RPM, rapamycin.

Figure 2 Effect of radiation plus RPM on cell viability of pancreatic cancer cells with MTT assay, in (A) PC-2 cells and (B) PANC-1 cells.

Notes: After 4 Gy X-ray irradiation, cell viability was determined by the MTT method. This assay was performed in triplicate. in the 10 nmol/L and the 15 nmol/L RPM treatment groups, cell survival was significantly inhibited compared with the control group. (P < 0.05, ANOVA analysis.)
Abbreviation: ANOVA, analysis of variance; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; RPM, rapamycin.

Figure 3 Survival fraction of pancreatic cancer cells treated by different dose of irradiation (A) PC-2 cells; (B) PANC-1 cells.

Notes: Pancreatic cancer cells were treated with different concentrations for 6 hours before radiation. The radiosensitivity of pancreatic cancer cells was determined by a colony formation assay. The multitarget click model in GraphPad Prism 5.0 (GraphPad Software Inc, San Diego, CA, USA) was used to fit the cell survival curves. This assay was performed in triplicate. The radiosensitizing effect was observed in the 10 nmol/L and 15 nmol/L RPM treatment groups.
Abbreviation: RPM, rapamycin.

Figure 4 MDC-labeled autophagic vacuoles in PC-2 cells by fluorescence microscope after RPM treatment. Autophagic vacuoles were labeled with 0.05 mmol/L MDC in PBS, at 37°C for 10 minutes. (A) 0 nmol/L RPM group; (B) 10 nmol/L RPM group; and (C) 15 nmol/L RPM group (×200).

Abbreviations: MDC, monodansylcadaverine; PBS, phosphate-buffered saline; RPM, rapamycin.

Figure 5 Effects of radiation plus RPM on the cell cycle of PC-2 cells, under FCM.

Notes: The cell cycle distributions in PC-2 cells were determined using PI staining and FCM analysis, after RPM treatment or radiation plus RPM for 24 hours. (1) blank control group; (2) 5 nmol/L RPM group; (3) 10 nmol/L RPM group; (4) 15 nmol/L RPM group; (5) radiation alone group; (6) radiation plus 5 nmol/L RPM group; (7) radiation plus 10 nmol/L RPM group; and (8) radiation plus 15 nmol/L RPM group. The results presented were representative of three independent experiments. *P < 0.05, **P < 0.01 versus the control group; ^#
P < 0.05, ^##
P < 0.01 versus the radiation alone group.
Abbreviations: FCM, flow cytometry; PI, propidium iodide; RPM, rapamycin.

Table 2 The DNA damage repair and transcription genes differentially expressed in PC-2 cells induced by RPM

Genbank ID	Gene description	Ratio (Cy5/Cy3)
NM_016931	Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4)	0.16
NM-001923	Damage-specific DNA binding protein (DDB1)	0.20
NM_018682	Myeloid/lymphoid or mixed-lineage leukemia 5 (trithorax homolog, Drosophila) (MLL5)	0.21
NM_013974	Dimethylarginine dimethylaminohydrolase 2 (DDAH2)	0.23
NM_001675	Activating transcription factor 4 (tax-responsive enhancer element B67) (ATF4)	0.27
NM_003489	Nuclear receptor interacting protein 1 (NRIP1)	0.28
BC049823	Ribosomal protein L22-like 1 (RPL22)	0.30
NM_175623	RAB3A interacting protein (rabin3) (RAB3IP), transcript variant alpha 2	0.32
BC022217	Chromosome 6 open reading frame 85(C6ORF85)	0.33
NM-005431	X-ray repair complementing defective repair in Chinese hamster cells 2 (XRCC2)	0.33
NM_000689	Aldehyde dehydrogenase 1 family, member A1 (ALDH1A1)	0.36
NM_002876	RAD51 homolog C (S. cerevisiae) (RAD51C), transcript variant 2	0.37
NM_001008712	RNA binding protein with multiple splicing (RBPMS), transcript variant 3	0.38
NM_139207	Nucleosome assembly protein 1-like 1 (NAP1L1)	0.39
NM_000075	Cyclin-dependent kinase 4 (CDK4)	0.40
NM_053277	Chloride intracellular channel 6 (CLIC6)	0.40
NM-021141	X-ray repair complementing defective repair in hamster cells 5 (XRCC5)	0.41
NM_001380	Dedicator of cytokinesis 1 (DOCK1)	0.42
NM_014515	CCR4-NOT transcription complex, subunit 2 (CNOT2)	0.42
NM_005655	Transforming growth factor-beta inducible early growth response (TGFBI EGR)	0.42
NM-003852	Transcriptional intermediary factor 1 (TIF 1)	0.43
NM_152721	Docking protein 6 (DOK6)	0.46
NM_139207	Nucleosome assembly protein 1-like 1 (NAP1L1)	0.46
NM_006312	Nuclear receptor corepressor 2 (NCOR2)	0.48
NM_000538	Regulatory factor X-associated protein (RFXAP)	0.49
NM_016545	Immediate early response 5 (IER5)	2.35
NM-002129	High-mobility group box 2 (HMGB2)	2.41
BC005107	Chromosome 21 open reading frame 105, mRNA (C21ORF10)	2.43
NM-006608	Putative homeodomain transcription factor 1 (PHTF 1)	2.57
NM_178820	F-box protein 27 (FBXO27)	2.61
NM_000927	ATP-binding cassette, subfamily B (MDR/TAP), member 1 (ABCB1)	2.73
NM_002592	Proliferating cell nuclear antigen (PCNA)	2.85
NM_001924	Growth arrest and DNA-damage-inducible, alpha (GADD45A)	2.91
NM_012153	Ets homologous factor (EHF)	3.13
NM_005845	ATP-binding cassette, subfamily C (CFTR/MRP), member 4 (ABCC4)	3.24
NM_199242	Unc-13 homolog D (C. elegans) (UNC13D)	3.24
NM_172366	F-box protein 16 (FBXO16)	3.28
NM_000107	Damage-specific DNA binding protein 2 (DDB2)	3.35
NM_000392	ATP-binding cassette, subfamily C (CFTR/MRP), member 2 (ABCC2)	3.38
NM_002439	MutS homolog 3 (MSH3)	3.42
NM_020165	RAD18 homolog (S. cerevisiae) (RAD18)	3.45
NM_006332	Interferon, gamma-inducible protein 30 (IFI30)	3.57
NM_018965	Triggering receptor expressed on myeloid cells 2 (TREM2)	4.35
BC009507	Interferon, alpha-inducible protein (clone IFI-15K)	6.59

Notes: Total RNA from PC-2 cells treated with 10 nmol/L RPM for 48 hours was extracted for microarray analysis. Representative genes were significantly upregulated or downregulated by RPM in PC-2 cells.

Abbreviation: RPM, rapamycin.

Figure 6 Scatter plot of the gene differential expression in PC-2 cells induced by RPM.

Notes: The Cy3 (RPM treated group) and Cy5 (untreated control) channel intensities from the two-color DNA microarray experiments were shown in the scatter plot. The variables appear in a linear relationship, and the linear correlation between them is 0.935. The red plots represented upregulated genes, and the green plots represented downregulated genes.
Abbreviation: RPM, rapamycin.

Figure 7 (A) RT-PCR analysis for the DNA-damage repair and transcription genes after the treatment with RPM; (B) Difference between the cDNA microarray and RT-PCR in DNA-damage repair and transcription genes.

Notes: The genes examined here are: (1) DDB1(NM_001923); (2) RAD51 (NM_002876); (3) XRCC5 (NM_021141); (4) PCNA (NM_002592); and (5) ABCC4 (NM_005845). The values represent the mean ± SD of the data from three independent experiments.
Abbreviations: RPM, rapamycin; RT-PCR, reverse transcription polymerase chain reaction; SD, standard deviation.