hCAIX Expression and Cell Cycle in Cytokine Stimulated Human Colon Cancer Cells

Background: At the aim of this study, we investigated the effects of different kind of cytokines and the combinations of which on human carbonic anhydrase IX ( hCAIX) expression in HT-29 cell selected as colon carcinoma model for different doses and time of exposure to determine role of cytokines for treatment of colon carcinoma cells. Results: To sum up, h CA9 expression in the levels of gene and protein increased in HT-29 cells when stimulated with 1000 U/mL TGF-β for 24 h. The stimulation of HT-29 cells with IL1 α alone and IL1α- TGF-β combination has not revealed any effect on hCA9 expression in both levels of gene and protein in contrast to, 1000 U/mL IL1α-TNFα and especially TGFβ-TNFα have reducing effect on h CA9 expression level in HT-29 cells for time ranges of 24 h, 48 h, 72 h. In addition to this data, it was observed that HT-29 cells at the phase of G0G1 are arrested in cell cycle when stimulated with either of 1000 U/mL IL1α-TNFα and TGFβ-TNFα. Moreover, it was observed that h CA9 expression level in HT-29 cells decreases at the phases of synthesis (S) and G2M. Discussion: We concluded that combination of TNFα-TGFβ has created antagonistic effect on hCA9 expression in HT-29 colon cancer cell model. On the other hand, combination of TNFα-IL1α caused sinergistic effect on h CA9 expression level in this cell model. When these results are demonstrated decrease in the cytokine exposed hCA9 expression is a finding to develop a novel approach for anticancer therapy. Conclusion: Our finding related to the change in the expression of hCA9 following cytokine stimulate to colon cancer cell line gives an idea about the effect of cytokine stimulation on the expression of this gene which will be a hot spot research in colon carcinogenesis. Methods: HT-29 colon cells were chosen as a colorectal adenocarcinoma model. hCA9 gene expression in the level of mRNA was measured in cytokines stimulated HT-29

3 Background Colorectal cancer is the most common third type of cancer observed in the world. In the world, colorectal cancer causes 395.000 people to die each year [1,2]. Therefore, enlightment of genes which are responsible for colorectal cancer is crucial to contribute a new dimension to conventional recruitment of colorectal cancer.
Human carbonic anhydrase IX (hCAIX), which is one of the popular transmembrane protein related with tumor progression and metastasis, is a member of transmembrane carbonic anhydrases. Expression of CAIX is so limited in normal cells. However, hypoxic conditions of the tumor cause to increased CAIX expression and hypoxia inducible factor alpha (HIF1α) in different tumor types. These data provide a hCAIX is a powerful marker in terms of tumor hypoxia [3,4]. Moreover, this hCAIX enzyme plays a critical function in tumor invasion and metastasis by the way of cell to cell adhesion [5]. Therefore, recent studies about especially hCAIX and various types of cancer attract the attention on this carbonic anhydrase subtype [6]. hCA9 is one of the important genes shown in colorectal tumorigenesis. hCAIX expression in colorectal tumors was first demonstrated by Saarnio and et al. along with Ki-67 antigen which is also a major marker for cell proliferation in colorectal adenocarcinoma [6,7].
Cytokines are secreted peptides which have multiple roles in regulation of cellular interaction and communication and cell proliferation, differentiation, invasion in cancerogenesis. On the side, since these molecules have been used in immunotherapy, studies about cytokines have been getting worse. The fact that these molecules have immunotherapy potential against malignant diseases is the focus of the researchers [8][9][10].
Immune effector cells capable of destruction of human tumors should be defined as in vitro [11]. IL-1α is the most potent pro-inflammatory cytokines that is generated by macrophages immediately after confronting the inflammatory stimuli. In addition, molecules from IL1 family are mostly expressed in tumor regions and is effective on each stage of tumorigenesis such as tumor invasion and connection between host and immune system in malign cells [12]. Like IL1α, TGFβ beta has also pleitropic roles in tumor cells.
TGFβ supports malign transformation and tumor progression in various types of cell. In studies about colorectal cancer, high level of expression of TGFβ in primary tumor depends on late stages. Divergent effect of TGFβ in carcinogenesis depends on differentiation status and proliferative capacity of epithelium cells. TGFβ has an inhibition effect on well differentiated primary colon carcinoma, TGFβ makes less differentiated cells invase and proliferate [13,14]. Therefore, the effect of cytokine on the modulation of cancer-related genes needs to be adressed. In this work, we focused on determining the effect of cytokine on hCAIX expressions. There is rather limited study available about cytokines regulation of hCAIX. Yildirim and Kockar in 2009 reported that TGFβ upregulates hCAIX expression in liver cell model, Hep3B. It is crucial to know that the knowledge of this type of interaction might be important for improvement of new therapeutic approaches [15].
We hypothesized that if cytokines acting through caix expression levels alteration and relation with cell cycle phases in HT-29 cancer cell line. To test this hypothesis, cell line treated with different cytokines and these combinations. Untreated cells were used as control. Our research group previously demonstrated that TNFα decreased the hCA9 mRNA anf protein expression along with cell cycle arrest in HT-29 cells [16]. Because of the insufficient research about the relation between cytokines and expression of hCAIX, this study was conducted in order to investigate contribution of the above mentioned cytokines in regulation of hCA9 which is a crucial marker in colorectal cancer.

Cell culture of HT-29 colon cells
HT-29 colon cell line provided by Sukran Yılmaz, Sap Institue, Ankara, Turkey. HT-29 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 % fetal bovine serum (FBS) and incubated at 37˚C in a humidified atmosphere of 95 air and 5% CO 2 incubator. Then, cells were seeded into a flask of 25 cm 2 and following 24 h, these cells were treated with double combinations of these cytokine (TGFβ, IL1α). TNFα cytokine was also included in combination protocol.

Cell cycle analysis of HT-29 colon cells by flow cytometry
The analysis of the cell cycle phase was performed as described previously [16]. Briefly, cells were washed in PBS and then stained with PI using a commercial kit (Beckman Coulter, USA). Cellular DNA content was measured and analyzed on a flow Cytometry System with CXP software (Beckman Coulter FC500 System, USA).

Annexin V staining apoptosis test
HT-29 colon cells were seeded into a 6-well plate. After 24 h incubation process, each one of 1000U/mL TGFβ, IL1α separately and 1000U/mL various combinations of IL1α-TNFα, IL1α-TGFβ, TNFα-TGFβ.were added to the cells. The follow-up protocols were carried out according to the manufacturer protocol of AnnexinV-FITC kit (Beckman Coulter, USA).
Samples were analysed by a flow cytometer and analyzed with CXP software (Beckman Coulter FC500 System, USA).

Total RNA isolation and cDNA synthesis
Total RNA isolation from the HT-29 cells was done with RNeasy mini kit (Qiagen GmBH., Germany). Reverse transcription for cDNA synthesis was performed in 20 μl final reaction volume including 50 U MuLV reverse transcriptase (Applied Biosystems, Foster City, CA), 10X PCR buffer, 5 pmol specific antisense primer, 4 U RNase inhibitor (Roche), 4 mmol/L of each dNTPs, 6.25 mmol/L MgCl 2 , and 2 μl (1ug) RNA. The RT step was performed as described previously on a Corbett Research Thermocycler at 42°C for 30 minutes followed by 94°C for 5 minutes [16] .
The reactions were performed with 5 µL cDNA template in a final volume of 25 µL, containing 12.5 μL Sybr Green master mix (Qiagen, Germany), with 15 pmoL primers for whole genes in a Rotor-Gene Real-time PCR instrument (Qiagen, Germany). Initial denaturation was 1 min at 94°C, 40 cycles of specific annealing temperature for 2 sec, extension at 72°C for 10 sec and denaturation at 94°C. Melting curve analysis was carried out in the temperature range of 55 to 95 °C. We calculated the relative quantification using the ratio between hCA9 and TBP mRNA. Arbitrary copy numbers were analyzed using Rotor-Gene v.5 software (Qiagen, Germany). A melt curve was obtained from 65-99 o C for the specificity of the reaction. Three technical and biological reactions were performed for each transcript and negative control was used.

h CAIX protein expression
Brifly, after cell culture procedures, cells were scraped with cell scraper and then 1x10 5 cells suspended in PBS. Human anti-CAIX antibody was added in each test tube for 45 minutes in ice bath and then washed with PBS to remove unbound antibodies. The assay was carried out in triplicate. The expression of h CAIX was monitored and analyzed on a Beckman Coulter FC500 Flow Cytometry System with CXP software (Beckman Coulter, USA).

Statistical analysis
Each data was the mean of three independent values. All data were expressed as mean ± S.D. and analyzed one-way ANOVA Test by SPSS 11.5 (SPSS Inc., Chicago, IL, USA) to determine the significance of differences between groups (untreated control cells and treated cells). P< 0.05 was considered statistically significant.

Protein expression
HT-29 colon cells were seeded into a 6-well plate and incubated for 24 h. After incubation process, either of 1000 U/mL TGFβ and IL1α and various combinations of 1000 U/mL of

mRNA expression of hCAIX
The effect of these cytokines on hCA9 mRNA expression level was evaluated relative to untreated control cell group. Firstly, HT-29 cells were exposed to TGFβ for 24 h, it was

Discussion
The expanding drugs and inhibitors have been used that inhibition of hCAIX against various cancer and metabolic disorders as a molecule [17][18][19]. This knowledge prompted us to investigate the effects of different cytokines that crucial proteins for immunotherapy on expression of hCAIX critical proliferation mechanism of cancer cells. HT-29 model colon cells are utilized at many studies about colorectal carcinoma. These cells are important in many studies since they have most of characteristic colonic epithelium [20]. Several investigators reported the hCAIX expression in colon cells. When normal mucosa in which hypoxic response is observed early hCAIX is compared with colorectal cancers, it is observed that there is an increase in hCAIX expression in colorectal cancers [6,21,22].
According to the study demonstrated by Adrián (2003), expression of carbonic anhydrase IX was increased under hypoxic conditions in xenograft nude mice model that were created a colorectal tumor using HT-29 colon carcinoma cells [23]. For this purpose, we selected HT-29 colon carcinoma cell line to point out relationship between hCA9 expression and cytokines. In this study, two cytokines (IL1α and TGFβ) were used since these cytokines have been implicated in cancer. TNFα, previously demonstrated decreasing effect on hCA9 gene expression, was also included into the study. This study, the association between cytokines and CA9 which is a gene related with tumor, was investigated due to the lack of information about this subject. The data obtained in our research attracts more attention because there was limited study conducted before about this topic. TGFβ, one of these cytokines, causes malign transformation and tumor progression in various types of cell. Moreover, production of TGFβ tends to increase in various cancer including colon cancer [24] . These cancer cells on which oncogenes are dominant activate transcription of TGF gene [25,26]. In studies about colon and types of oxygen and carcinogenesis [30]. There are so many investigation indicating the relation between cancer development and expressions of oncogenes. Besides this, existence of the relation between chronic inflammation and cytokine production brings additional mechanism in the carcinogenesis process. In cancer, NFKβ activation has essential role in various tumor [31]. This activation is also essential for regulation of genes acting on immune response. However, the association between expression of oncogene and pro-inflammatory cytokines could still not have been raised. Molecules from IL1 family are mostly expressed in tumor regions and which are potential cytokines affecting tumorigenesis. IL1 is effective on each stage of tumorigenesis such as tumor invasion and connection between host and immune system in malign cells [12]. Immune

Conclusions
This finding reflects the functionally multiple nature of these cytokines and represent a useful paradigm to study the complex cellular targets such as CAIX that regulate colon cancerogenesis. Therefore, immunotherapy potential of these cytokines against malign disease is always focus for researchers.