Cytotoxic and Apoptotic Effects of Teucrium Polium L Extract on Human Hepatocellular Carcinoma Cell Line SNU-449

Cytotoxic and Apoptotic Effects of Teucrium Polium L Extract on Human Hepatocellular Carcinoma Cell Line SNU-449 | 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 and Apoptotic Effects of Teucrium Polium L Extract on Human Hepatocellular Carcinoma Cell Line SNU-449 Ayshe Slocum, Basri Satilmis, Tevfik Tolga Sahin, Sezai Yilmaz, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8925333/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 Purpose : The current study aims to evaluate the effects of T. polium extract on the human HCC cell line SNU-449 in vitro and to determine possible cytotoxic, apoptotic and anti-cancer properties. Methods : The MTT assay was performed to determine the dose-dependent effects of T. polium on tumor cell proliferation and to calculate IC50. The colony formation assay was carried out using the IC50 dose of T. polium to evaluate its effects on colony formation. To study the effect of T. polium on in vitro migration of SNU-449 cells, the scratch assay was performed. Finally, Western blotting was performed for caspase-3 and cleaved caspase-3 to study possible apoptotic effects. Results : MTT analysis results demonstrated a dose-dependent decrease in cell viability. IC50 was calculated as 90 μg/mL using measurements from the 48th hour results. The colony formation assay showed that colony formation efficiency was reduced by 50% among SNU-449 cells in the T. polium treated group. With the wound healing assay results, half-gap times for the T. polium treated group and the control group were determined as 50.5 and 28.5 hours, respectively. Comparison of the caspase-3 and cleaved caspase-3 levels measured in T. polium treated cells and control cells by means of Western blotting did not demonstrate a significant difference between the two groups. Conclusion : Reducing proliferation and survival rates in SNU-449 cells, T. polium appears to be a potentially effective antineoplastic agent. However, the cytotoxic effect of T. polium on SNU-449 cells is not mediated by induction of apoptosis. Gastroenterology & Hepatology Hepatocellular carcinoma Teucrium polium SNU-449 MTT assay colony formation assay scratch assay Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Medicinal plants and their extracts have been used to treat various illnesses and ailments for centuries. Teucrium polium L. (Lamiaceae) (T. polium), one such medicinal plant, has a history of use that dates back over two thousand years. Although T. polium, also widely known as Golden/Felty germander, is a shrub native to the Mediterranean region, the Middle East and southwestern Asia, it is quite prevalent and approximately 300 subspecies of T. polium have been identified worldwide. T. polium has been employed for its diuretic, antipyretic, sudorific, spasmolytic, anti-inflammatory, antihypertensive, analgesic, antibacterial and antidiabetic properties [1, 2]. Nevertheless, more recent studies have indicated that T. polium also has beneficial effects when utilized in the treatment of nonalcoholic steatohepatitis and cancer [3]. The findings of these studies suggest that the anticancer and cytotoxic properties of T. polium are mainly attributed to the diterpenoid, flavonoid, iridoid and sterol compounds it contains [4]; however, among these, the polyphenolic compounds and specifically flavonoids are acknowledged as the most effective inducers of apoptosis [5]. Studies also indicate that administering T. polium alongside chemotherapeutics, such as vincristine, vinblastine, and doxorubicin, can enhance their efficacy [6]. The anticancer and cytotoxic properties of T. polium have been evaluated using various cancer cell lines, including those cell lines of cervical cancer (HeLa), colon cancer (SW480), melanoma (Skmel-3), breast cancer (MCF-7) [7], colon cancer (Cac0-2, HCT-116, LoVo, SW480), glioblastoma multiforme (GBM) (REYF-1) [8], osteosarcoma (Saos-2), lung cancer (COR-123) [9], prostate cancer (DU145, PC3) [10], bladder cancer (EL), laryngeal cancer (HEP-2), epidermoid carcinoma (A431), hepatoblastoma (HepG2), and chronic myeloid leukemia (K562) [11]. According to the findings of these studies, T. polium may be effective in treating various types of malignancies. However, the effects of T. polium extract on the human hepatocellular carcinoma (HCC) cell line SNU-449 have yet to be investigated. HCC, an aggressive and often insidious type of cancer, usually develops as a primary liver tumor in the setting of cirrhosis and chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infection [12]. The sixth most common cancer and the second leading cause of cancer-related death worldwide, HCC is difficult to diagnose [13]. Due to its variable manifestations clinically in terms of signs and symptoms, in addition to its non-specific radiological presentation, HCC is often diagnosed late in its course and may require multiple specific imaging modalities to be identified. Median survival following diagnosis is approximately 6 to 20 months, and many patients are untreatable at the time of diagnosis [14]. Treatment of HCC, similar to its diagnosis, is challenging. Surgical resection remains the mainstay of treatment; however, it is only effective during the early stages of HCC. Unfortunately, by the time HCC is diagnosed, most patients are ineligible for surgery due to severe liver failure or large tumor size. Other treatment options include liver transplantation, percutaneous ethanol or acetic acid ablation, radiofrequency, microwave or cryoablation, transarterial chemoembolization (TACE), transarterial radioembolization (TARE), radiation therapy or systemic treatment options such as chemotherapy, molecular targeted therapies and immunotherapy [15]. Sorafenib [16], a molecularly targeted tyrosine kinase inhibitor that inhibits RAF (Rapidly Accelerated Fibrosarcoma) kinase and vascular endothelial growth factor receptor (VEGFR), represents perhaps the most important component of systemic therapy for HCC. However, while sorafenib improves survival compared with best supportive care alone [17], it is generally not well tolerated by patients. The benefits of sorafenib and similar systemic therapies are often limited by the extent of liver dysfunction in patients. Therefore, the development and discovery of alternate agents in the treatment of HCC is of vital importance. Accordingly, the present study aims to evaluate the effects of T. polium extract on the human HCC cell line SNU-449 and to determine its possible cytotoxic, apoptotic and anti-cancer properties. 2. Materials and Methods 2.1 Cell lines The human HCC cell line SNU-449 utilized in this study was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). Southern blot hybridization examinations revealed the presence of HBV DNA in this cell line; however, HBV genomic RNA was not expressed. ATCC confirmed that this cell line contains HBV DNA sequences. Prior to initiation of the study, viable cells were cultured in 75 cm 2 tissue culture flasks in RPMI-1640 (Catalog No. 30-2001), which was formulated by ATCC for this specific cell line. They were maintained in an incubator at 37°C (Panasonic® MCO-18AC-PE incubator, Japan), with a humidified atmosphere of 5% CO2 and 95 % air. The cells cultured in RPMI-1640 were routinely supplemented with 5% heat-inactivated fetal bovine serum (FBS) (Sigma-Aldrich® F5724, USA) and %1 Penicillin-Streptomycin-Neomycin (5,000 units of penicillin, 5 mg streptomycin and 10 mg neomycin/mL) (Sigma-Aldrich® P4083, USA) to achieve a final concentration of 10%, promoting cell growth and proliferation and allowing for a doubling time of approximately 36 hours [18]. Following cultivation, the cells adherent to the flasks were separated from the flask bottom using 1 mL trypsin (Sigma-Aldrich® T3924, USA). Finally, after being mixed with 10 μL of trypan blue solution (Sigma-Aldrich® T8154, UK), the cells were counted using Luna-II Automated Cell Counter (Logos Biosystems®, South Korea). 2.2. Plant materials and Extract preparation 200 grams of T. polium plant material was obtained from the Inonu University, Department of Pharmacology in powder form. The ethanol extract of T. polium was prepared using the maceration method. The powdered plant material was soaked in 80% ethanol for 24 hours and then evaporated at 50°C with a rotary evaporator. The crude extract was subsequently fractionated using a solvent-solvent extraction method with petroleum ether [19, 20]. For the present study, 360 mg of T. polium extract was dissolved in 7.2 mL of dimethyl sulfoxide (DMSO) (Merck® 116743 Dimethyl Sulfoxide Emplura®, USA) to prepare a master stock solution at a concentration of 50 mg/mL. This master stock was then diluted with RPMI-1640 to create a second stock containing 500 µg/mL of T. polium extract. Before being applied to the cell cultures, further serial dilutions were performed using RPMI-1640 to obtain T. polium solutions with concentrations of 250 µg/mL, 100 µg/mL, 50 µg/mL, 40 µg/mL, 20 µg/mL, and 10 µg/mL, respectively. 2.3. Cell viability assay Cells in the logarithmic growth phase were trypsinized and resuspended in RPMI culture medium. For cytotoxicity assays, 10 4 viable cells in 100 microliters of RPMI-1640 were plated into each well of three 96-well plates, with one column of wells excluded in each plate. The plates were incubated overnight to allow for cell adhesion. The following day, the medium in each of the three plates for 24h, 48h, and 72h treatment was discarded and replaced by 100 microliters of T. polium at various concentrations (250 μg/mL, 100 μg/mL, 50 μg/mL, 40 μg/mL, 20 μg/mL, 10 μg/mL, 5 µg/mL, respectively). A total of 10 columns were used for each plate, including one control column containing 10 4 cells mixed with RPMI-1640, one DMSO column representing the highest DMSO concentration from the extract (1% DMSO in RPMI-1640), and one blank column that contained only RPMI-1640 without any cells. The first plate was incubated with T. polium for 24 hours, the second plate for 48 hours, and the third plate for 72 hours. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) analysis is a colorimetric technique used to assess cell viability. In this process, the yellow tetrazolium dye, MTT, is reduced to purple formazan by mitochondrial succinate dehydrogenase, an enzyme that is present only in viable cells. Therefore, this reaction serves as a reliable indicator of cellular metabolic function.The MTT (Sigma-Aldrich® M2128-5G, USA) used in this study was prepared by diluting it in PBS to a concentration of 5 mg/mL. After the incubation periods were completed, 10 μL of MTT solution was added to each well, including the blank wells. The plates were then incubated in the cell culture incubator for an additional 4 hours. At the end of 4 hours, the medium was aspirated and removed (BioTek® 50TM TS Microplate Washer 40-301, Germany). To solubilize the formazan, 100 μL of DMSO was added to each well. The plate was then incubated at 37 °C for 15 minutes. Following the incubation, spectrophotometric absorbance measurements were taken at 570 nm with a multimode microplate reader (BioTek® SynergyTM H1m Microplate Reader, USA). To determine the 50% inhibition concentration (IC50) value for T. polium extract in SNU-449 cells, firstly a calibration graph was created using Microsoft Excel® (Microsoft, Redmond, WA, USA) based on the MTT absorbance measurements obtained at different concentrations of T. polium administration. The IC50 value, which corresponds to 50% cell viability, was then calculated using an equation derived from the graph. 2.4. Colony Formation Assay Initially, 10 3 SNU-449 cells were seeded into each well of 6-well plates. The plates were then placed in an incubator at 37 °C overnight to allow for the adhesion of the cells to the surfaces. Following successful confirmation of cell seeding under the microscope, the existing medium was removed from the plates. RPMI-1640 was subsequently added to the control wells, while a sublethal dose of T. polium solution (IC50 = 90 μg/mL) diluted in RPMI-1640 was added to the T. polium wells. The cells were then incubated under standardized cell culture conditions. After 48 hours, both the T. polium solution and the RPMI-1640 medium were removed from the cells. During the final stage of the experiment, RPMI-1640 was added to all wells, including both T. polium and control wells. The medium was changed every 2-3 days and the cells were monitored carefully for approximately 9-11 days until the cells in the control wells formed sufficiently large colonies. Once an adequate number of colonies were cultivated, the medium was removed from all the wells, and the cells were carefully washed with PBS (phosphate-buffered saline). Afterwards the PBS was aspirated from the wells and Methanol: Acetic acid (3:1) was added to the wells instead. The plates were incubated with Methanol: Acetic acid (3:1) for 5 minutes. Thus, cell fixation was achieved. Following the removal of the fixation solution, a 0.5% crystal violet solution in methanol was added to the wells and left for 15 minutes to stain the cells. After the staining process, the plates were immersed in a container filled with water, and the excess crystal violet was rinsed away. Finally, the colonies were counted and measured using the calibrated eye-piece of an inverted microscope (Leica Dmi8, Germany). Plating efficiency (PE), is the ratio of the number of colonies that have formed to the number of cells seeded: PE = (number of colonies formed) / (number of cells seeded) x 100% The number of colonies formed after treatment is applied to the cells is called the survival fraction (SF) and is obtained with PE: SF = (number of colonies formed after treatment) / (number of seeded cells x PE) 2.5. In vitro cell scratch assay 10 6 SNU-449 cells were seeded in two 60 mm tissue culture petri dishes. They were then incubated at 37 °C overnight to ensure the adherence of the cells and formation of a confluent cell monolayer. After the confluence of the cell layers was confirmed using an inverted microscope, the monolayers in both petri dishes were scratched in a straight line using a 100 μL sterile pipette tip. The cell dishes were then washed once with 1 ml of PBS to remove the debris formed by the lysed cells. 5 mL of RPMI was added to the control petri dish, while 90 µg/mL of T. polium dissolved in 5 mL of RPMI was added to the other petri dish. Both cell layers were later placed in the incubator and incubated at 37 °C for 48 hours. Photos were taken of both cell layers every 4 hours using the Paula Smart Cell Imager (Leica Microsystems, Germany), in order to evaluate the cells' ability to repopulate the wound and study their migratory patterns. 2.6. Western Blotting for Caspase-3 and Cleaved Caspase-3 SDS-PAGE electrophoresis was performed using a 4–20% Mini-PROTEAN® TGX™ Precast Protein Gel (Bio-Rad, USA) on the Bio-Rad Mini-Protean electrophoresis system (Bio-Rad, USA). Equal amounts of protein samples were loaded into the wells, and electrophoresis was carried out for 30 minutes in an electrophoresis buffer containing 25 mM Tris, 192 mM glycine, and 0.1% SDS, pH 8.3 (Bio-Rad, USA), at a constant voltage of 200 V. At the end of the run, stain-free gel imaging was conducted using a gel imaging device (Bio-Rad ChemiDoc, USA) to visualize the proteins separated by gel electrophoresis between the cassettes. Proteins that were separated using SDS-PAGE electrophoresis were transferred to a membrane using the Trans-Blot® Turbo™ RTA Mini 0.2 μm PVDF Transfer Kit (BioRad 1704272, USA) and the Trans-Blot® Turbo™ Transfer System (BioRad 1704150, USA). At the end of the blotting process, stain-free blot imaging and stain-free gel imaging were performed on the imaging device to visualize the proteins that migrated from the gel to the PVDF membrane. The PVDF blotting membrane was blocked using the EveryBlot blocking solution (BioRad 12010020, USA) prior to the incubation with the primary antibodies. The blocked membrane was then incubated overnight at +4 °C with Caspase-3 Rabbit monoclonal antibody (mAb) (Cell Signaling 14220, USA) and Cleaved Caspase-3 Rabbit mAb (Cell Signaling 9664, USA) in EveryBlot solution at the recommended dilutions provided by the manufacturer. After incubation, the membrane was washed five times for five minutes each with Tris Buffered Saline (TBS) (BioRad 1706435, USA) containing Tween 20 (BioRad 1610781, USA) on a horizontal shaker (IKATM KS130). The washed membrane was subsequently incubated for one hour at room temperature with Anti-rabbit IgG, HRP-linked Antibody (Cell Signaling 7404, USA) in EveryBlot solution at the manufacturer’s recommended dilutions. Finally, the membrane was washed six times for five minutes each with Tris Buffered Saline containing Tween 20 on a horizontal shaker. The PVDF membrane, which had been incubated with primary and secondary antibodies, was treated with Clarity Max Western ECL Substrate (BioRad 1705062, USA) at the volume and for the duration recommended by the manufacturer. After the incubation period, chemiluminescence imaging of the blot was performed using a gel imaging device. Analysis was conducted with chemiluminescence blot images using Image Lab Software (BioRad, USA). 3. Results 3.1. Assessment of the Cytotoxic Effects of T. polium Extract on the SNU-449 Cell Line Absorbance measurement results from MTT assays, obtained spectrophotometrically using varying concentrations of T. polium, were converted into percentages indicating cell viability. Ultimately, each measurement demonstrated a dose-dependent decrease in cell viability (Figure 1). Calibration curves were created using Microsoft Excel based on MTT absorbance measurements obtained from different concentrations of T. polium applications. Using these values, line equations were created to represent cell viability corresponding to T. polium concentrations. The IC50 values were determined to be 110.935 μg/mL, 96.033 μg/mL, and 120.057 μg/mL at 24, 48, and 72 hours, respectively. For the colony formation and wound healing tests, the results at the 48-hour mark were selected, and the IC50 value of 90 μg/mL, which corresponds to 50% cell viability at this time point, was utilized. 3.2. Effect of T.Polium treatment on Colony Formation The effect of T. polium extract on the proliferation of the SNU-449 cell line was assessed using a colony formation assay. We examined the number of colonies formed after applying a sublethal dose of T. polium extract solution (IC50 = 90 μg/mL) diluted in RPMI-1640 at the 48-hour mark. Notable differences were observed between the treatment and control groups when comparing the number of colonies formed to the number of cells seeded. To evaluate these differences, we first calculated the plating efficiency (PE) for both control cells and those treated with the T. polium extract by dividing the number of colonies formed by the number of cells seeded. The surviving fraction (SF) was then calculated by dividing the PE values of the treated cell group by the PE values of the control cell group, which represents the proportion of colonies formed after treatment with the T. polium extract. Plating Efficiency (PE)= (Number of colonies formed / Number of cells seeded) × 100 Surviving Fraction (SF) = (PE of cells treated with 90 μg/mL T. polium / PE of control cells) × 100 Calculations yielded the following results: PE of control cells = ((62 + 14) / 2000) × 100 = 3.8% PE of cells treated with T. polium (90 μg/mL) = ((19 + 19) / 2000) × 100 = 1.9% Survival rate of cells treated with T. polium = (1.9 / 3.8) × 100 = 50% These results indicate that T. polium extract reduced colony formation efficiency by 50% compared to the control group (RPMI-1640) (Figure 2). 3.3. The ffect of T.Polium on In Vitro Scratch Assay Analysis The scratch test conducted to assess the effects of T. polium on the in vitro migration capabilities of SNU-449 cells, as well as on in vitro wound healing, revealed that the half-wound healing times were 50.5 hours for the group treated with T. polium and 28.5 hours for the control group that did not receive treatment, respectively. At the end of the test, it was observed that the scratch area in the group treated with 90 μg/mL of T. polium closed by 47.5%. For this T. polium-treated group, the average cell migration rate, representing the speed at which cells collectively migrated into the gap, was calculated to be 2.2 μm/hour, with a migration rate of 1% per hour. Additionally, the time required for 50% of the original surface area of the wound to close, referred to as the wound half-healing time (t1/2 gap), was determined to be approximately 50 hours and 31 minutes (or around 50.5 hours) ( Figure 3). In contrast to the T. polium group, in the control group, by the end of the test the scratch area closed by 96%. The average cell migration rate, or v migration , in the control group was measured at 2.6 μm/hour, resulting in a migration rate of 1.9% per hour. Additionally, the half-healing time for the wound, also known as t1/2 gap, was determined to be approximately 28 hours and 35 minutes (approximately 28.5 hours). In the independent samples t-test comparing the test group treated with T. polium to the control group, statistically significant differences were found in the cell density rates within the confluence region of interest in the scratched area. The gap fill percentages for the two groups also showed significant differences, with p-values of 0.004 and 0.001, respectively (with p ≤ 0.05 considered significant). However, no significant differences were noted in the cell migration rate (p = 0.928) or the gap fill area (p = 0.146) between the two groups. When comparing the hourly cell migration percentages (migration percentage per hour) of the T. polium test group and the control group using an independent samples t-test, a notable difference was observed, although it was not statistically significant (p = 0.058) . The effect of T. Polium treatment onCaspase-3 and Cleaved Caspase-3 expression In order to investigate how T. polium induces apoptosis in SNU-449 cells, Western blotting was conducted to measure the levels of caspase-3 and cleaved caspase-3. The results of the Western blotting showed that there was no statistically significant difference in caspase-3 and cleaved caspase-3 levels between the T. polium-treated cells and the control cells. The Mann-Whitney U test, performed using SPSS version 26.0, yielded significance levels of p = 0.827 for caspase-3 and p = 0.513 for cleaved caspase-3. Therefore, it was concluded that the cytotoxic effect of T. polium on SNU-449 cells was not mediated by the induction of caspase-dependent apoptosis. 4. Discussion This study was designed to investigate the potential cytotoxic and antiproliferative effects of T. polium extract on the human HCC cell line SNU-449. The findings revealed that T. polium extract exhibits a dose-dependent cytotoxic effect on SNU-449 cells, leading to a reduction in both proliferation and survival rates. Consequently, due to these observed cytotoxic and anti-proliferative effects, T. polium may serve as a potentially effective antineoplastic agent. However, it is important to note that the cytotoxic effect of T. polium on SNU-449 cells was not mediated by the induction of caspase-dependent apoptosis. Supporting the findings of the current study, numerous studies utilizing various human cancer cell lines have demonstrated that T. polium exhibits cytotoxic effects. For example, a study published by Nematollahi-Mahani et al. in 2007, which is similar to the present research, investigated the potential cytotoxic effects of an ethanol extract from T. polium in vitro. The results of this study, which used various cancer cell lines, including A549 (human lung adenocarcinoma), BT20 (human breast ductal carcinoma), MCF-7 (human breast adenocarcinoma), and PC12 (mouse pheochromocytoma). indicated that the T. polium extract exhibited cytotoxic effects on all the cell lines included in the study. Based on the WST-1 analysis, the IC50 value for the human lung adenocarcinoma cell line A549 was calculated to be 90 µg/mL. This finding is consistent with the value determined in the present study for SNU-449. Additionally, the IC50 values measured for the BT20, MCF-7, and PC-12 cell lines were reported to be 106 µg/mL, 140 µg/mL, and 120 µg/mL, respectively [11]. Similarly, in a 2016 study conducted by Nikodijević et al., the cytotoxic and pro-apoptotic effects of methanolic extracts from T. polium and T. montanum were investigated on breast cancer (MDA-MB-231) and colorectal carcinoma (SW-480) cell lines. The study reported that both plant extracts led to a dose- and time-dependent decrease in the viability of MDA-MB-231 cells, indicating cytotoxic effects. Specifically, for T. polium, the 24-hour IC50 was 429.37 µg/ml. [22]. In their 2020 study, Noumi et al. investigated the cytotoxic and potential anticancer effects of T. polium methanolic extract on breast ductal carcinoma (Walker 256/B) and prostate cancer (MatLyLu) cells. The results showed that T. polium methanolic extract exhibited cytotoxic and antiproliferative effects against both Walker 256/B and MatLyLu cells, which are known for their high metastatic potential. [5]. Several cytotoxicity studies have examined how T. polium affects proliferation in various human cancer cell lines and its potential antiproliferative effects, revealing results that align with those of the present study. For example, the study conducted by Eskandary et al. in 2007 investigated the effects of T. polium on the REYF-1 GBM cell line. The results of the colony formation experiment indicated that after applying T. polium methanolic extract at concentrations of 30, 60, 100, and 200 µg/mL, colony formation in REYF-1 cells decreased to 91%, 65%, 35%, and 0.006%, respectively, compared to the control group. The IC50 value was found to be 69 µg/mL [21]. In a study conducted by Haïdara et al. in 2011, researchers used two human lung cancer cell lines, referred to as H322 and A549, for a colony formation experiment. Likewise, the results indicated that, compared to the control group, the proliferation of both H322 and A549 cells was inhibited following the application of T. polium [9]. As discussed earlier, numerous publications illustrate similarities with the results of this study, supporting the cytotoxic and antiproliferative effects of T. polium. However, there are also some studies that dispute T. polium's anticancer properties. For example, in a study published by Ljubuncic et al. in 2005, the researchers investigated the antioxidant and cytotoxic effects of T. polium and seven different extracts on rat pheochromocytoma (PC12) cells and human hepatoblastoma (HepG2) cells.. The findings indicated that T. polium did not exhibit cytotoxic or inhibitory effects on either PC12 or HepG2 cells [25]. Upon examining the reasons for the differences in the results of the aforementioned studies, it is believed that these discrepancies primarily stem from the different methods used to obtain the T. polium extracts applied in the studies [21]. Some studies used ethanol or methanolic extracts of T. polium, while others utilized water-based crude extracts. Khader et al. reported in their 2007 study that the water-based crude extract of T. polium exhibited neither antimutagenic nor cytoprotective activity [26]. On the other hand, in their 2010 study, the authors reported that the ethanol extract of T. polium inhibited the mutagenicity of N-methyl-N'-nitro-N-nitrosoguanidine [27]. The variations observed among results from different studies can also be attributed to the use of different parts of the plant for extract preparation and geographical variations in the plant. Also, there are over 134 active components in various parts of T. polium subspecies, including neoklerodane diterpenoids, monoterpenes, sesquiterpenes, polyphenols, flavonoids, and fatty acids. Some studies suggest that terpenoids and flavonoids contribute to the anticancer effects of T. polium. However, further research is needed, particularly after fractionation, to identify which of these components are the active ingredients. It is also possible to argue that there may be synergistic or additive effects among the active components of T. polium, leading to strong cytotoxic effects and enhancing its overall anticancer properties [21]. Western blotting analysis conducted in this study revealed that the cytotoxic and anti-proliferative effects of T. polium on the SNU-449 cell line were not mediated by caspase-induced apoptosis. In comparison to these results, in their 2010 study, Kandouz et al. reported a limited induction of apoptosis in both cell lines after application of 100 µg/mL T. polium extract to PC3 and DU145 human prostate cancer cells for 1 to 4 days [10]. Similarly, in a study conducted by Hashem-Dabaghian et al. and published in 2020, where the HT29 human colon adenocarcinoma cell line was treated with T. polium and later assessed for sub-G1 apoptosis using Annexin V flow cytometry, results indicated morphological changes consistent with apoptosis [23]. In a study conducted by Nematollahi-Mahani et al. in 2012, the effects of T. polium on the U87 malignant glioblastoma (GBM) cell line were investigated. The researchers aimed to assess cell viability in 200 cells and identify the patterns of cell death (pre-apoptotic, apoptotic, and necrotic). To achieve this, they performed differential staining using the trypan blue staining test. Additionally, they used propidium iodide (20 mg/mL) and bisbenzamide (Hoechst, 1 µM) after applying T. polium for 48 hours. Both staining techniques revealed the presence of pre-apoptotic and apoptotic cell death patterns, as well as necrotic cell death in the cells treated with T. polium [20]. Similar to the present study's findings, there are also other studies that have indicated that T. polium does not induce apoptosis. In a study conducted by Nikodijević et al. in 2016, analyzing the levels of caspase 8 and 9 in human breast cancer (MDA-MB-231) and colorectal carcinoma (SW 480) cell lines to investigate the presence of apoptosis after the application of T. polium, results indicated that, in SW 480 cells, apoptosis occurred by means of a caspase-independent pathway [22]. Analysis of the mechanisms through which T. polium extract induces cytotoxic and anti-proliferative effects on the SNU-449 cell line in the present study indicates that these effects primarily result from the extract's impact on the migration of SNU-449 cells. Our in vitro cell migration analysis (scratch assay) revealed that T. polium significantly reduced the migration and motility of SNU-449 cells. In their in vitro analysis of cell migration, Kandouz et al. applied a 100 µl/ml of T. polium extract to PC3 cells and DU145 human prostate cancer cells for 24 and 48 hours. Their findings, consistent with the results of our study, indicated that T. polium extract inhibited the invasion and migration abilities of cells in both cell lines examined [10]. It is possible to suggest that the anticancer effects of T. polium on SNU-449 cells may result from various mechanisms. These could include cell cycle arrest, induction of a conversion to an epithelial phenotype in malignant SNU-449 cells through a process known as "mesenchymal-epithelial transition," or caspase-independent apoptosis,. These proteins play a crucial role in providing intercellular adhesion at the cell surface and within the intracellular membrane. Kandouz et al.demonstrated that T. polium extract induced cell cycle arrest in the S phase and led to a reduction in the duration of the G0–G1 phase, thereby inhibiting cell proliferation. Furthermore, it was determined that T. polium extract prompted differentiation into the epithelial phenotype through a process known as "mesenchymal-epithelial transition." This transition, which is crucial in the context of cell invasion and metastasis, resulted in a significant decrease in the invasion and motility abilities of PC3 and DU145 prostate cancer cells compared to untreated cells.. Nonetheless, further research and investigations are required to determine these effect mechanisms of T. polium in SNU-449 cells. 5. Conclusion This study demonstrates that T. polium exhibits significant antiproliferative and cytotoxic effects; however, it does not trigger caspase-mediated apoptosis in the SNU-449 human HCC cell line. Further in vitro and in vivo studies are necessary to investigate the mechanisms of action of T. polium as a potential anticancer agent against HCC and to identify its active components. Declarations Funding : This study was funded by the Scientific Research Projects Coordination Unit of Inonu University (IUBAP) (Project number: TTU-2021-2701) Conflict of Interests: The authors declare no conflict of interest. References Khazaei, M., S.N. Nematollahi-Mahani, T. Mokhtari and F. Sheikhbahaei. 2018. Review on Teucrium polium biological activities and medical characteristics against different pathologic situations . J Contemp Med Sci 4:1-6 Tariq, M., A.M. Ageel, M.A. Al-Yahya, J.S. Mossa, and M.S. Al-Said. 1989. Anti-inflammatory activity of Teucrium polium. Int J Tissue React 11(4):185-188 Nosrati, N., S. Aghazadeh, and R. Yazdanparast. 2010. Effects of Teucrium polium on insulin resistance in nonalcoholic steatohepatitis. J Acupunct Meridian Stud 3:104-110. https://doi.org/10.1016/S2005-2901(10)60019-2 Movahedi, A., R. Basir, A. Rahmat, M. Charaffedine, and F. Othman F. 2014. Remarkable anticancer activity of Teucrium polium on hepatocellular carcinogenic rats. Evid Based Complement Alternat Med 2014:726724. https://doi.org/10.1155/2014/726724 Noumi, E., M. Snoussi, E.H. Anouar, M. Alreshidi, V.N. Veettil, S. Elkahoui, M. Adnan, M. Patel, A. Kadri, K. Aouadi, and V. De Feo. 2020. HR-LCMS-based metabolite profiling, antioxidant, and anticancer properties of Teucrium polium L. methanolic extract: Computational and in vitro study. Antioxidants 9(11):1089. https://doi.org/10.3390/antiox9111089 Rajabalian, S. 2008. Methanolic extract of Teucrium polium L potentiates the cytotoxic and apoptotic effects of anticancer drugs of vincristine, vinblastine and doxorubicin against a panel of cancerous cell lines. Exp Oncol 30(2):133-138 Abu-Dahab, R., and F. Afifi. 2007. Antiproliferative activity of selected medicinal plants of Jordan against a breast adenocarcinoma cell line (MCF7). Sci Pharm 75:121-146. https://doi.org/10.3797/scipharm.2007.75.121 Line, M.C. 2007. Evaluation of cytotoxic effect of Teuerium polium on a new glioblastoma multiforme cell line (REYF-1) using MTT and soft agar clonogenic assays. Int J Pharmacol 3:435-437. https://doi.org/10.3923/ijp.2007.435.437 Haïdara, K., A. Alachkar, and A.E. Al Moustafa. 2011. Teucrium polium plant extract provokes significant cell death in human lung cancer cells. Health 3(6):366-369. https://doi.org/10.4236/health.2011.36062 Kandouz, M., A. Alachkar, L. Zhang, H. Dekhil, F. Chehna, A. Yasmeen, and A.E. Al Moustafa. 2010. Teucrium polium plant extract inhibits cell invasion and motility of human prostate cancer cells via the restoration of the E-cadherin/catenin complex. J Ethnopharmacol 129:410-415. https://doi.org/10.1016/j.jep.2009.10.035 Nematollahi-Mahani, S.N., M. Rezazadeh-Kermani, M. Mehrabani, and N. Nakhaee. 2007. Cytotoxic effects of Teucrium Polium on some established cell lines. Pharm Biol 45:295-298 Singal, A.G., P. Lampertico, and P. Nahon. 2020. Epidemiology and surveillance for hepatocellular carcinoma: New trends. J Hepatol 72(2):250-261. https://doi.org/10.1016/j.jhep.2019.08.025 Sung, H., J. Ferlay, R.L. Siegel, M. Laversanne, I. Soerjomataram, A. Jemal, and F. Bray. 2022. Global Cancer Observatory: Cancer Today 2020 . http://gco.iarc.fr/today/data/factsheets/cancers/11-Liver-fact-sheet.pdf. Accessed 24 May 2022 Cancer of the Liver Italian Program (CLIP) Investigators. A new prognostic system for hepatocellular carcinoma: a retrospective study of 435 patients. 1998. Hepatology 28:751-755. https://doi.org/10.1002/hep.510280322 Forner, A., M. Reig, and J. Bruix. 2018. Hepatocellular carcinoma. Lancet 391:1301-1314. https://doi.org/10.1016/S0140-6736(18)30010-2 Liu, L., Y. Cao, C. Chen, X. Zhang, A. McNabola, D. Wilkie, S. Wilhelm, M. Lynch, and C. Carter. 2006. Sorafenib blocks the RAF/MEK/ERK pathway, inhibits tumor-angiogenesis, and induces tumor cell apoptosis in hepatocellular carcinoma model PLC/PRF/5. Cancer Res 66:11851-11858. https://doi.org/10.1158/0008-5472.CAN-06-1377 Llovet, J.M., S. Ricci, V. Mazzaferro, P. Hilgard, E. Gane, J.F. Blanc, A.C. De Oliveira, A. Santoro, J.L. Raoul, A. Forner, and M. Schwartz M. 2008. Sorafenib in advanced hepatocellular carcinoma. N Engl J Med 359:378-390. https://doi.org/10.1056/NEJMoa0708857 American Tissue Culture Collection. 2022. https://www.atcc.org/products/crl-2234. Accessed 26 May 2022 Sheikhbahaei, F., M. Khazaei, and S.N.Nematollahi-Mahani. 2018. Teucrium polium extract enhances the anti-angiogenesis effect of tranilast on human umbilical vein endothelial cells. Adv Pharm Bull 8(1):131–139. https://doi.org/10.15171/apb.2018.016 Nematollahi-Mahani, S.N., Z. Mahdinia, R. Eftekharvaghefi, M. Mehrabani, V. Hemayatkhah-Jahromi, and F. Nabipour F. 2012. In vitro inhibition of the growth of glioblastoma by teucrium polium crude extract and fractions. Int J Phytomed 4(4):582-588 Eskandary, H., S. Rajabalian, T. Yazdi, M. Eskandari, K. Fatehi, and N.A. Ganjooei NA. 2007. Evaluation of the cytotoxic effects of Teucrium Polium on a new glioblastoma multiforme cell line (REYF-1) using MTT and soft agar clonogenic assays. Int J Pharmacol . 3(5):435-437. https://doi.org/10.3923/ijp.2007.435.437 Nikodijević, D., M. Milutinović, D. Cvetković, M. Stanković, M.N. Živanović, and S. Marković. 2016. Effects of Teucrium polium L. and Teucrium montanum L.: Extracts on mechanisms of apoptosis in breast and colon cancer cells. Kragujevac J Sci 38:147-159. https://doi.org/10.5937/KgJSci1638147N Hashem-Dabaghian, F., A. Shojaii, J. Asgarpanah, and M. Entezari. 2020. Anti-Mutagenicity and Apoptotic Effects of Teucrium polium L. Essential Oil in HT29 Cell Line. Jundishapur J Nat Pharm Prod 15(3)79559. https://doi.org/10.5812/jjnpp.79559 Menichini, F., F. Conforti, D. Rigano, C. Formisano, F. Piozzi, and F. Senatore F. 2009. Phytochemical composition, anti-inflammatory and antitumour activities of four Teucrium essential oils from Greece. Food Chem 115(2):679-686. https://doi.org/10.1016/j.foodchem.2008.12.067 Ljubuncic, P., H. Azaizeh, I. Portnaya, U. Cogan, O. Said, K.A. Saleh, and A. Bomzon. 2005. Antioxidant activity and cytotoxicity of eight plants used in traditional Arab medicine in Israel. J Ethnopharmacol 99(1):43-47. https://doi.org/10.1016/j.jep.2005.01.060 Khader, M., P.M. Eckl, and N. Bresgen N. 2007. Effects of aqueous extracts of medicinal plants on MNNG-treated rat hepatocytes in primary cultures. J Ethnopharmacol 112(1):199-202. https://doi.org/10.1016/j.jep.2007.01.027 Khader, M., N. Bresgen, and P.M. Eckl PM. 2010. Antimutagenic effects of ethanolic extracts from selected Palestinian medicinal plants. J Ethnopharmacol 127(2):319-24. https://doi.org/10.1016/j.jep.2009.11.001 Additional Declarations The authors declare no competing interests. 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. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8925333","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":594413141,"identity":"6be49550-770f-4d3d-810f-987b72db9bd0","order_by":0,"name":"Ayshe Slocum","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5UlEQVRIiWNgGAWjYHACNiC2ADEYHwAJHj4itUiAGMwGIC1spGhhk4Bx8QL+GenPHnz4IyHHPyM7rfJrjp0MGwPzw0c38GiRuJFjbjizTcJY4kbuttuy25KBDmMzNs7BZ82NHDZp3gaJxA0SQC2S25iBWnjYpPFpkb+R/kz6zx+IlmLJbfWEtRjcSDCTBnocrIXx47bDhLUYnnljJtkL8suZt5ulGbcd52FjJuAXuePpzyR+/LGR42/P3fjx57Zqe3725oeP8XpfIAHBZuYBk/iUgwD/AQSb8Qch1aNgFIyCUTAiAQBbzkPI5O4y9wAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-1612-6076","institution":"Inonu University, Department of Gastroenterology-Hepatology, Malatya, Turkey","correspondingAuthor":true,"prefix":"","firstName":"Ayshe","middleName":"","lastName":"Slocum","suffix":""},{"id":594414071,"identity":"1cafd129-a231-421d-8a77-4a480b4e23c5","order_by":1,"name":"Basri Satilmis","email":"","orcid":"https://orcid.org/0000-0002-2538-5774","institution":"Inonu University, Liver Transplant Institute, Malatya, Turkey","correspondingAuthor":false,"prefix":"","firstName":"Basri","middleName":"","lastName":"Satilmis","suffix":""},{"id":594414072,"identity":"350fa432-e11f-42a3-a7d4-569c3c728cb9","order_by":2,"name":"Tevfik Tolga Sahin","email":"","orcid":"https://orcid.org/0000-0002-9132-6115","institution":"Inonu University, Liver Transplant Institute, Malatya, Turkey","correspondingAuthor":false,"prefix":"","firstName":"Tevfik","middleName":"Tolga","lastName":"Sahin","suffix":""},{"id":594414823,"identity":"11ce1ff4-8ffe-4073-9e19-eb5fd3c547ca","order_by":3,"name":"Sezai Yilmaz","email":"","orcid":"https://orcid.org/0000-0002-8044-0297","institution":"Inonu University, Liver Transplant Institute, Malatya, Turkey","correspondingAuthor":false,"prefix":"","firstName":"Sezai","middleName":"","lastName":"Yilmaz","suffix":""},{"id":594414824,"identity":"24612c31-5f14-422b-b5eb-5100a3decd12","order_by":4,"name":"Muhsin Murat Muhip Harputluoglu","email":"","orcid":"https://orcid.org/0000-0002-9415-147X","institution":"nonu University, Department of Gastroenterology-Hepatology, Malatya, Turkey","correspondingAuthor":false,"prefix":"","firstName":"Muhsin","middleName":"Murat Muhip","lastName":"Harputluoglu","suffix":""}],"badges":[],"createdAt":"2026-02-20 11:23:44","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-8925333/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8925333/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103211741,"identity":"d3ed70f5-b3ef-4340-9273-d61a89ca6ba4","added_by":"auto","created_at":"2026-02-23 08:42:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":18734,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eCell viability of the SNU-499 cells which were treated with varying concentration of T.Polium during 24, 48, and 72 hours.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8925333/v1/ec6d775b58357ab327e5f459.png"},{"id":103211742,"identity":"fdc5b305-5b02-44e0-85a0-271719fd10a3","added_by":"auto","created_at":"2026-02-23 08:42:20","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":162526,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eColonies formed in the SNU-449 cell line control group and following the application of T. polium extract solution, a quantitative analysis of the number of colonies formed as a result of the colony formation experiment expressed as a percentage compared to the control cell group\u003c/em\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8925333/v1/a818c8d4aeae747430f65dee.png"},{"id":103211682,"identity":"e668244b-aa11-4fbc-8353-0d07d2bfdebf","added_by":"auto","created_at":"2026-02-23 08:42:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":378075,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eWound healing at the end of the 48th hour in the T. polium administered group versus the control group\u003c/em\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8925333/v1/8197c3bc1f8e0751e1591953.png"},{"id":103211746,"identity":"2662c01b-52dd-40d1-a2cb-c1b594a22dac","added_by":"auto","created_at":"2026-02-23 08:42:24","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":101827,"visible":true,"origin":"","legend":"\u003cp\u003eUnnumbered image in the Result section.\u003c/p\u003e","description":"","filename":"unnumberFigure.png","url":"https://assets-eu.researchsquare.com/files/rs-8925333/v1/2f60f7cb17173eb81d6769f3.png"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eCytotoxic and Apoptotic Effects of Teucrium Polium L Extract on Human Hepatocellular Carcinoma Cell Line SNU-449\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eMedicinal plants and their extracts have been used to treat various illnesses and ailments for centuries. \u0026nbsp;Teucrium polium L. (Lamiaceae) (T. polium), one such medicinal plant, has a history of use that dates back over two thousand years. Although T. polium, also widely known as Golden/Felty germander, is a shrub native to the Mediterranean region, the Middle East and southwestern Asia, it is quite prevalent and approximately 300 subspecies of T. polium have been identified worldwide. T. polium has been employed for its diuretic, antipyretic, sudorific, spasmolytic, anti-inflammatory, antihypertensive, analgesic, antibacterial and antidiabetic properties [1, 2]. Nevertheless, more recent studies have indicated that T. polium also has beneficial effects when utilized in the treatment of nonalcoholic steatohepatitis and cancer [3]. The findings of these studies suggest that the anticancer and cytotoxic properties of T. polium are mainly attributed to the diterpenoid, flavonoid, iridoid and sterol compounds it contains [4]; however, among these, the polyphenolic compounds and specifically flavonoids are acknowledged as the most effective inducers of apoptosis [5]. Studies also indicate that administering T. polium alongside chemotherapeutics, such as vincristine, vinblastine, and doxorubicin, can enhance their efficacy [6]. The anticancer and cytotoxic properties of T. polium have been evaluated using various cancer cell lines, including those cell lines of cervical cancer (HeLa), colon cancer (SW480), melanoma (Skmel-3), breast cancer (MCF-7) [7], colon cancer (Cac0-2, HCT-116, LoVo, SW480), glioblastoma multiforme (GBM) (REYF-1) [8], osteosarcoma (Saos-2), lung cancer (COR-123) [9], prostate cancer (DU145, PC3) [10], bladder cancer (EL), laryngeal cancer (HEP-2), epidermoid carcinoma (A431), hepatoblastoma (HepG2), and chronic myeloid leukemia (K562) [11]. According to the findings of these studies, T. polium may be effective in treating various types of malignancies. \u0026nbsp;However, the effects of T. polium extract on the human hepatocellular carcinoma (HCC) cell line SNU-449 have yet to be investigated.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHCC, an aggressive and often insidious type of cancer, usually develops as a primary liver tumor in the setting of cirrhosis and chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) infection [12]. \u0026nbsp;The sixth most common cancer and the second leading cause of cancer-related death worldwide, HCC is difficult to diagnose [13]. Due to its variable manifestations clinically in terms of signs and symptoms, in addition to its non-specific radiological presentation, HCC is often diagnosed late in its course and may require multiple specific imaging modalities to be identified. Median survival following diagnosis is approximately 6 to 20 months, and many patients are untreatable at the time of diagnosis [14].\u003c/p\u003e\n\u003cp\u003eTreatment of HCC, similar to its diagnosis, is challenging. Surgical resection remains the mainstay of treatment; however, it is only effective during the early stages of HCC. \u0026nbsp;Unfortunately, by the time HCC is diagnosed, most patients are ineligible for surgery due to severe liver failure or large tumor size. Other treatment options include liver transplantation, percutaneous ethanol or acetic acid ablation, radiofrequency, microwave or cryoablation, transarterial chemoembolization (TACE), transarterial radioembolization (TARE), radiation therapy or systemic treatment options such as chemotherapy, molecular targeted therapies and immunotherapy [15].\u003c/p\u003e\n\u003cp\u003eSorafenib [16], a molecularly targeted tyrosine kinase inhibitor that inhibits RAF (Rapidly Accelerated Fibrosarcoma) kinase and vascular endothelial growth factor receptor (VEGFR), represents perhaps the most important component of systemic therapy for HCC. However, while sorafenib improves survival compared with best supportive care alone [17], it is generally not well tolerated by patients. The benefits of sorafenib and similar systemic therapies are often limited by the extent of liver dysfunction in patients.\u003c/p\u003e\n\u003cp\u003eTherefore, the development and discovery of alternate agents in the treatment of HCC is of vital importance. Accordingly, the present study aims to evaluate the effects of T. polium extract on the human HCC cell line SNU-449 and to determine its possible cytotoxic, apoptotic and anti-cancer properties.\u003c/p\u003e"},{"header":"2.\tMaterials and Methods","content":"\u003cp\u003e2.1 Cell lines\u003c/p\u003e\n\u003cp\u003eThe human HCC cell line SNU-449 utilized in this study was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). Southern blot hybridization examinations revealed the presence of HBV DNA in this cell line; however, HBV genomic RNA was not expressed. \u0026nbsp;ATCC confirmed that this cell line contains HBV DNA sequences. Prior to initiation of the study, viable cells were cultured in 75 cm\u003csup\u003e2\u003c/sup\u003e tissue culture flasks in RPMI-1640 (Catalog No. 30-2001), which was formulated by ATCC for this specific cell line. They were maintained in an incubator at 37\u0026deg;C (Panasonic\u0026reg; MCO-18AC-PE incubator, Japan), with a humidified atmosphere of 5% CO2 and 95 % air. The cells cultured in RPMI-1640 were routinely supplemented with 5% heat-inactivated fetal bovine serum (FBS) (Sigma-Aldrich\u0026reg; F5724, USA) and %1 Penicillin-Streptomycin-Neomycin (5,000 units of penicillin, 5 mg streptomycin and 10 mg neomycin/mL) (Sigma-Aldrich\u0026reg; P4083, USA) to achieve a final concentration of 10%, promoting cell growth and proliferation and allowing for a doubling time of approximately 36 hours [18]. \u0026nbsp;Following cultivation, the cells adherent to the flasks were separated from the flask bottom using 1 mL trypsin (Sigma-Aldrich\u0026reg; T3924, USA). \u0026nbsp;Finally, after being mixed with 10 \u0026mu;L of trypan blue solution (Sigma-Aldrich\u0026reg; T8154, UK), the cells were counted using Luna-II Automated Cell Counter (Logos Biosystems\u0026reg;, South Korea).\u003c/p\u003e\n\u003cp\u003e2.2. Plant materials and Extract preparation\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e200 grams of T. polium plant material was obtained from the Inonu University, Department of \u0026nbsp;Pharmacology in powder form. The ethanol extract of T. polium was prepared using the maceration method. The powdered plant material was soaked in 80% ethanol for 24 hours and then evaporated at 50\u0026deg;C with a rotary evaporator. The crude extract was subsequently fractionated using a solvent-solvent extraction method with petroleum ether [19, 20]. For the present study, 360 mg of T. polium extract was dissolved in 7.2 mL of dimethyl sulfoxide (DMSO) (Merck\u0026reg; 116743 Dimethyl Sulfoxide Emplura\u0026reg;, USA) to prepare a master stock solution at a concentration of 50 mg/mL. This master stock was then diluted with RPMI-1640 to create a second stock containing 500 \u0026micro;g/mL of T. polium extract. Before being applied to the cell cultures, further serial dilutions were performed using RPMI-1640 to obtain T. polium solutions with concentrations of 250 \u0026micro;g/mL, 100 \u0026micro;g/mL, 50 \u0026micro;g/mL, 40 \u0026micro;g/mL, 20 \u0026micro;g/mL, and 10 \u0026micro;g/mL, respectively.\u003c/p\u003e\n\u003cp\u003e2.3. Cell viability assay\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCells in the logarithmic growth phase were trypsinized \u0026nbsp;and resuspended in RPMI culture medium. For cytotoxicity assays, 10\u003csup\u003e4\u003c/sup\u003e viable cells in 100 microliters of RPMI-1640 were plated into each well of three 96-well plates, with one column of wells excluded in each plate. The plates were incubated overnight to allow for cell adhesion. The following day, the medium in each of the three plates for 24h, 48h, and 72h treatment was discarded and replaced by 100 microliters of T. polium at various concentrations (250 \u0026mu;g/mL, 100 \u0026mu;g/mL, 50 \u0026mu;g/mL, 40 \u0026mu;g/mL, 20 \u0026mu;g/mL, 10 \u0026mu;g/mL, 5 \u0026micro;g/mL, respectively). A total of 10 columns were used for each plate, including one control column containing 10\u003csup\u003e4\u0026nbsp;\u003c/sup\u003ecells mixed with RPMI-1640, one DMSO column representing the highest DMSO concentration from the extract (1% DMSO in RPMI-1640), and one blank column that contained only RPMI-1640 without any cells. The first plate was incubated with T. polium for 24 hours, the second plate for 48 hours, and the third plate for 72 hours. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) analysis is a colorimetric technique used to assess cell viability. In this process, the yellow tetrazolium dye, MTT, is reduced to purple formazan by mitochondrial succinate dehydrogenase, an enzyme that is present only in viable cells. Therefore, this reaction serves as a reliable indicator of cellular metabolic function.The MTT (Sigma-Aldrich\u0026reg; M2128-5G, USA) used in this study was prepared by diluting it in PBS to a concentration of 5 mg/mL. After the incubation periods were completed, 10 \u0026mu;L of MTT solution was added to each well, including the blank wells. The plates were then incubated in the cell culture incubator for an additional 4 hours. At the end of 4 hours, the medium was aspirated and removed (BioTek\u0026reg; 50TM TS Microplate Washer 40-301, Germany). To solubilize the formazan, 100 \u0026mu;L of DMSO was added to each well. The plate was then incubated at 37 \u0026deg;C for 15 minutes. Following the incubation, spectrophotometric absorbance measurements were taken at 570 nm with a multimode microplate reader (BioTek\u0026reg; SynergyTM H1m Microplate Reader, USA). To determine the 50% inhibition concentration (IC50) value for T. polium extract in SNU-449 cells, firstly a calibration graph was created using Microsoft Excel\u0026reg; (Microsoft, Redmond, WA, USA) based on the MTT absorbance measurements obtained at different concentrations of T. polium administration. The IC50 value, which corresponds to 50% cell viability, was then calculated using an equation derived from the graph.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2.4. Colony Formation Assay\u003c/p\u003e\n\u003cp\u003eInitially, 10\u003csup\u003e3\u003c/sup\u003e SNU-449 cells were seeded into each well of 6-well plates. The plates were then placed in an incubator at 37 \u0026deg;C overnight to allow for the adhesion of the cells to the surfaces. Following successful confirmation of cell seeding under the microscope, the existing medium was removed from the plates. RPMI-1640 was subsequently added to the control wells, while a sublethal dose of T. polium solution (IC50 = 90 \u0026mu;g/mL) diluted in RPMI-1640 was added to the T. polium wells. The cells were then incubated under standardized cell culture conditions. After 48 hours, both the T. polium solution and the RPMI-1640 medium were removed from the cells. \u0026nbsp;During the final stage of the experiment, RPMI-1640 was added to all wells, including both T. polium and control wells. The medium was changed every 2-3 days and the cells were monitored carefully for approximately 9-11 days until the cells in the control wells formed sufficiently large colonies. Once an adequate number of colonies were cultivated, the medium was removed from all the wells, and the cells were carefully washed with PBS (phosphate-buffered saline). \u0026nbsp;Afterwards the PBS was aspirated from the wells and Methanol: Acetic acid (3:1) was added to the wells instead. The plates were incubated with Methanol: Acetic acid (3:1) for 5 minutes. Thus, cell fixation was achieved.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFollowing the removal of the fixation solution, a 0.5% crystal violet solution in methanol was added to the wells and left for 15 minutes to stain the cells. After the staining process, the plates were immersed in a container filled with water, and the excess crystal violet was rinsed away. Finally, the colonies were counted and measured using the calibrated eye-piece of an inverted microscope (Leica Dmi8, Germany). Plating efficiency (PE), is the ratio of the number of colonies that have formed to the number of cells seeded:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePE = (number of colonies formed) / (number of cells seeded) x 100%\u003c/p\u003e\n\u003cp\u003eThe number of colonies formed after treatment is applied to the cells is called the survival fraction (SF) and is obtained with PE:\u003c/p\u003e\n\u003cp\u003eSF = (number of colonies formed after treatment) / (number of seeded cells x PE)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2.5. \u0026nbsp;In vitro cell scratch assay\u003c/p\u003e\n\u003cp\u003e10\u003csup\u003e6\u003c/sup\u003e SNU-449 cells were seeded in two 60 mm tissue culture petri dishes. They were then incubated at 37 \u0026deg;C overnight to ensure the adherence of the cells and formation of a confluent cell monolayer. After the confluence of the cell layers was confirmed using an inverted microscope, the monolayers in both petri dishes were scratched in a straight line using a 100 \u0026mu;L sterile pipette tip. The cell dishes were then washed once with 1 ml of PBS to remove the debris formed by the lysed cells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e5 mL of RPMI was added to the control petri dish, while 90 \u0026micro;g/mL of T. polium dissolved in 5 mL of RPMI was added to the other petri dish. Both cell layers were later placed in the incubator and incubated at 37 \u0026deg;C for 48 hours. \u0026nbsp;Photos were taken of both cell layers every 4 hours using the Paula Smart Cell Imager (Leica Microsystems, Germany), in order to evaluate the cells\u0026apos; ability to repopulate the wound and study their migratory patterns.\u003c/p\u003e\n\u003cp\u003e2.6. Western Blotting for Caspase-3 and Cleaved Caspase-3\u003c/p\u003e\n\u003cp\u003eSDS-PAGE electrophoresis was performed using a 4\u0026ndash;20% Mini-PROTEAN\u0026reg; TGX\u0026trade; Precast Protein Gel (Bio-Rad, USA) on the Bio-Rad Mini-Protean electrophoresis system (Bio-Rad, USA). Equal amounts of protein samples were loaded into the wells, and electrophoresis was carried out for 30 minutes in an electrophoresis buffer containing 25 mM Tris, 192 mM glycine, and 0.1% SDS, pH 8.3 (Bio-Rad, USA), at a constant voltage of 200 V. At the end of the run, stain-free gel imaging was conducted using a gel imaging device (Bio-Rad ChemiDoc, USA) to visualize the proteins separated by gel electrophoresis between the cassettes. Proteins that were separated using SDS-PAGE electrophoresis were transferred to a membrane using the Trans-Blot\u0026reg; Turbo\u0026trade; RTA Mini 0.2 \u0026mu;m PVDF Transfer Kit (BioRad 1704272, USA) and the Trans-Blot\u0026reg; Turbo\u0026trade; Transfer System (BioRad 1704150, USA). At the end of the blotting process, stain-free blot imaging and stain-free gel imaging were performed on the imaging device to visualize the proteins that migrated from the gel to the PVDF membrane. The PVDF blotting membrane was blocked using the EveryBlot blocking solution (BioRad 12010020, USA) prior to the incubation with the primary antibodies. The blocked membrane was then incubated overnight at +4 \u0026deg;C with Caspase-3 Rabbit monoclonal antibody (mAb) (Cell Signaling 14220, USA) and Cleaved Caspase-3 Rabbit mAb (Cell Signaling 9664, USA) in EveryBlot solution at the recommended dilutions provided by the manufacturer. After incubation, the membrane was washed five times for five minutes each with Tris Buffered Saline (TBS) (BioRad 1706435, USA) containing Tween 20 (BioRad 1610781, USA) on a horizontal shaker (IKATM KS130). The washed membrane was subsequently incubated for one hour at room temperature with Anti-rabbit IgG, HRP-linked Antibody (Cell Signaling 7404, USA) in EveryBlot solution at the manufacturer\u0026rsquo;s recommended dilutions. Finally, the membrane was washed six times for five minutes each with Tris Buffered Saline containing Tween 20 on a horizontal shaker. The PVDF membrane, which had been incubated with primary and secondary antibodies, was treated with Clarity Max Western ECL Substrate (BioRad 1705062, USA) at the volume and for the duration recommended by the manufacturer. After the incubation period, chemiluminescence imaging of the blot was performed using a gel imaging device. Analysis was conducted with chemiluminescence blot images using Image Lab Software (BioRad, USA).\u0026nbsp;\u003c/p\u003e"},{"header":"3.\tResults","content":"\u003cp\u003e3.1. Assessment of the Cytotoxic Effects of T. polium Extract on the SNU-449 Cell Line\u003c/p\u003e\n\u003cp\u003eAbsorbance measurement results from MTT assays, obtained spectrophotometrically using varying concentrations of T. polium, were converted into percentages indicating cell viability. Ultimately, each measurement demonstrated a dose-dependent decrease in cell viability (Figure 1).\u003c/p\u003e\n\u003cp\u003eCalibration curves were created using Microsoft Excel based on MTT absorbance measurements obtained from different concentrations of T. polium applications. Using these values, line equations were created to represent cell viability corresponding to T. polium concentrations. The IC50 values were determined to be 110.935 μg/mL, 96.033 μg/mL, and 120.057 μg/mL at 24, 48, and 72 hours, respectively. For the colony formation and wound healing tests, the results at the 48-hour mark were selected, and the IC50 value of 90 μg/mL, which corresponds to 50% cell viability at this time point, was utilized.\u003c/p\u003e\n\u003cp\u003e3.2. Effect of T.Polium treatment on Colony Formation\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe effect of T. polium extract on the proliferation of the SNU-449 cell line was assessed using a colony formation assay. We examined the number of colonies formed after applying a sublethal dose of T. polium extract solution (IC50 = 90 μg/mL) diluted in RPMI-1640 at the 48-hour mark. Notable differences were observed between the treatment and control groups when comparing the number of colonies formed to the number of cells seeded. To evaluate these differences, we first calculated the plating efficiency (PE) for both control cells and those treated with the T. polium extract by dividing the number of colonies formed by the number of cells seeded. The surviving fraction (SF) was then calculated by dividing the PE values of the treated cell group by the PE values of the control cell group, which represents the proportion of colonies formed after treatment with the T. polium extract.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePlating Efficiency (PE)= (Number of colonies formed / Number of cells seeded) × 100 \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSurviving Fraction (SF) = (PE of cells treated with 90 μg/mL T. polium / PE of control cells) × 100\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCalculations yielded the following results:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePE of control cells = ((62 + 14) / 2000) × 100 = 3.8%\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePE of cells treated with T. polium (90 μg/mL) = ((19 + 19) / 2000) × 100 = 1.9%\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSurvival rate of cells treated with T. polium = (1.9 / 3.8) × 100 = 50%\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThese results indicate that T. polium extract reduced colony formation efficiency by 50% compared to the control group (RPMI-1640) (Figure 2). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e3.3. The ffect of T.Polium on In Vitro Scratch Assay Analysis\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe scratch test conducted to assess the effects of T. polium on the in vitro migration capabilities of SNU-449 cells, as well as on in vitro wound healing, revealed that the half-wound healing times were 50.5 hours for the group treated with T. polium and 28.5 hours for the control group that did not receive treatment, respectively. At the end of the test, it was observed that the scratch area in the group treated with 90 μg/mL of T. polium closed by 47.5%. For this T. polium-treated group, the average cell migration rate, representing the speed at which cells collectively migrated into the gap, was calculated to be 2.2 μm/hour, with a migration rate of 1% per hour. Additionally, the time required for 50% of the original surface area of the wound to close, referred to as the wound half-healing time (t1/2 gap), was determined to be approximately 50 hours and 31 minutes (or around 50.5 hours) ( Figure 3). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn contrast to the T. polium group, in the control group, by the end of the test the scratch area closed by 96%. The average cell migration rate, or v\u003csup\u003emigration\u003c/sup\u003e, in the control group was measured at 2.6 μm/hour, resulting in a migration rate of 1.9% per hour. Additionally, the half-healing time for the wound, also known as t1/2 gap, was determined to be approximately 28 hours and 35 minutes (approximately 28.5 hours).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the independent samples t-test comparing the test group treated with T. polium to the control group, statistically significant differences were found in the cell density rates within the confluence region of interest in the scratched area. The gap fill percentages for the two groups also showed significant differences, with p-values of 0.004 and 0.001, respectively (with p ≤ 0.05 considered significant). However, no significant differences were noted in the cell migration rate (p = 0.928) or the gap fill area (p = 0.146) between the two groups. When comparing the hourly cell migration percentages (migration percentage per hour) of the T. polium test group and the control group using an independent samples t-test, a notable difference was observed, although it was not statistically significant (p = 0.058) .\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe effect of T. Polium treatment onCaspase-3 and Cleaved Caspase-3 expression\u003c/p\u003e\n\u003cp\u003eIn order to investigate how T. polium induces apoptosis in SNU-449 cells, Western blotting was conducted to measure the levels of caspase-3 and cleaved caspase-3. The results of the Western blotting showed that there was no statistically significant difference in caspase-3 and cleaved caspase-3 levels between the T. polium-treated cells and the control cells. The Mann-Whitney U test, performed using SPSS version 26.0, yielded significance levels of p = 0.827 for caspase-3 and p = 0.513 for cleaved caspase-3. Therefore, it was concluded that the cytotoxic effect of T. polium on SNU-449 cells was not mediated by the induction of caspase-dependent apoptosis.\u0026nbsp;\u003c/p\u003e"},{"header":"4.\tDiscussion","content":"\u003cp\u003eThis study was designed to investigate the potential cytotoxic and antiproliferative effects of T. polium extract on the human HCC cell line SNU-449. The findings revealed that T. polium extract exhibits a dose-dependent cytotoxic effect on SNU-449 cells, leading to a reduction in both proliferation and survival rates. Consequently, due to these observed cytotoxic and anti-proliferative effects, T. polium may serve as a potentially effective antineoplastic agent. However, it is important to note that the cytotoxic effect of T. polium on SNU-449 cells was not mediated by the induction of caspase-dependent apoptosis.\u003c/p\u003e \u003cp\u003eSupporting the findings of the current study, numerous studies utilizing various human cancer cell lines have demonstrated that T. polium exhibits cytotoxic effects. For example, a study published by Nematollahi-Mahani et al. in 2007, which is similar to the present research, investigated the potential cytotoxic effects of an ethanol extract from T. polium in vitro. The results of this study, which used various cancer cell lines, including A549 (human lung adenocarcinoma), BT20 (human breast ductal carcinoma), MCF-7 (human breast adenocarcinoma), and PC12 (mouse pheochromocytoma). indicated that the T. polium extract exhibited cytotoxic effects on all the cell lines included in the study. Based on the WST-1 analysis, the IC50 value for the human lung adenocarcinoma cell line A549 was calculated to be 90 \u0026micro;g/mL. This finding is consistent with the value determined in the present study for SNU-449. Additionally, the IC50 values measured for the BT20, MCF-7, and PC-12 cell lines were reported to be 106 \u0026micro;g/mL, 140 \u0026micro;g/mL, and 120 \u0026micro;g/mL, respectively [11]. Similarly, in a 2016 study conducted by Nikodijević et al., the cytotoxic and pro-apoptotic effects of methanolic extracts from T. polium and T. montanum were investigated on breast cancer (MDA-MB-231) and colorectal carcinoma (SW-480) cell lines. The study reported that both plant extracts led to a dose- and time-dependent decrease in the viability of MDA-MB-231 cells, indicating cytotoxic effects. Specifically, for T. polium, the 24-hour IC50 was 429.37 \u0026micro;g/ml. [22]. In their 2020 study, Noumi et al. investigated the cytotoxic and potential anticancer effects of T. polium methanolic extract on breast ductal carcinoma (Walker 256/B) and prostate cancer (MatLyLu) cells. The results showed that T. polium methanolic extract exhibited cytotoxic and antiproliferative effects against both Walker 256/B and MatLyLu cells, which are known for their high metastatic potential. [5].\u003c/p\u003e \u003cp\u003eSeveral cytotoxicity studies have examined how T. polium affects proliferation in various human cancer cell lines and its potential antiproliferative effects, revealing results that align with those of the present study. For example, the study conducted by Eskandary et al. in 2007 investigated the effects of T. polium on the REYF-1 GBM cell line. The results of the colony formation experiment indicated that after applying T. polium methanolic extract at concentrations of 30, 60, 100, and 200 \u0026micro;g/mL, colony formation in REYF-1 cells decreased to 91%, 65%, 35%, and 0.006%, respectively, compared to the control group. The IC50 value was found to be 69 \u0026micro;g/mL [21]. In a study conducted by Ha\u0026iuml;dara et al. in 2011, researchers used two human lung cancer cell lines, referred to as H322 and A549, for a colony formation experiment. Likewise, the results indicated that, compared to the control group, the proliferation of both H322 and A549 cells was inhibited following the application of T. polium [9].\u003c/p\u003e \u003cp\u003eAs discussed earlier, numerous publications illustrate similarities with the results of this study, supporting the cytotoxic and antiproliferative effects of T. polium. However, there are also some studies that dispute T. polium's anticancer properties. For example, in a study published by Ljubuncic et al. in 2005, the researchers investigated the antioxidant and cytotoxic effects of T. polium and seven different extracts on rat pheochromocytoma (PC12) cells and human hepatoblastoma (HepG2) cells.. The findings indicated that T. polium did not exhibit cytotoxic or inhibitory effects on either PC12 or HepG2 cells [25].\u003c/p\u003e \u003cp\u003eUpon examining the reasons for the differences in the results of the aforementioned studies, it is believed that these discrepancies primarily stem from the different methods used to obtain the T. polium extracts applied in the studies [21]. Some studies used ethanol or methanolic extracts of T. polium, while others utilized water-based crude extracts. Khader et al. reported in their 2007 study that the water-based crude extract of T. polium exhibited neither antimutagenic nor cytoprotective activity [26]. On the other hand, in their 2010 study, the authors reported that the ethanol extract of T. polium inhibited the mutagenicity of N-methyl-N'-nitro-N-nitrosoguanidine [27]. The variations observed among results from different studies can also be attributed to the use of different parts of the plant for extract preparation and geographical variations in the plant. Also, there are over 134 active components in various parts of T. polium subspecies, including neoklerodane diterpenoids, monoterpenes, sesquiterpenes, polyphenols, flavonoids, and fatty acids. Some studies suggest that terpenoids and flavonoids contribute to the anticancer effects of T. polium. However, further research is needed, particularly after fractionation, to identify which of these components are the active ingredients. It is also possible to argue that there may be synergistic or additive effects among the active components of T. polium, leading to strong cytotoxic effects and enhancing its overall anticancer properties [21].\u003c/p\u003e \u003cp\u003eWestern blotting analysis conducted in this study revealed that the cytotoxic and anti-proliferative effects of T. polium on the SNU-449 cell line were not mediated by caspase-induced apoptosis. In comparison to these results, in their 2010 study, Kandouz et al. reported a limited induction of apoptosis in both cell lines after application of 100 \u0026micro;g/mL T. polium extract to PC3 and DU145 human prostate cancer cells for 1 to 4 days [10]. Similarly, in a study conducted by Hashem-Dabaghian et al. and published in 2020, where the HT29 human colon adenocarcinoma cell line was treated with T. polium and later assessed for sub-G1 apoptosis using Annexin V flow cytometry, results indicated morphological changes consistent with apoptosis [23]. In a study conducted by Nematollahi-Mahani et al. in 2012, the effects of T. polium on the U87 malignant glioblastoma (GBM) cell line were investigated. The researchers aimed to assess cell viability in 200 cells and identify the patterns of cell death (pre-apoptotic, apoptotic, and necrotic). To achieve this, they performed differential staining using the trypan blue staining test. Additionally, they used propidium iodide (20 mg/mL) and bisbenzamide (Hoechst, 1 \u0026micro;M) after applying T. polium for 48 hours. Both staining techniques revealed the presence of pre-apoptotic and apoptotic cell death patterns, as well as necrotic cell death in the cells treated with T. polium [20].\u003c/p\u003e \u003cp\u003eSimilar to the present study's findings, there are also other studies that have indicated that T. polium does not induce apoptosis. In a study conducted by Nikodijević et al. in 2016, analyzing the levels of caspase 8 and 9 in human breast cancer (MDA-MB-231) and colorectal carcinoma (SW 480) cell lines to investigate the presence of apoptosis after the application of T. polium, results indicated that, in SW 480 cells, apoptosis occurred by means of a caspase-independent pathway [22].\u003c/p\u003e \u003cp\u003eAnalysis of the mechanisms through which T. polium extract induces cytotoxic and anti-proliferative effects on the SNU-449 cell line in the present study indicates that these effects primarily result from the extract's impact on the migration of SNU-449 cells. Our in vitro cell migration analysis (scratch assay) revealed that T. polium significantly reduced the migration and motility of SNU-449 cells. In their in vitro analysis of cell migration, Kandouz et al. applied a 100 \u0026micro;l/ml of T. polium extract to PC3 cells and DU145 human prostate cancer cells for 24 and 48 hours. Their findings, consistent with the results of our study, indicated that T. polium extract inhibited the invasion and migration abilities of cells in both cell lines examined [10]. It is possible to suggest that the anticancer effects of T. polium on SNU-449 cells may result from various mechanisms. These could include cell cycle arrest, induction of a conversion to an epithelial phenotype in malignant SNU-449 cells through a process known as \"mesenchymal-epithelial transition,\" or caspase-independent apoptosis,. These proteins play a crucial role in providing intercellular adhesion at the cell surface and within the intracellular membrane. Kandouz et al.demonstrated that T. polium extract induced cell cycle arrest in the S phase and led to a reduction in the duration of the G0\u0026ndash;G1 phase, thereby inhibiting cell proliferation. Furthermore, it was determined that T. polium extract prompted differentiation into the epithelial phenotype through a process known as \"mesenchymal-epithelial transition.\" This transition, which is crucial in the context of cell invasion and metastasis, resulted in a significant decrease in the invasion and motility abilities of PC3 and DU145 prostate cancer cells compared to untreated cells.. Nonetheless, further research and investigations are required to determine these effect mechanisms of T. polium in SNU-449 cells.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study demonstrates that T. polium exhibits significant antiproliferative and cytotoxic effects; however, it does not trigger caspase-mediated apoptosis in the SNU-449 human HCC cell line. Further in vitro and in vivo studies are necessary to investigate the mechanisms of action of T. polium as a potential anticancer agent against HCC and to identify its active components.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/strong\u003e: This study was funded by the Scientific Research Projects Coordination Unit of Inonu University (IUBAP) (Project number: TTU-2021-2701)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConflict of Interests:\u0026nbsp;\u003c/em\u003e\u003c/strong\u003eThe authors declare no conflict of interest.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKhazaei, M., S.N. Nematollahi-Mahani, T. Mokhtari and F. Sheikhbahaei. 2018. Review on Teucrium polium biological activities and medical characteristics against different pathologic situations\u003cem\u003e. 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Sorafenib in advanced hepatocellular carcinoma. \u003cem\u003eN Engl J Med\u003c/em\u003e 359:378-390. https://doi.org/10.1056/NEJMoa0708857\u003c/li\u003e\n\u003cli\u003eAmerican Tissue Culture Collection. 2022. https://www.atcc.org/products/crl-2234. Accessed 26 May 2022\u003c/li\u003e\n\u003cli\u003eSheikhbahaei, F., M. Khazaei, and S.N.Nematollahi-Mahani. 2018. Teucrium polium extract enhances the anti-angiogenesis effect of tranilast on human umbilical vein endothelial cells. \u003cem\u003eAdv Pharm Bull\u003c/em\u003e 8(1):131\u0026ndash;139. https://doi.org/10.15171/apb.2018.016\u003c/li\u003e\n\u003cli\u003eNematollahi-Mahani, S.N., Z. Mahdinia, R. Eftekharvaghefi, M. Mehrabani, V. Hemayatkhah-Jahromi, and F. Nabipour F. 2012. In vitro inhibition of the growth of glioblastoma by teucrium polium crude extract and fractions. \u003cem\u003eInt J Phytomed\u003c/em\u003e 4(4):582-588\u003c/li\u003e\n\u003cli\u003eEskandary, H., S. Rajabalian, T. Yazdi, M. Eskandari, K. 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Conforti, D. Rigano, C. Formisano, F. Piozzi, and F. Senatore F. 2009. Phytochemical composition, anti-inflammatory and antitumour activities of four Teucrium essential oils from Greece. \u003cem\u003eFood Chem\u003c/em\u003e 115(2):679-686. https://doi.org/10.1016/j.foodchem.2008.12.067\u003c/li\u003e\n\u003cli\u003eLjubuncic, P., H. Azaizeh, I. Portnaya, U. Cogan, O. Said, K.A. Saleh, and A. Bomzon. 2005. Antioxidant activity and cytotoxicity of eight plants used in traditional Arab medicine in Israel. \u003cem\u003eJ Ethnopharmacol\u003c/em\u003e 99(1):43-47. https://doi.org/10.1016/j.jep.2005.01.060\u003c/li\u003e\n\u003cli\u003eKhader, M., P.M. Eckl, and N. Bresgen N. 2007. Effects of aqueous extracts of medicinal plants on MNNG-treated rat hepatocytes in primary cultures. \u003cem\u003eJ Ethnopharmacol\u003c/em\u003e 112(1):199-202. https://doi.org/10.1016/j.jep.2007.01.027\u003c/li\u003e\n\u003cli\u003eKhader, M., N. Bresgen, and P.M. Eckl PM. 2010. Antimutagenic effects of ethanolic extracts from selected Palestinian medicinal plants. \u003cem\u003eJ Ethnopharmacol\u003c/em\u003e 127(2):319-24. https://doi.org/10.1016/j.jep.2009.11.001\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"e9f2ab82-f521-4b8a-8964-c4a24f15c2bc","identifier":"10.13039/501100011576","name":"Inönü Üniversitesi","awardNumber":"TTU-2021-2701","order_by":0}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Inonu University","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":"Hepatocellular carcinoma, Teucrium polium, SNU-449, MTT assay, colony formation assay, scratch assay","lastPublishedDoi":"10.21203/rs.3.rs-8925333/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8925333/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003ePurpose\u003c/strong\u003e\u003c/em\u003e: The current study aims to evaluate the effects of T. polium extract on the human HCC cell line SNU-449 in vitro and to determine possible cytotoxic, apoptotic and anti-cancer properties.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/em\u003e: The MTT assay was performed to determine the dose-dependent effects of T. polium on tumor cell proliferation and to calculate IC50. The colony formation assay was carried out using the IC50 dose of T. polium to evaluate its effects on colony formation. To study the effect of T. polium on in vitro migration of SNU-449 cells, the scratch assay was performed. Finally, Western blotting was performed for caspase-3 and cleaved caspase-3 to study possible apoptotic effects.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/em\u003e: MTT analysis results demonstrated a dose-dependent decrease in cell viability. IC50 was calculated as 90 μg/mL using measurements from the 48th hour results. The colony formation assay showed that colony formation efficiency was reduced by 50% among SNU-449 cells in the T. polium treated group. With the wound healing assay results, half-gap times for the T. polium treated group and the control group were determined as 50.5 and 28.5 hours, respectively. Comparison of the caspase-3 and cleaved caspase-3 levels measured in T. polium treated cells and control cells by means of Western blotting did not demonstrate a significant difference between the two groups.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/em\u003e: Reducing proliferation and survival rates in SNU-449 cells, T. polium appears to be a potentially effective antineoplastic agent. However, the cytotoxic effect of T. polium on SNU-449 cells is not mediated by induction of apoptosis.\u003c/p\u003e","manuscriptTitle":"Cytotoxic and Apoptotic Effects of Teucrium Polium L Extract on Human Hepatocellular Carcinoma Cell Line SNU-449","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-23 08:39:48","doi":"10.21203/rs.3.rs-8925333/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":"7246c312-746d-4d59-a841-d9cd32a1b7b4","owner":[],"postedDate":"February 23rd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":63258147,"name":"Gastroenterology \u0026 Hepatology"}],"tags":[],"updatedAt":"2026-02-23T08:39:48+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-23 08:39:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8925333","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8925333","identity":"rs-8925333","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}