Antiproliferative activity of Hoslundia opposita leaf extract and fractions against a human glioblastoma cell line (U251)

Antiproliferative activity of Hoslundia opposita leaf extract and fractions against a human glioblastoma cell line (U251) | 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 Antiproliferative activity of Hoslundia opposita leaf extract and fractions against a human glioblastoma cell line (U251) Abosede Christiana Ajibare, Osaretin Albert Taiwo Ebuehi, Rahmat Adetutu Adisa, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4824462/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 BACKGROUND: The ineffectiveness of many known anticancer agents for treating several cancer types, especially glioblastoma (GMB), which affects the body's central nervous system, is highly important. GBM is highly invasive and recalcitrant and accountsfor 42% of all central nervous system tumors and 60% of all brain tumors in adults, with a median survival of 15 months. The limitationsencountered in GBM treatment necessitate the discovery and development of new drugs. METHODS: To investigate the anticancer activity of Hoslundia opposita leaf extracts and fractions against a human glioblastoma cell line (U251) and human keratinocyte HACAT cell line, standard methods, MTT, clonogenic and caspase3 and 7 assays were used to determine the viability of the cells and colony formation and apoptotic activities, respectively. The fluorescent probe dyes dihydrofluorescindiacetate (DCFH-DA) and tetramethylrhodamine (TMRE) were used to determinethe intracellular reactive oxygen species (ROS) concentration and mitochondrial membrane potential (MMP), respectively, in the cells. RESULTS: The crudemethanolic extracts and fractions of H. opposita leaves exhibited moderate cytotoxic and selective activity within the range of concentrations tested (25-100 µg/ml). The study revealed that crude AHO1 and specific fractions of AHO5 and AHO6 inhibitedmetastasis or colony formation, promoted apoptosis in the U251 cell line and depolarized the mitochondrial membrane potential, which was likelymediated by mitochondria-dependent ROS generation. Overall, the specificity and dose dependenceof the different treatments were observed for the U251 cell line. Conclusions: The antiproliferative activities of Hoslundia opposita Vahl demonstrated by the crude extract and specific fractions against U251 cells warrant further investigations todecipher its mechanism of action. General Biochemistry Cell Survival and Cell Death Cancer Biology Hoslundia opposita cancers cytotoxicity apoptosis gliomas Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION All tumors originating from glial cells are gliomas commonly affecting the body's central nervous system. According to the World Health Organization, gliomas are classified according to their presumed cell of origin as astrocytic tumors: astrocytoma grade I, astrocytoma grade II, astrocytoma grade III (anagal astrocytoma), astrocytoma IV (glioblastoma or GM), oligodendrogliomas, ependymomas and mixed gliomas ( 1 , 2 ). Glioblastoma is the most common, lethal and damaging type of primary brain tumor in humans and is characterized by an unavoidable propensity to relapse and poor prognosis ( 3 , 4 ). Glioblastoma accounts for 42% of all central nervous system tumors and 60% of all brain tumors in adults, with a median survival of 15 months ( 5 , 6 ). Glioblastoma multiforme (GBM) has no cure, and intertumoural or intratumour heterogeneity is one of the hallmarks of this cancer. The development of GBM is characterized by age, sex, race, genetic disorders, and ionizing radiation ( 7 ). In GBM, cellular heterogeneity is associated with therapeutic and drug resistance due to an array of genetic alterations involved in the control of cell cycle kinetics, cell growth, apoptosis, cell invasion and neovascularization ( 8 , 9 ). Despite surgical resection, irradiation and adjuvant chemotherapy, GBM remains a major therapeutic problem, as survival following diagnosis can reach 12 to 15 months, with less than 5% survival longer than five years ( 8 ). The clinical treatment of GBM has not been successful despite the knowledge and advances in understanding the associated complex biology. The genetic and epigenetic heterogeneity of GBM has helped but has not been fully translated to effective clinical ( 10 ) outcomes. Some conventional DNA-damaging anticancer drugs have failed in GBM, showing proven efficacy in other cancer types and hence limiting the treatment options for GBM. Hence, the failure of these agents necessitates the development of new drugs and new categories of therapeutics. Temozolomide (TMZ) was approved for GBM treatment for the first time, just as bevacizumab is used to treat recurrent GBM and was approved by the Food and Drug Administration. ( 11 , 12 ). Bevacizumab nitrosoureas and TMZ, which are the second-line agents, are the most common systemic agents for recurrent high-grade gliomas (HGGs), but there is no superiority of these agents over one another. Despite standard treatment and care centered on surgical resection, chemotherapy and radiation therapy, patients have a poor progression-free survival (PFS) of 7–8 months, a median survival of 14–16 months and a 5-year overall survival (OS) of 9.8% ( 13 – 15 ). Natural plant products, which possess similar molecular targets as pharmaceutical drugs in cancer treatment, remain an invaluable source of active components with therapeutic efficacy. Over 3000 medicinal plant species, with less than 10% of these species being analyzed for major bioactive molecules, are being used in cancer treatment ( 16 ). Compared with synthetic organic molecules, bioactive medicinal plants exhibit high potential and capacity for use in mechanism-based strategies. Curcumin, etoposide, camptothecin, and paclitaxel are plant-derived antineoplastic compounds that can effectively disrupt tumorigenic cell growth via apoptosis and inhibition of cancer cell proliferation. The Lamiaceae family includes an herbaceous perennial shrub called Hoslundia opposita Vahl ( H. opposita ) that is characteristically round and yellowish or orange. It is well distributed in tropical and subtropical lands of Africa ( 17 ), including Nigeria. Ethnobotanical use includes the treatment of various ailments, such as colds, sore throat, gonorrhea, convulsion, stomach pains, ringworms, parasitic skin infection, snake bites, and mental disorders ( 18 , 19 ). Scientific investigations have confirmed the antidiabetic, antispasmodic, expectorant, antimicrobial, anti-inflammatory and antibacterial effects of H. pylori ( 18 , 20 , 21 ). The reported anticancer activities of H. opposita in some cell lines, including human breast adenocarcinoma MCF-7 (ATCC No. HTB-22), BT-20 (ATCC No. HTB-19), and BT-549 (ATCC No. HTB-122); prostate adenocarcinoma PC-3 (ATCC No. CRL-1435); acute T-cell leukemia Jurkat (ATCC No. TIB-152); colon adenocarcinoma SW-480 (ATCC No. CCL-228) cells; and rhabdomyosarcoma cancer ( 22 , 23 ), are scant. Furthermore, no anticancer effects of H. opposita have been reported on GBM. In the present study, the cytotoxic, clonogenic, apoptotic, and reactive oxygen species effects and mitochondrial membrane potential of the crude extract and fractions of H. opposites were profiled in vitro in GBM cells. The results from these in vitro studies can be expanded upon in the future in animal models. MATERIALS AND METHODS Hoslundia opposita leaves were collected from Agbara ogun State (Nigeria) and were identified, authenticated, and deposited with voucher specimen number LUH 7433 at the Department of Botany, University of Lagos, in Lagos, Nigeria. 2.1 Preparation of crude extracts and fractions Crude extracts and fractions were obtained from previous studies by Ajibare and coworkers ( 24 ). Briefly, leaves were collected, air dried at room temperature for 20 days and milled using a laboratory grinder. Powdered samples were soaked in 80% methanol in distilled water for seven days. The filtrates were collected and filtered using Whatman N0.1 filter paper and evaporated using a rotary evaporator at 40°C to obtain crude methanol extracts (AHO1). AHO1 was subjected to vacuum liquid chromatography (VLC) with silica gel (0.063–0.2 mm mesh) using a gradient elution fractionation of solvent systems in order of increasing polarity, including hexane/ethyl acetate (90:10, 3 L) AHO2, (70:30, 8 L); AHO3, (50:50, 8.5 L) AHO4, (30:70, 3 L); ethyl acetate (100%, 4 L) AHO6; and ethyl acetate/methanol, (50:50, 3 L) AHO7. AHO2–7 represents the fractions obtained by various solvent systems. The collected fractions were evaporated using a rotary evaporator and stored at − 4°C. 2.2 Cytotoxic activities of H.opposita leaves 2.2.1 Hatching of Brine Shrimp A rectangular jar containing natural sea water obtained from the bar beach, Ikoyi, and Lagos serves as the habitat for the hatching of Artemia salina cysts. After hatching, the larvae were maintained under constant light for 48 h at 37°C to ensure survival and maturity ( 23 ). 2.2.2 Cell culture : The human glioma cell line (U251) and human keratinocyte (HACAT) cell line were obtained from the Department of Medical Sciences, University of Western Cape, South Africa. U251 cell growth was achieved in culture medium supplemented with Dulbecco’s modified Eagle’s medium (DMEM) supplemented with phenyl red, heat-inactivated fetal bovine serum (FBS, 10% (v/v), neutral red cell proliferation reagent and 100 unit/mL-streptomycin-amphotericin-B-mixture (PSA), which was maintained at 37°C in a 5% CO 2 humidified incubator (Labotech, South Africa). 2.2.3 Brine Shrimp Lethality Assay : The crude methanol extracts and fractions were weighed and used to prepare stock solutions (50, 500, 5000 µg/ml in seawater). Final concentrations of 10, 100, and 1000 µg/ml of each extract were further prepared in 5 ml of filtered sea water in test tubes as previously described ( 25 ). Test tubes were set up in replicate, 10 larvae were added, and sea water and liquid tubes served as controls. After 24 h, the set up was examined with a magnifying lens to determine the number of surviving larvae. The calculation of the LC 50 of the extract was performed using the probit method ( 26 ). 2.2.4 Cell viability assay : U251 cells were seeded at a density of 4000 cells per well in 96-well plates and incubated for 24 hrs under standard conditions. For 48 hours, the cells were exposed to the extracts at increasing concentrations (25–100 g/ml). Viability was evaluated by a colorimetric dye reduction assay [3-(4,5-dimethylthiazol-2yl) − 2,5-diphenyl tetrazolium bromide)] (MTT, Sigma‒Aldrich). The plant extract was removed, and 100 µL of PBS and fresh medium were added to the cells. The cells were incubated with 10 µL of MTT solution for 4 hours. The media of the cells were removed, and 100 L of DMSO was added to dissolve the purple formazan crystals. Using a microplate reader (BMG Labtech Omega® POLAR Star), optical density (OD) was read at 570 nm, and the mean cell proliferation in comparison to that of the control was determined. The IC 50 was calculated using GraphPad Prism 6 software (GraphPad®) from triplicate replicate measurements ( 24 ). 2.3 Clonogenic determination of the crude extracts and fractions A clonogenic assay was performed to assess the ability of cancer cells to divide and form colonies after treatment with the IC 50 of the crude product and fractions of H. opposita leaf using previously described methods ( 27 ). The cells were seeded in 6 cm dishes according to their density, treated for 48 h at their IC50, trypsinized, resuspended in 2 mL of culture media, counted, and reseeded at 500 cells/dish (35 mm). For 10 days, the untreated cells were monitored during incubation and stained. The cells were washed with PBS, and the cells were fixed with methanol and glacial acetic acid solution (3:1). The fixed cells were stained with 0.5% crystal violet in methanol, and the reaction was finally treated with both distilled water and PBS to obtain a clearer image of the cells in the dishes. Images of the dishes were taken, and areas covered by colonies were calculated using ImageJ software ( 24 ) and are expressed as a percentage of the control set to one hundred percent. 2.4 Evaluation of the cellular morphology Microphological changes related to treatment were established in U251 cell lines. Briefly, the cells were plated in 60 mm dishes, incubated for 24 h and treated with the indicated IC50 for 48 h. An inverted light microscope (Olympus, USA) was used to view the morphology, and images were obtained with a mounted Zeiss Axiocam camera (Germany). 2.5 Determination of apoptotic activity Caspase 3 and 7 activities were determined in the cancer cell line using a Caspase-Glo 3/7® Assay Kit (Promega, Madison, WI, USA). On 96-well plates with white walls, 4000 cells per well were plated. Together with the positive and negative controls, cells were treated with a portion of their IC50 values and cultured for 48 hours. The wells were filled with caspase-Glo 3/7 reagents at a final volume of 200 L, and the samples were then allowed to sit at room temperature for 30 minutes. Luminescence was measured at 520 nm using a microplate reader (BMG Labtech Omega® POLAR Star). The average apoptotic activity relative to that of the control group was calculated with GraphPad Prism 6 software (San Diego, CA, USA). 2.6 Determination of mitochondrial membrane potentials The fluorescent dye tetramethylrhodamine (TMRE) was used to determine the mitochondrial membrane potential (MMP). A total of 2000 cells per well were seeded in 96-well plates, after which the plates were subjected to 48 hours of treatment with Hoslundia opposita. As a positive control, the cells were exposed to carbonyl cyanide m-chlorophenyl hydrazine (CCP) for 10 minutes. The supernatant was then collected, and the cells were rinsed with 100 mL of PBS. The cell pellets were resuspended in 100 L of PBS, after which the fluorescence intensity was measured at 544 nm with a microplate reader. (BMG Labtech Omega® POLAR Star). The average percentage was calculated relative to the control from triplicate data from two different experiments using GraphPad Prism 6 software. 2.7 Evaluation of intracellular reactive oxygen species (ROS) Intracellular ROS levels were determined using the fluorescent probe dihydrofluorescin diacetate (DCFH-DA) with modifications ( 28 ). Cell lines (4000 cells/well) were seeded into 96-well plates, incubated for 24 h and treated with fractions of Hoslundia opposita for 48 h. As a positive control, control cells were treated with 250 mM H202 for 15 min. Cells were stained with 20 M DCFH-DA in the dark at 370°C for 60 min to detect ROS activity. The cells were then rinsed with PBS, 100 L of PBS was added to each well, and the fluorescence of DCFH-DA was measured using a POLAR Star Omega BMG. 2.8 Selectivity indices The cytotoxicities of the crude solution and the fractions of Hoslundia opposita leaves were evaluated against a normal malignant cell line for the purpose of determining the selectivity of the samples using the standard formula 2.9 Statistical analysis The statistical analysis of the data obtained for this study was conducted using Graph Pad Prism version 6 software, and the results are expressed as the mean ± standard error of the mean (SEM) of the triplicate repeat measurements. One-way analysis of variance (ANOVA) was used to determine the significance of the difference between the treated (intra) and control groups, and values were considered to be statistically significant at P ≤ 0.05. RESULTS 3.1 Cytotoxic effects of the crude extracts (AHO1) and fractions (AHO2-7) The preliminary inhibitory concentrations of the crude extracts (AHO1) and fractions (AHO2-7) responsible for killing 50% of the brine shrimp nauphli and U251 cancer cell lines are shown in Table 1 . AHO1, AHO5 and AHO6 were significantly cytotoxic toward the nauphli and U251 cell lines, although AHO6 (52.25 µg/ml) showed the best cytotoxic activity among all the other fractions evaluated. The selectivity index (SI) denotes the activity and safety of anticancer agents on cancer cells and normal cell lines. A SI greater than 2 indicates a potent cytotoxic effect on the cancer cell, and a SI less than 2 indicates that the anticancer agent is toxic to a normal cell line (selectivity). Table 1 also shows that AHO6 (2.11) was selective for U251 cancer cells and HaCaT normal cells. Figure 1 also shows significant (p < 0.05) cytotoxic effects on U251 cells in comparison to the positive control drug doxorubicin (DOX). Compared with other fractions of H. opposita leaves, fractions AHO5 and AHO6 were toxic to U251 cells, while AHO6 was more effective against the normal cell line HaCaT. Table 1 IC50s of fractions of H. opposita leaf on the brine shrimp line naupli and U251 and selectivity for the HaCaT cell line Plant Extracts BSL LD 50 (µg/ml) U251 CELLS IC 50 (µg/ml) HACAT IC 50 (µg/ml) SELECTIVITY INDEX AH01 (crude extract) 168.51 72.89 136.4 1.87 AH02 (Hex/Etoac 90:10) 156.19 156.7 NA NA AH03 (hex/Etoac 70;30) 143.68 234.9 NA NA AH04 (Hex/Etoac 50:50) 54.03 60.18 NA NA AH05 (Hex/EtoAc 30:70) 39.01 55.65 95.37 1.17 AH06 (EtoAc 100%) 40.42 52.25 111.3 2.11 AH07 (EtoAc/Met 50:50) 373.77 76.13 NA NA 3.2 Clonogenic effect of crude extracts (AHO1) and fractions (AHO5 and AHO6) on U251 cell lines The extent to which colonies formed after 48 h of treatment with the IC50 of AHO1, AHO5 and AHO6 is shown in Fig. 2 A. A significant difference in the number of colonies formed between the treated (fraction) and untreated (negative control) samples was also observed, as shown in Fig. 2 B. AHO5 and AHO6 had significant inhibitory effects on AHO5 compared to the control. 3.3 Effects of crude extracts (AHO1) and fractions (AHO5 and AHO6) on the cellular morphology of U251 cells The effects of AHO1, AHO5 and AHO6 were examined using an inverted microscope after the cells were treated with the indicated IC 50 for 48 h. Figure 3 shows that the treated cells exhibited different structural, confluence and morphological characteristics than did the untreated cells (control). The untreated cells exhibited a sustained structure, confluence and original morphology. 3.4 Apoptotic effects of the crude extract (AHO1) and the fractions (AHO5 and AHO6) on U251 cells Apoptotic activity was measured using caspase-3 and caspase-7 assays ( Fig. 4 ) after the cells were treated with the appropriate IC 50 for 48 hrs. AHO1, AHO5 and AHO6 significantly activated caspase 3/7 in U251 cells compared to negative control cells. Thus, the results of this study suggest that apoptotic activity occurs via the cascade cascade pathway. 3.5 Mitochondrial membrane potential and intracellular reactive oxygen species Mitochondria are known to be the main source of reactive species (ROS) in cells. To observe whether cell death occurred through ROS-mediated activity and loss of the MMP, the cells were stained with fluorescent dyes for 48 h and compared with their positive controls. As shown in Fig. 5 , AHO5 and AHO6 significantly increased ROS production, and a significant decrease in the MMP was observed. The observed cell death in this study might be due to ROS-induced activity. DISCUSSION The recalcitrant or aggressive nature of glioblastoma cells (GBMs) and limited treatment options underlie the overwhelmingly poor progression-free survival (PFS) and low overall survival (OS) in glioblastoma patients. Currently, there is no significant clinical improvement in the effectiveness of available anticancer drugs for GBM treatment. Therefore, there is urgency in drug discovery and development, which aims at identifying medicinal plant extracts, fractions and possible lead compounds that have great potential to reduce the metastatic potential of aggressive GBM cells and induce significant apoptosis. This study evaluated the cytotoxic effects of the crude extract and different fractions of Hoslundia opposita (HO) on the glioblastoma cell line U251. The biological or pharmacological activities of plants have been attributed to the array of phytoconstituents, which have no direct effect on plant growth ( 29 ). Ajibare et al. (2021) reported on the phytochemical screening of crude methanolic extract and the antioxidant capacity of different fractions of Hoslundia opposita (HO) (Ajibare et al., 2022). We evaluated the anticancer activity of Hoslundia opposita (HO) leaf extract, fractions and lead compound against human hepatoma cell lines (HepG2), breast cancer cell lines (MDA-MB-23), intestinal epithelial cell lines (Caco-2) and human keratinocyte HACAT cell lines. The study results showed that HO and hoslundin have significant anticancer effects on tumor cells by inducing apoptosis via mitochondrial-dependent reactive oxygen species generation and that normal cells tolerate potential antineoplastic agents (Ajibare et al., 2022). In the present study, because of the aggressive nature and relapse associated with GBM, we explored the preliminary investigations of the anticancer effects of crude methanolic extracts and fractions of HO on glioblastoma cell lines (U251). The anticancer or antiproliferative potential of plant extracts, fractions or compounds in any cancer study relies on preliminary cytotoxicity screening. The crude extracts of AHO1 and AHO2 to AHO7 showed varied IC50 values (52.25–234.90 µg/ml), indicating anticancer activity (Table 1 ). The percentage of cell viability illustrated (Fig. 1 ) by the MTT assay showed varied results (20–100 µg/ml) in U251 cells treated with the crude extract AHO1 and AHO2 to AHO7 fractions, which could be attributed to the aggressiveness and mutational pattern associated with these cell lines ( 30 ). Note that an inhibitory concentration of 50% inhibitory concentration (IC 50) at or less than 30 µg/ml, according to the American Cancer Institute (NCI), is the most potent cytotoxic or effective anticancer screening ( 31 ). This IC 50 value was not achievable at the set maximum concentration of 100 µg/ml; however, increasing the maximum concentration could or probably could produce an IC 50 at or less than 20 µg/ml for 72 hr, as reported in a past study ( 22 ). This implies that some plants might demonstrate more significant cytotoxicity at higher concentrations, which could be our focus in our subsequent study by extending the concentration range to 200 µg/ml. Previous studies have established the inhibitory dose-dependent cytotoxic effect of HO leaves on different human cancer cell lines (Ajibare et al.,2022). In this study, for the first time, we found that only the crude extracts AHO1, AHO5 and AHO6 (Table 1 ) had moderate cytotoxic effects, with IC 50 values of 72.89, 55.65 and 52.55 µg/ml, respectively, on U251 cells. This is supported by the findings of another study in which HO exhibited a similar cytotoxic effect against BT-549 cells, in which the IC 50 was very close to 76.4 µg/ml, and Byrsocarpus coccineus had an IC 50 value of 65.2 µg/ml against JURKAT ( 22 ). Overall, the selected crude extracts and the fractions elicited potent cytotoxic effects that inhibited U251 cells, resulting in a decrease in cell viability. Hence, the choice of crude extract or the two fractions was selected after the continuation of the other experimental assays in this study. The loss of cell viability indicated the cessation of metabolic activity in the U251 cell line after treatment with the extract AHO1, and the fractions AHO5 and AHO6 indicated some morphological distortions. Cytological observation under an inverted microscope (Fig. 2 ) revealed several key changes. These morphological changes are distinctive features of cell shrinkage, nuclear condensation, membrane shrinkage, and chromatin cleavage, among others, in the treated U251 cell line ( 32 , 33 ). In contrast, the visualization of the control (untreated cells) revealed an undistorted cell morphology because the cell content was intact and the cells adhered to the bottom of the culture plate. There were no noticeable echinoid spikes of apoptotic cells or apoptotic bodies or a decrease in cell number (Fig. 2 ) ( 33 ). Treatment of U251 cells with the extract AHO1 or fractions AHO5 and AHO6 resulted in early detachment from the basal membrane, referred to as anoikis ( 34 ), which is characteristic of apoptosis. An important promising strategy in developing antitumour agents requires the induction of apoptotic pathways ( 35 – 37 ). After treating the U251 cell line with the extract AHO1, fractions AHO5 and AHO6 had a significant increase in the activity of caspase-3/7, thus causing antiproliferation or inhibition of U251 cell lines ( 5 , 33 , 38 ) (Fig. 4 ). The ability of cancer cells to form colonies from single cells reveals the metastatic capacity or proliferative effect of cancer cells, thus increasing the difficulty of treatment or resulting in a poor prognosis. A colony formation assay was used to determine the proliferative pattern and survival of the cells. A significant increase in proliferation was observed upon treating U251 cells with the extract AHO1, the AHO5 fraction and the AHO6 fraction ( Fig. 4 ), which is in line with the findings of other studies ( 39 , 40 ). The metastatic mechanism enables firm anchorage of cell‒cell interactions of cancer cells with the endothelium, which colonizes and establishes secondary tumors elsewhere. Hence, the invasiveness and metastasis of most cancer cells are important for effective treatment ( 40 , 41 ). The clonogenic and morphological data showed that treatment with the crude extract of AHO1, fraction AHO5 or AHO6 significantly decreased the ability of U251 cells to form colonies and disrupted cancer cell integrity or integrity compared with that of the untreated cells. The continuous production of ROS could lead to the death of cancer cells, which is implicated in destroying the mitochondrial membrane and probably limiting the mitochondrial membrane potential, subsequently leading to the release of cytochrome c. Hence, the induction of caspase-3 leads to mitochondria/cytochrome-induced cancer cell death, which is evident in the release of cytochrome c (Ajibare et al.,2022). This study revealed that the crude extract of AHO1 and fractions AHO5 and AHO6 induced cell death via a mechanism similar to that of mitochondria and ROS (Fig. 5 A). Studies have revealed that the induction of ROS production is responsible for apoptosis in different types of cancer ( 42 , 43 ). CONCLUSION This study revealed that the crude extracts of AHO1, fractions AHO5 and AHO6, have relatively moderate cytotoxic effects on U251 cells. Compared to those of other fractions, a better safety profile was observed for the normal human cell line HACAT. The nature or composition of solvents has been shown to have a profound effect on the phytoconstituents that exert anticancer or antiproliferative effects. Further work depicting the underlying molecular mechanisms is recommended. Declarations Acknowledgment - Not applicable Author contribution statement AC, AOT, RA, MA, KA, TA, AO, HA and MP were involved in the data analysis, interpretation of the results and manuscript preparation. AC, AOT, RA and MA were involved in the research conception/design. AC, KA, TA, AO, HA and MP were involved in the data acquisition AC, AOT, RA, MA and KA were involved in critical revision of the manuscript. TA, AO, HA and MP were involved in the figure and/or table design. Competing interest All the authors declare that there are no potential conflicts of interest associated with this article. Funding The authors did not receive funding from anyone or institutions. 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J Inflamm Res 14:7065 Kooti W, Servatyari K, Behzadifar M, Asadi-Samani M, Sadeghi F, Nouri B et al (2017) Effective medicinal plant in cancer treatment, part 2: review study. J evidence-based Complement Altern Med 22(4):982–995 de Souza PO, Bianchi SE, Figueiró F, Heimfarth L, Moresco KS, Gonçalves RM et al (2018) Anticancer activity of flavonoids isolated from Achyrocline satureioides in gliomas cell lines. Toxicol In Vitro 51:23–33 Suffness M (1990) Assays related to cancer drug discovery. Methods in plant biochemistry: assays for bioactivity. ;6:71–133 Moongkarndi P, Kosem N, Kaslungka S, Luanratana O, Pongpan N, Neungton N (2004) Antiproliferation, antioxidation and induction of apoptosis by Garcinia mangostana (mangosteen) on SKBR3 human breast cancer cell line. J Ethnopharmacol 90(1):161–166 Syed Abdul Rahman SN, Abdul Wahab N, Abd Malek SN (2013) In vitro morphological assessment of apoptosis induced by antiproliferative constituents from the rhizomes of Curcuma zedoaria. Evidence-Based Complementary and Alternative Medicine. ;2013 Thuret G, Chiquet C, Herrag S, Dumollard J, Boudard D, Bednarz J et al (2003) Mechanisms of staurosporine induced apoptosis in a human corneal endothelial cell line. Br J Ophthalmol 87(3):346–352 Wang Y, Zhong J, Bai J, Tong R, An F, Jiao P et al (2018) The application of natural products in cancer therapy by targeting apoptosis pathways. Curr Drug Metab 19(9):739–749 Carneiro BA, El-Deiry WS (2020) Targeting apoptosis in cancer therapy. Nat reviews Clin Oncol 17(7):395–417 Hassan M, Watari H, AbuAlmaaty A, Ohba Y, Sakuragi N (2014) Apoptosis and molecular targeting therapy in cancer. BioMed research international. ;2014 Wang C, Lin D, Chen Q, Lin S, Shi S, Chen C (2018) Polysaccharide peptide isolated from grass-cultured Ganoderma lucidum induces anti-proliferative and pro-apoptotic effects in the human U251 glioma cell line. Oncol Lett 15(4):4330–4336 Popovici V, Bucur L, Vochita G, Gherghel D, Mihai CT, Rambu D et al (2021) In vitro anticancer activity and oxidative stress biomarkers status determined by Usnea barbata (L.) FH Wigg. dry extracts. Antioxidants 10(7):1141 Tavares-Carreón F, De la Torre-Zavala S, Arocha-Garza HF, Souza V, Galán-Wong LJ, Avilés-Arnaut H (2020) In vitro anticancer activity of methanolic extract of Granulocystopsis sp., a microalgae from an oligotrophic oasis in the Chihuahuan desert. PeerJ 8:e8686 Lee SH, Jaganath IB, Wang SM, Sekaran SD (2011) Antimetastatic effects of Phyllanthus on human lung (A549) and breast (MCF-7) cancer cell lines. PLoS ONE 6(6):e20994 Lin Y-T, Yang J-S, Lin S-Y, Tan T-W, Ho C-C, Hsia T-C et al (2008) Diallyl disulfide (DADS) induces apoptosis in human cervical cancer Ca Ski cells via reactive oxygen species and Ca2+-dependent mitochondria-dependent pathway. Anticancer Res 28(5A):2791–2799 Dhanyakrishnan R, Sunitha MC, Prakash Kumar B, Sandya S, Nevin KG (2018) Morphological and molecular effects of phenolic extract from coconut kernel on human prostate cancer cell growth in vitro. Mediterranean J Nutr Metabolism 11(1):21–36 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-4824462","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":333413862,"identity":"6d04d142-2384-40ca-8087-2ae7e8ae293c","order_by":0,"name":"Abosede Christiana Ajibare","email":"data:image/png;base64,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","orcid":"","institution":"Department of Biochemistry, Faculty of Basic Medical Sciences, College of Medicine, University of Lagos, Lagos, Nigeria","correspondingAuthor":true,"prefix":"","firstName":"Abosede","middleName":"Christiana","lastName":"Ajibare","suffix":""},{"id":333413863,"identity":"7eed4aa9-f82b-4a57-9a10-34f5294d6d9e","order_by":1,"name":"Osaretin Albert Taiwo Ebuehi","email":"","orcid":"","institution":"Department of Biochemistry, Faculty of Basic Medical Sciences, College of Medicine, University of Lagos, Lagos, Nigeria","correspondingAuthor":false,"prefix":"","firstName":"Osaretin","middleName":"Albert Taiwo","lastName":"Ebuehi","suffix":""},{"id":333413864,"identity":"519ca012-5f78-4174-ad50-4ea7966c0ab6","order_by":2,"name":"Rahmat Adetutu Adisa","email":"","orcid":"","institution":"Department of Biochemistry, Faculty of Basic Medical Sciences, College of Medicine, University of Lagos, Lagos, Nigeria","correspondingAuthor":false,"prefix":"","firstName":"Rahmat","middleName":"Adetutu","lastName":"Adisa","suffix":""},{"id":333413865,"identity":"f8cbcdcf-0dec-4671-a459-569259e696cb","order_by":3,"name":"Margaret Oluwatoyin Sofidiya","email":"","orcid":"","institution":"Department of Pharmacognosy, Faculty of Pharmacy, College of Medicine, University of Lagos, Lagos, Nigeria","correspondingAuthor":false,"prefix":"","firstName":"Margaret","middleName":"Oluwatoyin","lastName":"Sofidiya","suffix":""},{"id":333413866,"identity":"d9f69623-7c7e-45b5-8887-83414ebc41fa","order_by":4,"name":"Kolajo Adedamola Akinyede","email":"","orcid":"","institution":"Department of Medical Bioscience, Faculty of Natural Sciences, University of the Western Cape, Bellville, Cape Town 7530, South Africa","correspondingAuthor":false,"prefix":"","firstName":"Kolajo","middleName":"Adedamola","lastName":"Akinyede","suffix":""},{"id":333413867,"identity":"fa7b9837-f43a-4c28-8db8-541d111e53c1","order_by":5,"name":"Titilola Aderonke Samuel","email":"","orcid":"","institution":"Department of Biochemistry, Faculty of Basic Medical Sciences, College of Medicine, University of Lagos, Lagos, Nigeria","correspondingAuthor":false,"prefix":"","firstName":"Titilola","middleName":"Aderonke","lastName":"Samuel","suffix":""},{"id":333413868,"identity":"586875a6-9fc1-423b-9ab1-c4368689150b","order_by":6,"name":"Joseph A.O. Olugbuyiro","email":"","orcid":"","institution":"Department of Chemistry, Covenant University, PMB 1023 Ota, Ogun State, Nigeria.","correspondingAuthor":false,"prefix":"","firstName":"Joseph","middleName":"A.O.","lastName":"Olugbuyiro","suffix":""},{"id":333413869,"identity":"31b42545-c872-4f89-965a-fc15a68b6e09","order_by":7,"name":"Helen Adeola Iyiolaa","email":"","orcid":"","institution":"Department of Chemistry, Covenant University, PMB 1023 Ota, Ogun State, Nigeria.","correspondingAuthor":false,"prefix":"","firstName":"Helen","middleName":"Adeola","lastName":"Iyiolaa","suffix":""},{"id":333413870,"identity":"d3c192b8-20f7-4a8b-a40a-ca548a683803","order_by":8,"name":"Oluwagbemiga Mofolorunsho Phillips","email":"","orcid":"","institution":"Oluwagbemiga Mofolorunsho Phillips","correspondingAuthor":false,"prefix":"","firstName":"Oluwagbemiga","middleName":"Mofolorunsho","lastName":"Phillips","suffix":""}],"badges":[],"createdAt":"2024-07-29 21:28:34","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":true,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-4824462/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4824462/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":61405254,"identity":"7a70195a-3cb6-4a9c-931d-85cb7533deea","added_by":"auto","created_at":"2024-07-30 10:51:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":33380,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe percentage of cell viability in cancer (U251) and normal (HaCaT) cells treated with the negative control orincreasing concentrations of the positive control (DOX), crude extracts or fractions of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eH.opposita\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e after48 hrs was measured by MTT assay. The bars represent the mean percentage cell viability ±SEM relative to the control.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4824462/v1/2b8eced79dfb82552745f63d.png"},{"id":61405258,"identity":"19241d0a-7630-4ad0-ab0b-29a4b3abe2c0","added_by":"auto","created_at":"2024-07-30 10:51:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":334486,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eClonogenic view of the treated and untreated U251 cell lines (A) and bar graph showing the survival rate of the cells after 10 days(B). Treatment with the crude extract (AHO1) or the other fractions (AHO5 and AHO6) inhibited the growth of the cells compared to that of the control cells.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4824462/v1/5fca2a7f35c364b24b003f4c.png"},{"id":61405815,"identity":"84dbc5e3-484a-4384-bc19-49d03cb3767c","added_by":"auto","created_at":"2024-07-30 10:59:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":472073,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMicroscopic view of U251 cell lines treated with fractions of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eHoslundia opposita \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eleaf (AHO1, AHO5, AHO6) after 48 hrs of treatment. Arrows indicate dissociation of cells and a reduction in cell growth.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4824462/v1/bed6920726bf153386cd4113.png"},{"id":61405256,"identity":"184462af-98de-42e7-9a42-d5ba8d246b13","added_by":"auto","created_at":"2024-07-30 10:51:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":19389,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eApoptotic activity of crude extracts (AHO1) and fractions (AHO5 and AHO6) after 48 h of treatment in U251 cells. *, **, and *** indicate significant differences (P≤0.05) according to ANOVA and Tukey’s multiple comparison tests.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4824462/v1/1d8d0a5234c22233f5a2dbe2.png"},{"id":61405255,"identity":"a0d1893d-5e30-4f22-ba32-fa87cc852083","added_by":"auto","created_at":"2024-07-30 10:51:37","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":39939,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibition of reactive oxygen species generation A) and mitochondrial membrane potential(B) of U251 cellstreated with fractions of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eH. opposita\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eleaf. *, **, and *** indicate significant differences (P≤0.05) according to ANOVA and Tukey’s multiple comparison tests.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4824462/v1/8b4ce35cdad3b40605806a2e.png"},{"id":61406356,"identity":"6d9ee158-d198-4103-a440-1aa77dfe1590","added_by":"auto","created_at":"2024-07-30 11:07:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1909452,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4824462/v1/ddc28109-f94d-4fb5-9c2a-0069186c10f0.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eAntiproliferative activity of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eHoslundia opposita\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e leaf extract and fractions against a human glioblastoma cell line (U251)\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eAll tumors originating from glial cells are gliomas commonly affecting the body's central nervous system. According to the World Health Organization, gliomas are classified according to their presumed cell of origin as astrocytic tumors: astrocytoma grade I, astrocytoma grade II, astrocytoma grade III (anagal astrocytoma), astrocytoma IV (glioblastoma or GM), oligodendrogliomas, ependymomas and mixed gliomas (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Glioblastoma is the most common, lethal and damaging type of primary brain tumor in humans and is characterized by an unavoidable propensity to relapse and poor prognosis (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Glioblastoma accounts for 42% of all central nervous system tumors and 60% of all brain tumors in adults, with a median survival of 15 months (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGlioblastoma multiforme (GBM) has no cure, and intertumoural or intratumour heterogeneity is one of the hallmarks of this cancer. The development of GBM is characterized by age, sex, race, genetic disorders, and ionizing radiation (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). In GBM, cellular heterogeneity is associated with therapeutic and drug resistance due to an array of genetic alterations involved in the control of cell cycle kinetics, cell growth, apoptosis, cell invasion and neovascularization (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Despite surgical resection, irradiation and adjuvant chemotherapy, GBM remains a major therapeutic problem, as survival following diagnosis can reach 12 to 15 months, with less than 5% survival longer than five years (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe clinical treatment of GBM has not been successful despite the knowledge and advances in understanding the associated complex biology. The genetic and epigenetic heterogeneity of GBM has helped but has not been fully translated to effective clinical (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e) outcomes. Some conventional DNA-damaging anticancer drugs have failed in GBM, showing proven efficacy in other cancer types and hence limiting the treatment options for GBM. Hence, the failure of these agents necessitates the development of new drugs and new categories of therapeutics. Temozolomide (TMZ) was approved for GBM treatment for the first time, just as bevacizumab is used to treat recurrent GBM and was approved by the Food and Drug Administration. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Bevacizumab nitrosoureas and TMZ, which are the second-line agents, are the most common systemic agents for recurrent high-grade gliomas (HGGs), but there is no superiority of these agents over one another. Despite standard treatment and care centered on surgical resection, chemotherapy and radiation therapy, patients have a poor progression-free survival (PFS) of 7\u0026ndash;8 months, a median survival of 14\u0026ndash;16 months and a 5-year overall survival (OS) of 9.8% (\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNatural plant products, which possess similar molecular targets as pharmaceutical drugs in cancer treatment, remain an invaluable source of active components with therapeutic efficacy. Over 3000 medicinal plant species, with less than 10% of these species being analyzed for major bioactive molecules, are being used in cancer treatment (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Compared with synthetic organic molecules, bioactive medicinal plants exhibit high potential and capacity for use in mechanism-based strategies. Curcumin, etoposide, camptothecin, and paclitaxel are plant-derived antineoplastic compounds that can effectively disrupt tumorigenic cell growth via apoptosis and inhibition of cancer cell proliferation.\u003c/p\u003e \u003cp\u003eThe Lamiaceae family includes an herbaceous perennial shrub called \u003cem\u003eHoslundia opposita\u003c/em\u003e Vahl (\u003cem\u003eH. opposita\u003c/em\u003e) that is characteristically round and yellowish or orange. It is well distributed in tropical and subtropical lands of Africa (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e), including Nigeria. Ethnobotanical use includes the treatment of various ailments, such as colds, sore throat, gonorrhea, convulsion, stomach pains, ringworms, parasitic skin infection, snake bites, and mental disorders (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Scientific investigations have confirmed the antidiabetic, antispasmodic, expectorant, antimicrobial, anti-inflammatory and antibacterial effects of \u003cem\u003eH. pylori\u003c/em\u003e (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). The reported anticancer activities of \u003cem\u003eH.\u003c/em\u003e opposita in some cell lines, including human breast adenocarcinoma MCF-7 (ATCC No. HTB-22), BT-20 (ATCC No. HTB-19), and BT-549 (ATCC No. HTB-122); prostate adenocarcinoma PC-3 (ATCC No. CRL-1435); acute T-cell leukemia Jurkat (ATCC No. TIB-152); colon adenocarcinoma SW-480 (ATCC No. CCL-228) cells; and rhabdomyosarcoma cancer (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e), are scant. Furthermore, no anticancer effects of \u003cem\u003eH. opposita\u003c/em\u003e have been reported on GBM. In the present study, the cytotoxic, clonogenic, apoptotic, and reactive oxygen species effects and mitochondrial membrane potential of the crude extract and fractions of H. opposites were profiled in vitro in GBM cells. The results from these in vitro studies can be expanded upon in the future in animal models.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e \u003cem\u003eHoslundia opposita\u003c/em\u003e leaves were collected from Agbara ogun State (Nigeria) and were identified, authenticated, and deposited with voucher specimen number LUH 7433 at the Department of Botany, University of Lagos, in Lagos, Nigeria.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Preparation of crude extracts and fractions\u003c/h2\u003e \u003cp\u003eCrude extracts and fractions were obtained from previous studies by Ajibare and coworkers (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Briefly, leaves were collected, air dried at room temperature for 20 days and milled using a laboratory grinder. Powdered samples were soaked in 80% methanol in distilled water for seven days. The filtrates were collected and filtered using Whatman N0.1 filter paper and evaporated using a rotary evaporator at 40\u0026deg;C to obtain crude methanol extracts (AHO1). AHO1 was subjected to vacuum liquid chromatography (VLC) with silica gel (0.063\u0026ndash;0.2 mm mesh) using a gradient elution fractionation of solvent systems in order of increasing polarity, including hexane/ethyl acetate (90:10, 3 L) AHO2, (70:30, 8 L); AHO3, (50:50, 8.5 L) AHO4, (30:70, 3 L); ethyl acetate (100%, 4 L) AHO6; and ethyl acetate/methanol, (50:50, 3 L) AHO7. AHO2\u0026ndash;7 represents the fractions obtained by various solvent systems. The collected fractions were evaporated using a rotary evaporator and stored at \u0026minus;\u0026thinsp;4\u0026deg;C.\u003c/p\u003e \u003cp\u003e \u003cb\u003e2.2 Cytotoxic activities of\u003c/b\u003e \u003cb\u003eH.opposita\u003c/b\u003e \u003cb\u003eleaves\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2.1 Hatching of Brine Shrimp\u003c/h2\u003e \u003cp\u003eA rectangular jar containing natural sea water obtained from the bar beach, Ikoyi, and Lagos serves as the habitat for the hatching of \u003cem\u003eArtemia salina\u003c/em\u003e cysts. After hatching, the larvae were maintained under constant light for 48 h at 37\u0026deg;C to ensure survival and maturity (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003e2.2.2 Cell culture\u003c/b\u003e: The human glioma cell line (U251) and human keratinocyte (HACAT) cell line were obtained from the Department of Medical Sciences, University of Western Cape, South Africa.\u003c/p\u003e \u003cp\u003eU251 cell growth was achieved in culture medium supplemented with Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM) supplemented with phenyl red, heat-inactivated fetal bovine serum (FBS, 10% (v/v), neutral red cell proliferation reagent and 100 unit/mL-streptomycin-amphotericin-B-mixture (PSA), which was maintained at 37\u0026deg;C in a 5% CO\u003csub\u003e2\u003c/sub\u003e humidified incubator (Labotech, South Africa).\u003c/p\u003e\u003cp\u003e \u003cb\u003e2.2.3 Brine Shrimp Lethality Assay\u003c/b\u003e: The crude methanol extracts and fractions were weighed and used to prepare stock solutions (50, 500, 5000 \u0026micro;g/ml in seawater). Final concentrations of 10, 100, and 1000 \u0026micro;g/ml of each extract were further prepared in 5 ml of filtered sea water in test tubes as previously described (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Test tubes were set up in replicate, 10 larvae were added, and sea water and liquid tubes served as controls. After 24 h, the set up was examined with a magnifying lens to determine the number of surviving larvae. The calculation of the LC\u003csub\u003e50\u003c/sub\u003e of the extract was performed using the probit method (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003e2.2.4 Cell viability assay\u003c/b\u003e: U251 cells were seeded at a density of 4000 cells per well in 96-well plates and incubated for 24 hrs under standard conditions. For 48 hours, the cells were exposed to the extracts at increasing concentrations (25\u0026ndash;100 g/ml). Viability was evaluated by a colorimetric dye reduction assay [3-(4,5-dimethylthiazol-2yl)\u0026thinsp;\u0026minus;\u0026thinsp;2,5-diphenyl tetrazolium bromide)] (MTT, Sigma‒Aldrich). The plant extract was removed, and 100 \u0026micro;L of PBS and fresh medium were added to the cells. The cells were incubated with 10 \u0026micro;L of MTT solution for 4 hours. The media of the cells were removed, and 100 L of DMSO was added to dissolve the purple formazan crystals. Using a microplate reader (BMG Labtech Omega\u0026reg; POLAR Star), optical density (OD) was read at 570 nm, and the mean cell proliferation in comparison to that of the control was determined. The IC\u003csub\u003e50\u003c/sub\u003e was calculated using GraphPad Prism 6 software (GraphPad\u0026reg;) from triplicate replicate measurements (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" height=\"45\" width=\"576\"\u003e\u003c/p\u003e\u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Clonogenic determination of the crude extracts and fractions\u003c/h2\u003e \u003cp\u003eA clonogenic assay was performed to assess the ability of cancer cells to divide and form colonies after treatment with the IC\u003csub\u003e50\u003c/sub\u003e of the crude product and fractions of H. opposita leaf using previously described methods (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). The cells were seeded in 6 cm dishes according to their density, treated for 48 h at their IC50, trypsinized, resuspended in 2 mL of culture media, counted, and reseeded at 500 cells/dish (35 mm). For 10 days, the untreated cells were monitored during incubation and stained. The cells were washed with PBS, and the cells were fixed with methanol and glacial acetic acid solution (3:1). The fixed cells were stained with 0.5% crystal violet in methanol, and the reaction was finally treated with both distilled water and PBS to obtain a clearer image of the cells in the dishes. Images of the dishes were taken, and areas covered by colonies were calculated using ImageJ software (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e) and are expressed as a percentage of the control set to one hundred percent.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Evaluation of the cellular morphology\u003c/h2\u003e \u003cp\u003eMicrophological changes related to treatment were established in U251 cell lines. Briefly, the cells were plated in 60 mm dishes, incubated for 24 h and treated with the indicated IC50 for 48 h. An inverted light microscope (Olympus, USA) was used to view the morphology, and images were obtained with a mounted Zeiss Axiocam camera (Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Determination of apoptotic activity\u003c/h2\u003e \u003cp\u003eCaspase 3 and 7 activities were determined in the cancer cell line using a Caspase-Glo 3/7\u0026reg; Assay Kit (Promega, Madison, WI, USA). On 96-well plates with white walls, 4000 cells per well were plated. Together with the positive and negative controls, cells were treated with a portion of their IC50 values and cultured for 48 hours. The wells were filled with caspase-Glo 3/7 reagents at a final volume of 200 L, and the samples were then allowed to sit at room temperature for 30 minutes. Luminescence was measured at 520 nm using a microplate reader (BMG Labtech Omega\u0026reg; POLAR Star). The average apoptotic activity relative to that of the control group was calculated with GraphPad Prism 6 software (San Diego, CA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Determination of mitochondrial membrane potentials\u003c/h2\u003e \u003cp\u003eThe fluorescent dye tetramethylrhodamine (TMRE) was used to determine the mitochondrial membrane potential (MMP). A total of 2000 cells per well were seeded in 96-well plates, after which the plates were subjected to 48 hours of treatment with Hoslundia opposita. As a positive control, the cells were exposed to carbonyl cyanide m-chlorophenyl hydrazine (CCP) for 10 minutes. The supernatant was then collected, and the cells were rinsed with 100 mL of PBS. The cell pellets were resuspended in 100 L of PBS, after which the fluorescence intensity was measured at 544 nm with a microplate reader. (BMG Labtech Omega\u0026reg; POLAR Star). The average percentage was calculated relative to the control from triplicate data from two different experiments using GraphPad Prism 6 software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Evaluation of intracellular reactive oxygen species (ROS)\u003c/h2\u003e \u003cp\u003eIntracellular ROS levels were determined using the fluorescent probe dihydrofluorescin diacetate (DCFH-DA) with modifications (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Cell lines (4000 cells/well) were seeded into 96-well plates, incubated for 24 h and treated with fractions of \u003cem\u003eHoslundia opposita\u003c/em\u003e for 48 h. As a positive control, control cells were treated with 250 mM H202 for 15 min. Cells were stained with 20 M DCFH-DA in the dark at 370\u0026deg;C for 60 min to detect ROS activity. The cells were then rinsed with PBS, 100 L of PBS was added to each well, and the fluorescence of DCFH-DA was measured using a POLAR Star Omega BMG.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Selectivity indices\u003c/h2\u003e \u003cp\u003eThe cytotoxicities of the crude solution and the fractions of Hoslundia opposita leaves were evaluated against a normal malignant cell line for the purpose of determining the selectivity of the samples using the standard formula\u003c/p\u003e \u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" height=\"46\" width=\"238\"\u003e\u003c/p\u003e\u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Statistical analysis\u003c/h2\u003e \u003cp\u003eThe statistical analysis of the data obtained for this study was conducted using Graph Pad Prism version 6 software, and the results are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM) of the triplicate repeat measurements. One-way analysis of variance (ANOVA) was used to determine the significance of the difference between the treated (intra) and control groups, and values were considered to be statistically significant at P\u0026thinsp;\u0026le;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e "},{"header":"RESULTS","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e3.1 Cytotoxic effects of the crude extracts (AHO1) and fractions (AHO2-7)\u003c/h2\u003e \u003cp\u003eThe preliminary inhibitory concentrations of the crude extracts (AHO1) and fractions (AHO2-7) responsible for killing 50% of the brine shrimp nauphli and U251 cancer cell lines are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. AHO1, AHO5 and AHO6 were significantly cytotoxic toward the nauphli and U251 cell lines, although AHO6 (52.25 \u0026micro;g/ml) showed the best cytotoxic activity among all the other fractions evaluated.\u003c/p\u003e \u003cp\u003eThe selectivity index (SI) denotes the activity and safety of anticancer agents on cancer cells and normal cell lines. A SI greater than 2 indicates a potent cytotoxic effect on the cancer cell, and a SI less than 2 indicates that the anticancer agent is toxic to a normal cell line (selectivity). Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e also shows that AHO6 (2.11) was selective for U251 cancer cells and HaCaT normal cells. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e also shows significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) cytotoxic effects on U251 cells in comparison to the positive control drug doxorubicin (DOX). Compared with other fractions of H. opposita leaves, fractions AHO5 and AHO6 were toxic to U251 cells, while AHO6 was more effective against the normal cell line HaCaT.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003csub\u003eIC50s\u003c/sub\u003e of fractions of \u003cem\u003eH. opposita\u003c/em\u003e leaf on the brine shrimp line naupli and U251 and selectivity for the HaCaT cell line\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePlant Extracts\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBSL LD\u003csub\u003e50\u003c/sub\u003e(\u0026micro;g/ml)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eU251 CELLS IC\u003csub\u003e50\u003c/sub\u003e (\u0026micro;g/ml)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHACAT IC\u003csub\u003e50\u003c/sub\u003e (\u0026micro;g/ml)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSELECTIVITY INDEX\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAH01 (crude extract)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e168.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e72.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e136.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.87\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAH02 (Hex/Etoac 90:10)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e156.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e156.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAH03 (hex/Etoac 70;30)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e143.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e234.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAH04 (Hex/Etoac 50:50)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e54.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e60.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAH05 (Hex/EtoAc 30:70)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e39.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e55.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e95.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAH06 (EtoAc 100%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e40.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e52.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e111.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAH07 (EtoAc/Met 50:50)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e373.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e76.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.2 Clonogenic effect of crude extracts (AHO1) and fractions (AHO5 and AHO6) on U251 cell lines\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe extent to which colonies formed after 48 h of treatment with the IC50 of AHO1, AHO5 and AHO6 is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA. A significant difference in the number of colonies formed between the treated (fraction) and untreated (negative control) samples was also observed, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB. AHO5 and AHO6 had significant inhibitory effects on AHO5 compared to the control.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.3 Effects of crude extracts (AHO1) and fractions (AHO5 and AHO6) on the cellular morphology of U251 cells\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe effects of AHO1, AHO5 and AHO6 were examined using an inverted microscope after the cells were treated with the indicated IC\u003csub\u003e50\u003c/sub\u003e for 48 h. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e shows that the treated cells exhibited different structural, confluence and morphological characteristics than did the untreated cells (control). The untreated cells exhibited a sustained structure, confluence and original morphology.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\u003cp\u003e \u003cb\u003e3.4 Apoptotic effects of the crude extract (AHO1) and the fractions (AHO5 and AHO6) on U251 cells\u003c/b\u003e \u003c/p\u003e \u003cp\u003eApoptotic activity was measured using caspase-3 and caspase-7 \u003cb\u003eassays (\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) after the cells were treated with the appropriate IC\u003csub\u003e50\u003c/sub\u003e for 48 hrs. AHO1, AHO5 and AHO6 significantly activated caspase 3/7 in U251 cells compared to negative control cells. Thus, the results of this study suggest that apoptotic activity occurs via the cascade cascade pathway.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Mitochondrial membrane potential and intracellular reactive oxygen species\u003c/h2\u003e \u003cp\u003eMitochondria are known to be the main source of reactive species (ROS) in cells. To observe whether cell death occurred through ROS-mediated activity and loss of the MMP, the cells were stained with fluorescent dyes for 48 h and compared with their positive controls. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, AHO5 and AHO6 significantly increased ROS production, and a significant decrease in the MMP was observed. The observed cell death in this study might be due to ROS-induced activity.\u003c/p\u003e \u003c/div\u003e "},{"header":"DISCUSSION","content":"\u003cp\u003eThe recalcitrant or aggressive nature of glioblastoma cells (GBMs) and limited treatment options underlie the overwhelmingly poor progression-free survival (PFS) and low overall survival (OS) in glioblastoma patients. Currently, there is no significant clinical improvement in the effectiveness of available anticancer drugs for GBM treatment. Therefore, there is urgency in drug discovery and development, which aims at identifying medicinal plant extracts, fractions and possible lead compounds that have great potential to reduce the metastatic potential of aggressive GBM cells and induce significant apoptosis. This study evaluated the cytotoxic effects of the crude extract and different fractions of \u003cem\u003eHoslundia opposita\u003c/em\u003e (HO) on the glioblastoma cell line U251.\u003c/p\u003e \u003cp\u003eThe biological or pharmacological activities of plants have been attributed to the array of phytoconstituents, which have no direct effect on plant growth (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Ajibare et al. (2021) reported on the phytochemical screening of crude methanolic extract and the antioxidant capacity of different fractions of \u003cem\u003eHoslundia opposita\u003c/em\u003e (HO) (Ajibare et al., 2022). We evaluated the anticancer activity of \u003cem\u003eHoslundia opposita\u003c/em\u003e (HO) leaf extract, fractions and lead compound against human hepatoma cell lines (HepG2), breast cancer cell lines (MDA-MB-23), intestinal epithelial cell lines (Caco-2) and human keratinocyte HACAT cell lines. The study results showed that HO and hoslundin have significant anticancer effects on tumor cells by inducing apoptosis via mitochondrial-dependent reactive oxygen species generation and that normal cells tolerate potential antineoplastic agents (Ajibare et al., 2022). In the present study, because of the aggressive nature and relapse associated with GBM, we explored the preliminary investigations of the anticancer effects of crude methanolic extracts and fractions of HO on glioblastoma cell lines (U251).\u003c/p\u003e \u003cp\u003eThe anticancer or antiproliferative potential of plant extracts, fractions or compounds in any cancer study relies on preliminary cytotoxicity screening. The crude extracts of AHO1 and AHO2 to AHO7 showed varied IC50 values (52.25\u0026ndash;234.90 \u0026micro;g/ml), indicating anticancer activity (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The percentage of cell viability illustrated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) by the MTT assay showed varied results (20\u0026ndash;100 \u0026micro;g/ml) in U251 cells treated with the crude extract AHO1 and AHO2 to AHO7 fractions, which could be attributed to the aggressiveness and mutational pattern associated with these cell lines (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Note that an inhibitory concentration of 50% inhibitory concentration (IC\u003csub\u003e50)\u003c/sub\u003e at or less than 30 \u0026micro;g/ml, according to the American Cancer Institute (NCI), is the most potent cytotoxic or effective anticancer screening (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). This IC\u003csub\u003e50\u003c/sub\u003e value was not achievable at the set maximum concentration of 100 \u0026micro;g/ml; however, increasing the maximum concentration could or probably could produce an IC\u003csub\u003e50\u003c/sub\u003e at or less than 20 \u0026micro;g/ml for 72 hr, as reported in a past study (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). This implies that some plants might demonstrate more significant cytotoxicity at higher concentrations, which could be our focus in our subsequent study by extending the concentration range to 200 \u0026micro;g/ml. Previous studies have established the inhibitory dose-dependent cytotoxic effect of HO leaves on different human cancer cell lines (Ajibare et al.,2022). In this study, for the first time, we found that only the crude extracts AHO1, AHO5 and AHO6 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e had moderate cytotoxic effects, with IC\u003csub\u003e50\u003c/sub\u003e values of 72.89, 55.65 and 52.55 \u0026micro;g/ml, respectively, on U251 cells. This is supported by the findings of another study in which HO exhibited a similar cytotoxic effect against BT-549 cells, in which the IC\u003csub\u003e50\u003c/sub\u003e was very close to 76.4 \u0026micro;g/ml, and \u003cem\u003eByrsocarpus coccineus\u003c/em\u003e had an IC\u003csub\u003e50\u003c/sub\u003e value of 65.2 \u0026micro;g/ml against JURKAT (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Overall, the selected crude extracts and the fractions elicited potent cytotoxic effects that inhibited U251 cells, resulting in a decrease in cell viability. Hence, the choice of crude extract or the two fractions was selected after the continuation of the other experimental assays in this study.\u003c/p\u003e \u003cp\u003eThe loss of cell viability indicated the cessation of metabolic activity in the U251 cell line after treatment with the extract AHO1, and the fractions AHO5 and AHO6 indicated some morphological distortions. Cytological observation under an inverted microscope (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) revealed several key changes. These morphological changes are distinctive features of cell shrinkage, nuclear condensation, membrane shrinkage, and chromatin cleavage, among others, in the treated U251 cell line (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). In contrast, the visualization of the control (untreated cells) revealed an undistorted cell morphology because the cell content was intact and the cells adhered to the bottom of the culture plate. There were no noticeable echinoid spikes of apoptotic cells or apoptotic bodies or a decrease in cell number (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Treatment of U251 cells with the extract AHO1 or fractions AHO5 and AHO6 resulted in early detachment from the basal membrane, referred to as anoikis (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e), which is characteristic of apoptosis. An important promising strategy in developing antitumour agents requires the induction of apoptotic pathways (\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). After treating the U251 cell line with the extract AHO1, fractions AHO5 and AHO6 had a significant increase in the activity of caspase-3/7, thus causing antiproliferation or inhibition of U251 cell lines (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe ability of cancer cells to form colonies from single cells reveals the metastatic capacity or proliferative effect of cancer cells, thus increasing the difficulty of treatment or resulting in a poor prognosis. A colony formation assay was used to determine the proliferative pattern and survival of the cells. A significant increase in proliferation was observed upon treating U251 cells with the extract AHO1, the AHO5 fraction and the AHO6 \u003cb\u003efraction (\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u003cb\u003e), which\u003c/b\u003e is in line with the findings of other studies (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). The metastatic mechanism enables firm anchorage of cell‒cell interactions of cancer cells with the endothelium, which colonizes and establishes secondary tumors elsewhere. Hence, the invasiveness and metastasis of most cancer cells are important for effective treatment (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). The clonogenic and morphological data showed that treatment with the crude extract of AHO1, fraction AHO5 or AHO6 significantly decreased the ability of U251 cells to form colonies and disrupted cancer cell integrity or integrity compared with that of the untreated cells.\u003c/p\u003e \u003cp\u003eThe continuous production of ROS could lead to the death of cancer cells, which is implicated in destroying the mitochondrial membrane and probably limiting the mitochondrial membrane potential, subsequently leading to the release of cytochrome c. Hence, the induction of caspase-3 leads to mitochondria/cytochrome-induced cancer cell death, which is evident in the release of cytochrome c (Ajibare et al.,2022). This study revealed that the crude extract of AHO1 and fractions AHO5 and AHO6 induced cell death via a mechanism similar to that of mitochondria and ROS (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Studies have revealed that the induction of ROS production is responsible for apoptosis in different types of cancer (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e).\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThis study revealed that the crude extracts of AHO1, fractions AHO5 and AHO6, have relatively moderate cytotoxic effects on U251 cells. Compared to those of other fractions, a better safety profile was observed for the normal human cell line HACAT. The nature or composition of solvents has been shown to have a profound effect on the phytoconstituents that exert anticancer or antiproliferative effects. Further work depicting the underlying molecular mechanisms is recommended.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003cstrong\u003e- \u003c/strong\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAC, AOT, RA, MA, KA,\u0026nbsp;TA, AO, HA and\u0026nbsp;MP were involved in\u0026nbsp;the\u0026nbsp;data analysis, interpretation\u0026nbsp;of the results\u0026nbsp;and manuscript preparation.\u003c/p\u003e\n\u003cp\u003eAC, AOT, RA and MA were\u0026nbsp;involved\u0026nbsp;in\u0026nbsp;the\u0026nbsp;research conception/design.\u003c/p\u003e\n\u003cp\u003eAC, KA,\u0026nbsp;TA, AO, HA and\u0026nbsp;MP were involved\u0026nbsp;in the\u0026nbsp;data acquisition\u003c/p\u003e\n\u003cp\u003eAC, AOT, RA, MA and\u0026nbsp;KA\u0026nbsp;were involved\u0026nbsp;in\u0026nbsp;critical revision of the manuscript.\u003c/p\u003e\n\u003cp\u003eTA, AO, HA and\u0026nbsp;MP were involved\u0026nbsp;in the\u0026nbsp;figure and/or table design.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll\u0026nbsp;the\u0026nbsp;authors\u0026nbsp;declare\u0026nbsp;that there\u0026nbsp;are\u0026nbsp;no potential\u0026nbsp;conflicts\u0026nbsp;of interest\u0026nbsp;associated with\u0026nbsp;this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors did not receive funding from anyone or institutions.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSchwartzbaum JA, Fisher JL, Aldape KD, Wrensch M (2006) Epidemiology and molecular pathology of glioma. 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Mediterranean J Nutr Metabolism 11(1):21\u0026ndash;36\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"University of Lagos","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":"Hoslundia opposita, cancers, cytotoxicity, apoptosis, gliomas","lastPublishedDoi":"10.21203/rs.3.rs-4824462/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4824462/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBACKGROUND: \u003c/strong\u003eThe ineffectiveness of many known anticancer agents for treating several cancer types, especially glioblastoma (GMB), which affects the body's central nervous system, is highly important. GBM is highly invasive and recalcitrant and accountsfor 42% of all central nervous system tumors and 60% of all brain tumors in adults, with a median survival of 15 months. The limitationsencountered in GBM treatment necessitate the discovery and development of new drugs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMETHODS: \u003c/strong\u003eTo investigate the anticancer activity of \u003cem\u003eHoslundia opposita\u003c/em\u003eleaf extracts and fractions against a human glioblastoma cell line (U251) and human keratinocyte HACAT cell line, standard methods, MTT, clonogenic and caspase3 and 7 assays were used to determine the viability of the cells and colony formation and apoptotic activities, respectively. The fluorescent probe dyes dihydrofluorescindiacetate (DCFH-DA) and tetramethylrhodamine (TMRE) were used to determinethe intracellular reactive oxygen species (ROS) concentration and mitochondrial membrane potential (MMP), respectively, in the cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRESULTS: \u003c/strong\u003eThe crudemethanolic extracts and fractions of H. opposita leaves exhibited moderate cytotoxic and selective activity within the range of concentrations tested (25-100 µg/ml). The study revealed that crude AHO1 and specific fractions of AHO5 and AHO6 inhibitedmetastasis or colony formation, promoted apoptosis in the U251 cell line and depolarized the mitochondrial membrane potential, which was likelymediated by mitochondria-dependent ROS generation. Overall, the specificity and dose dependenceof the different treatments were observed for the U251 cell line.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eThe antiproliferative activities of \u003cem\u003eHoslundia opposita\u003c/em\u003e Vahl demonstrated by the crude extract and specific fractions against U251 cells warrant further investigations todecipher its mechanism of action.\u003c/p\u003e","manuscriptTitle":"Antiproliferative activity of Hoslundia opposita leaf extract and fractions against a human glioblastoma cell line (U251)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-30 10:51:33","doi":"10.21203/rs.3.rs-4824462/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":"f3f5a0fe-4883-4d4e-a5f5-4d223cea3f47","owner":[],"postedDate":"July 30th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":35307779,"name":"General Biochemistry"},{"id":35307780,"name":"Cell Survival and Cell Death"},{"id":35307781,"name":"Cancer Biology"}],"tags":[],"updatedAt":"2024-08-05T05:33:12+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-30 10:51:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4824462","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4824462","identity":"rs-4824462","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}