4th INTERNATIONAL AZERBAIJAN CONGRESS ON LIFE, ENGINEERING, AND APPLIED SCIENCESEPTEMBER 15-18, 2023 ONLINE & IN-PERSON PARTICIPATION BAKU, AZERBAIJAN

CONGRESS ID

CONGRESS NAME

4TH INTERNATIONAL AZERBAIJAN CONGRESS ON LIFE, ENGINEERING, AND APPLIED SCIENCES

DATE AND VENUE

SEPTEMBER 15-18, 2023

ONLINE & IN-PERSON PARTICIPATION (BAKU, AZERBAIJAN) (FROM 13 DIFFERENT COUNTRIES)Azerbaijan, United States of America, Nigeria, Pakistan, India, Morocco, Philippines, Kosovo,

Portugal, Hungary, Qatar, Türkiye, Ethiopia

ORGANIZERS & PARTICIPANT INSTITUTIONS

BZT Academy, TürkiyeAzerbaijan University of Languages, AzerbaijanPorto Higher Institute of Engineering, Portugal Bharathidasan University, IndiaPresident of Turkic Culture and Heritage Foundation, Azerbaijan Azerbaijan Presidency HSDM Branch Manager, Azerbaijan Baku International Multiculturalism Centre, Azerbaijan

Presidents of Organization Committee

PROF. DR. KAMAL MEHDİ OĞLU ABDULLAYEV / Rector

President of Scientific Committee

PROF. DR. İBRAHİM KAZIMBƏYLİ

President of Coordinators’ / General Coordinator

Dr. Baha Ahmet YILMAZ

ACCEPTED PAPERS

27 Turkish Participants36 Foreign Participants %54 Foreign Participant Rate %46 Turkish Participant Rate

REFUSED PAPERS

2 Turkish Participants

EVALUATION PROCESS

All Submissions Have Passed a Double-Blind Referee Evaluation Process All Responsibility for the Works Belongs to the Author

INVESTIGATION OF CHANGES IN GENE AND PROTEIN EXPRESSIONS OF MESOBUTHUS EUPEUS SCORPION VENOM IN HUMAN COLON CANCER

Havva Nur CANAK*, Ozgün SIPAHIOGLU, Serdar KARAKURT

Faculty of Science, Department of Biochemistry, Selcuk University, Konya, Türkiye

ORCID Code: 0009-0005-4453-6936 0000-0001-7160-8775 0000-0002-4449-6103

ABSTRACT

Colon cancer, which occurs as a result of environmental relations as well as genetic factors, ranks third in the world. Different treatment methods can be used, but these methods not only damage healthy cells, but also wear out the individual physically and psychologically. For this reason, studies have been carried out on scorpion venom, which has recently been seen as a natural medicine source as an alternative treatment method. In this study, the effects of Mesobuthus eupeus scorpion venom(MEV) belonging to the Buthidae family on gene and protein expressions in human colon cancer cell lines were investigated. DLD-1 and HT-29 cells were grown in RPMI-1640 and McCoy’s5A growth media, respectively. RNA isolation by trizol method for qRT-PCR in vitro and protein isolation with RIPA buffer for western blot were performed. Changes in mRNA level in BcL-2, BAX, NF-ĸB, TP53, Caspase-3, Caspase-9, Caspase-12, BRAF, KRAS, Smad-4, MLH-1 and APC genes in both cell lines are determined by qRT-PCR method. Western blot studies were performed using BcL-2, BAX, NF-ĸB and TP53 proteins for changes in protein level. Compared to the control group in qRT-PCR studies; Expression levels of BcL-2, BAX, Braf and APC genes were decreased in DLD-1 cell line and NF-ĸB and Caspase-12 expression levels were decreased in HT-29 cell line. As a result of protein expression, the expression of BcL-2 decreased in both cell lines, while the expression level of TP53 was decreased in DLD-1 it was also increased in HT-29. BAX expression level increased in DLD-1 and decreased in HT-29. As a result of the study, it was seen that changes in gene and protein expression levels in MEV cell lines promoted apoptosis. As a result, it was predicted that MEV could be used as an alternative therapeutic agent in the treatment of colon cancer.

Keywords: Apoptosis, Colon cancer, Mesobuthus eupeus, Scorpion venom, qRT-PCR, Western blot. INTRODUCTION

Cancer, which occurs as a result of uncontrolled proliferation, spread and metastasis of cells, is an important health problem that occurs after cardiovascular diseases. Different types of cancer occur

in women and men under the influence of genetic, environmental, behavioral and biological factors. (Forman et al., 2014). Colon cancer is the third leading cause of cancer-related death in women and men. (Wild, Stewart et al. 2014). In addition to genetic factors, the causes of colon cancer include a low-fiber and high-fat diet or processed meat consumption, alcohol and cigarette use. Conventional methods such as chemotherapy, surgery and radiotherapy are used as treatment methods. However, these treatment methods are both inadequate and cause undesirable side effects. (Qureshı et al., 2018). That’s why alternative treatment methods have been sought. Active compounds obtained from these alternative treatment methods are included. Today, studies are being conducted on the role of active compounds obtained from animals, especially snake and scorpion venoms, in cancer treatment. Various bioactive compounds, peptides and proteins found in the structure of scorpion venoms show that they can be used as effective treatment methods against many diseases such as cancer. At the same time, scorpion venoms are programmed to have anticancer activities, ranging from inhibition of cell proliferation and storage of cells to induction of apoptosis and reduction of cell migration and invasion (Ding et al., 2014). When we look at the studies in the literature, we come across studies that examine either the total effects of scorpion venoms or the cytotoxic effects of a few active components on various types of cancer, but it can be seen that studies explaining the molecular mechanisms at both the gene and protein levels are quite limited. In this study, the venom of the scorpion species Mesobuthus eupeus, belonging to the Buthidae family, known as the eastern yellow scorpion, was used. Although this scorpion species is found in Southeastern Anatolia in Turkey, it is also found in regions such as Syria and Iran. The bodies of these scorpions are yellow-brown; legs are light yellowish brown. Their length is between 3.5-5.0 cm. The shell parts on their back are blackish brown and have five (5) stripes (Kovařík et al., 2011). In this study, the effects of the scorpion species Mesobuthus eupeus, whose molecular effects on cancer have not been determined before, on human colon cancer were elucidated at the molecular level.

MATERIALS AND METHODS

1. Growing Cell Lines

Colon cancer cell lines DLD-1 and HT-29 cell lines were grown in growth media containing 10% fetal bovine serum (FBS) and 2 mM L-glutamine in RPMI-1640 and McCoy’s 5A Medium, respectively, in an incubator containing 37 °C and 5% CO2.

2. Determining the Changes in the Expression of Genes Playing a Key Role in the Colon Cancer Pathway by qRT-PCR Method

2.1. Total RNA Isolation

Total RNA from samples obtained from cell lines was isolated by the trizol method. Cells were grown in 24-well plates and treated with 1 mL Trizol and transferred to 1.5 mL eppendorf tubes. All samples were then centrifuged by adding 250 μL chloroform. The resulting transparent liquid phase was taken, isopropanol was added and centrifuged again. After the RNA was precipitated, the RNA was cleaned with the help of 75% cooled ethanol. After removing the ethanol, the RNA was dried at room temperature and converted to soluble form by adding ddH2O. The amount, integrity and purity of template RNA were measured with the help of Agilent 2100 model Bioanalyzer. Samples were stored at -80°C for use in subsequent studies.

2.2. cDNA synthesis

cDNA was synthesized from the RNAs obtained using the 18-nucleotide-long oligo d(T) primer and Moloney-Murine Leukemia Virus Reverse Transcriptase. For cDNA synthesis, 2 ng of total RNA and 1

μL of oligo (dT) primer were mixed and the final volume was completed with RNAse-free water to 11 μL. After the mixture was incubated at 70oC for 5 minutes, 5X reaction buffer and dNTP mixture were added. After incubation for 5 minutes at 37°C, 50 units of Moloney-Murine Leukemia Virus Reverse Transcriptase was added and the final volume was completed to 20 μl. After adding the enzyme, the final mixture was first incubated at 42°C for 1 hour for cDNA synthesis and at the end of this period, it was kept at 70°C for 10 minutes to inhibit the enzyme. The amount of cDNA obtained was measured and controlled on the Nanodrop device. cDNAs were stored at -20°C for use in subsequent studies.

2.3. Determination of mRNA Level Expression of Genes Playing a Role in the Colon Cancer Molecular Pathway

Quantitative Real Time PCR was performed using the Bio-Rad CFX96 device (Bio-Rad, USA). The reaction was performed with 1X Maxima® SYBR Green qPCR Master mix. cDNA, RNase-free water, gene specific primer, SYBR green qRT-PCR mixture were added to the reaction tube. GAPDH was used as the internal standard gene. The primers to be used in qRT-PCR were designed using Primer 3 software and their gene specificity was checked with NCBI blast software. Then, the reaction mixture (tube) was first subjected to denaturation at 95oC for 10 minutes for the PCR process, and the amplification process was carried out after a total of 45 cycles, including 20 seconds at 95oC, 30 seconds at 54-60oC, and 30 seconds at 72oC, at 49oC ıt was performed with a final extension of 30 seconds. The change in gene expressions was found by calculating the ratio of specific gene mRNA expressions to GAPDH mRNA expression.

3. Determination of Expression Changes of Proteins Playing a Key Role in the Colon Cancer Pathway and Proteins Playing a Role in the Apoptosis Pathway by Western Blotting Method

3.1. Protein Expression Studies

The cells, which were planted at 3x105 cells in 100 mm3 petri dishes, were treated with scorpion venom and incubated for 48 hours at 37 °C and 5% CO2. At the end of the incubation, the cells were washed 3 times with cold PBS, and after the cells were scraped mechanically with the help of a scraper, cold PBS was added and the cells were precipitated by centrifugation. After the cells were precipitated, the supernatant was discarded and ripa buffer mixture was added to each eppendorf tube and incubated on ice for 5 minutes. After centrifugation at 14000 g for 10 minutes, the supernatant was transferred to clean 1.5 ml centrifuge tubes. The obtained proteins were stored in a -80 oC deep freezer for use in later studies. To determine the concentration of the resulting proteins, protein fractions diluted at a ratio of 50:1, with BSA (bovine serum albumin) used as a standard, were added to the tubes, and 200 μL of the diluted protein fractions were added to the multiplate reader in the spectrophotometer, and 25 μL of the sample was added on top, and measurements were taken at 660 nm.

3.2. Determination of the Effects of Scorpion Venom on Protein Expression

After determining the effects of scorpion venom on the genes that play an active role in the formation of colon cancer, its effects on the expression of the proteins encoded by these genes were determined by the western blot method by applying the vertical electrophoresis technique. After the proteins were separated from each other by the SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) method, the differences in protein expressions were determined using protein-specific proteins. Polyacrylamide gel electrophoresis (PAGE) was performed on 8.5% separation and 4% compression gels in a batch buffer system defined by the presence of the ionic detergent sodium dodecyl sulfate (SDS).

Combs were placed on top of the separating gel. After the wells were formed, proteins and markers were loaded into the wells. After the gels were prepared, electrophoresis was performed for execution. Following the SDS-PAGE process, proteins were transferred electrophoretically at 25 volts for 30 minutesAfter the transfer was completed, the proteins transferred to the membrane were shaken in a shaker for 15 minutes with TBST, which is a block solution. At the end of the period, the membranes were left to shake with blocking solution for 1 hour. After 1 hour, the membranes were shaken with the relevant primary antibodies for 15 minutes and incubated in the primary antibodies overnight. At the end of the incubation, the membranes were left to shake with the relevant secondary antibodies for 1 hour. After shaking, the membranes were washed with TBST. Then, ECL solutions were prepared and added to the membranes and imaging was performed.

RESULTS

1. Gene Expression Results

Gene expression study was carried out by qRT-PCR to determine the effect of Mesobuthus eupues scorpion venom on the expression of genes involved in apoptosis and cancer signaling pathways on colon cancer cell linesThese genes are divided into three different groups, namely apoptotic genes (BAX, BCL-2, Caspase 3, Caspase 9 and Caspase 12), genes related to apoptosis (NF-ĸB, TP53) and genes involved in cancer signaling pathways (Braf, Kras, Smad -4, APC, MLH-1) genes were examined. As a result of this study, it was observed that Mesobuthus eupues caused changes in gene expression levels in the HT-29 cell line compared to the control group (Figure 1).

Figure 1 Graph of changes in the expression levels of apoptotic genes, genes related to apoptosis and genes involved in cancer signaling pathways on the HT-29 cell line

2. Protein Expression Studies

The changes caused by Mesobuthus eupues scorpion venom in the expression of proteins involved in the apoptosis pathway on colon cancer cell lines were determined by western blot experiment. The resulting band intensities were calculated with imageJ software. In the DLD-1 cell line, the expression levels of BAX, NF-ĸB, MLH-1 proteins were 1.39, respectively; 3.50; While it increases by 2.30; The levels of BCL-2 and p53 proteins decreased by 0.74 and 0.54, respectively (Figure 2).

Figure 2 Graph of expression levels of Mesobuthus eupues scorpion venom against the reference protein GAPDH on the DLD-1 cell line

DISCUSSION

Cancer, whose incidence and mortality rate has increased rapidly recently; ıt occurs as a result of behavioral disorders such as smoking and alcohol, as well as age, gender and genetic factors. Along with these factors, mutations occur in tumor suppressor genes and oncogenes. Chemotherapy, radiotherapy and surgical procedures used as treatment methods are inadequate. That’s why an alternative treatment method has been sought. Nowadays, research is being carried out on alternative methods and scorpion venoms containing various bioactive compounds, peptides and proteins. In the literature, Salem et al. In a study conducted in 2016, they administered the poison obtained from Androctonus amoreuxi and Androctonus crassicauda, which are in the Buthidae family, to the human breast cancer MCF-7 cell line and examined the toxicity of these poisons in cancer cells and their ability to induce necrosis or apoptosis. These poisons have been found to increase the expression of caspase-3, which is involved in apoptosis (Salem et al., 2016). As a result of our study, the expression level of Bax, which promotes apoptosis, increased and the expression level of BcL-2 decreased in the HT-29 cell line. These changes have been proven by the changes in caspases, and it has been observed that the poison promotes apoptosis. It also caused toxic changes in the TGFβ signaling pathway, in which SMAD-4 and MLH-1 play a role, promoting the metastasis and proliferation of cells. Expression of both is increased in the HT-29 cell line (Chaudhury et al., 2009). The expression level of APC, which has an important role in colon cancer and is involved in cell differentiation, increased. (Van den Broek et al., 2016). TP53, a tumor suppressor gene, plays a fundamental role in cancer because it controls the transcription of genes in metastasis. (Toyooka et al., 2003). Expression levels of NF-κB., which plays a role in the immune system, were also examined (Barkett et al., 1999). The expression of these two genes decreased in HT-29. At the same time, activation of growth factors such as EGFR is important to become independent of growth signals. Because as a result of this activation, tumor cells can proliferate and survive. The increase in the expression of BRAF and KRAS genes, which play a role in this signaling pathway, prevents this situation (Gurdal et al., 2019). It was found that the levels of both genes increased in the HT-29 cell line thanks to the poison we used in the study. Changes in the protein expressions of Bax, NF-Κb, BcL-2 and p53 were also examined by Western blot method. In the DLD-1 cell line, the expression level of Bax and NF-κB. increased and the expression level of BcL-2 and p53 decreased compared to the control group.

Looking at the results of these studies in the literature and considering the suitability of the experimental studies in the literature, it is thought that Mesobuthus eupeus scorpion venom may be a promising drug candidate in the treatment of colon cancer.

CONCLUSION

As a result of gene and protein studies carried out with Mesobuthus eupeus scorpion venom on colon cancer cell lines, changes in expression levels have been observed that the venom promotes apoptosis. In line with these results, it is thought that Mesobuthus eupeus scorpion venom can be used as an alternative treatment method according to the colon cancer cell line.

ACKNOWLEDGEMENTS

This study was supported by TÜBİTAK. (Project No:120Z942). We thank TÜBİTAK.

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