Cytotoxic Potential of Crataegus orientalis Fruit Extract: Expression Profiles of Apoptosis - Related Genes (Bax, Bcl2, p53)

Cytotoxic Potential of Crataegus orientalis Fruit Extract: Expression Profiles of Apoptosis - Related Genes (Bax, Bcl2, p53) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Cytotoxic Potential of Crataegus orientalis Fruit Extract: Expression Profiles of Apoptosis - Related Genes (Bax, Bcl2, p53) Lütfiye Kadıoğlu Dalkılıç, Semih Dalkılıç, Şemse Nur Akbalık, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8270980/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Scientists are becoming more interested in the possibility of natural bioactive chemicals as supplemental medicines in cancer treatment. Investigating the biological effects of Crataegus orientalis fruit extract on different human cell lines was the goal of this investigation. HepG2 (liver), MCF-7 (breast), HEK293 (embryonic kidney), HCT-116 (colon), and A549 (lung) cell lines were used to evaluate cytotoxic characteristics using the MTT test. The extract was made with a variety of solvents and tested at different doses to assess dose-dependent effects. The results demonstrated that the cytotoxic effects were varied by the cell line, concentration, and extraction solvent. Notably, the extract's low IC₅₀ value demonstrated high cytotoxicity against the HCT-116 cell line. The mRNA expression levels of important apoptosis-related genes, specifically Bcl2, Bax, and p53, were quantitatively examined using qRT-PCR in human HepG2 and MCF-7 cancer cell lines in order to obtain additional understanding of apoptotic mechanisms. Gene expression was found to have decreased statistically significantly in HepG2 cells (p 0.0002 for Bcl2, p 0.0344 for Bax, and p 0.0035 for p53). All of these results suggest that the fruit extract of C. orientalis may have anticancer properties via influencing the expression of genes linked to apoptosis in cancer cells. Crataegus orientalis Cytotoxicity Apoptosis qRT-PCR IC₅₀ HepG2-MCF-7-HCT-116-HEK293-A549 cell lines Figures Figure 1 Figure 2 Highlights • The cytotoxic effects of C. orientalis fruit extract were evaluated in five human cell lines • HCT-116 cells showed the highest sensitivity with a low IC₅₀ value • The extract was prepared using different solvents and tested at various doses • Apoptosis-related genes Bcl2, Bax, and p53 were analyzed by qRT-PCR • Significant downregulation of these genes was observed in HepG2 cells. INTRODUCTION Serious health issues include hypertension, liver-related disorders, blood sugar abnormalities, and cardiovascular disease affect a large number of people worldwide. The most dangerous of these illnesses is cancer, for which there is now no known cure [ 1 ]. According to the World Health Organization’s 2019 report, cancer ranks among the primary causes of premature mortality in 112 out of 183 countries, affecting individuals before the age of 70. In 23 countries, it is identified as the third or fourth leading cause of early death [ 2 ]. It is anticipated that 15.5 million individuals will be battling cancer globally by 2030, and 11.5 million will lose their lives to the illness. It is anticipated that 15.5 million individuals will be battling cancer globally by 2030, and 11.5 million will lose their lives to the illness [ 3 ]. Even though there are many medications available to treat cancer, a totally safe and effective therapeutic approach has not yet been created [ 4 ]. Among the most popular methods for combating cancer are include radiotherapy, bone marrow transplants, surgery, immunotherapy, and hormone therapy [5, 6 ]. Cancer treatment with chemotherapy can harm healthy cells and result in severe side effects like anemia, hair loss, and exhaustion. Therefore, creating novel methods to minimize side effects and lower treatment expenses is crucial. Patients frequently resort to traditional therapy approaches in areas with limited access to modern care [ 4 ]. For ages, herbal items have been used extensively in various countries and have served as the foundation for traditional medical systems [ 7 ]. According to recent studies, 63% of cancer medications are sourced from plants, and traditional herbal extracts have anti-cancer properties. It has been discovered that certain herbal ingredients have a high response to treatment and few adverse effects. Thus, research into the potential of herbal remedies in the battle against cancer is still ongoing. According to recent studies, 63% of cancer medications are sourced from plants, and traditional herbal extracts have anti-cancer properties. It has been discovered that certain herbal ingredients have a high response to treatment and few adverse effects. Thus, research into the potential of herbal remedies in the battle against cancer is still ongoing [ 8 , 9 , 10 ]. According to the World Health Organization, herbal medicines account for almost 80% of the economies of developing nations [ 11 ]. Additionally, since about 20% of current medications are derived from traditional medicinal plants, there is growing interest in researching herbal items for the treatment of cancer [2, 12 ]. The fact that 80% of synthetic medications used globally are plant-based further supports this tendency [ 13 ]. Therefore, many plants have been found to have anti-cancer capabilities in addition to their beneficial effects as medical plants [14, 15 ]. Crataegus , a member of the Rosaceae family, is regarded by some as a prickly tree or shrub [16, 17 ]. The other names of this plant, which is native to the Mediterranean, Crimea, Turkey and Western Iran, are carpets, nuts, hawthorn, wild roses and aktiken [17, 18 ]. Since ancient times, extracts made from hawthorn leaves, blossoms, and fruits have been used to treat respiratory issues, diarrhea, abdominal pain, palpitations, cardiovascular illnesses, and hypertension [19, 20 ]. Because of its historical use, Hawthorn has also caught the attention of scientists [ 21 ]. In addition to being a strong source of vitamin C, hawthorn fruit is said to include sugars and carbs and is especially rich in important minerals including calcium, potassium, phosphorus, magnesium, and iron [ 22 ]. Flavonoids, proanthocyanidins ,organic acids, and amines are its primary constituents [ 23 ]. It is well known that phytochemicals derived from a variety of plants can effectively treat cancer. Research is being conducted worldwide to find novel plant extracts with potent antibacterial and anticancer effects [ 24 ]. In the context of cancer research, oncogenes and tumor suppressor genes have recently drawn a lot of attention for their functions in regulating the cell cycle and apoptosis. Since resistance to apoptosis is a significant treatment issue for cancer, a thorough understanding of apoptosis, a type of programmed cell death, is crucial for the development of effective anticancer medicines. [12, 25 ]. There have been some studies on C.orientalis 's antibacterial and antioxidant properties, but none that have looked closely at how it affects human cells. With an emphasis on apoptosis-related gene expression, this work intends to examine the cytotoxic and pro-apoptotic effects of C. orientalis fruit extract on HepG2 and MCF-7 cell lines. The extract was also tested in other human-derived cell lines, such as A549, HCT-116 and HEK293 to investigate its effects across a variety of cellular models and deepen our understanding of its biological activity. Glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) was employed as a housekeeping gene to normalize the expression data and ensure analytical accuracy. Quantitative real-time PCR (qRT-PCR) was conducted to measure the mRNA expression levels of apoptosis-related genes, including p53, Bax , and Bcl2 . MATERIALS AND METHODS Obtaining C.orientalis and cell lines used in the experiment C.orientalis species collected from Cip town in Elazığ province of Turkey in September and November. Assoc. Prof. Dr. Semih Dalkılıç, one of the faculty members of the Molecular Biology and Genetics Programme of the Department of Biology, Fırat University that has HepG2, MCF-7, HEK293, HCT-116 and A549 cell lines in his laboratory. Preparation of the extract C. orientalis fruits were dried at room temperature and ground to obtain a homogeneous particle size. For the extraction process, 1 gram of dry plant sample was weighed on a precision balance, and the process was carried out using two different solvents. In the first step, extraction was performed with 10 mL hexane, followed by 10 mL methanol. The extraction process was carried out in a shaking oven at 37°C for 72 hours, after which the extracts were filtered with Whatman No. 1 filter paper to remove the solvents. After drying the extracts, yield calculation was performed, and extraction efficiency was determined. When evaluated on a solvent basis, the yield of hexane extract was 31.3%, and the yield of methanol extract was 29.4%, as shown in Table 1. It was dissolved in 10 mL dimethylsulfoxide (Dimethyl Sulfoxide Merck 116743.1000) The extracts were stored at +4 °C for the duration of the study. Hexane and methanol solvents, which are widely preferred for the identification of phenolic compounds, were chosen for the cytotoxicity evaluation of C. orientalis extract in this study. Cell culture and determination of cytotoxic activity MCF-7, HepG2, A549, HCT-116 and HEK 293 cell lines were cultured in 75 cm² flasks at 37°C with 5% CO₂ in RPMI 1640 Medium, with L-Glutamine (Cat-No: RPMI-A) medium containing 10% FBS (Cat-No: FBS-11A) and 1% Penicillin-Streptomycin (Cat-No:PS-B ) . MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay is a technique used to evaluate cytotoxicity, proliferation and cell viability. The basis of this technique is that MTT and active mitochondria in living cells combine to form water-insoluble purple formazan crystals [26]. MTT method was used to evaluate the effect on C.orientalis cell viability. After dissolving formazan crystals in appropriate solvents, the amount of viable cells was counted using spectrophotometry. 90% confluent cells were removed from the medium in 75 cm² flasks and washed with 5 ml sterile PBS. After adding 1 ml trypsin-EDTA (Lot: 325043240, Cat:325-043-EL) to the flasks, they were incubated at 37˚C for two minutes in an incubator containing 5% CO₂. Trypsin-EDTA was inactivated and 5 ml RPMI was added after the cells were detached from the surface. After the cells were removed from the flask, the supernatant was discarded after centrifugation at 2000 rpm for five minutes. Countess II automated cell counter was used to count the cell pellet after resuspension with 1000 µL RPMI. After cell dilution was calculated, 96-well plates containing 5x10³ cells and 100 µL RPMI per well were applied. The positive control was 2.5 µg/mL doxorubicin, while blank and negative controls were media only. Cells were incubated at 37.0 C with 5% CO 2 for 24 hours. Methanol and hexane extracts of C.orientalis fruit produced in RPMI were administered to the cells in six replicates at four different concentrations (100, 200, 400 and 800 µg/mL) after the medium was removed after incubation. Cells were incubated at 37˚C with 5% CO₂ for 72 hours. After completion, 10 μL of MTT solution (5 mg/mL) was added to each well and incubated at 37˚C with 5% CO₂ for 4 hours. Once incubation was over, the medium was taken out, formazan crystals were dissolved in 100 μL DMSO, and an ELISA microplate reader (Allsheng) set to 450 nm wavelength was used to measure the anticipated color change [27]. The control wells' absorbance readings were calculated, averaged, and approved as representing a 100% viable cell ratio. The viability % was calculated by comparing the absorbance values from the plant extracts with the absorbance value of the control group. This is how cytotoxicity was computed: % Live cell = (Sample absorbance) / (Control absorbance) x100 RNA isolation from cell and cDNA synthesis The apoptotic pathway effect of C.orientalis on HepG2 and MCF-7 cell lines was evaluated by looking at the expression levels of Bcl2, Bax and p53 genes. Based on the cytotoxicity results of C.orientalis , the best extract and dosage for the two cell lines were determined. HepG2 and MCF-7 cells were exposed to the highest concentration of C.orientalis hexane extract (800 μg/ml) for 48 hours. Following the manufacturer's instructions, total RNA was extracted from these cells using the Total RNA Mini Kit (Generaid-Cat. No. RB300). The quality of the extracted RNA was analyzed using the iBright FL 1000 gel imaging system. The quantity and quality of RNA molecules determine how reliable gene expression analysis is [11]. Following the manufacturer's instructions, the quality and quantity of total RNA isolated was assessed using a Qubit™ RNA HS Assay Kit (REF: Q32852, LOT 1913928) and a Thermo Fisher Scientific Invitrogen instrument. RNA concentration was measured in ng/ml. RNA samples determined to be optimal in both quality and quantity were used to generate cDNA. Following the manufacturer's instructions, cDNA was generated by reverse transcription using the WizScript™ cDNA Synthesis Kit (REF: W2211, LOT 4E0420-02). Two microliters of reverse transcription buffer, 0.8 μl 25x dNTPs, 2 μl 10x random primers, one microliter MultiScribe Reverse Transcriptase, 4.2 μl nuclease-free water and 500 ng total RNA (50 μl) were included in 20 microliters of reaction mix. Using a SimpliAmp Thermal Cycler (Applied Biosystems, Singapore), reaction mixtures were incubated at 25°C for 10 min, 37°C for 120 min and 85°C for 5 min for each sample. Quantitative real-time polymerase chain reaction (qRT-PCR) The expression levels of four different genes ( Bcl2, Bax, p53 and GAPDH ) were evaluated using quantitative RT-PCR. As indicated by the manufacturer, reactions were performed using Amplifyme SYBR Universal Mix (AM02-020) and Applied Biosystems Step One Plus Real-Time PCR System. The following components were present in 10 µl of reaction mix for each sample: Each sample was run in duplicate using 5 µl 2X Amplifyme SG Universal Mix, 0.3 µl forward primer, 0.3 µl reverse primer, 0.2 µl 50× High Rox solution, 2.2 µl PCR Grade Water and 2 µl cDNA sample. The following are the steps of the PCR cycling program: 3 minutes at 95°C followed by 40 cycles of 95°C for 5 seconds, 58°C for 30 seconds, 95°C for 15 seconds, 60°C for 1 minute and finally 95°C for 15 seconds. Untreated cells were used as an external control in qRT-PCR, while GAPDH was used as an internal control. ( Table 2) lists the primer sequences used. Statistical analysis SPSS (version 22) and GraphPad Prism (version 8.0.2) software were used for experimental design and statistical analysis of the data. As a result of the analysis, the statistical difference between the groups was evaluated by ANOVA test and significance levels were determined as p < 0.05. RESULTS Extract yield Fruits of C. orientalis were gathered in September and November from the Cip district in the Elazığ region of Turkey. The HepG2, MCF-7, HEK293, HCT-116, and A549 cell lines utilized in this investigation were acquired from the lab of Assoc. Prof. Dr. Semih Dalkılıç, a professor in the Department of Biology's Molecular Biology and Genetics Program at Fırat University. The following formula was used to determine the extraction efficiency [28]. Percent yield (w/w) = (weight of the dried extract (g))/ (weight of the dry seed material before extraction (g)) x100 Cytotoxic activity The cytotoxic potential of MCF-7 and HepG2 methanol and hexane extracts obtained from Crataegus orientalis fruit. The MTT method was used to assess the cancer cell lines HCT-116 and A549 as well as the healthy cell model HEK293 cell line. Fruit extracts' cytotoxic activity on C. orientalis in vitro, based on dosage and solvent type (Figure 1(A-E)). Cell viability in the MCF-7 cell line with the methanol extract was between 59-64%, while with the hexane extract it was between 57-66%. HepG2 cells show that both extracts have moderate cytotoxic effects. HCT-116 cell line showed the highest cytotoxic effect. With 400 g/mL hexane extract, cell viability decreased by 19%, while with 800 g/mL methanol extract it decreased by 15%. These results confirm that both extracts have strong antiproliferative effects in a dose-dependent manner. A more limited cytotoxic effect was observed in A549 cells compared to other cell lines; the highest efficacy was obtained with 29% viable cell ratio in 400 µg/mL hexane extract treatment. In contrast, HEK293 cells were used as a reference to evaluate the possible toxic effects of the extracts on healthy cells and no significant cytotoxic effect was observed in this cell line. Therefore, the IC₅₀ value for HEK293 cells was not calculated. This suggests that C. orientalis fruit extracts have low toxicity on healthy cells and have selective anticancer potential. These data were further supported by IC₅₀ analyses, revealing that methanol and hexane extracts exhibited cytotoxic effects that varied depending on the cell type but generally showed anticancer potential (Table 3). Effects of C. orientalis extract on apoptotic pathway To assess the impact of the C. orientalis hexane extract on apoptosis-related genes in HepG2 and MCF-7 cell lines, gene expression analysis was carried out using quantitative real-time PCR (qRT-PCR). Following a 24-hour treatment period, there were observable changes in the expression levels of the p53 , Bax , and Bcl2 genes when compared to the control group. Notably, treatment led to a modest shift toward apoptosis, particularly in MCF-7 cells, as reflected by the calculated Bax/Bcl2 ratio of 0.83 in MCF-7 and 0.67 in HepG2 cells. As shown in Figure 2, Bcl2 expression increased more markedly in HepG2 cells, whereas Bax expression exhibited a relative increase in both cell lines post-treatment. Similarly, p53 expression showed a moderate elevation in treated groups. These results suggest that the C. orientalis hexane extract may modulate apoptotic gene expression patterns, though the overall pro-apoptotic shift appears limited. In addition, the specificity of qRT-PCR amplification was confirmed by melting curve analysis, which revealed single and distinct peaks for all target genes, indicating the absence of non-specific products or primer-dimer formation. DISCUSSION Apoptosis, a regulated form of programmed cell death, plays a vital role in preserving cellular homeostasis and is a central mechanism in cancer biology. The intrinsic apoptotic pathway, which is mitochondria-mediated, is tightly controlled by the relative expression of pro-apoptotic ( Bax ) and anti-apoptotic ( Bcl2 ) genes. This balance determines the release of cytochrome c from mitochondria into the cytosol, a critical event that triggers the activation of downstream apoptotic signaling cascades [ 29 , 30 ]. A crucial stage in the intrinsic apoptotic process, the Bax protein stimulates the release of cytochrome c from the mitochondria. By blocking this release, Bcl2 , which is attached to the outer mitochondrial membrane, stops apoptosis. Consequently, it is commonly acknowledged that a key factor influencing a cell's propensity to undergo apoptosis is the Bax/Bcl2 expression ratio [ 31 ]. In this study, C. orientalis fruit extracts showed significant cytotoxic effects on various cancer cell lines. In particular, dose-dependent cytotoxic effects were observed in HCT-116 (colorectal cancer) and MCF-7 (breast cancer) cell lines. Methanol extract stood out as the most effective fraction with lower IC 50 values in most cell lines. On the other hand, since no toxic effect was observed in HEK293 (human embryonic kidney) cells, IC 50 values could not be calculated for these cells. This suggests that the extracts have low toxicity on healthy cells and may have a selective effect against cancer cells. When the data obtained are evaluated in general, it can be said that C.orientalis has anticancer potential especially against colorectal and breast cancer cells. The effect of the hexane extract on the expression of apoptosis-related genes ( p53, Bax , and Bcl2 ) was further evaluated in MCF-7 and HepG2 cells. The results demonstrated an induction of apoptosis, characterized by a significant upregulation of p53 and Bax , and a concurrent downregulation of the anti-apoptotic gene Bcl2 . This pattern indicates activation of the mitochondrial (intrinsic) apoptotic pathway. Moreover, the increased Bax/Bcl2 ratio, commonly considered a key marker for apoptosis initiation, reinforces this conclusion [ 31 , 32 ]. Hexane extract from C. orientalis fruit was used to evaluate the expression of apoptosis-related genes in MCF-7 and HepG2 cells. The calculated Bax/Bcl2 ratios were found to be 0.83 for MCF-7 cells and 0.67 for HepG2 cells. The fact that these ratios were below 1 in both cell lines indicates that the anti-apoptotic effect was dominant and the apoptotic response was limited. However, the up-regulation observed in the Bax gene and the increase in the p53 gene reveal the potential of the extract to direct the cell to apoptosis. Indeed, the increase in the Bax/Bcl2 ratio in our study supports that the extract activates the apoptotic process. The p53 tumor suppressor gene, a critical regulator of apoptosis, becomes activated under cellular stress conditions, particularly those involving genomic instability [ 33 ]. In this context, increased p53 expression is expected to trigger Bax transcription and initiate cell death [ 31 ]. In the literature, it is reported that some compounds of Crataegus species, especially triterpenoid ursolic and oleanolic acids, stimulate apoptosis via mitochondrial pathway and accelerate cell death by increasing caspase-3 and caspase-9 activation [ 34 , 35 ]. The gene expression profile found in this investigation is consistent with earlier findings. In particular, the idea that triterpenoid chemicals in C. orientalis fruit may trigger apoptotic pathways is supported by the overexpression of p53 and Bax and the downregulation of Bcl2 . It is also noteworthy that compared to previous studies on the anticancer effects of nearby species such as Crataegus aronia , C. orientalis showed a higher cytotoxic effect at lower concentrations. For instance, it has been observed that extracts from C. aronia only significantly affect HepG2 cells at 500 µg/mL [ 36 ]. In this respect, it can be said that C. orientalis has a stronger anticancer potential. CONCLUSION According to the study's findings, fruit extracts from C. orientalis have cytotoxic effects on cancer cell lines like MCF-7 and HepG2, and they may also alter the expression of genes linked to apoptosis. The methanol extract's increased bioactivity emphasizes how important the extraction technique and solvent choice are in affecting the effectiveness of chemicals produced from plants. The Bax, Bcl2 , and p53 gene expression levels did not approach statistical significance, but the trends particularly in the MCF-7 cell line indicate a pro-apoptotic response. This possible effect may also be reflected molecularly in the rise in the Bax/Bcl2 ratio. Important hints that C. orientalis might be a potential natural anticancer drug are provided by these early results. However, more thorough in vitro testing, mechanistic research, and in vivo investigations are required to completely illustrate this effect. Whether this plant represents a pharmacologically evaluable therapeutic resource will be made clear by more thorough research in the future. Declarations CONFLICT OF INTEREST The authors declare any conflicts of interest. F INANCIAL SUPPORT The Scientific and Technological Research Council of Turkey (TÜBİTAK) provided funding for this study under project number 1919B012217792 as part of the TÜBİTAK 2209-A Undergraduate Research Support Program. ACKNOWLEDGEMENT This research was supported by the Scientific and Technological Research Council of Turkey (TÜBİTAK) under the 2209-A Undergraduate Research Support Program (Project No: 1919B012217792). We would like to express our sincere gratitude to the Department of Parasitology, Faculty of Medicine, Fırat University, for providing laboratory facilities and their valuable contributions throughout the study. We also extend our special thanks to Mr. Mustafa Kaplan for his technical assistance and support. The findings of this study were presented in English as an oral presentation at the 9th International Conference on Contemporary Scientific Studies in the Middle East, held on March 13–15, 2024, at the Islamic University in Beirut, Lebanon. AUTHOR CONTRIBUTIONS LKD contributions to the manuscript's final editing and conceptual design. S D Contributed to the manuscript's structural revision and content development. ŞA experimental research, writing, data analysis, and manuscript preparation. EA contributions to the manuscript's revision. References Ahmed, F., Ijaz, B., Ahmad, Z., Farooq, N., Sarwar, M. B., & Husnain, T. Phytomedicine , 68 , 153168. (2020). Assouyouti, A. Master's thesis, Bursa Uludag University (Turkey) , (2022). Shin, S. A., Moon, S. Y., Kim, W. Y., Paek, S. M., Park, H. H., & Lee, C. S. International journal of molecular sciences , 19 (9), 2651, (2018). Majolo, F., Delwing, L. K. D. O. B., Marmitt, D. J., Bustamante-Filho, I. C., & Goettert, M. I. Phytochemistry Letters , 31 , 196-207, (2019). Kayabaşı, Ç., Avcı, Ç. B., Süslüer, S. Y., Okcanoğlu, T. B., Yelken, B. Ö., Kurt, C. Ç., ... & Gündüz, C. Ege Medical Journal, 61 (2), 232-243, (2022). Pekmezci, H., Köse, B. G., Akbal, Y., Özdemir, V. A., & Çol, B. K, Journal of Health Academics, 9( 3). (2022). Top, R., Erden, Y., & Tekin, S. Bitlis Eren University Journal of Science and Technology , 8 (2), 435-442, (2019). Gezici, S., Kocum, D., Yayla, F., & Şekeroğlu, N. Kahramanmaraş Sütçü İmam University Journal of Agriculture and Nature , 25 (5), 974-985, (2022). Pekdemir, S., Çiftci, M., & Karatepe, M. European Journal of Science and Technology, (18) , 368-378, (2020). Saraç, H., Daştan, T., Demirbaş, A., Daştan, S. D., Karaköy, T., & Durukan, H. Journal of the Faculty of Agriculture, 340-347 , (2018). Dalkılıç, S., Korkmaz, İ., Dalkılıç, L. K., Akay, G., & Fidan, S. Food Bioscience , 46, 101501. (2022). Özgüç, S., Kayalar, H., & Zeybek, U. Journal of Faculty of Pharmacy of Ankara University, 42 (2), 42-62. (2018). Thomford, N. E., Senthebane, D. A., Rowe, A., Munro, D., Seele, P., Maroyi, A., & Dzobo, K. International journal of molecular sciences , 19 (6), 1578. (2018). Aiello, P., Sharghi, M., Mansourkhani, S. M., Ardekan, A. P., Jouybari, L., Daraei, N., ... & Kooti, W. Oxidative Medicine and Cellular Longevity , 2019 (1), 2075614, (2019). Aydoğmuş-öztürk, F. Journal of the Institute of Science and Technology , 11 (1), 645-653, (2021). Murathan, Z. T., Erbil, N., & Arslan, M. Bitlis Eren University Journal of Science and Technology, 11 (1), 218-226, (2016). Rocchetti, G., Senizza, B., Zengin, G., Mahomodally, M. F., Senkardes, I., Lobine, D., & Lucini, L. Journal of the Science of Food and Agriculture , 100 (5), 1998-2006 ,(2020). Murathan, Z. T., Erbil, N., & Arslan, M. Bitlis Eren Üniversitesi Fen Bilimleri Dergisi , 11 (1), 218-226, (2016). Hatipoğlu, M., Sağlam, M., Köseoğlu, S., Köksal, E., Keleş, A., & Esen, H. H. PLoS One, 10 (6), e0128134, (2015). T.Ülger, T., Oçkun, M. A., Guzelmeric, E., Sen, N. B., Sipahi, H., Özhan, Y., ... & Yesilada, E. Molecules , 28 (18), 6520, (2023). Özderin, S., Fakir, H., & Dönmez, İ. Süleyman Demirel Üniversitesi Fen Bilimleri Enstitüsü Dergisi , 19 (1), 120-123, (2015). Sülüşoğlu, M., & Çavusoglu, A . Journal of the Faculty of Agriculture, 9 (1), 77-84, (2014). Arslan, R., Bor, Z., Bektas, N., Meriçli, A. H., & Ozturk, Y. Thrombosis Research , 127 (3), 210-213, (2011). Demirkaya, A. K., Gündoğdu, G., Dodurga, Y., Seçme, M., & Gündoğdu, K. Atatürk University Journal of Veterinary Sciences , 14 (1), 29-37, (2019). Güneş, H., Oktay, M., Çelebi, F., & Tül, B. Journal of Applied Biological Sciences, 8 (2), 16-21 (2014). Medhasi, S., Sriwarom, A., Permpalung, N., Torvorapanit, P., Plongla, R., Chindamporn, A., & Worasilchai, N. Human Vaccines & Immunotherapeutics , 20 (1), 2304372, (2024). Dalkılıç, L. K., Dalkılıç, S., & Uygur, L. International Journal of Plant Based Pharmaceuticals , 3 (1), 62-67, (2023). Ateşşahin, D. A., Dalkılıç, L. K., Özeren, Y., Dalkılıç, S., Çakmak, K., & Çiçek, T. A. International Journal of Nature and Life Sciences , 7 (2), 129-135, (2023). Harandi, H., Falahati-Pour, S. K., Mahmoodi, M., Faramarz, S., Maleki, H., Nasab, F. B., ... & Nematollahi, M. H. (2022). Nanoliposomal formulation of pistachio hull extract: preparation, characterization and anti-cancer evaluation through Bax/Bcl2 modulation. Molecular biology reports , 1-9. Opferman, J. T., & Kothari, A. (2018). Anti-apoptotic BCL-2 family members in development. Cell Death & Differentiation , 25 (1), 37-45. Oltval, Z. N., Milliman, C. L., & Korsmeyer, S. J. (1993). Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programed cell death. cell, 74( 4), 609-619. Adams, J. M., & Cory, S. (2007). The Bcl-2 apoptotic switch in cancer development and therapy. Oncogene, 26 (9), 1324-1337. Aubrey, B. J., Kelly, G. L., Janic, A., Herold, M. J., & Strasser, A. (2018). How does p53 induce apoptosis and how does this relate to p53-mediated tumour suppression? . Cell death & differentiation, 25 (1), 104-113. Li, T., Zhu, J., Guo, L., Shi, X., Liu, Y., & Yang, X. (2013). Differential effects of polyphenols-enriched extracts from hawthorn fruit peels and fleshes on cell cycle and apoptosis in human MCF-7 breast carcinoma cells. Food Chemistry, 141 (2), 1008-1018. Jiang, X., Li, T., & Liu, R. H. (2016). 2α-Hydroxyursolic acid inhibited cell proliferation and induced apoptosis in MDA-MB-231 human breast cancer cells through the p38/MAPK signal transduction pathway . Journal of Agricultural and Food Chemistry, 64 (8), 1806-1816. Kmail, A., Lyoussi, B., Zaid, H., & Saad, B. (2015). In vitro assessments of cytotoxic and cytostatic effects of Asparagus aphyllus, Crataegus aronia, and Ephedra alata in monocultures and co-cultures of Hepg2 and THP-1-derived macrophages. Pharmacogn. Commun, 5, 165-172. Tables Tables 1 to 3 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Tables.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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17:34:22","extension":"xml","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":90420,"visible":true,"origin":"","legend":"","description":"","filename":"1b3295fdb2a04d8ba52c068f719e273e1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8270980/v1/4af5753b35dc624eb2a6fcfb.xml"},{"id":98451940,"identity":"cc8ed4ed-41df-47fe-b22a-352579a89d55","added_by":"auto","created_at":"2025-12-17 17:34:19","extension":"html","order_by":26,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":103568,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8270980/v1/010c1cfcb9511a0d44f8158b.html"},{"id":98451934,"identity":"471b53ce-982a-41e2-b907-326cbe69ba5b","added_by":"auto","created_at":"2025-12-17 17:34:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":217296,"visible":true,"origin":"","legend":"\u003cp\u003eCytotoxic effects of different concentrations of hexane and methanol extracts of \u003cem\u003eC. orientalis\u003c/em\u003e on (A) MCF-7, (B) HepG2, (C) HCT-116, (D) A549 and (E) HEK293 cell lines. Statistical analysis was performed using one-way ANOVA followed by Tukey's post hoc test. p \u0026lt; 0.05 was considered statistically significant. PC = Doxorubicin (2.5 μg/ml); NC= Untreated cells (DMEM).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8270980/v1/bb14ab63e75bb853d14657ea.png"},{"id":98451955,"identity":"504f5dc6-891f-4619-8a93-c0fa7616d794","added_by":"auto","created_at":"2025-12-17 17:34:22","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":25638,"visible":true,"origin":"","legend":"\u003cp\u003eMCF-7 and HepG2 cell lines were treated with gypsum extract at a concentration of 800 µg/mL. Gene expression levels of (a) \u003cem\u003e\u003cstrong\u003eBcl2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e,\u003c/strong\u003e (b) \u003cem\u003e\u003cstrong\u003eBax\u003c/strong\u003e\u003c/em\u003e, and (c) \u003cem\u003e\u003cstrong\u003ep53 \u003c/strong\u003e\u003c/em\u003ewere evaluated after 48 hours using the quantitative real-time PCR method. The results were compared with untreated cells, and gene expression levels were normalized to the \u003cem\u003e\u003cstrong\u003eGAPDH\u003c/strong\u003e\u003c/em\u003e\u003cem\u003e \u003c/em\u003egene. \u003cstrong\u003eStatistical significance: p \u0026lt; 0.0002 for \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eBcl2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, p \u0026lt; 0.0344 for \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eBax\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, and p \u0026lt; 0.0035 for \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ep53\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8270980/v1/8d94aef82d7299951a2059c4.png"},{"id":100730101,"identity":"080badd4-6c85-47d4-b168-48382c05e95a","added_by":"auto","created_at":"2026-01-20 21:19:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":717459,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8270980/v1/95ee9cff-16eb-4e8b-90d0-4d738c6fab2e.pdf"},{"id":98451869,"identity":"8e5a1ed5-35af-43c3-b4e1-eca7cfb2acd5","added_by":"auto","created_at":"2025-12-17 17:33:54","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":60703,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-8270980/v1/cf769a2d5a129e3cded1247d.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Cytotoxic Potential of Crataegus orientalis Fruit Extract: Expression Profiles of Apoptosis - Related Genes (Bax, Bcl2, p53)","fulltext":[{"header":"Highlights","content":"\u003cp\u003e\u0026bull; The cytotoxic effects of C. orientalis fruit extract were evaluated in five human cell lines\u003c/p\u003e\u003cp\u003e\u0026bull; HCT-116 cells showed the highest sensitivity with a low IC₅₀ value\u003c/p\u003e\u003cp\u003e\u0026bull; The extract was prepared using different solvents and tested at various doses\u003c/p\u003e\u003cp\u003e\u0026bull; Apoptosis-related genes Bcl2, Bax, and p53 were analyzed by qRT-PCR\u003c/p\u003e\u003cp\u003e\u0026bull; Significant downregulation of these genes was observed in HepG2 cells.\u003c/p\u003e"},{"header":"INTRODUCTION","content":"\u003cp\u003eSerious health issues include hypertension, liver-related disorders, blood sugar abnormalities, and cardiovascular disease affect a large number of people worldwide. The most dangerous of these illnesses is cancer, for which there is now no known cure [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. According to the World Health Organization\u0026rsquo;s 2019 report, cancer ranks among the primary causes of premature mortality in 112 out of 183 countries, affecting individuals before the age of 70. In 23 countries, it is identified as the third or fourth leading cause of early death [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. It is anticipated that 15.5\u0026nbsp;million individuals will be battling cancer globally by 2030, and 11.5\u0026nbsp;million will lose their lives to the illness. It is anticipated that 15.5\u0026nbsp;million individuals will be battling cancer globally by 2030, and 11.5\u0026nbsp;million will lose their lives to the illness [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Even though there are many medications available to treat cancer, a totally safe and effective therapeutic approach has not yet been created [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Among the most popular methods for combating cancer are include radiotherapy, bone marrow transplants, surgery, immunotherapy, and hormone therapy [5, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Cancer treatment with chemotherapy can harm healthy cells and result in severe side effects like anemia, hair loss, and exhaustion. Therefore, creating novel methods to minimize side effects and lower treatment expenses is crucial. Patients frequently resort to traditional therapy approaches in areas with limited access to modern care [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. For ages, herbal items have been used extensively in various countries and have served as the foundation for traditional medical systems [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. According to recent studies, 63% of cancer medications are sourced from plants, and traditional herbal extracts have anti-cancer properties. It has been discovered that certain herbal ingredients have a high response to treatment and few adverse effects. Thus, research into the potential of herbal remedies in the battle against cancer is still ongoing. According to recent studies, 63% of cancer medications are sourced from plants, and traditional herbal extracts have anti-cancer properties. It has been discovered that certain herbal ingredients have a high response to treatment and few adverse effects. Thus, research into the potential of herbal remedies in the battle against cancer is still ongoing [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. According to the World Health Organization, herbal medicines account for almost 80% of the economies of developing nations [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Additionally, since about 20% of current medications are derived from traditional medicinal plants, there is growing interest in researching herbal items for the treatment of cancer [2, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The fact that 80% of synthetic medications used globally are plant-based further supports this tendency [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Therefore, many plants have been found to have anti-cancer capabilities in addition to their beneficial effects as medical plants [14, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. \u003cem\u003eCrataegus\u003c/em\u003e, a member of the \u003cem\u003eRosaceae\u003c/em\u003e family, is regarded by some as a prickly tree or shrub [16, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The other names of this plant, which is native to the Mediterranean, Crimea, Turkey and Western Iran, are carpets, nuts, hawthorn, wild roses and aktiken [17, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Since ancient times, extracts made from hawthorn leaves, blossoms, and fruits have been used to treat respiratory issues, diarrhea, abdominal pain, palpitations, cardiovascular illnesses, and hypertension [19, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Because of its historical use, Hawthorn has also caught the attention of scientists [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In addition to being a strong source of vitamin C, hawthorn fruit is said to include sugars and carbs and is especially rich in important minerals including calcium, potassium, phosphorus, magnesium, and iron [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Flavonoids, proanthocyanidins ,organic acids, and amines are its primary constituents [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. It is well known that phytochemicals derived from a variety of plants can effectively treat cancer. Research is being conducted worldwide to find novel plant extracts with potent antibacterial and anticancer effects [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In the context of cancer research, oncogenes and tumor suppressor genes have recently drawn a lot of attention for their functions in regulating the cell cycle and apoptosis. Since resistance to apoptosis is a significant treatment issue for cancer, a thorough understanding of apoptosis, a type of programmed cell death, is crucial for the development of effective anticancer medicines. [12, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. There have been some studies on \u003cem\u003eC.orientalis\u003c/em\u003e's antibacterial and antioxidant properties, but none that have looked closely at how it affects human cells.\u003c/p\u003e \u003cp\u003eWith an emphasis on apoptosis-related gene expression, this work intends to examine the cytotoxic and pro-apoptotic effects of \u003cem\u003eC. orientalis\u003c/em\u003e fruit extract on HepG2 and MCF-7 cell lines. The extract was also tested in other human-derived cell lines, such as A549, HCT-116 and HEK293 to investigate its effects across a variety of cellular models and deepen our understanding of its biological activity. Glyceraldehyde-3-phosphate dehydrogenase (\u003cem\u003eGAPDH\u003c/em\u003e) was employed as a housekeeping gene to normalize the expression data and ensure analytical accuracy. Quantitative real-time PCR (qRT-PCR) was conducted to measure the mRNA expression levels of apoptosis-related genes, including \u003cem\u003ep53, Bax\u003c/em\u003e, and \u003cem\u003eBcl2\u003c/em\u003e.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cem\u003eObtaining C.orientalis and cell lines used in the experiment \u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eC.orientalis\u003c/em\u003e species collected from Cip town in Elazığ province of Turkey in September and November. Assoc. Prof. Dr. Semih Dalkılı\u0026ccedil;, one of the faculty members of the Molecular Biology and Genetics Programme of the Department of Biology, Fırat University that has HepG2, MCF-7, HEK293, HCT-116 and A549 cell lines in his laboratory.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePreparation of the extract \u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e C. orientalis\u003c/em\u003e fruits were dried at room temperature and ground to obtain a homogeneous particle size. For the extraction process, 1 gram of dry plant sample was weighed on a precision balance, and the process was carried out using two different solvents. In the first step, extraction was performed with 10 mL hexane, followed by 10 mL methanol. The extraction process was carried out in a shaking oven at 37\u0026deg;C for 72 hours, after which the extracts were filtered with Whatman No. 1 filter paper to remove the solvents. After drying the extracts, yield calculation was performed, and extraction efficiency was determined. When evaluated on a solvent basis, the yield of hexane extract was 31.3%, and the yield of methanol extract was 29.4%, as shown in Table 1. It was dissolved in 10 mL dimethylsulfoxide (Dimethyl Sulfoxide Merck 116743.1000) The extracts were stored at +4 \u0026deg;C for the duration of the study. Hexane and methanol solvents, which are widely preferred for the identification of phenolic compounds, were chosen for the cytotoxicity evaluation of \u003cem\u003eC. orientalis \u003c/em\u003eextract in this study.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCell culture and determination of cytotoxic activity\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eMCF-7, HepG2, A549, HCT-116 and HEK 293 cell lines were cultured in 75 cm\u0026sup2; flasks at 37\u0026deg;C with 5% CO₂ in RPMI 1640 Medium, with L-Glutamine (Cat-No: RPMI-A) medium containing 10% FBS (Cat-No: FBS-11A) and 1% Penicillin-Streptomycin (Cat-No:PS-B\u003cstrong\u003e)\u003c/strong\u003e. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay is a technique used to evaluate cytotoxicity, proliferation and cell viability. The basis of this technique is that MTT and active mitochondria in living cells combine to form water-insoluble purple formazan crystals [26]. MTT method was used to evaluate the effect on \u003cem\u003eC.orientalis\u003c/em\u003e cell viability. After dissolving formazan crystals in appropriate solvents, the amount of viable cells was counted using spectrophotometry. 90% confluent cells were removed from the medium in 75 cm\u0026sup2; flasks and washed with 5 ml sterile PBS. After adding 1 ml trypsin-EDTA (Lot: 325043240, Cat:325-043-EL) to the flasks, they were incubated at 37˚C for two minutes in an incubator containing 5% CO₂. Trypsin-EDTA was inactivated and 5 ml RPMI was added after the cells were detached from the surface. After the cells were removed from the flask, the supernatant was discarded after centrifugation at 2000 rpm for five minutes. Countess II automated cell counter was used to count the cell pellet after resuspension with 1000 \u0026micro;L RPMI. After cell dilution was calculated, 96-well plates containing 5x10\u0026sup3; cells and 100 \u0026micro;L RPMI per well were applied. The positive control was 2.5 \u0026micro;g/mL doxorubicin, while blank and negative controls were media only. Cells were incubated at 37.0 C with 5% CO\u003csub\u003e2\u003c/sub\u003e for 24 hours. Methanol and hexane extracts of \u003cem\u003eC.orientalis \u003c/em\u003efruit produced in RPMI were administered to the cells in six replicates at four different concentrations (100, 200, 400 and 800 \u0026micro;g/mL) after the medium was removed after incubation. Cells were incubated at 37˚C with 5% CO₂ for 72 hours. After completion, 10 \u0026mu;L of MTT solution (5 mg/mL) was added to each well and incubated at 37˚C with 5% CO₂ for 4 hours. Once incubation was over, the medium was taken out, formazan crystals were dissolved in 100 \u0026mu;L DMSO, and an ELISA microplate reader (Allsheng) set to 450 nm wavelength was used to measure the anticipated color change [27]. The control wells\u0026apos; absorbance readings were calculated, averaged, and approved as representing a 100% viable cell ratio. The viability % was calculated by comparing the absorbance values from the plant extracts with the absorbance value of the control group. This is how cytotoxicity was computed:\u003c/p\u003e\n\u003cp\u003e% Live cell = (Sample absorbance) / (Control absorbance) x100\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eRNA isolation from cell and cDNA synthesis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe apoptotic pathway effect of \u003cem\u003eC.orientalis \u003c/em\u003eon HepG2 and MCF-7 cell lines was evaluated by looking at the expression levels of \u003cem\u003eBcl2, Bax\u003c/em\u003e and \u003cem\u003ep53\u003c/em\u003e genes. Based on the cytotoxicity results of \u003cem\u003eC.orientalis \u003c/em\u003e, the best extract and dosage for the two cell lines were determined. HepG2 and MCF-7 cells were exposed to the highest concentration of \u003cem\u003eC.orientalis\u003c/em\u003e hexane extract (800 \u0026mu;g/ml) for 48 hours. Following the manufacturer\u0026apos;s instructions, total RNA was extracted from these cells using the Total RNA Mini Kit (Generaid-Cat. No. RB300). The quality of the extracted RNA was analyzed using the iBright FL 1000 gel imaging system. The quantity and quality of RNA molecules determine how reliable gene expression analysis is [11]. Following the manufacturer\u0026apos;s instructions, the quality and quantity of total RNA isolated was assessed using a Qubit\u0026trade; RNA HS Assay Kit (REF: Q32852, LOT 1913928) and a Thermo Fisher Scientific Invitrogen instrument. RNA concentration was measured in ng/ml. RNA samples determined to be optimal in both quality and quantity were used to generate cDNA. Following the manufacturer\u0026apos;s instructions, cDNA was generated by reverse transcription using the WizScript\u0026trade; cDNA Synthesis Kit (REF: W2211, LOT 4E0420-02). Two microliters of reverse transcription buffer, 0.8 \u0026mu;l 25x dNTPs, 2 \u0026mu;l 10x random primers, one microliter MultiScribe Reverse Transcriptase, 4.2 \u0026mu;l nuclease-free water and 500 ng total RNA (50 \u0026mu;l) were included in 20 microliters of reaction mix. Using a SimpliAmp Thermal Cycler (Applied Biosystems, Singapore), reaction mixtures were incubated at 25\u0026deg;C for 10 min, 37\u0026deg;C for 120 min and 85\u0026deg;C for 5 min for each sample.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eQuantitative real-time polymerase chain reaction (qRT-PCR)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe expression levels of four different genes (\u003cem\u003eBcl2, Bax,\u003c/em\u003e \u003cem\u003ep53\u003c/em\u003e and \u003cem\u003eGAPDH\u003c/em\u003e) were evaluated using quantitative RT-PCR. As indicated by the manufacturer, reactions were performed using Amplifyme SYBR Universal Mix (AM02-020) and Applied Biosystems Step One Plus Real-Time PCR System. The following components were present in 10 \u0026micro;l of reaction mix for each sample: Each sample was run in duplicate using 5 \u0026micro;l 2X Amplifyme SG Universal Mix, 0.3 \u0026micro;l forward primer, 0.3 \u0026micro;l reverse primer, 0.2 \u0026micro;l 50\u0026times; High Rox solution, 2.2 \u0026micro;l PCR Grade Water and 2 \u0026micro;l cDNA sample. The following are the steps of the PCR cycling program: 3 minutes at 95\u0026deg;C followed by 40 cycles of 95\u0026deg;C for 5 seconds, 58\u0026deg;C for 30 seconds, 95\u0026deg;C for 15 seconds, 60\u0026deg;C for 1 minute and finally 95\u0026deg;C for 15 seconds. Untreated cells were used as an external control in qRT-PCR, while\u003cem\u003e GAPDH\u003c/em\u003e was used as an internal control. (\u003cstrong\u003eTable 2)\u003c/strong\u003e lists the primer sequences used.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eStatistical analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eSPSS (version 22) and GraphPad Prism (version 8.0.2) software were used for experimental design and statistical analysis of the data. As a result of the analysis, the statistical difference between the groups was evaluated by ANOVA test and significance levels were determined as p \u0026lt; 0.05.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cem\u003eExtract yield\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFruits of C. orientalis were gathered in September and November from the Cip district in the Elazığ region of Turkey. The HepG2, MCF-7, HEK293, HCT-116, and A549 cell lines utilized in this investigation were acquired from the lab of Assoc. Prof. Dr. Semih Dalkılı\u0026ccedil;, a professor in the Department of Biology\u0026apos;s Molecular Biology and Genetics Program at Fırat University. The following formula was used to determine the extraction efficiency [28]. \u003c/p\u003e\n\u003cp\u003ePercent yield (w/w) = (weight of the dried extract (g))/ (weight of the dry seed material before extraction (g)) x100\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCytotoxic activity\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe cytotoxic potential of MCF-7 and HepG2 methanol and hexane extracts obtained from Crataegus orientalis fruit. The MTT method was used to assess the cancer cell lines HCT-116 and A549 as well as the healthy cell model HEK293 cell line. Fruit extracts\u0026apos; cytotoxic activity on \u003cem\u003eC. orientalis\u003c/em\u003e in vitro, based on dosage and solvent type (Figure 1(A-E)).\u003c/p\u003e\n\u003cp\u003eCell viability in the MCF-7 cell line with the methanol extract was between 59-64%, while with the hexane extract it was between 57-66%. HepG2 cells show that both extracts have moderate cytotoxic effects. HCT-116 cell line showed the highest cytotoxic effect. With 400 g/mL hexane extract, cell viability decreased by 19%, while with 800 g/mL methanol extract it decreased by 15%. These results confirm that both extracts have strong antiproliferative effects in a dose-dependent manner.\u003c/p\u003e\n\u003cp\u003eA more limited cytotoxic effect was observed in A549 cells compared to other cell lines; the highest efficacy was obtained with 29% viable cell ratio in 400 \u0026micro;g/mL hexane extract treatment. In contrast, HEK293 cells were used as a reference to evaluate the possible toxic effects of the extracts on healthy cells and no significant cytotoxic effect was observed in this cell line. Therefore, the IC₅₀ value for HEK293 cells was not calculated. This suggests that \u003cem\u003eC. orientalis\u003c/em\u003e fruit extracts have low toxicity on healthy cells and have selective anticancer potential.\u003c/p\u003e\n\u003cp\u003eThese data were further supported by IC₅₀ analyses, revealing that methanol and hexane extracts exhibited cytotoxic effects that varied depending on the cell type but generally showed anticancer potential (Table 3).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEffects of \u003c/em\u003e\u003cem\u003eC.\u003c/em\u003e\u003cem\u003e \u003c/em\u003e\u003cem\u003eorientalis \u003c/em\u003e\u003cem\u003eextract on apoptotic pathway\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the impact of the \u003cem\u003eC. orientalis\u003c/em\u003e hexane extract on apoptosis-related genes in HepG2 and MCF-7 cell lines, gene expression analysis was carried out using quantitative real-time PCR (qRT-PCR). Following a 24-hour treatment period, there were observable changes in the expression levels of the \u003cem\u003ep53\u003c/em\u003e, \u003cem\u003eBax\u003c/em\u003e, and \u003cem\u003eBcl2 \u003c/em\u003egenes when compared to the control group. Notably, treatment led to a modest shift toward apoptosis, particularly in MCF-7 cells, as reflected by the calculated \u003cem\u003eBax/Bcl2\u003c/em\u003e ratio of 0.83 in MCF-7 and 0.67 in HepG2 cells.\u003c/p\u003e\n\u003cp\u003eAs shown in Figure 2, \u003cem\u003eBcl2 \u003c/em\u003eexpression increased more markedly in HepG2 cells, whereas \u003cem\u003eBax \u003c/em\u003eexpression exhibited a relative increase in both cell lines post-treatment. Similarly, \u003cem\u003ep53 \u003c/em\u003eexpression showed a moderate elevation in treated groups. These results suggest that the \u003cem\u003eC. orientalis\u003c/em\u003e hexane extract may modulate apoptotic gene expression patterns, though the overall pro-apoptotic shift appears limited.\u003c/p\u003e\n\u003cp\u003eIn addition, the specificity of qRT-PCR amplification was confirmed by melting curve analysis, which revealed single and distinct peaks for all target genes, indicating the absence of non-specific products or primer-dimer formation.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eApoptosis, a regulated form of programmed cell death, plays a vital role in preserving cellular homeostasis and is a central mechanism in cancer biology. The intrinsic apoptotic pathway, which is mitochondria-mediated, is tightly controlled by the relative expression of pro-apoptotic (\u003cem\u003eBax\u003c/em\u003e) and anti-apoptotic (\u003cem\u003eBcl2\u003c/em\u003e) genes. This balance determines the release of cytochrome c from mitochondria into the cytosol, a critical event that triggers the activation of downstream apoptotic signaling cascades [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. A crucial stage in the intrinsic apoptotic process, the \u003cem\u003eBax\u003c/em\u003e protein stimulates the release of cytochrome c from the mitochondria. By blocking this release, \u003cem\u003eBcl2\u003c/em\u003e, which is attached to the outer mitochondrial membrane, stops apoptosis. Consequently, it is commonly acknowledged that a key factor influencing a cell's propensity to undergo apoptosis is the \u003cem\u003eBax/Bcl2\u003c/em\u003e expression ratio [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, \u003cem\u003eC. orientalis\u003c/em\u003e fruit extracts showed significant cytotoxic effects on various cancer cell lines. In particular, dose-dependent cytotoxic effects were observed in HCT-116 (colorectal cancer) and MCF-7 (breast cancer) cell lines. Methanol extract stood out as the most effective fraction with lower IC\u003csub\u003e50\u003c/sub\u003e values in most cell lines. On the other hand, since no toxic effect was observed in HEK293 (human embryonic kidney) cells, IC\u003csub\u003e50\u003c/sub\u003e values could not be calculated for these cells. This suggests that the extracts have low toxicity on healthy cells and may have a selective effect against cancer cells. When the data obtained are evaluated in general, it can be said that \u003cem\u003eC.orientalis\u003c/em\u003e has anticancer potential especially against colorectal and breast cancer cells.\u003c/p\u003e \u003cp\u003eThe effect of the hexane extract on the expression of apoptosis-related genes (\u003cem\u003ep53, Bax\u003c/em\u003e, and \u003cem\u003eBcl2\u003c/em\u003e) was further evaluated in MCF-7 and HepG2 cells. The results demonstrated an induction of apoptosis, characterized by a significant upregulation of \u003cem\u003ep53\u003c/em\u003e and \u003cem\u003eBax\u003c/em\u003e, and a concurrent downregulation of the anti-apoptotic gene \u003cem\u003eBcl2\u003c/em\u003e. This pattern indicates activation of the mitochondrial (intrinsic) apoptotic pathway. Moreover, the increased \u003cem\u003eBax/Bcl2\u003c/em\u003e ratio, commonly considered a key marker for apoptosis initiation, reinforces this conclusion [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHexane extract from \u003cem\u003eC. orientalis\u003c/em\u003e fruit was used to evaluate the expression of apoptosis-related genes in MCF-7 and HepG2 cells. The calculated \u003cem\u003eBax/Bcl2\u003c/em\u003e ratios were found to be 0.83 for MCF-7 cells and 0.67 for HepG2 cells. The fact that these ratios were below 1 in both cell lines indicates that the anti-apoptotic effect was dominant and the apoptotic response was limited. However, the up-regulation observed in the \u003cem\u003eBax\u003c/em\u003e gene and the increase in the \u003cem\u003ep53\u003c/em\u003e gene reveal the potential of the extract to direct the cell to apoptosis. Indeed, the increase in the \u003cem\u003eBax/Bcl2\u003c/em\u003e ratio in our study supports that the extract activates the apoptotic process. The \u003cem\u003ep53\u003c/em\u003e tumor suppressor gene, a critical regulator of apoptosis, becomes activated under cellular stress conditions, particularly those involving genomic instability [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In this context, increased \u003cem\u003ep53\u003c/em\u003e expression is expected to trigger \u003cem\u003eBax\u003c/em\u003e transcription and initiate cell death [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In the literature, it is reported that some compounds of \u003cem\u003eCrataegus\u003c/em\u003e species, especially triterpenoid ursolic and oleanolic acids, stimulate apoptosis via mitochondrial pathway and accelerate cell death by increasing caspase-3 and caspase-9 activation [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The gene expression profile found in this investigation is consistent with earlier findings. In particular, the idea that triterpenoid chemicals in \u003cem\u003eC. orientalis\u003c/em\u003e fruit may trigger apoptotic pathways is supported by the overexpression of \u003cem\u003ep53\u003c/em\u003e and \u003cem\u003eBax\u003c/em\u003e and the downregulation of \u003cem\u003eBcl2\u003c/em\u003e. It is also noteworthy that compared to previous studies on the anticancer effects of nearby species such as \u003cem\u003eCrataegus aronia\u003c/em\u003e, \u003cem\u003eC. orientalis\u003c/em\u003e showed a higher cytotoxic effect at lower concentrations. For instance, it has been observed that extracts from \u003cem\u003eC. aronia\u003c/em\u003e only significantly affect HepG2 cells at 500 \u0026micro;g/mL [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In this respect, it can be said that \u003cem\u003eC. orientalis\u003c/em\u003e has a stronger anticancer potential.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eAccording to the study's findings, fruit extracts from \u003cem\u003eC. orientalis\u003c/em\u003e have cytotoxic effects on cancer cell lines like MCF-7 and HepG2, and they may also alter the expression of genes linked to apoptosis. The methanol extract's increased bioactivity emphasizes how important the extraction technique and solvent choice are in affecting the effectiveness of chemicals produced from plants. The \u003cem\u003eBax, Bcl2\u003c/em\u003e, and \u003cem\u003ep53\u003c/em\u003e gene expression levels did not approach statistical significance, but the trends particularly in the MCF-7 cell line indicate a pro-apoptotic response. This possible effect may also be reflected molecularly in the rise in the \u003cem\u003eBax/Bcl2\u003c/em\u003e ratio. Important hints that \u003cem\u003eC. orientalis\u003c/em\u003e might be a potential natural anticancer drug are provided by these early results. However, more thorough in vitro testing, mechanistic research, and in vivo investigations are required to completely illustrate this effect. Whether this plant represents a pharmacologically evaluable therapeutic resource will be made clear by more thorough research in the future.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCONFLICT OF INTEREST\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare any conflicts of interest.\u003c/p\u003e\n\u003cp\u003eF\u003cstrong\u003eINANCIAL SUPPORT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Scientific and Technological Research Council of Turkey (T\u0026Uuml;BİTAK) provided funding for this study under project number 1919B012217792 as part of the T\u0026Uuml;BİTAK 2209-A Undergraduate Research Support Program.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the Scientific and Technological Research Council of Turkey (T\u0026Uuml;BİTAK) under the 2209-A Undergraduate Research Support Program (Project No: 1919B012217792). We would like to express our sincere gratitude to the Department of Parasitology, Faculty of Medicine, Fırat University, for providing laboratory facilities and their valuable contributions throughout the study. We also extend our special thanks to Mr. Mustafa Kaplan for his technical assistance and support. The findings of this study were presented in English as an oral presentation at the 9th International Conference on Contemporary Scientific Studies in the Middle East, held on March 13\u0026ndash;15, 2024, at the Islamic University in Beirut, Lebanon.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLKD\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003econtributions to the manuscript\u0026apos;s final editing and conceptual design. \u003cstrong\u003eS\u003c/strong\u003e\u003cstrong\u003eD\u003c/strong\u003e Contributed to the manuscript\u0026apos;s structural revision and content development. \u003cstrong\u003eŞA\u003c/strong\u003e experimental research, writing, data analysis, and manuscript preparation. \u003cstrong\u003eEA\u0026nbsp;\u003c/strong\u003econtributions to the manuscript\u0026apos;s revision.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAhmed, F., Ijaz, B., Ahmad, Z., Farooq, N., Sarwar, M. B., \u0026amp; Husnain, T. \u003cem\u003ePhytomedicine\u003c/em\u003e, \u003cstrong\u003e68\u003c/strong\u003e, 153168. (2020). \u003c/li\u003e\n\u003cli\u003eAssouyouti, A. \u003cem\u003eMaster\u0026apos;s thesis, Bursa Uludag University (Turkey)\u003c/em\u003e, (2022). \u003c/li\u003e\n\u003cli\u003eShin, S. A., Moon, S. Y., Kim, W. Y., Paek, S. M., Park, H. H., \u0026amp; Lee, C. S. \u003cem\u003eInternational journal of molecular sciences\u003c/em\u003e, \u003cstrong\u003e19\u003c/strong\u003e(9), 2651, (2018).\u003c/li\u003e\n\u003cli\u003eMajolo, F., Delwing, L. K. D. O. B., Marmitt, D. J., Bustamante-Filho, I. C., \u0026amp; Goettert, M. I. \u003cem\u003ePhytochemistry Letters\u003c/em\u003e, \u003cstrong\u003e31\u003c/strong\u003e, 196-207, (2019). \u003c/li\u003e\n\u003cli\u003eKayabaşı, \u0026Ccedil;., Avcı, \u0026Ccedil;. B., S\u0026uuml;sl\u0026uuml;er, S. Y., Okcanoğlu, T. B., Yelken, B. \u0026Ouml;., Kurt, C. \u0026Ccedil;., ... \u0026amp; G\u0026uuml;nd\u0026uuml;z, C. \u003cem\u003eEge Medical Journal,\u003c/em\u003e\u003cstrong\u003e 61\u003c/strong\u003e(2), 232-243, (2022).\u003c/li\u003e\n\u003cli\u003ePekmezci, H., K\u0026ouml;se, B. G., Akbal, Y., \u0026Ouml;zdemir, V. A., \u0026amp; \u0026Ccedil;ol, B. K, \u003cem\u003eJournal of Health Academics, \u003c/em\u003e\u003cstrong\u003e9(\u003c/strong\u003e3). (2022).\u003c/li\u003e\n\u003cli\u003eTop, R., Erden, Y., \u0026amp; Tekin, S. \u003cem\u003eBitlis Eren University Journal of Science and Technology\u003c/em\u003e, \u003cstrong\u003e8\u003c/strong\u003e(2), 435-442, (2019).\u003c/li\u003e\n\u003cli\u003eGezici, S., Kocum, D., Yayla, F., \u0026amp; Şekeroğlu, N.\u003cem\u003e Kahramanmaraş S\u0026uuml;t\u0026ccedil;\u0026uuml; İmam University Journal of Agriculture and Nature\u003c/em\u003e, \u003cstrong\u003e25\u003c/strong\u003e(5), 974-985, (2022).\u003c/li\u003e\n\u003cli\u003ePekdemir, S., \u0026Ccedil;iftci, M., \u0026amp; Karatepe, M. \u003cem\u003eEuropean Journal of Science and Technology,\u003c/em\u003e \u003cstrong\u003e(18)\u003c/strong\u003e, 368-378, (2020).\u003c/li\u003e\n\u003cli\u003eSara\u0026ccedil;, H., Daştan, T., Demirbaş, A., Daştan, S. D., Karak\u0026ouml;y, T., \u0026amp; Durukan, H.\u003cem\u003e Journal of the Faculty of Agriculture, \u003c/em\u003e\u003cstrong\u003e340-347\u003c/strong\u003e\u003cem\u003e, (2018).\u003c/em\u003e\u003c/li\u003e\n\u003cli\u003eDalkılı\u0026ccedil;, S., Korkmaz, İ., Dalkılı\u0026ccedil;, L. K., Akay, G., \u0026amp; Fidan, S. \u003cem\u003eFood Bioscience\u003c/em\u003e, \u003cstrong\u003e46,\u003c/strong\u003e 101501. (2022).\u003c/li\u003e\n\u003cli\u003e\u0026Ouml;zg\u0026uuml;\u0026ccedil;, S., Kayalar, H., \u0026amp; Zeybek, U. \u003cem\u003eJournal of Faculty of Pharmacy of Ankara University,\u003c/em\u003e \u003cstrong\u003e42\u003c/strong\u003e(2), 42-62. (2018).\u003c/li\u003e\n\u003cli\u003eThomford, N. E., Senthebane, D. A., Rowe, A., Munro, D., Seele, P., Maroyi, A., \u0026amp; Dzobo, K. \u003cem\u003eInternational journal of molecular sciences\u003c/em\u003e, \u003cstrong\u003e19\u003c/strong\u003e(6), 1578. (2018).\u003c/li\u003e\n\u003cli\u003eAiello, P., Sharghi, M., Mansourkhani, S. M., Ardekan, A. P., Jouybari, L., Daraei, N., ... \u0026amp; Kooti, W. \u003cem\u003eOxidative Medicine and Cellular Longevity\u003c/em\u003e, \u003cstrong\u003e2019\u003c/strong\u003e(1), 2075614, (2019). \u003c/li\u003e\n\u003cli\u003eAydoğmuş-\u0026ouml;zt\u0026uuml;rk, F.\u003cem\u003e Journal of the Institute of Science and Technology\u003c/em\u003e, \u003cstrong\u003e11\u003c/strong\u003e(1), 645-653, (2021).\u003c/li\u003e\n\u003cli\u003eMurathan, Z. T., Erbil, N., \u0026amp; Arslan, M. \u003cem\u003eBitlis Eren University Journal of Science and Technology, \u003c/em\u003e\u003cstrong\u003e11\u003c/strong\u003e(1), 218-226, (2016). \u003c/li\u003e\n\u003cli\u003eRocchetti, G., Senizza, B., Zengin, G., Mahomodally, M. F., Senkardes, I., Lobine, D., \u0026amp; Lucini, L. \u003cem\u003eJournal of the Science of Food and Agriculture\u003c/em\u003e, \u003cstrong\u003e100\u003c/strong\u003e(5), 1998-2006 ,(2020).\u003c/li\u003e\n\u003cli\u003eMurathan, Z. T., Erbil, N., \u0026amp; Arslan, M. \u003cem\u003eBitlis Eren \u0026Uuml;niversitesi Fen Bilimleri Dergisi\u003c/em\u003e, \u003cstrong\u003e11\u003c/strong\u003e(1), 218-226, (2016).\u003c/li\u003e\n\u003cli\u003eHatipoğlu, M., Sağlam, M., K\u0026ouml;seoğlu, S., K\u0026ouml;ksal, E., Keleş, A., \u0026amp; Esen, H. \u003cem\u003eH. PLoS One,\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e(6), e0128134, (2015).\u003c/li\u003e\n\u003cli\u003eT.\u0026Uuml;lger, T., O\u0026ccedil;kun, M. A., Guzelmeric, E., Sen, N. B., Sipahi, H., \u0026Ouml;zhan, Y., ... \u0026amp; Yesilada, E. \u003cem\u003eMolecules\u003c/em\u003e\u003cem\u003e,\u003c/em\u003e \u003cstrong\u003e28\u003c/strong\u003e(18), 6520, (2023).\u003c/li\u003e\n\u003cli\u003e\u0026Ouml;zderin, S., Fakir, H., \u0026amp; D\u0026ouml;nmez, İ. \u003cem\u003eS\u0026uuml;leyman Demirel \u0026Uuml;niversitesi Fen Bilimleri Enstit\u0026uuml;s\u0026uuml; Dergisi\u003c/em\u003e, \u003cstrong\u003e19\u003c/strong\u003e(1), 120-123, (2015).\u003c/li\u003e\n\u003cli\u003eS\u0026uuml;l\u0026uuml;şoğlu, M., \u0026amp; \u0026Ccedil;avusoglu, A\u003cem\u003e. Journal of the Faculty of Agriculture,\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e(1), 77-84, (2014).\u003c/li\u003e\n\u003cli\u003eArslan, R., Bor, Z., Bektas, N., Meri\u0026ccedil;li, A. H., \u0026amp; Ozturk, Y. \u003cem\u003eThrombosis Research\u003c/em\u003e, \u003cstrong\u003e127\u003c/strong\u003e(3), 210-213, (2011).\u003c/li\u003e\n\u003cli\u003eDemirkaya, A. K., G\u0026uuml;ndoğdu, G., Dodurga, Y., Se\u0026ccedil;me, M., \u0026amp; G\u0026uuml;ndoğdu, K. \u003cem\u003eAtat\u0026uuml;rk University\u003c/em\u003e \u003cem\u003eJournal of Veterinary Sciences\u003c/em\u003e, \u003cstrong\u003e14\u003c/strong\u003e(1), 29-37, (2019).\u003c/li\u003e\n\u003cli\u003eG\u0026uuml;neş, H., Oktay, M., \u0026Ccedil;elebi, F., \u0026amp; T\u0026uuml;l, B.\u003cem\u003e \u003c/em\u003e\u003cem\u003eJournal of Applied Biological Sciences,\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e(2), 16-21 (2014).\u003c/li\u003e\n\u003cli\u003eMedhasi, S., Sriwarom, A., Permpalung, N., Torvorapanit, P., Plongla, R., Chindamporn, A., \u0026amp; Worasilchai, N. \u003cem\u003eHuman Vaccines \u0026amp; Immunotherapeutics\u003c/em\u003e, \u003cstrong\u003e20\u003c/strong\u003e(1), 2304372, (2024).\u003c/li\u003e\n\u003cli\u003eDalkılı\u0026ccedil;, L. K., Dalkılı\u0026ccedil;, S., \u0026amp; Uygur, L. \u003cem\u003eInternational Journal of Plant Based Pharmaceuticals\u003c/em\u003e, \u003cstrong\u003e3\u003c/strong\u003e(1), 62-67, (2023). \u003c/li\u003e\n\u003cli\u003eAteşşahin, D. A., Dalkılı\u0026ccedil;, L. K., \u0026Ouml;zeren, Y., Dalkılı\u0026ccedil;, S., \u0026Ccedil;akmak, K., \u0026amp; \u0026Ccedil;i\u0026ccedil;ek, T. A. \u003cem\u003eInternational Journal of Nature and Life Sciences\u003c/em\u003e, \u003cstrong\u003e7\u003c/strong\u003e(2), 129-135, (2023). \u003c/li\u003e\n\u003cli\u003eHarandi, H., Falahati-Pour, S. K., Mahmoodi, M., Faramarz, S., Maleki, H., Nasab, F. B., ... \u0026amp; Nematollahi, M. H. (2022). \u003cem\u003eNanoliposomal formulation of pistachio hull extract: preparation, characterization and anti-cancer evaluation through Bax/Bcl2 modulation. Molecular biology reports\u003c/em\u003e, 1-9.\u003c/li\u003e\n\u003cli\u003eOpferman, J. T., \u0026amp; Kothari, A. (2018).\u003cem\u003e Anti-apoptotic BCL-2 family members in development. \u003c/em\u003eCell\u003cem\u003e \u003c/em\u003eDeath \u0026amp; Differentiation\u003cem\u003e, \u003c/em\u003e\u003cstrong\u003e25\u003c/strong\u003e(1), 37-45.\u003c/li\u003e\n\u003cli\u003eOltval, Z. N., Milliman, C. L., \u0026amp; Korsmeyer, S. J. (1993). \u003cem\u003eBcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programed cell death. cell,\u003c/em\u003e \u003cstrong\u003e74(\u003c/strong\u003e4), 609-619. \u003c/li\u003e\n\u003cli\u003eAdams, J. M., \u0026amp; Cory, S. (2007). \u003cem\u003eThe Bcl-2 apoptotic switch in cancer development and therapy. \u003c/em\u003eOncogene, \u003cstrong\u003e26\u003c/strong\u003e(9), 1324-1337.\u003c/li\u003e\n\u003cli\u003eAubrey, B. J., Kelly, G. L., Janic, A., Herold, M. J., \u0026amp; Strasser, A. (2018).\u003cem\u003e How does p53 induce apoptosis and how does this relate to p53-mediated tumour suppression?\u003c/em\u003e. Cell death \u0026amp; differentiation, \u003cstrong\u003e25\u003c/strong\u003e(1), 104-113.\u003c/li\u003e\n\u003cli\u003eLi, T., Zhu, J., Guo, L., Shi, X., Liu, Y., \u0026amp; Yang, X. (2013).\u003cem\u003e Differential effects of polyphenols-enriched extracts from hawthorn fruit peels and fleshes on cell cycle and apoptosis in human MCF-7 breast carcinoma cells.\u003c/em\u003e Food Chemistry, \u003cstrong\u003e141\u003c/strong\u003e(2), 1008-1018.\u003c/li\u003e\n\u003cli\u003eJiang, X., Li, T., \u0026amp; Liu, R. H. (2016). \u003cem\u003e2\u0026alpha;-Hydroxyursolic acid inhibited cell proliferation and induced apoptosis in MDA-MB-231 human breast cancer cells through the p38/MAPK signal transduction pathway\u003c/em\u003e. Journal of Agricultural and Food Chemistry, \u003cstrong\u003e64\u003c/strong\u003e(8), 1806-1816.\u003c/li\u003e\n\u003cli\u003eKmail, A., Lyoussi, B., Zaid, H., \u0026amp; Saad, B. (2015).\u003cem\u003e In vitro assessments of cytotoxic and cytostatic effects of Asparagus aphyllus, Crataegus aronia, and Ephedra alata in monocultures and co-cultures of Hepg2 and THP-1-derived macrophages. \u003c/em\u003ePharmacogn. Commun, 5, 165-172.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 3 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Crataegus orientalis, Cytotoxicity, Apoptosis, qRT-PCR, IC₅₀, HepG2-MCF-7-HCT-116-HEK293-A549 cell lines","lastPublishedDoi":"10.21203/rs.3.rs-8270980/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8270980/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cdiv class=\"SectionHeadings\"\u003e\u003cdiv class=\"SectionHeading\"\u003e\u003cdiv class=\"Paragraphs\"\u003e\u003cp\u003eScientists are becoming more interested in the possibility of natural bioactive chemicals as supplemental medicines in cancer treatment. Investigating the biological effects of Crataegus orientalis fruit extract on different human cell lines was the goal of this investigation. HepG2 (liver), MCF-7 (breast), HEK293 (embryonic kidney), HCT-116 (colon), and A549 (lung) cell lines were used to evaluate cytotoxic characteristics using the MTT test. The extract was made with a variety of solvents and tested at different doses to assess dose-dependent effects. The results demonstrated that the cytotoxic effects were varied by the cell line, concentration, and extraction solvent. Notably, the extract's low IC₅₀ value demonstrated high cytotoxicity against the HCT-116 cell line. The mRNA expression levels of important apoptosis-related genes, specifically Bcl2, Bax, and p53, were quantitatively examined using qRT-PCR in human HepG2 and MCF-7 cancer cell lines in order to obtain additional understanding of apoptotic mechanisms. Gene expression was found to have decreased statistically significantly in HepG2 cells (p 0.0002 for Bcl2, p 0.0344 for Bax, and p 0.0035 for p53). All of these results suggest that the fruit extract of C. orientalis may have anticancer properties via influencing the expression of genes linked to apoptosis in cancer cells.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e","manuscriptTitle":"Cytotoxic Potential of Crataegus orientalis Fruit Extract: Expression Profiles of Apoptosis - Related Genes (Bax, Bcl2, p53)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-17 17:31:14","doi":"10.21203/rs.3.rs-8270980/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8c529c61-06d7-4001-931a-f73d2bd1f290","owner":[],"postedDate":"December 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-20T19:01:17+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-17 17:31:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8270980","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8270980","identity":"rs-8270980","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}