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HIF-1 inhibitors: differential effects of Acriflavine and Echinomycin on tumor associated CA-IX enzyme and VEGF in melanoma

  • Beyza Ecem Öz Bedir ORCID logo , Emine Terzi ORCID logo , Ender Şimşek ORCID logo , İbrahim Karakuş ORCID logo , Tuğba Kevser Uysal ORCID logo , Elif Ercan ORCID logo and Özen Özensoy Güler ORCID logo EMAIL logo
Published/Copyright: June 29, 2021
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Turkish Journal of Biochemistry
From the journal Turkish Journal of Biochemistry Volume 46 Issue 6

Abstract

Objectives

To determine the effects of hypoxia-inducible factors (HIF)-1 inhibitors on carbonic anhydrase-IX (CA-IX) enzyme and vascular endothelial growth factor (VEGF) in melanoma. The HIF-1 pathway induces tumor growth and metastasis by stimulating the expression of CA-IX enzyme and VEGF proteins.

Methods

We evaluated the inhibition effects of Acriflavine and Echinomycin on CA-IX enzyme and VEGF in WM115 (primary) and SKMEL30 (metastatic) cell lines in normoxic and hypoxic conditions with Enzyme Linked Immunosorbent Assay. The cytotoxic activity of HIF-1 inhibitors was performed by using WST-1 assay. All experiments were performed at 450 nm using Epoch™ Microplate Spectrophotometer.

Results

IC50 values were observed with a concentration of 3 μmol/L for Echinomycin and Acriflavine in the WST-1 assay. Decreased CA-IX and VEGF levels were determined in both normoxia and hypoxia after inhibitors’ treatment with WM115 and SKMEL30 cell lines (p<0.05). Inhibitory effect of HIF-1 inhibitors on CA-IX and VEGF proteins was observed in cell lines WM115 and SKMEL30.

Conclusions

Due to the importance of our study, using HIF-1 inhibitors may be an alternative treatment for melanoma. Also to design new HIF-1 inhibitor derivatives is a promising approach for further studies targeting CA-IX enzyme and VEGF.

Öz

Amaç

Bu çalışmanın amacı, HIF-1 inhibitörlerinin karbonik anhidraz-IX (CA-IX) enzimi ve vasküler endotelyal büyüme faktörünün (VEGF) üzerindeki inhibisyon etkilerini melanomda incelemektir. HIF-1 yolağının, CA-IX enzimi ve VEGF proteinlerinin ekspresyonunu uyararak tümör büyümesini ve metastazı indüklediği bilinmektedir.

Gereç ve Yöntem

Akriflavin’in ve Ekinomisin’in CA-IX enzimi ve VEGF proteini üzerindeki inhibisyon etkileri WM115 (primer) ve SKMEL30 (metastatik) hücre hatlarında Enzim Bağlı İmmünosorbent Testi ile normoksik ve hipoksik koşullarda değerlendirilmiştir. HIF-1 inhibitörlerinin sitotoksik aktivitesi, WST-1 yöntemi kullanılarak gerçekleştirilmiştir. Tüm ölçümler, Epoch™ Microplate Spektrofotometre kullanılarak 450 nm’de yapılmıştır.

Bulgular

Ekinomisin ve Akriflavin için IC50 değeri WST-1 testi ile 3 μmol/L olarak belirlenmiştir. Normoksik ve hipoksik koşullarda bu inhibitörlerin her iki hücre hattında CA-IX enzimi ve VEGF protein düzeylerinde azalmaya neden olduğu belirlenmiştir (p<0.05). HIF-1 inhibitörlerinin CA-IX enzimi ve VEGF proteinleri üzerine inhibisyon etkisi gözlemlenmiştir.

Sonuçlar

HIF-1 inhibitörlerinin melanom tedavisinde alternatif bir yaklaşım olacağı düşünülmektedir. HIF-1 inhibitör türevlerinin sentezi CA-IX enzimi ve VEGF proteinlerini hedefleyen başka çalışmalar için umut verici bir yaklaşımdır.

Keywords: Acriflavine; CA-IX; Echinomycin; hypoxia; melanoma; VEGF
Anahtar Kelimeler: Akriflavin; CA-IX; Ekinomisin; Hipoksi; Melanom; VEGF

Introduction

Melanoma, a most common type of cancer, is derived from melanocyte cells and the field has seen an unprecedented set of clinical advances in recent years [1]. Hypoxia stimulates melanoma survival and its tumor development by reducing the amount of oxygen radicals in the permissive skin microenvironment contributes a great sense to tumor progression [2, 3].

Tumor hypoxia is related to cancer prognosis also it is associated with aggressive growth and metastasis [4]. In hypoxia, hypoxia-inducible factors’ (HIF) activity is controlled primarily through post-translational modification and stabilization of the HIFs (HIF-1α and HIF-2α subunits). Thus, HIF-1α protein levels are stimulated by overall HIF transcriptional activity. HIF-1α is responsible for the prognosis in many tumors such as gastric, bladder, breast, colorectal and melanoma [5]. Therefore, they could be candidates as a potential therapeutic key in the progression and aggressiveness of cancer comparing with conventional therapies such as chemotherapy or radiation therapy [6]. Genes that are up-regulated by HIFs include pH regulation enzyme (carbonic anhydrase-IX [CA-IX]) and angiogenic growth factor (vascular endothelial growth factor [VEGF]) [4, 7]. CA-IX promotes metastasis by stimulating the acidification in the tumor microenvironment in melanoma and VEGF-mediated angiogenesis is managed to promote the progression of melanoma by hypoxia [8].

CA family is responsible for catalyzing the reaction of CO2 to produce H+ and HCO3 − ions [9]. Based on the literature, the α-CAs family is classified into 16 different members such as cytosolic; CA-I, CA-II, CA-III, CA-VII, CA-XIII and membrane-associated; CA-IV, CA-IX, CA-XII, CA-XIV and CA-XV. CA-VA/VB are located in mitochondria and one of them is distributed in saliva as presented CA-VI. There are carbonic anhydrase related proteins (CARPs): VIII, X and XI proteins also the members of the CA family as catalytically inactive forms of CA [10, 11]. CAs are identified as key regulators of metabolism, especially for tumor formation and pH/CO2 homeostasis [12]. CO2 is directly responsible for acidemia in metabolism so CA acts as a pH modulator in metabolism. Acidic extracellular pH (pHe) has the ability to contribute to tumor cell migration and invasion. According to this contribution, CA enzyme inhibition is crucial to preventing the acidity of the tumor microenvironment [8]. The main role of regulating pH, it is well known that tumor associated CA-IX enzyme correlates cell aggressiveness and cell migration with a poor prognosis in cancer patients [13, 14]. CA-IX is a hypoxia-inducible component of the tumoral pH regulatory system [15].

Another important factor affected by hypoxia is VEGF, an important mediator in cancer, contributes to the ability of tumor angiogenesis, has a great expression in most of the tumors [16, 17]. In this regard, it increases vascular permeability and stimulates both apoptosis and cell migration [16]. VEGF regulates the angiogenesis in cell signaling as a powerful angiogenic cytokine in carcinogenesis [7].

All of these processes are controlled under hypoxic conditions, suggesting as a key mediator in carcinogenesis [15]. Thus, HIF-1 inhibitors have potential applications targeting hypoxic cell signaling for cancer treatment [18, 13]. Acriflavine and Echinomycin are the best-known inhibitors of HIF-1. They inhibit HIF-1 activity by affecting different steps of the HIF pathway. Acriflavine contains trypaflavine and proflavine groups and prevents dimerization of HIF subunits [19]. Echinomycin, a natural cyclic peptide, prevents the attachment of HIF-1α and HIF-1β proteins to the HRE domain [19].

Hypoxia is associated with a poor prognosis of cancers including melanoma. This led us to focus on HIF pathway targets especially CA-IX and VEGF. In this study, we aim to investigate the effect of HIF-1 pathway inhibition on CA-IX and VEGF by using Acriflavine and Echinomycin in melanoma.

Material and methods

Cell lines

Primary melanoma cell line WM115 (WM115-01-0001) and metastatic melanoma cell line SKMEL30 (03010901) were purchased from Rockland (USA) and the Ministry of Agriculture and Forestry SAP Institute Cell Bank, the Republic of Turkey, respectively. WM115 and SKMEL30 were incubated in DMEM (Capricorn, DMEM/HPA) and RPMI 1640 (Diagnovum, D840), respectively, including 10% Fetal bovine serum (FBS) (Capricorn, FBS-HI-11A), 1% Streptomycin/Penicillin (Capricorn, PS-B) at optimum conditions (37 °C, 5% CO2). WM115 and SKMEL30 cell lines were used to compare the effects of Acriflavine and Echinomycin on melanoma cell lines, thus primary melanoma cell line WM115 was evaluated as a control group.

Hypoxic condition and inhibitor treatment

The density of 0.3 × 106 cells/well in a six-well plate of the confluent cells were trypsinized and subcultured. To create a hypoxic environment, CoCl2, a chemical inducer of hypoxia-inducible factor, was added to the cell cultures as 10% FBS-CoCl2 (Sigma–Aldrich, Turkey). Hypoxia was induced by adding CoCl2 at the final concentration of 100 μmol/L in cell culture media. The cell cultures treated with CoCl2 were incubated in an incubator (37 °C; 5% CO2) for 24 h.

Cytotoxicity assay

The IC50 values of Echinomycin (Sigma–Aldrich, SML0477, Turkey) and Acriflavine (Sigma–Aldrich, 01673, Turkey) were determined as 3 μmol/L by using a WST-1 assay (Cayman Chemical). Using 96-well plates, all cells were cultured with DMEM supplemented with 10% FBS. The cells were treated with 0, 2.5, 5, 10, 25, 50, 100 μmol/L concentrations of Echinomycin and Acriflavine for 24 h, in triplicate in a humidified 5% CO2 atmosphere. WST-1 reagent was added to the wells and incubated at 37 °C for 2 h. The plates were then read at 450 nm on an Epoch™ Microplate Spectrophotometer.

Determination of CA-IX and VEGF

CA-IX and VEGF levels were measured by using CK-bio-10734, CK-bio-13937 ELISA kits (Seoul, Korea). Firstly, the collected cells were diluted in PBS to reach a level of one million/mL. The damaged cells were performed with prepared freeze-thaw cycles. After centrifugation (+4 °C, 1,500 g, 10 min), the supernatants were collected carefully. The protein concentrations were calculated using the NanoDrop™. Protein concentration is a range of 0.048–0.082 ng/40 µL in ELISA for each well.

Briefly, 40 µL of the supernatant, 10 µL primer antibody and 50 µL Streptavidin-Horseradish peroxidase were mixed together to reach a 100 µL final volume at 37 °C for 60 min. After incubation, chromogen solutions of A and B were added into the wells. After the incubation at 37 °C for 10 min we used a stop solution to end the reaction. All experiments were performed three times (n=3) and read at 450 nm by an Epoch™ Microplate Spectrophotometer (BioTek, USA).

Statistical analysis

The groups (no inhibitor, Acriflavine and Echinomycin) were compared with two-way ANOVA, and post hoc Tukey test. Normoxia-hypoxia comparison of CA-IX and VEGF levels was performed with Student’s t-test. Statistical significance level was accepted as p<0.05. Statistical analyzes and calculations were carried out in GraphPad Prism 9.1.0.

Results

CA-IX ELISA assay

In hypoxic conditions, CA-IX levels were increased in WM115 (p<0.0001) while decreased in SKMEL30 (p>0.05). In Figure 1, the enzyme levels of CA-IX were decreased in SKMEL30 cell lines and in WM115 cell lines by Acriflavine and Echinomycin. Inhibitors reduced the enzyme levels of CA-IX in normoxia and hypoxia for both SKMEL30 and WM115 cell lines. These results were statistically significant (p<0.05). The analyses were performed by using GraphPad Prism 9.1.

Figure 1: Effect of Acriflavine and Echinomycin on CA-IX levels in melanoma cell lines. ELISA assays were performed as three independent times. Data were expressed as ± SEM. (A) Effect of HIF-1 inhibitors on CA-IX levels in SKMEL30 cells. Acriflavine and Echinomycin decreased CA-IX levels in normoxic and hypoxic conditions (p<0.05). (B) Effect of HIF-1 inhibitors on CA-IX levels in WM115. Acriflavine and Echinomycin decreased CA-IX levels in normoxic and hypoxic conditions (p<0.05). CA-IX, Carbonic anhydrase-IX.
Figure 1:

Effect of Acriflavine and Echinomycin on CA-IX levels in melanoma cell lines. ELISA assays were performed as three independent times. Data were expressed as ± SEM.

(A) Effect of HIF-1 inhibitors on CA-IX levels in SKMEL30 cells. Acriflavine and Echinomycin decreased CA-IX levels in normoxic and hypoxic conditions (p<0.05). (B) Effect of HIF-1 inhibitors on CA-IX levels in WM115. Acriflavine and Echinomycin decreased CA-IX levels in normoxic and hypoxic conditions (p<0.05). CA-IX, Carbonic anhydrase-IX.

VEGF ELISA assay

In hypoxic conditions, VEGF levels were both increased in SKMEL-30 and WM115 cell lines compared to normoxia (SKMEL30, p<0.001 and WM115, p<0.05). In Figure 2, VEGF levels were reduced by Acriflavine and Echinomycin in normoxic and hypoxic conditions for both cell lines and these results were also statistically significant (p<0.05). The analyses were performed by using GraphPad Prism 9.1.

Figure 2: Effect of Acriflavine and Echinomycin on VEGF levels in melanoma cell lines. ELISA assay were performed as three independent times. Data were expressed as ± SEM. (A) Effect of HIF-1 inhibitors on VEGF levels in SKMEL30 cells. Acriflavine and Echinomycin decreased VEGF levels in normoxic and hypoxic conditions (p<0.05). (B) Effect of HIF-1 inhibitors on VEGF levels in WM115. Acriflavine and Echinomycin decreased VEGF levels in normoxic and hypoxic conditions (p<0.05). VEGF, Vascular endothelial growth factor.
Figure 2:

Effect of Acriflavine and Echinomycin on VEGF levels in melanoma cell lines. ELISA assay were performed as three independent times. Data were expressed as ± SEM.

(A) Effect of HIF-1 inhibitors on VEGF levels in SKMEL30 cells. Acriflavine and Echinomycin decreased VEGF levels in normoxic and hypoxic conditions (p<0.05). (B) Effect of HIF-1 inhibitors on VEGF levels in WM115. Acriflavine and Echinomycin decreased VEGF levels in normoxic and hypoxic conditions (p<0.05). VEGF, Vascular endothelial growth factor.

Discussion

In this study, the effects of HIF-1 inhibitors on CA-IX and VEGF protein levels were investigated in melanoma. According to our study, we demonstrated that HIF-1 inhibitors decreased CA-IX and VEGF levels both in primary and metastatic melanoma cell lines.

Melanoma is the major reason of the death in skin cancer. Standard treatment methods used in melanoma, are surgery, radiotherapy, chemotherapy and biological therapy [20]. New approaches towards anti-cancer targets in melanoma are so crucial because of melanoma invasion [21]. Targeted anti-cancer drugs have important results for melanoma patients [22]. A key role of targeted melanoma treatment is related to hypoxia. Since hypoxia maintains reduced oxygen to encourage tumor development, HIF-1 is a remarkable phenomenon for cancer treatment approaches. CA-IX and VEGF are proteins expressed by the HIF-1α pathway [23].

Due to the importance of hypoxia in cancer, recent studies in this area have a popular topic [24]. Mills et al. showed that an increased HIF-1α expression contributes to a malignant phenotype in human melanoma cells and it indicates the importance of HIF-1α in melanoma biology [17].

Cheng et al. indicated higher HIF-1α and VEGF levels in melanoma patients. Based on their study data, a positive correlation between both parameters was indicated. They also persuaded HIF-1α can act as a relative partner of VEGF [25]. Kim et al. indicated a reduced VEGF expression with HIF-1α inhibition in the mouse models [26]. Jensen et al. suggested that VEGF secretion in malignant gliomas reduced by HIF-1α inhibition [27]. Semenza had postulated that using HIF-1 inhibitors can be acted as applicable tumor agents for focusing on VEGF [28]. Milesi-Hallé et al. also showed that Echinomycin is a potent inhibitor of hepatocyte cells which is capable of reducing the VEGF production [29]. A study designed with Echinomycin was stated that this inhibitor is also promoted to decrease VEGF expression [30]. Marti-Díaz et al. showed that Acriflavine increases VEGF expression under non-hypoxic conditions in melanoma cells. They stated that HIF-1α is not completely required for VEGF expression under normoxic conditions [31]. Mangraviti et al. have also postulated the effects of hypoxia to determine the potent inhibition of Acriflavine reducing VEGF expression in breast cancer cells. According to the inhibition results of Acriflavine, Mangraviti et al.’s study indicated a HIF-1α pathway crucial status in the regulation of VEGF [32]. In our study, under normoxic and hypoxic conditions, VEGF levels were significantly decreased by HIF-1 inhibitors. The efficacy of HIF-1 inhibitors on VEGF relation focuses on a potential therapeutic targeting of angiogenesis in melanoma under hypoxic and normoxic conditions.

pH regulation is related to acidification in tumor microenvironment and CA-IX is responsible for carcinogenesis. That is why the inhibition of tumor associated CA-IX enzyme may be the main target for cancer treatment to prevent the acidic microenvironment of the tumor [14]. The only study in the literature on HIF inhibitors related to CA-IX was designed by Dekervel et al. that explained the inhibitor effects of Acriflavine on CA-IX levels [33]. In this preliminary study, we indicate the efficacy of HIF-1 inhibitors on the CA-IX in both primary and metastatic melanoma cell lines under normoxia/hypoxia. Our results also confirmed that the HIF-1α pathway had a potential effect on the regulation of CA-IX. Decreasing the CA-IX enzyme levels with these inhibitors could be used as a new alternative treatment approach in melanoma.

As a consequence, there are limited studies in the literature that demonstrate the effect of HIF-1 inhibitors on CA-IX and VEGF. According to their ability and their roles in the tumor environment we aimed to understand the effects of these inhibitors on VEGF and CA-IX in melanoma. In conclusion, we determined that these inhibitors show effective inhibition on CA-IX and VEGF in melanoma. For further studies, designing new HIF-1 inhibitor derivatives may be an alternative treatment approach. Also, we aim to examine mRNA levels of CA-IX and VEGF in our future studies.

Corresponding author: Özen Özensoy Güler, Medical Biology Department, Faculty of Medicine, Ankara Yildirim Beyazit University, Ankara, Turkey, E-mail:

Funding source: Ankara Yildirim Beyazit University Research Foundation 10.13039/501100014801

Award Identifier / Grant number: 4206

  1. Research funding: This work was financed by Ankara Yildirim Beyazit University Research Foundation Project 147 Award Number 4206.

  2. Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.

  3. Competing interests: The authors declare that there is no conflict of interest of this paper.

  4. Çıkar Çatışması: Bu çalışmanın herhangi bir çıkar çatışması yoktur.

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Received: 2021-04-20
Accepted: 2021-05-26
Published Online: 2021-06-29

© 2021 Beyza Ecem Öz Bedir et al., published by De Gruyter, Berlin/Boston

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

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https://doi.org/10.1515/tjb-2021-0085
Keywords for this article
Acriflavine; CA-IX; Echinomycin; hypoxia; melanoma; VEGF
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Articles in the same Issue

  1. Frontmatter
  2. Review Article
  3. Overview of COVID-19’s relationship with thrombophilia proteins
  4. Research Articles
  5. Is it possible to determine antibiotic resistance of E. coli by analyzing laboratory data with machine learning?
  6. Detection of circulating prostate cancer cells via prostate specific membrane antigen by chronoimpedimetric aptasensor
  7. Two approaches for measurement uncertainty estimation: which role for bias? Complete blood count experience
  8. Is there any relationship between C-reactive protein/albumin ratio and clinical severity of childhood community-acquired pneumonia
  9. Comparison of nitric oxide and adrenomedullin levels of children with attention deficit hyperactivity disorder and anxiety disorder
  10. Performance evaluation of internal quality control rules, EWMA, CUSUM, and the novel machine learning model
  11. Are serum molecular markers more effective than the invasive methods used in the diagnosis of breast cancers?
  12. HIF-1 inhibitors: differential effects of Acriflavine and Echinomycin on tumor associated CA-IX enzyme and VEGF in melanoma
  13. Evaluation of BD Vacutainer Eclipse and BD Vacutainer Ultra-Touch butterfly blood collecting sets in laboratory testing
  14. The effects of various strength training intensities on blood cardiovascular risk markers in healthy men
  15. Is there any relationship between LGALS3 gene variations and histopathological criteria in laryngeal squamous cell carcinoma (LSCC)?
  16. Expression levels of BAP1, OGT, and YY1 genes in patients with eyelid tumors
  17. Association of bitter and sweet taste gene receptor polymorphisms with dental caries formation
  18. Case Report
  19. Glucose-6-phosphate dehydrogenase gene Ala365Thr mutation in an Iraqi family with confusing clinical differences
  20. Acknowledgment
  21. Acknowledgment
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