Objective: The present study aimed to determine whether a single-fraction of gamma radiation could induce the expression of specific genes involved in apoptosis signaling pathways, which helps us in better understanding cancer dynamics and treatment targets for cancer.

Materials and methods: C-4 I cells were treated with a single fraction of gamma radiation at various doses (0, 2, 8, 16, 32 and 64 Gy) and investigated after incubation for five time periods (0, 24, 48, 60 and 72 h). The proliferation of C-4 I cells was measured by MTT assay, but apoptotic index (AI) and apoptotic morphological features were assessed by fluorescent microscopy. Moreover, the expression of the apoptotic genes was evaluated using microarray and qRT-PCR molecular processes. In addition, gene ontology and pathway analysis were performed.

Results: Gamma irradiation inhibits proliferation of C-4 I cells in a dose- and timedependent manner, and 16 Gy was identified as the IC50 and AI dose. Microarray and qRT-PCR results monitored the expression of some factors that are known apoptosis activators were up regulated by gamma radiation treatment, whereas some anti-apoptosis members were down regulated.

Pathway analysis identified that significant pathways related to apoptosis, cell cycle and P53 were significantly enriched.

Conclusions: These results provide evidence that gamma radiation directly induces anti-proliferative effects by altering the expression of genes associated with cell proliferation and apoptosis pathways in C-4 I cells.

•    Apoptosis
•    C-4 I cells
•    Gamma radiation
•    Microarray
•    qRT-PCR

Khalilia WM, Özcan G, Karaçam S (2017) Gene Expression and Pathway Analysis of Radiation-Induced Apoptosis in C-4 I Cervical Cancer Cells. J Autoimmun Res 4(1): 1014.

IR: Ionizing Radiation; TNF: Tumor Necrosis Factor; MTT: 3-[4, 5-Dimethylthiazol-2-yl]-2, 5-Diphenyltetrazolium bromide; DAPI: 4,6-Diamidino-2-phenylindole; AI: Apoptotic Index; FC: Fold Change; GO: Gene Ontology; mCt: Mean threshold cycle.

Radiation therapy is one of the most common treatments for cancer, used in more than half of all cancer cases, in which highenergy rays used to destroy cancer cells in the body. Radiotherapy is the most effective therapy for cervical cancer in advanced stages [1]. Ionizing radiation (IR) leads to many cellular changes like activates signaling pathways in the nucleus as a result of DNA damage, and signaling pathways initiated at the level of the plasma membrane. DNA damage result in a coordinate network of signal transduction pathways involved in cell cycle arrest, apoptosis, stress response and the activation of DNA repair processes. However, the plasma membrane signaling pathways through activation of some receptors results in activation of the initiator caspase-8 which can propagate the apoptosis signal by direct cleavage of downstream effectors caspases. IR can also induce apoptosis via the generation of free radical oxygen species, which gives rise to a variety of cellular lesions including both DNA and membrane damage [2]. Previous studies suggest that changes in membrane fluidity, superoxide dismutase (SOD) and calcium level were involved in the mechanism of radiation induced cervical cells apoptosis. Moreover, apoptotic sensitivity of these cells after the first dose of radiation treatment showed a direct correlation with the radiation treatment outcome in patients after completion of radiotherapy protocol (70 Gy) [3].

Apoptosis is the major mode of programmed cell death, and is characterized by a series of morphological hallmarks, including cell shrinkage, nuclear DNA condensation and fragmentation, as well as plasma membrane blebbing, which formed the apoptotic bodies. There are two main apoptotic pathways: the extrinsic initiated by death receptors and the intrinsic pathway initiated by mitochondrial events. However, there is evidence that the two pathways are linked and that molecules in one pathway can affect the other. Although, there is an additional apoptotic pathway under studies yet [4].

Apoptosis is a genetically regulated biological process. Changes on apoptotic cells occur after a series of events regulated by caspases and various cell signals that regulate pro-apoptotic and anti-apoptotic proteins. Extrinsic and intrinsic pathways can be activated separately, but most caspases activation is common in an apoptotic pathway. Ligand-bound tumor necrosis factor (TNF) receptors initiate apoptosis by recruiting fas associated death domain (FADD) and other death domain adaptor proteins that then recruit and activate caspases [5,6]. Environmental stresses trigger BCL2 protein oligomerization and insertion into the mitochondrial membrane [7,8], releasing apoptotic protease activating factor 1 (APAF1) and other caspase activation and recruitment domain (CARD) family members that also oligomerize to recruit and activate caspases [4,9,10]. Caspases promote a proteolysis cascade that degrades cellular protein targets, while the inhibitor of apoptosis protein (IAP) family directly inhibits caspases. One of the major apoptosis signaling pathways involves the P53 tumor suppressor. Tumor protein P53 is a nuclear transcription factor that regulates the expression of a wide variety of genes involved in apoptosis in response to genotoxic or cellular stress [2,11]. Understanding apoptosis is often considered a key to understanding the genesis of tumors and to devising innovative strategies for cancer treatment [12].

Therefore, the present study aimed to determine whether gamma radiation could induce the expression of specific genes involved in apoptosis signaling pathways. Identifying up-regulated or down-regulated genes helps us in better understanding the cancer dynamics and helps identify markers and treatment targets for cervical cancer.

Cell culture and γ-60co irradiation

Cervical cancer C-4 I cell line (ATCC), maintained at 37°C in Waymouth’s MB 752/1 (Sigma) supplemented with 10% fetal bovine serum (Gibco Lab.), 100 IU/ml penicillin (Pronapen, Pfizer) and 100 go/ml streptomycin (streptomycin sulfate, ?.E. Ulagay) in a humidified atmosphere of 5% CO2 . Cells were “passaged” every 2 to 3 days using 0.25% trypsin (Sigma) to detach the cells from the flasks [13]. Before the cell proliferation and apoptosis assays, and prior to evaluating genes expression, C-4 I cells were placed in a 6-well plate at a density of 2.5×105 cells per well. After overnight incubation, cells were irradiated at the Istanbul University, Faculty of Medicine, Radiation Oncology, Turkey (Cirus, CCR CisBio, Canada) by different dozes of gamma radiation (2, 8, 16, 32 and 64 Gy) with a dose rate of 100 cGy/min. and incubated for 24, 48, 60 and 72 h, control group (0 Gy) were not irradiated. After irradiation, cell cultures were replaced in the incubator and maintained at 37°C under 5% CO2 . For the irradiation groups, the time when the irradiation was finished was set as time 0 [14,15].

Cytotoxicity and cell proliferation

Cell proliferation was evaluated using the 3-[4, 5-Dimethylthiazol-2-yl]-2, 5-Diphenyltetrazolium bromide (MTT; Sigma) cell viability assay, as previously described [16].

Observation of morphological changes

The cellular morphological changes were observed using phase contrast microscopy and fluorescent microscopy [13].

Apoptotic index (AI)

After irradiation and incubation, C-4 I cells were trypsinized, washed with mixture of 200 µl methanol: farnesyl thiosalicylic acid (FTS) (1:1), fixed in iced pure methanol, stained with 50μg/ mL of 4,6-diamidino-2-phenylindole (DAPI, Sigma) in dark for 20 minutes, and then the slides were washed in phosphate buffered saline (PBS). DAPI is a blue-fluorescent DNA stain that exhibits ~20-fold enhancement of fluorescence upon binding to AT regions of dsDNA. It is commonly used as a nuclear counter stain in fluorescence microscopy. Because of its high affinity for DNA, it is also frequently used for counting cells, measuring apoptosis, sorting cells based on DNA content.

The nuclear morphology was observed under a fluorescent microscope (λex. 358 nm, λem. 461 nm). And apoptotic cells were identified by their characteristic fragmented chromatin masses. Small groups of apoptotic bodies were counted as remnants of one apoptotic cell. Apoptotic cells were counted among 100 cells randomly. Apoptosis was expressed as the number of apoptotic nuclei per number of total nuclei counted in the same microscopic field. This AI was a mean of three independent experiments [12,16].

Isolation and quantification of total RNA

C-4 I cells were plated in a 6-well plate at a density of 2.5×105 cells per well. After overnight incubation cells were irradiated by 16 Gy single dose of γ-60Co radiation and incubated for 60 hours, control group (0 Gy) were not irradiated. After wash with PBS, 6x106 cells were harvested from samples and total RNA was extracted using High Pure RNA Isolation kit (Roche) according to the manufacturer’s instructions. RNA concentrations were determined with visible spectrophotometer (GBC Cintra 20). RNA purity was checked by measurement of the A 260/280 nm ratio, which was routinely in the range of 1.8-2.0. Purified total RNA samples were stored frozen at -80°C until needed for subsequent gene expression profiling [17].

Bead array technology

Human HT-12 Expression Bead Chips with the Illumina Whole-Genome Gene Expression Direct Hybridization Assay (Illumina) system were used to evaluate the expression patterns of more than 48,000 transcripts in C-4 I cells, according to the manufacturer’s instructions. Complementary DNA (cDNA) was produced following reverse transcription from 100 ng of total RNA and then fluorescent cRNA was performed according to the TargetAmp™-Nano Labeling Kit (Illumina). 750 ng cRNA per sample, which was then hybridized to Human HT-12 Illumina® Expression BeadChip® (Illumina) [18].

Bead Chip scanning and data analysis

Microarray slides were then scanned using the Illumina iScan System (Illumina). For gene expression analysis and the generation of gene lists for fold change (FC), functional annotation and pathway analysis, microarray data were processed in a Genome Studio module (V2011.1; Illumina). The signal was taken as the measure of mRNA abundance derived from the level of gene expression. Raw data were adjusted by using Box Plots normalization. Significance levels of differences between the groups were calculated for each probe set using FC and P-values (p < 0.05 and FC ≥ |2|) [19,20].

Bioinformatics analysis

Differentially expressed genes were used as input for a network analyses that were performed with Gene Ontology (GO) network building tools (PANTHER, http://www.pantherdb.org/ pathway/). GO and pathway analysis were performed on the up-regulated and down-regulated genes. Significant functions were identified based on p-value (p < 0.05) [21].

qRT-PCR technology

A Real Time Ready Apoptosis Panel (Human Apoptosis Panel, 96, Roche) was used to assess expression patterns of apoptosisrelated genes in C-4 I cells, according to the manufacturer’s instructions. The panel is designed for expression profiling of genes that play a major role in apoptotic processes in human cells. On the plate, assays for the 84 apoptosis-related genes are grouped to reflect the various protein families and pathways involved in apoptosis.

cDNA synthesis and qRT-PCR set up

First-strand cDNA was synthesized from 3 μg total RNA and a primer was performed using the cDNA synthesis kit (Transcriptor High Fidelity cDNA Synthesis kit, Roche) following manufacturer instructions [22].

Eighty-four apoptosis-related genes were quantified using a Real Time Ready Apoptosis Panel (Human Apoptosis Panel, 96, Roche) according to manufacturer instructions. PCR mix for one reaction (20 μl) that was done by adding 2 μl of primer-probe mix and 10 μl probe and 3 μl dH2O to a total volume of 15 μl in a 1.5 ml reaction tube on ice. After mixing carefully, the 15μl mixture was pipetted into each well of the 96 well plate (Light Cycler 480® Multiwell plate, Roche). After that a 5 μl cDNA template was added to each well, and the plate was sealed with sealing foil and centrifuged for 2 minutes at 1500 xg followed by loading the multi well plate into the instrument (Light Cycler 480® Instrument II, Roche). All reactions were run for one cycle of PCR that consisted of enzyme activation and template denaturation for 10 minutes at 95o C, followed by 40 cycles of PCR. Each cycle consisted of 30 second annealing at 60o C and an extension phase at 72o C for 1 second followed by 20 seconds at 85°C to denature the doublehelix PCR product. Fluorescence data was acquired after each annealing and extension step. Results were expressed using the comparative threshold (Ct) method [23].

Quantitative data analysis of qRT-PCR

Calculation of gene expression was carried out using Ct cycle. The mean threshold cycle (mCt) was obtained from triplicate amplifications during the exponential phase. The mCt value of reference genes was then subtracted from mCt value of the target genes to obtain the ΔCt and ΔΔCt values of each sample, which were calculated from corresponding Ct values; where ΔΔCt= [mCt target – mCt reference] (treated sample) - [mCt target – mCt reference] (untreated sample). Finally, a target gene expression/ reference gene expression ratio was calculated using the ratio formula (ratio = 2 -ΔΔCt), which was used for gene expression analysis and the generation of gene lists for FC (FC ≥ |2|) [24].

Statistical analysis

For the experimental cell proliferation and AI data sets, statistical significance was determined using one-way ANOVA and unpaired Students’t test (p < 0.05).

Initial studies on the treatment of a cervical cancer adenocarcinoma C-4 I cells with 2, 8, 16, 32 and 64 Gy of γ-60Co radiation for a duration of 0-72 hours indicated tha tradiation inhibits C-4 I cells proliferation and apoptosis was elevated by time and γ-60Co radiation dose rate, with maximum levels observed 60 hours after irradiation.

Cell proliferation

MTT assay showed that radiation inhibited proliferation of C-4 I cells. Our results showed that C-4 I cell survival rates of all irradiated groups at 0-hour post-irradiation time were nearly the same at more than 70%, while the cell survival at 24 hours postirradiation time in the 16, 32 and 64 Gy groups were much lower, less than 70%. Our experiments showed that a radiation dose of 16 Gy induced cell death in 50% of C-4 I cells at 24 hours postirradiation time compared with control group (0 Gy). A such a 16 Gy γ-60Co radiation dose was determined as the IC50 dose (Figure 1).

Figure 1: Effect of γ-60Co radiation on survival of C-4 I cells at 0 and 24 hours post-irradiation time. Cell survival was determined by MTT assay. Results are presented as a percent of control. Control is 100% in all time points. The experiment was repeated triple with similar results.

Microscopy and apoptotic index (AI)

Fluorescent images of the nuclear morphology of C-4 I cells in control and in all irradiated groups (2, 8, 16, 32 and 64 Gy) at 60 hours post-irradiation time after DAPI staining. The nuclei were normal in the control group; whereas the nuclei became condensed or fragmented in the all irradiated groups (2, 8, 16, 32 and 64 Gy) (Figure 2).

Figure 2: Representative fluorescent images of the nuclear morphology of C-4 I cells in the control and all irradiated (2, 8, 16, 32 and 64 Gy) groups at 60 hours post-irradiation time, following DAPI staining. In control group, cellular nuclear chromatin homogeneous distribution; whereas the nuclei became condensed or fragmented in the all irradiated groups observed by a fluorescence microscope (X1000). Arrows represent apoptotic cells. The experiment was repeated triple with similar results.

AI was induced by γ-60Co radiation, and increased in a radiation dose and time-dependent manner (Figure 3).

Figure 3: Measure of apoptotic index by counting 100 of C-4 I cells in the control and all irradiated (2, 8, 16, 32 and 64 Gy) groups at 24, 48, 60, and 72 hours post-irradiation time, following DAPI staining under fluorescence microscope (×1000). The data shown represents mean of three independent experiments.

The highest AI values (at 60 and 72 hours post-irradiation time) were reached more than 50% for 64 Gy and 32 Gy. While, at 0 and 24 hours post-irradiation time the lowest AI values, less than 20% for all irradiated groups. Apoptosis levels remained nearly stable for durations ranging from 60 to72 hours post-irradiation time (Figure 3).

Figure 3: Measure of apoptotic index by counting 100 of C-4 I cells in the control and all irradiated (2, 8, 16, 32 and 64 Gy) groups at 24, 48, 60, and 72 hours post-irradiation time, following DAPI staining under fluorescence microscope (×1000). The data shown represents mean of three independent experiments.

16 Gy γ-60Co radiation dose was determined to be the optimum AI value in our current study.

Gamma radiation cytotoxicity and AI considering 16 Gy as the IC50 and optimum AI dose, based on these data, 16 Gy group at 60-hour time point was chosen for further studies.

In the present study, C-4 I cell growth was depressed by large doses of γ-60Co radiation (8, 16, 32 and 64 Gy), but not by low doses (2) at 24 hours post-irradiation time. These results are in agreement with those of previous studies [25]. In addition to decreasing cell proliferation and viability, a single dose of γ-60Co radiation induced apoptosis in C-4 I cells. From our results the percentage of condensed or fragmented nuclei increased with time and dose of radiation. These results are consistent with those of previous studies [26].

The percentages of apoptotic cells in AI assay was showed that the level of apoptosis increased with γ-60Co radiation dose. These results indicate that irradiation primarily affects the C-4 I cell proliferation by inducting cell apoptosis. These results are consistent with those of previous studies [12].

Gene expressions by microarray and pathway analysis

For statistical analysis of microarray data, the study began with 47 231probes, which filtered based on the expression levels from the normalized data. Probes received radiation under 20% was filtered and the study was continued with 47 088 total probe. If any sample received radiation above 20%was allowed to pass through the filter and there by minimize loss of probes. Genes with a standard deviation greater than 0.1 were removed from further analysis. Finally, from the total 47 088 probes 47 071 probes were filtered, and the study were continued with this number of probes [27].

At 60 hours after exposure to 16 Gy of γ-60Co radiation in C-4 I cells, the microarray showed that, 105 of the probes expression increased according to the control group (had 2-fold up regulation), and 210 probes expression was lower than the control group (had 2-fold down regulation) (Table 1). In particular, ITPR1 and KREMEN2 had significant up regulation, whereas expressions of MAPK3, MKNK2, SNF and CAP2P were down regulated.

Cellular response to γ-60Co radiation is mediated via genes that control complex regulatory pathways such as cell cycle progression, apoptosis, or DNA repair. The relative contribution of changes in the expression of these genes on signaling pathways is unknown [28].

In this study ITPR1, KREMEN2 gene members expression triggers the death of C-4 I cells by WNT and PDGF signaling pathways in γ-60Co radiation treated C-4 I cells compared to control cells. The type 1 inositol-1,4,5-trisphosphate receptor (ITPR1) mediates calcium release from the endoplasmic reticulum (ER). ITPR1receptor located on the ER membrane that is critical to calcium homeostasis, was reported to be cleaved during apoptosis in Jurkat cells [29].

Mapk3, Mknk2, Snf ve Cap2p gene members of the cell Glycolysis,TGF-β, WNT, PDGF, P38 MAPK, Oxidative stress response andP53signaling pathwayswere significantly downregulated in γ-60Co radiation treated C-4 I compared to control C-4 I cells.

MAPK3 regulates various cellular processes such as proliferation, differentiation, and cell cycle progression in response to a variety of extracellular signals [30]. ERK-1 MAP kinase prevents TNF-induced apoptosis through bad phosphorylation and inhibition of Bax translocation in HeLa Cells [ 31].

MKNK2 protein is one of the downstream kinases activated by mitogen-activated protein (MAP) kinases. It playing important roles in the initiation of mRNA translation, oncogenic transformation and malignant cell proliferation [32].

GO and pathways analysis identified the cell glycolysis related pathways were significantly down-regulated in gamma radiation treated C-4 I cells compared to control cells. Several gene members of the cell glycolysis related pathways were significantly down-regulated in gamma radiation treated C-4 I cells compared to control cells. Most cancer cells exhibit increased glycolysis and use this metabolic pathway for generation of ATP as a main source of their energy supply. Thus, combination of glycolytic inhibitors and DNA-damaging agents seems to be an attractive therapeutic strategy to effectively kill cancer cells [33].

Gene expressions by qRT-PCR

To gain insights into radiation response, qRT-PCR was used to assess expression patterns of many apoptosis-related genes, grouped to reflect the various protein families and pathways involved in apoptosis, in C-4 I cells at 60 hours after exposure to 16 Gy single dose of γ-60Co radiation (Figure 4).

Figure 4: Pie charts showing pathways altered in C-4 I cells at 60 hours after exposure to 16 Gy single dose of gamma radiation, as compared to control cells. Chart was generated from the microarray data analysis according to PANTHER pathway enrichment analysis. (A) were performed on up-regulated genes. (B) Were performed on down-regulated genes.

In this part of the present study, it was hypothesized that apoptosis-related gene expression levels in C-4 I cells were affected by γ-60Co radiation. Figure (5) shows that the apoptosisrelated genes Bid, Bik, Pmi?p1, Traf2, Traf3, Traf6, Tnf, Tnfrsf10b, Tnfrsf10d, Tp53?3, Socs2, Socs3, Nf-kb1, Nf-kb2, Cradd, Pten,Dffa, Fam96a and Fam96b were up-regulated (FC ≥2). While other apoptosis-related Bcl2l2, Bcl2l13, Bag1, Bad, Cad, Hrk, Fadd, Traf7, Tp53, Stat5b and Relb gene expression levels were down regulated (FC ≥ -2) (Figure 5).

Figure 5: Fold changes of genes involved in apoptosis in C-4 I cells at 60 hours after exposure to 16 Gy single dose of gamma radiation.

There are two main apoptotic pathways: The extrinsic apoptotic pathway involves engagement of particular death receptors that belong to the TNFR (Tumor Necrosis Factor Receptor) family and, through the formation of the DISC (Death Inducing-Signaling-Complex), leads to a cascade of activation of Caspases, including Caspase 8 and Caspase 3, which in turn induce apoptosis. Our results were in agreement with previous results that found the death-domain-containing receptor for TRAIL (TNF-Related Apoptosis-Inducing Ligand) was induced by P53 in response to DNA damage and in turn promotes cell death through Caspase 8. Thus, the extrinsic apoptotic pathway was activated by inducing the expression of TRAIL death receptor in response to γ-60Co radiation of C-4 I cells [34,35].

The Intrinsic apoptotic pathway is dominated by the BCL2 family of proteins, which governs the release of Cytochrome-C (CytoC) from the mitochondria. BCL2 family comprises antiapoptotic (pro-survival) and pro-apoptotic members. From our results radiation induced a pro-apoptotic BID and BIK genes expression in C-4 I cells. BID, a critical link between the extrinsic apoptosis pathway with the mitochondrial pathway in certain cell types [36]. This gene encodes a death agonist that hetero dimerizes with either agonist BAX or antagonist BCL2. It is a mediator of mitochondrial damage induced by caspase-8 [37,38].

BIK, as a BH-only pro-apoptotic member, binds with BCL-2, BCL2L1 or MCL-1 to replace BAX or BAK, which forms BAX/ BAK oligomerization and then triggers mitochondrial outer membrane permeabilization, cytochrome c into cytoplasm, caspase-9 activation and at last cell apoptosis [39]. Hur et al. [40], suggest that expression of BIK in human breast cancer cells is regulated at the mRNA level by a mechanism involving a non transcriptional activity of P53 by gamma radiation and by proteasomal degradation of BIK protein. Jiao et al. [41], generated a novel mutant form of BIK, as a therapeutic gene for breast cancer. Therefore, BIK and its mutant forms can be a novel therapeutic gene in cervix cancer targeted gene therapy.

Our results also suggested that radiation decreased gene expression of the BCL-2 family anti-apoptotic members. In a study conducted by Loriot et al. [42], radiotherapy has a critical role in the treatment of small-cell lung cancer (SCLC). The effectiveness of radiation in SCLC remains limited as resistance results from defects in apoptosis. Their results show the inhibition of BCL-2/ BCL-XL can enhance radiosensitivity of SCLC lung cancer cells in vitro and in vivo.

In another study Wu et al. [43], support the combination of radiation and pro-survival Bcl-2 family inhibitor as a potential novel therapeutic strategy in the local-regional management of breast cancer.

Table 1: Differentially expressed genes in 16 Gy C-4 I compared to C-4 I control groups from microarray data analysis.

This in vitro study presents the altering in gene expression and pathway analysis of gamma irradiation in cervical cancer C-4 I cells. This study reveals that gamma radiation induced the proapoptosis Bid, Bik, Traf2, Traf3, Traf6, Tnf, Tnfrsf10b, Tnfrsf10d and other gene expressions in C-4 I cells presumably and it may be a down-regulator of some anti-apoptotic gene expression levels. These results indicate that single dose of γ-60Co irradiation primarily affects C-4 I cells proliferation by inducing the intrinsic and extrinsic apoptotic pathways. Identification of specific genes may allow the determination of pathways important in radiation responses, that it may be beneficial in novel treatment strategy to increase the cancer cell sensitivity to radiotherapy by modulation of many genes expression.

This study was supported by project number 25190 of the Research Fund at University of ?stanbul.

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