Investigation of the effect of CA IX enzyme inhibition on the EZH2 gene and histone 3 modifications

İbrahim Karakus; Özen Özensoy Guler

doi:10.1515/tjb-2023-0066

Article Open Access

Investigation of the effect of CA IX enzyme inhibition on the EZH2 gene and histone 3 modifications

İbrahim Karakus and Özen Özensoy Guler

Published/Copyright: October 12, 2023

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From the journal Turkish Journal of Biochemistry Volume 48 Issue 6

Abstract

Objectives

Colon cancer is the most common gastrointestinal cancer worldwide with high morbidity and mortality rates. The main purpose of our study is to elucidate the interaction mechanism of the H⁺ ion concentration effect in the CO₂/HCO₃ ⁻ buffer system of tumor-associated carbonic anhydrase IX (CA IX) enzyme inhibition in the HT-29 colon cancer cell line on cell epigenetic modifications.

Methods

Cell culture was performed using the human colon cancer cell line HT-29. CA IX enzyme inhibitor Acetazolamide (AZA) was administered. The results of the cell viability test and inhibition were evaluated. Extracellular pH measurements were performed. Total histone protein isolation was performed and Histone H3 modifications were analyzed by ELISA method. After RNA isolation, complementary DNA (cDNA) synthesis was carried out. RT-PCR was performed to determine the gene expression levels of hypoxia-inducible factor 1A (HIF1A), enhancer of zeste homolog 2 (EZH2) and CA IX.

Results

CA IX enzyme inhibition in the HT-29 cell line decreased the expression of CA IX (p<0.05) and HIF1A (p<0.01) genes and increased the expression of the EZH2 (p<0.05). There was a significant decrease in the expression of CA IX (p<0.05) and HIF1A genes as a result of inhibition with AZA performed under hypoxic conditions. It was observed that CA IX enzyme inhibition increases the expression of the EZH2 gene by more than 3 times (p<0.01). As a result of AZA inhibition, methylation levels were observed to increase in normoxic conditions, while methylation levels were observed to decrease in hypoxic conditions.

Conclusions

Observing the changes in the H3 modifications and changes in the expression of CA IX, HIF1A and EZH2 genes in this study supports that CA IX enzyme inhibition plays an active role in epigenetic modifications.

Keywords: CA IX; H3 modifications; colon cancer; EZH2 ; HIF1A

Introduction

Colorectal cancer (CRC) is the third deadliest and fourth most frequently diagnosed cancer in the world [1]. The incidence and mortality of CRC vary significantly around the world. This geographical difference is thought to be caused by diet, environmental factors, genetic predisposition, and other risk factors [2]. Colon cancer accounts for 70 % of intestinal cancers. It is known that approximately 95 % of CRCs develop from adenomatous polyps. Various genetic and molecular changes occur as it transforms from a benign to a malignant form [3]. Loss of genomic stability due to genetic changes (gene mutations, gene amplification, etc.) and epigenetic changes (aberrant DNA methylation, chromatin modifications, etc.) that transform colonic epithelial cells into colon adenocarcinoma cells (gene mutations, gene amplification, etc.) and consequent gene changes are key molecular pathogenic steps that occur early in tumorigenesis [4].

The tumour microenvironment (TME) largely influences the tumorigenesis process. In many tumours, rapidly proliferating cancer cells form hypoxic areas. In response to tumour hypoxia, cancer cells regulate gene expression to fit the needs of the changing microenvironment [5]. The ability to sense and respond to changes in oxygen is essential for survival. Oxygen sensing mechanisms have been developed to maintain cell and tissue homeostasis as well as adapt to the chronic low oxygen conditions found in diseases such as cancer [6]. The carbonic anhydrase enzymes family (CA, EC 4.2.1.1) is a metalloenzyme that contains zinc (Zn²⁺) in its active centre and catalyses the conversion between CO₂ and bicarbonate [7]. To avoid the intracellular accumulation of acidic metabolic products that are incompatible with survival and proliferation, tumour cells activate the molecular mechanism that regulates pH by activating ion fluxes. CAs form carbonic acid (H₂CO₃) by hydrating CO₂. In this mechanism, carbonic anhydrase IX (CA IX) acts as a hypoxia-dependent catalytic component of HCO₃ ⁻ uptake [7]. It ensures the transport of HCO₃ ⁻ across the plasma membrane, neutralizes the intracellular pH (pH_i), and provides cancer cells with a survival advantage in the hypoxic/acidic microenvironment. Overexpression of CA IX in cancer tissues is regulated by hypoxia-inducible factor 1A (HIF1A)-mediated transcription. Therefore, the enzyme is considered to be an indicator of tumour hypoxia [8]. Under hypoxic conditions, the rate of change in pH_i due to lactate reveals an increase in lactate/H⁺ flux [9].

Chemical modifications to DNA and histone proteins create a complex regulatory network that modulates chromatin structure and genome function [10]. Histone acetylation has also been shown to play a role in regulating pH [11]. DNA methylation constitutes an important step in epigenetic programming. It has been reported that hypoxia induces histone methylation in several human culture cells and that hypoxia causes rapid changes in histone methylation, which reprograms chromatin in the cellular response [12]. Trimethylation (H3K27me3) at histone H3 lysine 4 (H3K4me3) and lysine 27 controls gene activity during development and differentiation [13]. There is very little information about whether H3K27me3 dynamically changes in response to environmental conditions in tumours under low oxygen conditions and an altered microenvironment. It is thought that demethylation of H3K27me3 may affect the histone trimethylation of hypoxia mediated by oxygen and 2-oxoglutarate dioxygenase enzymes [13]. Enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase that suppresses gene expression by methylating histone 3 lysine 27 and is the catalytic subunit of polycomb suppressor complex 2 (PRC2). EZH2-mediated methylation has been identified as an independent mechanism for epigenetic silencing of tumour suppressor genes in cancer [14]. The role of carbonic anhydrase enzymes in cancer as a prognostic marker or as a potential drug target is an extremely important research topic [15]. When the studies in the literature are examined, it has been found that there is a significant relationship between pH regulation and tumour cell proliferation and survival [16]. In these studies, the fact that the CA enzyme family is responsible for acidification in the tumour microenvironment and directly affects tumour prophylaxis and survival processes is an important indicator for anti-cancer therapies based on pH interactions [17]. The advantages of molecular techniques developed in recent years have been described or proposed for many new molecules that may be a treatment option in response to hypoxia, but it has been observed that all of them have different limitations [18]. Hypoxia has been identified as the main inducer of the CA IX enzyme, but up to now, there have not been enough studies in the literature on how the tumour microenvironment affects the CA IX expression and activity of extracellular acidosis [19]. Increased expression of CA IX has been observed in many solid tumours in a hypoxic and acidic microenvironment [20].

It is known that the determination of the effects of epigenetic changes in the tumour microenvironment, invasion, and metastasis has important potential in cancer management. There are also studies in the literature that show that epigenetic modifications are affected by intracellular and extracellular pH in hypoxic conditions [21]. However, the uncertainty about what effect hypoxia-induced CA IX has on the tumour microenvironment and epigenetic modifications has directed us to seek answers to the questions on this subject. Accordingly, our study aimed to investigate the relationship between epigenetic mechanisms and CA IX, a tumour hypoxia marker and pH regulator.

Methods

Cell culture and CA IX inhibition

The human colon cancer cell line HT-29 was obtained from the Ankara Gülhane Training and Research Hospital Physiology Laboratory. The cell lines are cultured as described by Tülüce et al. [22], in the medium containing 4,5 glucose, L-glutamine and sodium pyruvate (Dulbecco’s Modified Eagle’s Medium/DMEM, Cambrex Bio Science, Bergamo, Italy), to which 10 % Fetal Bovine Serum (FBS-Capricorn CP17-1756 Germany) and 1 % penicillin-streptomycin (Capricorn CP17-1828 Germany) were added. Hypoxic culture conditions (2 % O₂) were generated in a humidified incubator supplied with 95 % N₂ and 10 % CO₂ (Panasonic, Japan).

Acetazolamide (AZA) (Sigma, BCBZ9159-USA) was used in the study for CA IX enzyme inhibition. In the study, the IC50 value was calculated for the CA IX inhibitor AZA. The IC50 value of AZA was calculated with the detected cytotoxicity percentages. For AZA, the IC50 value in the HT29 cell line was determined to be 35.2 μM. After the calculated inhibition doses, culturing was carried out in normoxic and hypoxic conditions, with and without inhibitor, in such a way that each well had 10⁷ cells/mL.

Analysis of total histone protein isolation and histone H3 modifications

In our study, the EpiQuik Nuclear Extraction Kit (Epigentek, USA) was used to isolate nuclear proteins from cells based on the principle of preserving enzymatic activity [23] according to the study of Liu et al. in 2019. After treatment with pre-cleavage, cleavage, and equilibration buffers in the experiment, total core histones were isolated according to the manufacturer’s protocol for immediate use. For pre-lysis in the experiment, cells were centrifuged in 2 mL Eppendorf tubes at 4 °C and 112 rcf (g) for 5 min. They were suspended in pre-lysis buffer and mixed on ice for 10 min. For lysis, approximately 200 μL of cell pellet was incubated with Lysis buffer for 30 min on ice. The supernatant was centrifuged at 16.100 rcf (g) for 5 min at 4 °C and transferred to a new tube. 0.3 mL of Balance-DTT Buffer was added to the isolated proteins. Due to the different amounts of carbon-carbon double bonds in nucleic acids and proteins, nucleic acids have a maximum absorption at 260 nm, and proteins have a maximum absorption at 280 nm. After isolation, using a micro-volume spectrophotometer (Maestrogen, Taiwan) capable of measuring the absorption value at 280 nm wavelength, the extracted histone3 proteins were measured.

The EpiQuik Histone H3 Modification Multiplex Assay Kit (Epigentek, USA) was used to determine the variation levels of H3 modifications [24]. In a 96-well plate, the appropriate antibody was used to coat the wells for each histone H3 modification. The samples were studied in accordance with the experimental protocol. Modified histone ratios were calculated by measuring the absorbance density at 450 nm with the ELISA reader (Biotek Epoch-2, USA).

RNA isolation and cDNA synthesis

In order to observe the expression changes of HIF1A, EZH2, and CA IX genes, RNA isolation was performed after creating a normoxic and hypoxic environment [25]. The miRNeasy Mini Kit (Qiagen, Cat: 217084, Germany) was used for RNA isolation. The purity and amount of the isolated RNAs were calculated with the QIAxpert (Qiagen, Germany) device based on the absorption values (A260nm/A280nm) at 260 and 280 nm wavelengths [26]. In order to determine the expression of target genes, the isolated RNAs were first converted into cDNA. A Reverse Transcriptase Kit (QuantiNova, Cat: 205411, Germany) was used for this process [27]. Before the cDNA synthesis process, genomic DNA inhibition was performed to avoid DNA contamination.

Determination of gene expression level

RT-PCR Kit (Mix) (QuantiNova, Cat: 208252, Germany) and the QuantiNova LNA PCR Assay protocol were used in our study for quantitative real-time PCR to determine gene expression levels. Primer and locked nucleic acid probes (LNA probes) linked to the relevant gene region were used to determine the gene expression levels of EZH2, HIF1A, and CA IX under normoxic and hypoxic conditions [28]. The reaction was carried out in the Rotor Gene Q (Qiagen, Germany) device by adding 5 μL of cDNA sample to the tube content after adding the mastermix to the 0.1 mL PCR tubes. Primers used in the study were HIF1Α (UPFH0476121-HS_597202, QuantiNova, Germany), EZH2 (UPFH0128337-HS_249394, QuantiNova, Germany), CA IX (UPFH1132903-HS_2476193, QuantiNova, Germany), GAPDH (UPFH0007663-HS_Nova, Germany) as reference genes. Each sample was studied three times.

Statistical analysis

After the qPCR reaction, mRNA expression levels were calculated with the delta CT method and the fold change = 2^−ΔΔCT value from the formula ΔΔCT=ΔCT treatment – ΔCT control. For the statistical significance of the expression change between the test and control groups, the p-value was analysed with the t-test.

Results

Cell culture results

AZA inhibition of the CA IX enzyme caused a decrease in cell proliferation in cell culture after its application (Figure 1). At the same time, it has created an alkaline environment at pH value. It was also observed that the cells came together and clustered, and the cell-cell adhesion decreased.

Figure 1:

HT-29 cell culture microscopy images. (A) Normoxic state (without inhibitor), (B) normoxic state (with inhibitor), (C) hypoxic state (no inhibitor), (D) hypoxic state (with inhibitor).

The effect of the CA IX enzyme inhibitor AZA has been demonstrated in both normoxic and hypoxic conditions (Figure 1). As a result of the inhibition of HT-29 cells by AZA under normoxic conditions, it was observed that the cells lost their adhesion ability and invaded in small clusters (Figure 1A and B).

It was observed that under hypoxic conditions, HT-29 cells invaded more in large clusters, both with and without inhibitor. More morphological changes occurred in HT-29 cells that were exposed to hypoxia and administered inhibitors (Figure 1C and D).

Effect of CA IX enzyme inhibition on pH

While the pH value of the cells in the extracellular matrix before the AZA inhibition was applied to the HT-29 cell line was 6.29, it caused a decrease in the acidification of the cells in normoxic conditions (pH-6.65) after the inhibition of CA IX. Similarly, it was observed that acidification decreased in HT-29 cells in hypoxic conditions after CA IX enzyme inhibition (pH-6.39) (Figure 2).

Figure 2:

Effect of CA IX enzyme inhibition on pH.

Histone modifications

In the HT-29 cell line, it was determined that the amount of total histone protein decreased by 23 % in the cells treated with AZA and CA IX inhibitors under normoxic conditions in the measurements made after the isolation of total histone protein. However, in hypoxic conditions, this resulted in a 32 % increase in the amount of total histone protein in cells treated with the AZA inhibitor. Under normoxic conditions, after inhibition of CA IX by AZA, H3 modifications increased except for H3K27me3 (Table 1). It showed a decrease in H3 modifications after inhibition of CA IX by AZA under hypoxic conditions (Table 2).

Table 1:

Change in H3 modifications after AZA with CA IX inhibition under normoxic conditions.

Histone modification	Normoxic (AZA−) ng/µg protein	Normoxic (AZA+) ng/µg protein	Alteration, %
Total H3	10.89	12.57	15 %
H3K4me1	12.55	15.69	25 %
H3K4me2	11.54	15,85	37 %
H3K4me3	11.93	15.74	32 %
H3K9me1	11.44	15.95	39 %
H3K9me2	12.97	15.97	23 %
H3K9me3	8.83	15.55	76 %
H3K27me1	12.70	15.53	22 %
H3K27me2	10.65	16.01	50 %
H3K27me3	12.98	11.81	−9%
H3K36me1	9.27	15.72	69 %
H3K36me2	13.16	15.80	20 %
H3K36me3	9.08	9.35	3 %
H3K79me1	11.88	15.65	32 %
H3K79me2	10.84	15.94	47 %
H3K79me3	13.13	15.75	20 %
H3K9ac	11.63	15.50	33 %
H3K14ac	13.05	15.44	18 %
H3K18ac	9.95	9.33	−6%
H3K56ac	12.42	15.03	21 %
H3ser10PE	11.19	15.78	41 %
H3ser28P	11.56	15.96	38 %

Table 2:

Change in H3 modifications after AZA with CA IX inhibition under hypoxic conditions.

Histone modification	Hypoxic (AZA−) ng/µg protein	Hypoxic (AZA+) ng/µg protein	Alteration, %
Total H3	10.20	8.28	−19 %
H3K4me1	12.39	10.14	−18 %
H3K4me2	13.75	10.31	−25 %
H3K4me3	12.41	7.32	−41 %
H3K9me1	13.62	10.55	−23 %
H3K9me2	12.57	10.35	−18 %
H3K9me3	13.42	10.44	−22 %
H3K27me1	12.27	9.02	−27 %
H3K27me2	14.04	10.39	−26 %
H3K27me3	12.27	10.03	−18 %
H3K36me1	12.95	10.84	−16 %
H3K36me2	12.35	10.26	−17 %
H3K36me3	14.06	10.55	−25 %
H3K79me1	12.04	10.02	−17 %
H3K79me2	14.24	10.63	−25 %
H3K79me3	12.28	9.32	−24 %
H3K9ac	14.21	10.33	−27 %
H3K14ac	11.95	8.52	−29 %
H3K18ac	10.78	10.50	−3%
H3K56ac	11.99	8.99	−25 %
H3ser10P	14.08	10.40	−26 %
H3ser28P	12.49	10.19	−18 %

Gene expression results

Effect of CA IX enzyme inhibition in normoxic conditions

In our experimental studies, the HT-29 cell line without the AZA inhibitor was used as the control group. The second group is the HT-29 cell line with an inhibitor, in which AZA inhibition was applied under normoxic conditions.

When these groups were compared, there was a decrease in the expression of the HT-29 cell line CA IX (p<0.05) and HIF1A (p<0.01) genes under normoxic conditions as a result of AZA inhibition. CA IX inhibition associated with this condition was observed to increase the expression of the EZH2 gene 2.5-fold (p<0.05) (Figure 3A and B).

Figure 3:

Effect of CA IX enzyme inhibition in normoxic conditions. (A and B) EZH2, HIF1Α, CA IX gene expression levels as a result of CA IX inhibition in normoxic conditions. *Expresses statistical significance. *CA IX (p<0.05). *HIF1A (p<0.01). *EZH2 (p<0.05).

Effect of CA IX inhibition in hypoxic conditions

In our studies, the HT-29 cell line without the AZA inhibitor was used as the control group. The second group is the HT-29 cell line with an inhibitor, in which AZA inhibition is applied under hypoxic conditions. When these groups were compared, there was a significant decrease in the expression of CA IX (p<0.05) and HIF1A genes as a result of inhibition with AZA performed under hypoxic conditions. It was observed that CA IX inhibition increased the expression of the EZH2 gene more than 3 times (p<0.01) (Figure 4A and B).

Figure 4:

Effect of CA IX inhibition in hypoxic conditions. (A and B) EZH2, HIF1A, CA IX gene expression levels as a result of CA IX inhibition in hypoxic conditions. *Expresses statistical significance. *CA IX (p<0.05). *EZH2 (p<0.01).

Discussion

All these results show that AZA, a CA inhibitor, changes the acid-base balance by affecting intracellular and extracellular H⁺ ion concentrations in the CO₂/HCO₃ ⁻ buffer system of CA IX enzyme inhibition in colon cancer, and this ionic balance difference affects EZH2 gene expression and histone 3 modifications. However, we experimentally demonstrated that epigenetic mechanisms in tumour cells behave differently in normoxic and hypoxic conditions. The observed decrease in methylation and acetylation levels in histone modifications of the CA IX enzyme inhibition in HT-29 cells in our study is consistent with these findings. Also, the increased expression of EZH2 showed that this gene showed different expression patterns in both normoxic and hypoxic conditions and acted as both an oncogene and a tumour suppressor gene.

In the CA IX enzyme inhibition study of Sansone et al. in 2009, they showed that the survival ability of CRC cells was lost [29]. Both the morphological change and the numerical decrease of the cells, especially in hypoxic conditions, observed in our study are similar to this study. Similarly, in a study conducted by Koyuncu et al. in 2018, it was shown that inhibition of the CA IX enzyme in tumour cells reduces cell proliferation and induces apoptosis in cancer cells [30]. Many new molecule identification studies are carried out in cancer treatment, and it is thought that response to hypoxia may be an important treatment option. The acidic microenvironment facilitates poor prognosis and active tumour progression [31]. In the study conducted by Parks et al. in 2017, they showed that inhibition of the whole genomic CA IX enzyme inhibits intracellular pH decrease, cell growth, and progression [32]. In this context, the decrease in acidification in our study was evaluated as a good prognosis.

The metabolic rate and angiogenesis resulting from hypoxia cause phenotypic diversity, and the tumour cell shows its own specific gene expression. Gene expression is directly affected by factors such as histone modifications and DNA methylation [33]. In their 2019 study, Batie et al. reported that hypoxia stimulated histone methylation independent of HIF [12]. This feature of hypoxia indicates that it causes a decrease or increase in active histone methylation. These observations have directed us to focus on and clarify the relationship between CA IX and acetylation and methylation levels.

In our study showing the change in histone 3 modifications after CA IX enzyme inhibition, an increase in H3K4, H3K9, H3K27, H3K36, and H3K79 methylation was observed under normoxic conditions. On the other hand, H3K4, H3K9, H3K27, H3K36, and H3K79 methylation decreased under hypoxic conditions. It supports our hypothesis that inhibition of the CA IX enzyme may play a suppressive or stimulatory role in specific genes. The decrease in methylation levels after CA IX enzyme inhibition, especially in cells under hypoxia in our study, suggests that CA IX may be the right candidate for the treatment of over-methylated genes.

Hypoxia affects HIF activation and modulates the epigenetic environment as a result. A general decrease in histone acetylation [34] as a result of hypoxia is shown in many studies. In another study by McBrain et al., it was shown that histone acetylation regulates the intracellular pH value [9]. It has been shown that under these conditions, a decrease in acetyl-CoA, which is a co-substrate in histone acetylation, can cause significant changes in cellular metabolism [35]. CA IX enzyme inhibition, which is a hypoxia indicator, showed improvement in acetylation levels under normoxic conditions, while it was found to be insufficient under hypoxic conditions. It is known that hyperacetylation is associated with the activation of gene transcription and leads to increased gene expression, while hypoacetylation means suppression of gene expression [36]. In this case, it is thought that inhibition of the CA IX enzyme will provide a relative improvement in improving oxygen deficiency and acidification, especially in the tumour microenvironment resulting from a high metabolic rate.

It was observed that the inhibition of the CA IX enzyme caused an increase in histone 3 phosphorylation in normoxic conditions, whereas phosphorylation decreased in hypoxic conditions. In particular, it suggests that the decrease in the level of phosphorylation occurs by inhibition of the overexpression of the CA IX enzyme, which has a hypoxia response domain (HRE) in the promoter region under hypoxic conditions.

We showed that the expression of HIF1A decreased with the decrease in ambient acidification as a result of CA IX enzyme inhibition. However, our findings demonstrated that EZH2 expression increased concomitantly with a decrease in cellular acidification induced by the CA IX enzyme. The reduction in cellular acidification reveals an inverse correlation between CA IX and EZH2. Thus, the known role of EZH2 is to act as a tumour suppressor gene by catalysing the methylation of H3K27me3.

The literature shows that EZH2 has a H3K27me3-independent function as a transcriptional coactivator and plays a critical role as an oncogene in cancer initiation, development, and progression [37]. This supports the fact that EZH2 is involved in other pathways independent of PRC2, together with the reduction of acidification. In a study by Bremer et al. in 2021, they showed that EZH2 gene expression and activity were associated with colorectal carcinogenesis, and strong expression of EZH2 was associated with a significantly positive prognosis in patients with CRC [38]. In our study, the increase in EZH2 expression after CA IX enzyme inhibition, especially the morphological shrinkage and loss of adhesion in the tumour phenotype, show that the CA IX enzyme is an important molecular player. In a study by Hoffmann et al. in 2020, it was associated with the regulation of H3K27me3 expression of EZH2, which acts as the histone methyl transferase enzyme, in tumour progression, metastasis, and resistance to immune checkpoint blockade [39]. In another study, Liu et al. stated in 2016 that EZH2 is closely related to the development and progression of CRC in CRC patients and therefore can be used as a tumour biomarker that can show prognosis [40].

In line with the data we obtained as a result of CA IX enzyme inhibition using the colorectal cancer cell line HT-29 within the scope of our study, our recommendations are as follows: In future studies, an experimental design that includes measurements of intracellular and extracellular pH should be carried out. Histone H3 total methylation levels were measured for the relationship between CA IX enzyme inhibition and epigenetic regulators. Measuring HAT and HDAC family members and methyltransferase DNMT1, DNMT3A, and DNMT3B change levels will play an important role in elucidating how the tumour microenvironment and epigenetic indicators are related. We believe that our study is of high value, with our study revealing new strategies for treatment in colon cancer and their applicability to other cancer types. The data in the literature, the change in H3 modifications, and the observation of expression changes in our study strongly support that CA IX enzyme inhibition may play an active regulatory role in tumour progression and treatment. However, these data are not sufficient to explain the relationship between CA IX enzyme inhibition and the epigenetic mechanism. Therefore, this mechanism can be better understood if the expression of CA IX in vitro and in vivo, in which there are regulatory genes such as the proton transporter MCT, vascular endothelial growth factor (VEGF), and MMP (matrix-metalloproteinase), which play an active role in the tumour microenvironment, is examined together with epigenetic biomarkers.

Corresponding Author: İbrahim Karakus, Ministry of Health, Examination and Diagnostic Services Department, Ankara, Türkiye, Phone: 0312 471 79 32, E-mail: karakus1833@gmail.com

Research ethics: Not applicable.
Informed consent: Not applicable.
Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.
Competing interests: The authors have no conflicts of interests to disclose.
Research funding: This research was supported by Ankara Yıldırım Beyazıt University Scientific Research Projects Units with project number: 5563.

References

1. Rawla, P, Sunkara, T, Barsouk, A. Epidemiology of colorectal cancer: incidence, mortality, survival and risk factors. Gastroenterol 2019;14:89–103. https://doi.org/10.5114/pg.2018.81072.Search in Google Scholar PubMed PubMed Central

2. Aydın, İ, Şehitoğlu, İ, Özer, E, Yücel, AF, Pergel, A, Bedir, R, et al.. The evaluation of patients operated due to colorectal cancer. Kocatepe Med J 2015;16:102–9.10.18229/ktd.73484Search in Google Scholar

3. Manne, U, Shanmugam, C, Katkoori, V. Development and progression of colorectal neoplasia. Cancer Biomarkers 2010;9:235–65. https://doi.org/10.3233/cbm-2011-0160.Search in Google Scholar

4. Grady, WM, Carethers, JM. Genomic and epigenetic instability in colorectal cancer pathogenesis. Gastroenterology 2008;135:1079–99. https://doi.org/10.1053/j.gastro.2008.07.076.Search in Google Scholar PubMed PubMed Central

5. Benej, M, Pastorekova, S, Pastorek, J. Carbonic anhydrase IX: regulation and role in cancer. Subcell Biochem 2014;75:199–219. https://doi.org/10.1007/978-94-007-7359-2_11.Search in Google Scholar PubMed

6. Giaccia, AJ, Simon, MC, Johnson, R. The biology of hypoxia: the role of oxygen sensing in development, normal function and disease. Genes Dev 2004;18:2183–94. https://doi.org/10.1101/gad.1243304.Search in Google Scholar PubMed PubMed Central

7. Sedlakova, O, Svastova, E, Takacova, M. Carbonic anhydrase IX, a hypoxia-induced catalytic component of the pH regulating machinery in tumors. Front Physiol 2014;4:400. https://doi.org/10.3389/fphys.2013.00400.Search in Google Scholar PubMed PubMed Central

8. Ambrosio, MR, Di Serio, C, Danza, G. Carbonic anhydrase IX is a marker of hypoxia and correlates with higher Gleason scores and ISUP grading in prostate cancer. Diagn Pathol 2016;11:45. https://doi.org/10.1186/s13000-016-0495-1.Search in Google Scholar PubMed PubMed Central

9. McBrian, MA, Behbahan, IS, Ferrari, R. Histone acetylation regulates intracellular pH. Mol Cell 2012;49:310–21. https://doi.org/10.1016/j.molcel.2012.10.025.Search in Google Scholar PubMed PubMed Central

10. Jamali, S, Klier, M, Ames, S. Hypoxia-induced carbonic anhydrase IX facilitates lactate flux in human breast cancer cells by non-catalytic function. Sci Rep 2015;5:13605. https://doi.org/10.1038/srep13605.Search in Google Scholar PubMed PubMed Central

11. Bernstein, BE, Meissner, A, Lander, ES. The mammalian epigenome. Cell 2007;128:669–81. https://doi.org/10.1016/j.cell.2007.01.033.Search in Google Scholar PubMed

12. Batie, M, Frost, J, Frost, M. Hypoxia induces rapid changes to histone methylation and reprograms chromatin. Science 2019;363:1222–6. https://doi.org/10.1126/science.aau5870.Search in Google Scholar PubMed

13. Prickaerts, P, Adriaens, ME, Beucken, TVD. Hypoxia increases genome-wide bivalent epigenetic marking by specific gain of H3K27me3. Epigenet Chromatin 2016;9:46. https://doi.org/10.1186/s13072-016-0086-0.Search in Google Scholar PubMed PubMed Central

14. Tan, JZ, Yan, Y, Wang, XX. EZH2: biology, disease, and structure-based drug discovery. Acta Pharmacol Sin 2014;35:161–74. https://doi.org/10.1038/aps.2013.161 Feb.Search in Google Scholar PubMed PubMed Central

15. Ozensoy Guler, O, Supuran, CT, Capasso, C. Carbonic anhydrase IX as a novel candidate in liquid biopsy. J Enzym Inhib Med Chem 2020;35:255–60. https://doi.org/10.1080/14756366.2019.1697251.Search in Google Scholar PubMed PubMed Central

16. Parks, SK, Chiche, J, Pouyssegur, J. pH control mechanisms of tumor survival and growth. J Cell Physiol 2011;226:299–308. https://doi.org/10.1002/jcp.22400.Search in Google Scholar PubMed

17. Mboge, MY, Mahon, BP, McKenna, R. Carbonic anhydrases: role in pH control and cancer. Metabolites 2018;8:19. https://doi.org/10.3390/metabo8010019.Search in Google Scholar PubMed PubMed Central

18. Sørensen, BS, Horsman, MR. Tumor hypoxia: impact on radiation therapy and molecular pathways. Front Oncol 2020;10:562. https://doi.org/10.3389/fonc.2020.00562.Search in Google Scholar PubMed PubMed Central

19. Andreucci, E, Peppicelli, S, Carta, F. Carbonic anhydrase IX inhibition affects viability of cancer cells adapted to extracellular acidosis. J Mol Med (Berl) 2017;95:1341–53. https://doi.org/10.1007/s00109-017-1590-9.Search in Google Scholar PubMed

20. Kazokaitė, J, Aspatwar, A, Parkkila, S, Matulis, D. An update on anticancer drug development and delivery targeting carbonic anhydrase IX. PeerJ 2017;5:e4068. https://doi.org/10.7717/peerj.4068.Search in Google Scholar PubMed PubMed Central

21. Chano, T, Avnet, S, Kusuzaki, K, Bonuccelli, G, Sonveaux, P, Rotili, D, et al.. Tumour-specific metabolic adaptation to acidosis is coupled to epigenetic stability in osteosarcoma cells. Am J Cancer Res 2016;6:859–75.Search in Google Scholar

22. Tülüce, Y, Ahmed, BA, Koyuncu, I. The cytotoxic, apoptotic and oxidative effects of carbonic anhydrase IX inhibitor on colorectal cancer cells. J Bioenerg Biomembr 2018;50:107–16. https://doi.org/10.1007/s10863-018-9749-9.Search in Google Scholar PubMed

23. Liu, W, Cui, Y, Ren, W. Epigenetic biomarker screening by FLIM-FRET for combination therapy in ER+ breast cancer. Clin Epigenet 2019;11:16.10.1186/s13148-019-0620-6Search in Google Scholar PubMed PubMed Central

24. Peixoto, P, Etcheverry, A, Aubry, M. EMT is associated with an epigenetic signature of ECM remodeling genes. Cell Death Dis 2019;10:205. https://doi.org/10.1038/s41419-019-1397-4.Search in Google Scholar PubMed PubMed Central

25. Brown, RAM, Epis, MR, Horsham, JL, Kabir, TD, Richardson, KL, Leedman, PJ. Total RNA extraction from tissues for microRNA and target gene expression analysis: not all kits are created equal. BMC Biotechnol 2018;18:16. https://doi.org/10.1186/s12896-018-0421-6.Search in Google Scholar PubMed PubMed Central

26. Vildmyren, I, Cao, HJV, Haug, LB. Daily intake of protein from cod residual material lowers serum concentrations of nonesterified fatty acids in overweight healthy adults: a randomized double-blind pilot study. Mar Drugs 2018;16:197. https://doi.org/10.3390/md16060197.Search in Google Scholar PubMed PubMed Central

27. Browne, DJ, Brady, JL, Waardenberg, AJ, Loiseau, C, Doolan, DL. An analytically and diagnostically sensitive RNA extraction and RT-qPCR protocol for peripheral blood mononuclear cells. Front Immunol 2020;11:402. https://doi.org/10.3389/fimmu.2020.00402.Search in Google Scholar PubMed PubMed Central

28. Senamela, T, Kock, M, Becker, P, Potgieter, JJC. Detection of the Janus kinase 2 V617F mutation using a locked nucleic-acid, real-time polymerase chain reaction assay. Afr J Lab Med 2018;7:658. https://doi.org/10.4102/ajlm.v7i1.658.Search in Google Scholar PubMed PubMed Central

29. Sansone, P, Piazzi, G. Cyclooxygenase-2/carbonic anhydrase-IX up- regulation promotes invasive potential and hypoxia survival in colorectal cancer cells. J Cell Mol Med 2009;13:3876–87. https://doi.org/10.1111/j.1582-4934.2008.00580.x.Search in Google Scholar PubMed PubMed Central

30. Koyuncu, I, Gonel, A, Kocyigit, A. Selective inhibition of carbonic anhydrase-IX by sulphonamide derivatives induces pH and reactive oxygen species-mediated apoptosis in cervical cancer HeLa cells. J Enzym Inhib Med Chem 2018;33:1137–49. https://doi.org/10.1080/14756366.2018.1481403.Search in Google Scholar PubMed PubMed Central

31. Peppicelli, S, Bianchini, F, Calorini, L. Extracellular acidity, a "reappreciated" trait of tumor environment driving malignancy: perspectives in diagnosis and therapy. Cancer Metastasis Rev 2014;33:823–32. https://doi.org/10.1007/s10555-014-9506-4.Search in Google Scholar PubMed

32. Parks, SK, Cormerais, Y, Durivault, J. Genetic disruption of the pHi-regulating proteins Na+/H+ exchanger 1 (SLC9A1) and carbonic anhydrase 9 severely reduces growth of colon cancer cells. Oncotarget 2017;8:10225–37. https://doi.org/10.18632/oncotarget.14379.Search in Google Scholar PubMed PubMed Central

33. Miller, JL, Grant, PA. The role of DNA methylation and histone modifications in transcriptional regulation in humans. Subcell Biochem 2013;61:289–317. https://doi.org/10.1007/978-94-007-4525-4_13.Search in Google Scholar PubMed PubMed Central

34. Kindrick, JD, Mole, DR. Hypoxic regulation of gene transcription and chromatin: cause and effect. Int J Mol Sci 2020;21:8320. https://doi.org/10.3390/ijms21218320.Search in Google Scholar PubMed PubMed Central

35. Platt, JL, Salama, R, Smythies, J. Capture-C reveals preformed chromatin interactions between HIF-binding sites and distant promoters. EMBO Rep 2016;17:1410–21. https://doi.org/10.15252/embr.201642198.Search in Google Scholar PubMed PubMed Central

36. Gräff, J, Tsai, L. Histone acetylation: molecular mnemonics on the chromatin. Nat Rev Neurosci 2013;14:97–111. https://doi.org/10.1038/nrn3427.Search in Google Scholar PubMed

37. Huang, J, Gou, H, Yao, J. The noncanonical role of EZH2 in cancer. Cancer Sci 2021;112:1376–82. https://doi.org/10.1111/cas.14840.Search in Google Scholar PubMed PubMed Central

38. Bremer, SCB, Conradi, LC, Mechie, NC. Enhancer of zeste homolog 2 in colorectal cancer development and progression. Digestion 2021;102:227–35. https://doi.org/10.1159/000504093.Search in Google Scholar PubMed

39. Hoffmann, F, Niebel, D, Aymans, P. H3K27me3 and EZH2 expression in melanoma: relevance for melanoma progression and response to immune checkpoint blockade. Clin Epigenet 2020;12:24. https://doi.org/10.1186/s13148-020-0818-7.Search in Google Scholar PubMed PubMed Central

40. Liu, X, Liu, H, Gu, L. EZH2 expression and its correlation with clinicopathological features in patients with colorectal carcinoma. Open Life Sci 2016;11:287–92. https://doi.org/10.1515/biol-2016-0042.Search in Google Scholar

Received: 2023-03-24

Accepted: 2023-06-16

Published Online: 2023-10-12

This work is licensed under the Creative Commons Attribution 4.0 International License.

Articles in the same Issue

https://doi.org/10.1515/tjb-2023-0066

Keywords for this article

CA IX; H3 modifications; colon cancer; EZH2; HIF1A

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BY 4.0