# The Effect of Phototherapy on Cancer Predisposition Genes of Diabetic and Normal Human Skin Fibroblasts

Authors: Pongsathorn Chotikasemsri; Boonsin Tangtrakulwanich; Surasak Sangkhathat
Journal: BioMed Research International
 (2017)

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## Abstract

The purpose of this study was to investigate whether LED light at different wavelengths affects the expression profile of 143 cancer predisposition genes in both diabetic and normal human fibroblasts. In this study, both diabetic and normal fibroblast cell lines were cultured and irradiated with red (635 nm), green (520 nm), and blue (465 nm) LED light for 10 minutes at 0.67 J/cm2 each. After that, mRNA from all cell lines was extracted for microarray analysis. We found that green light activates EPHB2, KIT, ANTXR2, ESCO2, MSR1, EXT1, TSC1, KIT, NF1, BUB1B, FANCD2, EPCAM, FANCD2, NF, DIS3L2, and RET in normal fibroblast cells, while blue and red light can upregulate RUNX1, PDGFRA, EHBP1, GPC3, AXIN2, KDR, GLMN, MSMB, EPHB2, MSR1, KIT, FANCD2, BMPR1A, BUB1B, PDE11A, and RET. Therefore, genetic screening before phototherapy treatment may be required.


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## Body

## 1. Introduction

Nowadays, there are a number of light-assisted treatments for medical application, such as UV-B treatment for psoriasis, red light for acne vulgaris, daylight spectrum for seasonal affected disorder, Levulan blue light for skin psoriasis or jaundice, and GreenLight Laser PVP (Photoselective Vaporization of the Prostate) for benign prostatic hyperplasia. However, there have been problems noted with light treatments; for example, Valejo Coelho and Apetato [1] did a study on adverse effects of phototherapy which could lead to certain skin cancers. In addition, other studies from Wickremasinghe et al. [2] and Frazier et al. [3] have noted a significant increased risk of cancer in infants after neonatal phototherapy.There are 143 well-known hereditary cancer predisposition genes (Table1) that have been documented for genetic analysis. The mutation of these genes serves as markers of cancer cells after pathological and biopsy analysis. Therefore, the question arises; if any patients who have been previously diagnosed with mutation of such genes need phototherapy or light-assisted treatments, will there be any effect on these mutant gene expression levels? As a number of wavelengths have been used, how certain wavelengths affect the whole genes and their expression has not been clarified.Table 1
There are 143 predisposition genes that have been widely used for SNP detection of various types of cancer.
AIP
APC
BAP1
BRCA2
AKT1
ASCC1
BARD1
BRIP1
ALK
ATM
BLM
BUB1B
ANTXR1
ATR
BMPR1A
CD96
ANTXR2
AXIN2
BRCA1
CDC73
CDH1
FANCI
MUTYH
RUNX1
CDK4
FANCL
NBN
SBDS
CDKN1B
FANCM
NDUFA13
SDHA
CDKN2A
FH
NF1
SDHAF2
CHEK1
FLCN
NF2
SDHB
CHEK2
GALNT12
NTRK1
SDHC
CYLD
GATA2
PALB2
SDHD
CYP21A2
GL13
PALLD
SLX4
DDB2
GLMN
PDE11A
SMAD4
DICER1
GPC3
PDGFRA
SMARCA4
DIS3L2
HFE
PIK3CA
SMARCB1
DKC1
HRAS
PMS2
STK11
EHBP1
KDR
POLD1
SUFU
EPCAM
KIF1B
POLE
TERT
EPHB2
KIT
POLH
TGFBR1
ERCC2
KLHDC8B
POU6F2
TINF2
ERCC3
LIG4
PRKAR1A
TMC6
ERCC4
LYST
PTCH1
TMC8
ERCC5
MAX
PTCH2
TMEM127
ERCC6
MC1R
PTEN
TP53
ESCO2
MEN1
RAD50
TSC1
EXT1
MET
RAD51B
TSC2
EXT2
MITE
RAD51C
UROD
FAH
MLH1
RAD51D
VHL
FANCA
MLH3
RB1
WAS
FANCB
MRE11A
RECQL4
WRN
FANCC
MSH2
RET
WT1
FANCD2
MSH6
RHBDF2
XPA
FANCE
MSMB
RNASEL
XPC
FANCF
MSR1
RSPO1
XRCC3
FANCG
MTAP
RTEL1This study aimed to evaluate the changes of expression levels of cancer predisposition genes after irradiation with red (635 nm), green (520 nm), and blue (465 nm) LED light by using microarray and cell culture techniques.


## 2. Materials and Methods

### 2.1. LED Light Source

An apparatus equipped with blue (465 nm), green (520 nm), and red (635 nm) light emitting diodes with a 1 ampere power supply was constructed for this experiment. Calibrating analysis of the light’s emission spectra and power was provided by a monochrome meter and a power meter from the Department of Physics, Prince of Songkla University. The LED diode array (15 cm by 15 cm), assembled by a technician from the Department of Electrical Engineering, Prince of Songkla University, consisted of three different wavelength array panels that could be placed over and perfectly cover a 10 cm cell culture plate. In this array, the blue LED emitted light between 440 and 500 nm with peak emission at 465 nm, the green LED emitted light between 495 and 575 nm with peak emission at 520 nm, and the red LED emitted light between 610 and 660 nm with peak emission at 635 nm.


### 2.2. Cell Culture Assay

Both the type 2 diabetic fibroblast cell line from a donor diagnosed with type II diabetes (untransformed, Caucasian, MODY, aged 23 years, Cat. #AG06083) and healthy normal fibroblast cell line (male, leg skin, untransformed, Caucasian, Cat. #GM03440) were obtained from the Coriell Institute for Medical Research and cultured at 37°C 5% CO2 in DMEM (supplemented with 10% FBS and 100 μg/ml streptomycin Penn Strep) from GIBCO in 10 cm plates with 2,000,000 cells of initial seeding density. During the entire experiment, these cells were maintained and used in not more than five passages. The type 2 diabetic fibroblast cell line and healthy normal fibroblast cell line were cultured at the regular normal glucose level under the assumption that they were from glycemic well-controlled patients. The type 2 diabetic fibroblast cell line and healthy normal fibroblast cell line served as treated groups and control groups, respectively.


### 2.3. LED Irradiation Assay and mRNA Isolation

Before LED-light exposure, the cells were cultured until 90% confluence and a number of artificial wounds were randomly created with a 1 ml plastic pipette tip in order to reduce bias of the result and simulate the real wounded shape. Then, in the treatment groups, 3 groups each of diabetic fibroblasts and normal fibroblast cells were exposed to the light at 0.67 J/cm2 for 10 minutes at different wavelengths. According to our preliminary experiment, the light irradiating energy at 0.67 J/cm2 was the optimum level for this in vitro experiment. One of the diabetic fibroblasts and one healthy normal fibroblast received no light exposure to serve as controls. The details of the treated groups are shown in Table 2. The energy power of the light that the cells received was carefully calibrated by an optical power meter. The irradiation distance from the LED diode array to the media surface was 10 cm. The apparatus had been placed in a laminar flow (ESCO class II), and a cooling airflow fan constantly maintained a constant temperature during the light exposure. After that, all cells were suddenly trypsinized and their mRNA extracted. All extractions were performed with a GeneJet RNA purification kit from Thermo Scientific. Each condition was pentaplicated for reliable and valid results.Table 2
Assigned abbreviations for each condition.
Untreated
Treated with red light
Treated with green light
Treated with blue light
NCL control
NCL red
NCL green
NCL blue
DMCL control
DMCL red
DMCL green
DMCL blue


### 2.4. mRNA Microarray Assay

All mRNA samples were shipped to a certified Agilent microarray service in India and the purity of the mRNA samples was tested by Bioanalyzer with an RNA integrity number (RIN) higher than 8.0 before carrying on to hybridize with the microarray chips. SurePrint G3 human gene expression 8x60k V2 chips were used for this experiment.


### 2.5. Statistical and Microarray Data Analysis

All raw data were analyzed by GeneSpring 13. All gene expression profiles with a 2-fold or greater difference were selected and followed by differential gene expression analysis (fold change analysis), gene ontology analysis, and pathway analysis. For differential gene expression analysis (fold change analysis), it was calculated by the absolute ratio of normalized intensities (no log scale) between the average intensities of the samples grouped. For gene ontology (GO) analysis, it was an analysis to observe how significantly such genes can affect certain function of a cell. This analysis was calculated by using Benjamini-Yekutieli procedure with correctedp values. Finally, for pathway analysis, curated pathways from the WikiPathways.org were imported to GeneSpring 13 software in order to visualize every pathway that such genes had been affected. The cut-off level for all analyses was a p value less than 0.05. The raw data were also submitted to the NCBI database for other GEO researchers to use.


## 2.1. LED Light Source

An apparatus equipped with blue (465 nm), green (520 nm), and red (635 nm) light emitting diodes with a 1 ampere power supply was constructed for this experiment. Calibrating analysis of the light’s emission spectra and power was provided by a monochrome meter and a power meter from the Department of Physics, Prince of Songkla University. The LED diode array (15 cm by 15 cm), assembled by a technician from the Department of Electrical Engineering, Prince of Songkla University, consisted of three different wavelength array panels that could be placed over and perfectly cover a 10 cm cell culture plate. In this array, the blue LED emitted light between 440 and 500 nm with peak emission at 465 nm, the green LED emitted light between 495 and 575 nm with peak emission at 520 nm, and the red LED emitted light between 610 and 660 nm with peak emission at 635 nm.


## 2.2. Cell Culture Assay

Both the type 2 diabetic fibroblast cell line from a donor diagnosed with type II diabetes (untransformed, Caucasian, MODY, aged 23 years, Cat. #AG06083) and healthy normal fibroblast cell line (male, leg skin, untransformed, Caucasian, Cat. #GM03440) were obtained from the Coriell Institute for Medical Research and cultured at 37°C 5% CO2 in DMEM (supplemented with 10% FBS and 100 μg/ml streptomycin Penn Strep) from GIBCO in 10 cm plates with 2,000,000 cells of initial seeding density. During the entire experiment, these cells were maintained and used in not more than five passages. The type 2 diabetic fibroblast cell line and healthy normal fibroblast cell line were cultured at the regular normal glucose level under the assumption that they were from glycemic well-controlled patients. The type 2 diabetic fibroblast cell line and healthy normal fibroblast cell line served as treated groups and control groups, respectively.


## 2.3. LED Irradiation Assay and mRNA Isolation

Before LED-light exposure, the cells were cultured until 90% confluence and a number of artificial wounds were randomly created with a 1 ml plastic pipette tip in order to reduce bias of the result and simulate the real wounded shape. Then, in the treatment groups, 3 groups each of diabetic fibroblasts and normal fibroblast cells were exposed to the light at 0.67 J/cm2 for 10 minutes at different wavelengths. According to our preliminary experiment, the light irradiating energy at 0.67 J/cm2 was the optimum level for this in vitro experiment. One of the diabetic fibroblasts and one healthy normal fibroblast received no light exposure to serve as controls. The details of the treated groups are shown in Table 2. The energy power of the light that the cells received was carefully calibrated by an optical power meter. The irradiation distance from the LED diode array to the media surface was 10 cm. The apparatus had been placed in a laminar flow (ESCO class II), and a cooling airflow fan constantly maintained a constant temperature during the light exposure. After that, all cells were suddenly trypsinized and their mRNA extracted. All extractions were performed with a GeneJet RNA purification kit from Thermo Scientific. Each condition was pentaplicated for reliable and valid results.Table 2
Assigned abbreviations for each condition.
Untreated
Treated with red light
Treated with green light
Treated with blue light
NCL control
NCL red
NCL green
NCL blue
DMCL control
DMCL red
DMCL green
DMCL blue


## 2.4. mRNA Microarray Assay

All mRNA samples were shipped to a certified Agilent microarray service in India and the purity of the mRNA samples was tested by Bioanalyzer with an RNA integrity number (RIN) higher than 8.0 before carrying on to hybridize with the microarray chips. SurePrint G3 human gene expression 8x60k V2 chips were used for this experiment.


## 2.5. Statistical and Microarray Data Analysis

All raw data were analyzed by GeneSpring 13. All gene expression profiles with a 2-fold or greater difference were selected and followed by differential gene expression analysis (fold change analysis), gene ontology analysis, and pathway analysis. For differential gene expression analysis (fold change analysis), it was calculated by the absolute ratio of normalized intensities (no log scale) between the average intensities of the samples grouped. For gene ontology (GO) analysis, it was an analysis to observe how significantly such genes can affect certain function of a cell. This analysis was calculated by using Benjamini-Yekutieli procedure with correctedp values. Finally, for pathway analysis, curated pathways from the WikiPathways.org were imported to GeneSpring 13 software in order to visualize every pathway that such genes had been affected. The cut-off level for all analyses was a p value less than 0.05. The raw data were also submitted to the NCBI database for other GEO researchers to use.


## 3. Results

### 3.1. Differential Gene Expression

After the mRNA samples of the fibroblast cells were analyzed by GeneSpring 13, all results were categorized and compared as shown in Figures1 and 2. Total gene expression profiles and raw data can be accessed through the GEO database of the NCBI at GSE78017 and GSE78018. Only 143 predisposition cancer genes were selected and further analyzed, comparing them with untreated normal human fibroblasts (control group). Figure 1 shows 37 genes from normal human fibroblast cells of each treatment group which were significantly 2-fold or more up or down (p<0.05). In the NCL blue group, BUB1B, MSR1, FANCD2, and ERCC5 were significantly upregulated, while DKC1, HFE, MSR1, FLCN, MSH6, CD96, RAD51B, ERCC5, and TP53 genes were significantly downregulated. In the NCL green group, the EPHB2, KIT, ANTXR2, ESCO2, MSR1, EXT1, TSC1, BUB1B, EPCAM, FANCD2, NF1, DIS3L2, and RET genes were significantly expressed at higher levels, while 6 genes, DKC1, ERCC5, GPC3, MSH6, PDE11A, and TP53, showed significantly lower expression. In the NCL red group, FANCB, POLH, KIT, POLE, BUB1B, DIS3L2, FANCD2, SUFU, TMC8, MSR1, and RET had significantly high expression, while NF1, NTRK1, MSR1, ANTXR1, ERCC5, FLCN, and TP53 were expressed at significantly lower levels.Figure 1
37 out of 143 genes were significantly up- or downregulated with more than 2-fold differences after irradiation with red, green, or blue light in normal skin fibroblast cells (p<0.05).Figure 2
58 out of 143 genes were significantly up- and downregulated for 2-fold differences after irradiation with red, green, or blue light in diabetic fibroblast cells (p<0.05).Figure2 shows 54 genes from diabetic human fibroblast cells of each treatment group which were significantly over- or downexpressed 2-fold or more (p<0.05). In the DMCL blue group, RUNX1, FANCD2, BMPR1A, BUB1B, EPHB2, EHBP1, PDE11A, RET, and KDR were significantly upregulated, while NF1, KIT, GALNT12, TMC6, ERCC5, DKC1, and TP53 were significantly downregulated. In the DMCL green group, only EPHB2, SDHC, and NF1 genes were significantly expressed at higher levels, while 12 genes, CD96, PTCH2, AXIN2, NF1, EPCAM, MSH6, GALNT12, ERCC5, KIT, NF1, DKC1, and TP53, had significantly lower expression. And in the DMCL red group, RUNX1, PDGFRA, EHBP1, GPC3, AXIN2, KDR, GLMN, MSMB, EPHB2, MSR1, and KIT had significantly high expression, and 5 genes, GPC3, TMC6, PTCH1, DKC1, and TP53 genes, were expressed at significantly lower levels.


### 3.2. Gene Ontology Analysis of the Blue Light Irradiation Group

For the groups which were irradiated with blue light and compared with NCL, only 4 genes, BUB1B, MSR1, FANCD2, and ERCC5, were upregulated after blue light irradiation in normal fibroblast cells. However, there were no significant changes to gene ontology. In contrast, 9 genes were detected with significantly lower expression, DKC, HFE, MSR, FLCN, MSH6, CD96, RAD51B, ERCC5, and TP53. These suppressed genes are known to have a direct impact on lowering DNA repair activity during the replication fork process.In addition, in the DMCL blue group, the 9 genes shown in Table3 (RUNX1, FANCD2, BMPR1A, BUB1B, EPHB2, EHBP1, PDE11A, RET, and KDR) were highly activated and all of them can significantly affect embryonic hemopoiesis, transferase activities, and protein phosphorylation. On the other hand, 7 genes were clearly observed to have lower expression, NF, KIT, GALNT12, TMC6, ERCC5, DKC, and TP53. These genes were significantly downregulated, and downregulation of these genes has a negative effect on the regulation of neuroblast proliferation.Table 3
Summary of genes that were affected from each treatment (p<0.05).
Blue
Green
Red
NCL
Up
BUB1B
FANCD2
EPHB2
BUB1B
FANCB
FANCD2
MSR1
ERCC5
KIT
EPCAM
POLH
SUFU
ANTXR2
FANCD2
KIT
TMC8
ESCO2
NF1
POLE
MSR1
MSR1
DIS3L2
BUB1B
RET
EXT1
RET
DIS3L2
TSC1
Down
DKC1
CD96
DKC1
MSH6
NF1
ERCC5
HFE
RAD51B
ERCC5
PDE11A
NTRK1
FLCN
MSR1
ERCC5
GPC3
TP53
MSR1
TP53
FLCN
TP53
ANTXR1
MSH6
DMCL
Up
RUNX1
PDE11A
EPHB2
RUNX1
GLMN
FANCD2
RET
SDHC
PDGFRA
MSMB
BMPR1A
KDR
NF1
EHBP1
EPHB2
BUB1B
GPC3
MSR1
EPHB2
AXIN2
KIT
EHBP1
KDR
Down
NF1
ERCC5
CD96
GALNT12
GPC3
DKC1
KIT
DKC1
PTCH2
ERCC5
TMC6
TP53
GALNT12
TP53
AXIN2
KIT
PTCH1
TMC6
NF1
NF1
EPCAM
DKC1
MSH6
TP53


### 3.3. Gene Ontology Analysis of Green Light Irradiation

Sixteen genes were upregulated in the NCL green group after green light irradiation in normal fibroblast cells. This set of genes could significantly affect transmembrane receptor protein tyrosine kinase activities. In contrast, 6 genes (DKC1, ERCC5, GPC3, MSH6, PDE11A, and TP53) were detected with significantly lower expression (Table3). These suppressed genes directly affected structure-specific DNA secondary structure binding for DNA repair, replication, and DNA metabolic process.In addition, in the DMCL green group, only 3 genes, EPHB2, SDHC, and NF, were highly activated; all of these genes are significantly involved in the regulation of neuronal synaptic plasticity. On the other hand, the other 12 genes, CD96, PTCH2, AXIN2, NF1, EPCAM, MSH6, GALNT12, ERCC5, KIT, NF1, DKC1, and TP53, had lower expression (Table3). These 12 genes were significantly downregulated which affected the cellular immune response, DNA metabolic process, the negative regulation of neuroblast proliferation, response to light stimulus, epithelium development, and maintenance of DNA repeat elements.


### 3.4. Gene Ontology Analysis of Red Light Irradiation

In the NCL red group in which the cell lines were irradiated with red light and compared with NCL, there were no significant positive changes to gene ontology (12 genes, Table3). In contrast, 7 genes, NF1, NTRK1, MSR1, ANTXR1, ERCC5, FLCN, and TP53, showed significantly lower expression. These suppressed genes are known to have a direct impact on RAS protein signal transduction, the negative regulation of neuroblast proliferation, sympathetic nervous system development, and the positive regulation of adenylate cyclase activities.In addition, in the DMCL red group, 12 genes (RUNX1, PDGFRA, EHBP1, GPC3, AXIN2, KDR, GLMN, MSMB, EPHB2, MSR1, and KIT, Table3) were upregulated in normal fibroblast cells. These highly active genes directly affected protein kinase activities, vascular endothelial growth factor binding, ovarian follicle development, and hematopoietic progenitor cell differentiation (immune system development). On the other hand, only 5 genes in this group were clearly observed to have lower expression, GPC3, TMC6, PTCH1, DKC1, and TP53. This set of genes is known to have a direct effect on the process of cell proliferation involved in metanephros development of the kidney.


## 3.1. Differential Gene Expression

After the mRNA samples of the fibroblast cells were analyzed by GeneSpring 13, all results were categorized and compared as shown in Figures1 and 2. Total gene expression profiles and raw data can be accessed through the GEO database of the NCBI at GSE78017 and GSE78018. Only 143 predisposition cancer genes were selected and further analyzed, comparing them with untreated normal human fibroblasts (control group). Figure 1 shows 37 genes from normal human fibroblast cells of each treatment group which were significantly 2-fold or more up or down (p<0.05). In the NCL blue group, BUB1B, MSR1, FANCD2, and ERCC5 were significantly upregulated, while DKC1, HFE, MSR1, FLCN, MSH6, CD96, RAD51B, ERCC5, and TP53 genes were significantly downregulated. In the NCL green group, the EPHB2, KIT, ANTXR2, ESCO2, MSR1, EXT1, TSC1, BUB1B, EPCAM, FANCD2, NF1, DIS3L2, and RET genes were significantly expressed at higher levels, while 6 genes, DKC1, ERCC5, GPC3, MSH6, PDE11A, and TP53, showed significantly lower expression. In the NCL red group, FANCB, POLH, KIT, POLE, BUB1B, DIS3L2, FANCD2, SUFU, TMC8, MSR1, and RET had significantly high expression, while NF1, NTRK1, MSR1, ANTXR1, ERCC5, FLCN, and TP53 were expressed at significantly lower levels.Figure 1
37 out of 143 genes were significantly up- or downregulated with more than 2-fold differences after irradiation with red, green, or blue light in normal skin fibroblast cells (p<0.05).Figure 2
58 out of 143 genes were significantly up- and downregulated for 2-fold differences after irradiation with red, green, or blue light in diabetic fibroblast cells (p<0.05).Figure2 shows 54 genes from diabetic human fibroblast cells of each treatment group which were significantly over- or downexpressed 2-fold or more (p<0.05). In the DMCL blue group, RUNX1, FANCD2, BMPR1A, BUB1B, EPHB2, EHBP1, PDE11A, RET, and KDR were significantly upregulated, while NF1, KIT, GALNT12, TMC6, ERCC5, DKC1, and TP53 were significantly downregulated. In the DMCL green group, only EPHB2, SDHC, and NF1 genes were significantly expressed at higher levels, while 12 genes, CD96, PTCH2, AXIN2, NF1, EPCAM, MSH6, GALNT12, ERCC5, KIT, NF1, DKC1, and TP53, had significantly lower expression. And in the DMCL red group, RUNX1, PDGFRA, EHBP1, GPC3, AXIN2, KDR, GLMN, MSMB, EPHB2, MSR1, and KIT had significantly high expression, and 5 genes, GPC3, TMC6, PTCH1, DKC1, and TP53 genes, were expressed at significantly lower levels.


## 3.2. Gene Ontology Analysis of the Blue Light Irradiation Group

For the groups which were irradiated with blue light and compared with NCL, only 4 genes, BUB1B, MSR1, FANCD2, and ERCC5, were upregulated after blue light irradiation in normal fibroblast cells. However, there were no significant changes to gene ontology. In contrast, 9 genes were detected with significantly lower expression, DKC, HFE, MSR, FLCN, MSH6, CD96, RAD51B, ERCC5, and TP53. These suppressed genes are known to have a direct impact on lowering DNA repair activity during the replication fork process.In addition, in the DMCL blue group, the 9 genes shown in Table3 (RUNX1, FANCD2, BMPR1A, BUB1B, EPHB2, EHBP1, PDE11A, RET, and KDR) were highly activated and all of them can significantly affect embryonic hemopoiesis, transferase activities, and protein phosphorylation. On the other hand, 7 genes were clearly observed to have lower expression, NF, KIT, GALNT12, TMC6, ERCC5, DKC, and TP53. These genes were significantly downregulated, and downregulation of these genes has a negative effect on the regulation of neuroblast proliferation.Table 3
Summary of genes that were affected from each treatment (p<0.05).
Blue
Green
Red
NCL
Up
BUB1B
FANCD2
EPHB2
BUB1B
FANCB
FANCD2
MSR1
ERCC5
KIT
EPCAM
POLH
SUFU
ANTXR2
FANCD2
KIT
TMC8
ESCO2
NF1
POLE
MSR1
MSR1
DIS3L2
BUB1B
RET
EXT1
RET
DIS3L2
TSC1
Down
DKC1
CD96
DKC1
MSH6
NF1
ERCC5
HFE
RAD51B
ERCC5
PDE11A
NTRK1
FLCN
MSR1
ERCC5
GPC3
TP53
MSR1
TP53
FLCN
TP53
ANTXR1
MSH6
DMCL
Up
RUNX1
PDE11A
EPHB2
RUNX1
GLMN
FANCD2
RET
SDHC
PDGFRA
MSMB
BMPR1A
KDR
NF1
EHBP1
EPHB2
BUB1B
GPC3
MSR1
EPHB2
AXIN2
KIT
EHBP1
KDR
Down
NF1
ERCC5
CD96
GALNT12
GPC3
DKC1
KIT
DKC1
PTCH2
ERCC5
TMC6
TP53
GALNT12
TP53
AXIN2
KIT
PTCH1
TMC6
NF1
NF1
EPCAM
DKC1
MSH6
TP53


## 3.3. Gene Ontology Analysis of Green Light Irradiation

Sixteen genes were upregulated in the NCL green group after green light irradiation in normal fibroblast cells. This set of genes could significantly affect transmembrane receptor protein tyrosine kinase activities. In contrast, 6 genes (DKC1, ERCC5, GPC3, MSH6, PDE11A, and TP53) were detected with significantly lower expression (Table3). These suppressed genes directly affected structure-specific DNA secondary structure binding for DNA repair, replication, and DNA metabolic process.In addition, in the DMCL green group, only 3 genes, EPHB2, SDHC, and NF, were highly activated; all of these genes are significantly involved in the regulation of neuronal synaptic plasticity. On the other hand, the other 12 genes, CD96, PTCH2, AXIN2, NF1, EPCAM, MSH6, GALNT12, ERCC5, KIT, NF1, DKC1, and TP53, had lower expression (Table3). These 12 genes were significantly downregulated which affected the cellular immune response, DNA metabolic process, the negative regulation of neuroblast proliferation, response to light stimulus, epithelium development, and maintenance of DNA repeat elements.


## 3.4. Gene Ontology Analysis of Red Light Irradiation

In the NCL red group in which the cell lines were irradiated with red light and compared with NCL, there were no significant positive changes to gene ontology (12 genes, Table3). In contrast, 7 genes, NF1, NTRK1, MSR1, ANTXR1, ERCC5, FLCN, and TP53, showed significantly lower expression. These suppressed genes are known to have a direct impact on RAS protein signal transduction, the negative regulation of neuroblast proliferation, sympathetic nervous system development, and the positive regulation of adenylate cyclase activities.In addition, in the DMCL red group, 12 genes (RUNX1, PDGFRA, EHBP1, GPC3, AXIN2, KDR, GLMN, MSMB, EPHB2, MSR1, and KIT, Table3) were upregulated in normal fibroblast cells. These highly active genes directly affected protein kinase activities, vascular endothelial growth factor binding, ovarian follicle development, and hematopoietic progenitor cell differentiation (immune system development). On the other hand, only 5 genes in this group were clearly observed to have lower expression, GPC3, TMC6, PTCH1, DKC1, and TP53. This set of genes is known to have a direct effect on the process of cell proliferation involved in metanephros development of the kidney.


## 4. Discussion

We found that phototherapy can significantly affect the expression of certain brain, lung, breast, ovarian, and prostate cancer “predisposition genes” in nondiabetic patients and also increase the risk of juvenile intestinal polyposis, hemangioma, multiple endocrine neoplasia, endometrial cancer, and chronic myeloid leukemia in diabetic patients.Green light irradiation may significantly increase the chances for a number of cancers in nondiabetic patients (Table4). For example, irradiated patients are at increased risk of developing hyaline fibromatosis, neurofibromatosis, brain cancer, lung cancer, prostate cancer, breast cancer, Wilms tumor, multiple endocrine neoplasia, Barrett’s esophagus/esophageal adenocarcinoma, and endometrial cancer in patients with an existing gastrointestinal stromal tumor. In contrast, the green light irradiation could develop only a few types of cancers, such as brain cancer, prostate cancer, neurofibromatosis, and paragangliomas in diabetic patients (Table 5). Thus, patients who require prostate surgery with a green light laser should be carefully checked to see whether they have mutated predisposition genes before their surgery. In addition, using excessive blue light for skin psoriasis and jaundice treatment may increase the occurrences of particular cancers in newborns who have been genetically diagnosed with these mutated genes.Table 4
Cancer-related predisposition genes that were overexpressed after blue, green, or red light irradiation in normal healthy fibroblast cells (nondiabetic patients) (p<0.05).
Light condition
Overexpressed genes after irradiation
Possible cancers affected
Blue
BUB1B
Brain and lung cancer
ERCC5
Ovarian cancer
FANCD2
Breast cancer and Fanconi anemia
MSR1
Prostate cancer and Barrett’s esophagus/esophageal adenocarcinoma
Green
ANTXR2
Hyaline fibromatosis syndrome and hyalinosis, inherited systemically (its related pathways are infectious disease and uptake and actions of bacterial toxins)
BUB1B
Brain and lung cancers
DIS3L2
Perlman syndrome and Wilms’ tumor susceptibility-5
EPCAM
Endometrial cancer, biliary tract cancer, and skin cancer
ESCO2
Roberts syndrome and SC phocomelia syndrome
EPHB2
Prostate cancer/brain cancer susceptibility, somatic and prostate cancer
EXT1
Chondrosarcoma and exostoses, multiple, type 1
FANCD2
Breast cancer and Fanconi anemia
KIT
Endometrial cancer as a gastrointestinal stromal tumor
MSR1
Prostate cancer and Barrett’s esophagus/esophageal adenocarcinoma
NF1
Neurofibromatosis-Noonan syndrome and neurofibromatosis, type 1
RET
Multiple endocrine neoplasia iia and medullary thyroid carcinoma, familial
TSC1
Tuberous sclerosis-1 and lymphangioleiomyomatosis
Red
BUB1B
Brain and lung cancer
DIS3L2
Perlman syndrome and Wilms’ tumor susceptibility-5
FANCB
Fanconi anemia, complementation group B, and Fanconi anemia, complementation group A
FANCD2
Breast cancer and Fanconi anemia
KIT
Endometrial cancer as a gastrointestinal stromal tumor
MSR1
Prostate cancer and Barrett’s esophagus/esophageal adenocarcinoma
POLE
FILS syndrome and colorectal cancer 12
POLH
Xeroderma pigmentosum, variant type and POHL-related xeroderma pigmentosum
RET
Multiple endocrine neoplasia iia and medullary thyroid carcinoma, familial
SUFU
Medulloblastoma and basal cell nevus syndrome
TMC8
Epidermodysplasia verruciformis and superficial mycosisTable 5
Cancer-related predisposition genes that were overexpressed after blue, green, or red light irradiation in diabetic fibroblast cells (p<0.05).
Light condition
Overexpressed genes after irradiation
Possible cancers affected
Blue
BUB1B
Brain and lung cancers
BMPR1A
Polyposis syndrome, hereditary mixed, 2 and polyposis, juvenile intestinal
EHBP1
Prostate cancer, hereditary, 12 and prostate cancer
EPHB2
Prostate cancer/brain cancer susceptibility, somatic and prostate cancer
FANCD2
Breast cancer and Fanconi anemia
KDR
Hemangioma, capillary infantile and hemangioma
PDE11A
Pigmented nodular adrenocortical disease, primary, 2 and primary pigmented nodular adrenocortical disease
RET
Multiple endocrine neoplasia iia and medullary thyroid carcinoma, familial
RUNX1
Platelet disorder, familial, with associated myeloid malignancy and isolated delta-storage pool disease (among its related pathways are endometrial cancer and chronic myeloid leukemia)
Green
EPHB2
Prostate cancer/brain cancer susceptibility, somatic and prostate cancer
NF1
Neurofibromatosis-Noonan syndrome and neurofibromatosis, type 1
SDHC
Paragangliomas 3 and paraganglioma and gastric stromal sarcoma (among its related pathways are Alzheimer’s disease and carbon metabolism)
Red
AXIN2
Oligodontia-colorectal cancer syndrome and colorectal cancer
EHBP1
Prostate cancer, hereditary, 12 and prostate cancer
EPHB2
Prostate cancer/brain cancer susceptibility, somatic and prostate cancer
GLMN
Glomuvenous malformations and glomangioma
GPC3
Simpson-Golabi-Behmel syndrome, type 1 and Wilms tumor susceptibility-5
KDR
Hemangioma, capillary infantile and hemangioma
KIT
Endometrial cancer in gastrointestinal stromal tumor
MSMB
Prostate cancer, hereditary, 13 and prostate cancer
MSR1
Prostate cancer and Barrett esophagus/esophageal adenocarcinoma
PDGFRA
Gastrointestinal stromal tumor and hypereosinophilic syndrome, idiopathic, resistant to imatinib
RUNX1
Platelet disorder, familial, with associated myeloid malignancy and isolated delta-storage pool disease (among its related pathways are endometrial cancer and chronic myeloid leukemia)For red light irradiation, prostate cancer, breast cancer, brain cancer, lung cancer, Wilms’ tumor, Fanconi anemia, Barrett’s esophagus/esophageal adenocarcinoma, xeroderma pigmentosum, medulloblastoma, epidermodysplasia verruciformis, colorectal cancer, and endometrial cancer in gastrointestinal stromal tumor could develop in nondiabetic patients (Table4). On the other hand, for diabetic patients considered for red light irradiation, the high expression of such genes may increase the rates of colorectal cancer, prostate cancer, Barrett’s esophagus/esophageal adenocarcinoma, glomangioma, Wilms’ tumor, hemangioma, and myeloid malignancy (Table 5). Hence, a red light phototherapy for acne vulgaris and other cosmetic purposes could be a potential threat that the doctors and diabetic patients should be aware of.In short, according to the results of this experiment, any wavelength can either increase or lower expression of particular mutated predisposition cancer genes. Therefore, awareness of potential side effects should be considered before any proposed laser operation. In addition, one can take advantage of this phototherapy to either be aware of the side effects on how light can induce the expression of certain predisposition cancer genes or suppress certain mutated genes to reduce causes of cancers. In contrast, one may take advantage of how phototherapy downregulates or suppresses the expression of certain predisposition genes which may reduce the progression of cancer cells to develop further. For example, the expression of mutated TP53 gene, a cancer marker gene causing Li-Fraumeni syndrome, breast cancer, soft tissue sarcoma, osteosarcoma (bone cancer), leukemia, brain tumors, and adrenocortical carcinoma, can be reduced by irradiating with blue, green, or red light. In addition, both ERCC5 and TP53 genes were found with significantly lower expression in all treated groups. These genes work as tumor suppressor genes; therefore, intense phototherapy could definitely decrease the DNA repair function. Moreover, lower expression of DKC1 was also found, which is important as DKC1 is able to control the length of telomerases after the cell cycle. Eventually, either more abnormal cells would be produced or more apoptosis could occur. These hypotheses are supported by previous studies from Płonka et al. [4] and Acedo and Zawacka-Pankau [5]. These studies found that the expression of the P53 (also called TP53) gene was lower after phototherapy, which led to the initiation of the apoptosis process.In addition, for patients with neurofibromatosis type 1 (a mutated NF1 gene), intense pulsed-radio frequency (IPL-RF) in combination with topical application of vitamin D3 ointment is one of the current treatments. What if we use red light to suppress the expression of this mutated NF1? Could it improve the quality of their lives? Our study suggests that red light irradiation could significantly reduce the expression of the NF1 gene in nondiabetic patients and blue light irradiation significantly lowers the expression of NF1 gene in diabetic patients.Therefore, gene screening for these 143 predisposition genes should be implemented before any phototherapy treatment and further investigations are strongly recommended to explore the benefits of phototherapy for cancer treatment.


## 5. Conclusion

Phototherapy increases the expression of cancer predisposing genes in both normal and diabetic cell lines. Green light activates EPHB2, KIT, ANTXR2, ESCO2, MSR1, EXT1, TSC1, KIT, NF1, BUB1B, FANCD2, EPCAM, FANCD2, NF, DIS3L2, and RET in normal fibroblast cells, whereas blue and red light can upregulate RUNX1, PDGFRA, EHBP1, GPC3, AXIN2, KDR, GLMN, MSMB, EPHB2, MSR1, KIT, FANCD2, BMPR1A, BUB1B, PDE11A, and RET. Genetic screening before applying phototherapy may be warranted.



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*Source: 7604861-2017-03-12.xml*