Lepidium sativum or garden cress (family Brassicaceae) has been widely used worldwide for its wide therapeutic application, including anticancer and chemoprotective agents. Plant tissue culture techniques hold great promise for natural product enhancement without any climatic boundaries. In this study, glucosinolates and petroleum ether fractions were isolated from in vitro cell cultures and used against different carcinoma cell lines to investigate their anticancer potential.Finding natural products with anticancer activity is an effective strategy to fight this disease. In this respect, In this study, callus cultures from leaf and root explants were initiated, cell suspension cultures were established, and cell growth and viability profiles were characterized. Different amino acids were added as precursors to the cell suspension cultures to enhance glucosinolates accumulation. Gas chromatography–mass spectrometric analysis (GC–MS) of glucosinolates and petroleum ether fractions was performed, and all fractions were tested against different carcinoma cell lines.50 of GL extract (47.5 ug/ml) upregulates pro-apoptotic BAX and downregulates anti-apoptotic BCL2, which disrupts the BAX/BCL2 ratio, leading to activation of caspase 3 inside treated HepG2 cells.The findings clarified that the maximum callus initiation percentage was obtained in the medium containing 1.0 mg/l 2,4-dichlorophenoxy acetic acid  + 1.0 mg/l kinetin (Kin) (C1). The viable cell number of cell suspension cultures from leaves and roots increased until it reached the maximum values on day 15. Adding tyrosine and methionine to the cell suspension cultures was the most influential and recorded high glucosinolate percentages. 1H-Cyclopenta (b) pyridine-3-carbonitrile-4,5,6,7-tetrahydro-2-methylthio-4-spirocyclohexane was the main glucosinolate compound found in tyrosine-treated leaf suspension (GLT). Fifteen compounds were detected in the petroleum ether fraction in both cell suspensions initiated from the leaf and root (OL and OR). The major compounds were benzene-1,3,5-trimethyl (12.99%) in root cell suspension (OR), and benzene-2-ethyl-1,4-dimethyl (10.66%) in leaf cell suspension (OL). All glucosinolate extracts demonstrated significant anticancer activity against the prostate (PC3), lung (A-549), colorectal (caco2), and liver (HepG2) cell lines. Glucosinolates extracted from leaf cell suspension (GL) were the most active on the hepatocellular carcinoma cell line (HepG2) among all remaining glucosinolate extracts. Treated hepatocellular carcinoma with an ICThe anticancer action of the GL extract was validated by the cell cycle study of its glucosinolates, which successfully promoted apoptosis and reduced hepatocellular growth by causing S-phase arrest. Lepidium sativum, or garden cress (family Brassicaceae), is an edible herb cultivated in various parts of the world. It is considered a standing herbaceous annual plant that grows from 15 to 45 cm in height and has its own small white flowers along with broad or obovate pods emarginated at the apex and winged [d winged  and is ad winged . This isd winged .Garden cress has been declared to have various clinical values in traditional medicine, including for inflammatory diseases such as diabetes mellitus, arthritis, and hepatitis , 8. CancArabidopsis thaliana cell cultures [Plant biotechnology techniques fulfill the output of plant material and natural products to some extent if all factors that achieve this goal are controlled. Plant cell culture introduces many optimization strategies, such as precursor feeding, media, and cultivation modifications . Severalcultures .In the previous study, glucosinolate compounds were detected in callus cultures, and these extracts were effective against three human carcinoma cell lines . The curMS-medium, gelrite, 2,4-dichlorophenoxy acetic acid , kinetin, benzyladenine, trypan blue, L-phenylalanine, L-tyrosine, L- methionine, and L-cysteine purchased from Biotech Serve Co. Potassium penicillin, RPMI 1640 medium, streptomycin sulfate, amphotericin B, L-glutamine, doxorubicin, MTT salt, sodium dodecyl sulfate (SDS), biotin-conjugated antibody, streptavidin-HRP, streptavidin conjugated to horseradish peroxidase, and propidium iodide (PI) were brought from Sigma, Aldrich. DMSO, petroleum ether, ethanol, Tween 20, and dichloromethane were purchased from Omega for Trading of Solvents. L. sativum were immersed in 70% ethanol for 2–3 min, then rinsed three times in sterile distilled water. The seeds were then sterilized for 20 min in 20% commercial Clorox (5% NaOCl) containing 0.5% Tween 20. After rinsing three times with sterile distilled water, seeds were cultured on MS medium  containing 3% (w/v) sucrose and solidified with 0.2% (w/v) gelrite. The culture medium was adjusted to pH 5.8. The cultures were incubated in a growth room at 26 ± 2 °C and kept under a 16-h photoperiod of fluorescent, 45 μmol cool white light tubes, and an 8-h dark.Seeds of−1) and kin (1.0 and 2.0 mg L−1). After 30 days of culturing, the callus initiation percentage was recorded and calculated based on the following equation:Explants of leaf and root (approximately 0.5 cm) were taken from 30-day-old seedlings, then cultured on solidified MS medium containing different combinations of 2,4-dichlorophenoxy acetic acid  + kinetin (kin). The concentrations of two plant growth regulators were as follows: 2,4-D . Cell growth and viable cell number were evaluated using the trypan blue exclusion method [The friable calli (0.2 g) produced after two sub-cultures on the most convenient medium were transferred into fresh liquid media containing 1.0 mg Ln method . Aliquot−1 of different amino acids  were added. After that, the callus tissues were isolated from the media, collected, and oven-dried for 24 h at 65 °C. Then glucosinolates and oil fractions were extracted and detected by gas chromatography–mass spectrometry (GC–MS).The amino acids used in the experiments were filter-sterilized and added to the autoclaved medium for four weeks. In the second passage of cell suspension, 1.0 g LThe extraction method of glucosinolate derivatives was carried out according to Al-Gendy and Lockwood . Oil ext−1. The injector and MS transfer line temperature was set at 280 °C. The sample injection volume was 2 μl. The quantification of identified components was investigated using a relative peak area (%). Tentative identification of the compounds was performed based on the comparison of their relative retention time and mass spectra with those of the NIST, WILLY library data of the GC–MS system.The extracts were analyzed in the National Research Center using gas chromatography–mass spectrometry with the following specifications. The GC–MS analysis was performed using a Thermo Scientific, Trace GC Ultra/ISQ Single Quadrupole MS, TG-5MS fused silica capillary column . An electron ionization system with an ionization energy of 70 eV was used for GC–MS detection. Helium gas was used as the carrier gas at a constant flow rate of 1 ml minHuman breast cancer cells (MCF-7 cell line), human lung cancer cells (A-549 cell line), human colorectal cancer cells (HCT-116 cell line), human liver cancer cells (HepG2), human colon cancer cells (caco2 cell line), human prostate cancer cells (PC3 cell line), human melanoma cancer (A-375 cell line), and a normal human cell line (BJ-1); “immortalized telomerase normal foreskin fibroblast cell line” were obtained from Karolinska Center, Department of Oncology and Pathology, Karolinska Institute and Hospital, Stockholm, Sweden.The procedure was carried out in a sterile area using a laminar airflow cabinet. The culture was maintained in RPMI 1640 medium with 1% antibiotic–antimycotic mixture ,1% L-glutamine, and supplemented with 10% heat-inactivated fetal bovine serum. Doxorubicin was used as a positive control. A negative control, composed of DMSO, was also used. Culturing and sub-culturing were carried out according to Thabrew et al. .3 cells per well in the case of MCF-7 and PC3 and HepG2; 20 × 103 cells per well in case of A-549, HCT-116, caco2, and A-375 cell lines; and 35–45 × 103cells/well in case of BJ-1 using 96-well plates at 37 °C. After 48 h of incubation, the medium was aspirated and 40 uL MTT salt (2.5 mg/mL) was added and further incubated for 4 h. Then, 200 uL of 10% sodium dodecyl sulfate (SDS) was added. The absorbance was measured at 595 nm.This was done according to Mounier et al. . The cel50 values were calculated using probit analysis and the SPSS computer program .ICQuantitative identification of human CASP3 (Caspase 3) using an ELISA Kit from Invitrogen INC. Catalog # KHO1091 (96 tests) . The procedure was done according to the manufacturer’s instructions.BCL-2 in the samples and standards were estimated according to Barbareschi et al. . A biotiBax protein levels were evaluated according to Onur et al. . A monoc4 and incubated at 37 °C, 5% CO2 overnight. After treatment with the tested compound C, for 24 h, cell pellets were collected and centrifuged . Cell pellets were fixed with 70% ethanol on ice for 15 min for cell cycle analysis and collected again [Apoptosis detection and cell cycle analysis were carried out using flow cytometry. HepG2 cells were seeded at 1–5 × 10ed again . The colCallus initiation (%) was presented in Fig. C1 medium  was the most convenient treatment that obtained the maximum callus initiation (%) from two types of explants, recording 84% from the leaf and 62% from the root. Whereas C4  showed the lowest callus initiation (%), leaf explants recorded 72%, and no calli was initiated from root explants. It was noted that initiating calli from two types of explants required lower concentrations of 2,4-D + kin than higher concentrations. The highest 2,4-D + kin concentrations found in the C4 medium resulted in no calli initiating from root explants accordingly. Moreover, leaf explants respond more strongly to initiating calli than root explants. This could be due to different internal hormone levels in different plant parts.There is also no significant difference in the leaf or root explant response to varying concentrations of 2,4-D + kin, but there is a significant difference in the level of explant response.Other concentrations of growth regulators have been used to induce calli, such as different concentrations of naphthalene acetic acid  + benzyl adenine (0.5 mg/l), and they were not effective in callus initiation (data not shown). Callus was grown on a C1 medium and subcultured twice to the same 2,4-D + kin concentration to be used later in cell suspension cultures. Figure 3/ml and 85 × 103/ml for both root and leaf cell suspension cultures, respectively. On day 18, the viable cell number decreased . This could indicate a change in cellular metabolism as multiplying cells stop with nutrient decrease. It is clear from the results that the viability of cell suspension cultures produced from leaf explants was higher than that produced from root explants. Also, the fifteenth day of growth recorded the highest vitality in both cell suspension cultures produced from leaf or root. In general, callus growth was appropriate in the suspension cultures. This enabled us to add some precursors that could enhance the active compounds of interest in the study.Cell suspension culture was established after two sub-cultures of callus on the solid medium. Both growth curves Fig. a and b c1.0 g of different commonly used amino acids (precursors) was added to cell suspension cultures derived from both root and leaf. After 1 month, the cells were harvested for glucosinolate (GLSs) extraction and detection. Table Lepidum sativum. Significant differences exist between treatments and control, as well as within treatments.In terms of the leaf cell suspension, glucosinolates were detected in the cell suspensions treated with L-tyrosine, L-methionine, and control. In contrast, the glucosinolates disappeared with L-phenylalanine and L-cysteine treatments. A maximum percentage of GLSs was recorded with L-tyrosine treatment 3.95%, Fig. %, Fig. 5Petroleum ether extracts of cell suspension obtained from root and leaf were analyzed using gas chromatography–mass spectrometry and are shown in Table Lepidium sativum, and it has a variety of biological effects). This highlights the necessity of creating plant cell cultures from different explants and shows that the range of chemicals that can be recovered in the produced extracts may vary depending on the choice of grown explant. While in the extract of leaf cell suspension, the major compounds were benzene-2-ethyl-1,4-dimethyl 10.66%), followed by 4-ethyl-2-methyl pyrrole-3-carbonitrile (10.45%), 9-methyl-6-(1-naphthyl methyloxy)-4-ethyl-á-carboline-3-carboxylic acid ethyl ester (9.66%), naphthalene-2-methyl (9.23%), and naphthalene (8.51%). Figure 6%, folloEight samples were tested for their anti-proliferative activity against seven human cancer cell lines at 100 ug/ml, as shown in Table According to our findings, all glucosinolates from the leaf and root cell suspension were safe on the tested normal human cells, in contrast to the petroleum ether fractions, which were toxic on the tested normal cells.50 was calculated using a second assay in which each active extract was screened at four additional concentrations  as shown in Table 50 value on HepG2 with no toxicity on normal cell tested. The apoptotic mode of action of GL on HepG2 was studied. Also, different apoptotic parameters were studied, including BAX, BCL2, Caspase3, and cell cycle analysis.To distinguish between all glucosinolate extracts that showed 60% cytotoxicity on the tested cell lines and determine their order in cytotoxicity, IC50 value) to determine the level of BCL2 and BAX compared to the non-treated HepG2 cells, as shown in Fig. HepG2 carcinoma cell line was treated with 47.5 ug/ml GL extract  Fig. . Also, G-1) and BAP (2 mg L-1) was used to boost the formation of callus and secondary metabolites from L. sativum [The current study’s findings were supported by a prior study that found leaf explants more capable of developing calli than hypocotyl and root explants . Additio sativum .Escherichia coli into A. thaliana resulted in increased levels of benzyl GLSs. Additionally, more aliphatic GLSs result from more L-methionine [Several studies found that the availability of free amino acids, such as L-phenylalanine, was increased by introducing L-phenylalanine biosynthetic genes from thionine , 23.As an alternative to growing plants in vivo, plant cell suspension culture offers a viable method for producing high-value plant metabolites. Safety and regulatory criteria are introduced with this production technique. Additionally, it is likely to create novel compounds that are not found in native plants , 25. AddTropaeolum majus) cell cultures produced the most GLSs after substrate feeding, which resulted in 44 mmol/g DW [Tropaeolum majus, L-phenylalanine and L-cysteine feeding increased the concentration of GLSs (85.8 mmol/g FW) [Nasturtium montanum and Cleome chelidonii [Numerous experiments have attempted to produce GLSs in cell cultures. Indian cress (mol/g DW . In hairol/g FW) ; howeverelidonii .Lepidium sativum plants contained thiocyanate-forming protein (TFP) and glucosinolate hydrolysis products [L. sativum cell culture in an attempt to produce glucosinolates. The current study’s findings support the viability of this production, although more research is required to produce more accurate findings. Lepidium sativum also contains a variety of active substances in its seed, leaves, and aerial portions [products . No prioportions .L. sativum seeds and fresh herbs. They detected 2-ethyl butyl glucosinolate, methyl glucosinolate, and glucotropaeolin in the fresh herbs, in addition to dimeric imidazole alkaloids such as lepidine that were also detectable [The previous studies isolated and fractionated glucosinolates from tectable .Cancer is characterized by cellular changes that result in uncontrolled growth. Even though several treatments and interventions, including chemotherapy, radiation, gene therapy, hormone therapy, immunotherapy, and targeted therapy, have been successfully used to combat cancer, it is still a significant health issue. Even with the most cutting-edge anticancer therapies, several problems remain, such as side effects, cancer resistance, metastasis, and recurrences. Therefore, it is essential to look for new chemotherapeutic agents . PreviouLepidium sativum were investigated for their cytotoxic potential against seven human cancer cell lines, including; MCF-7 cell line, A-549 cell line, HCT-116 cell line, HepG2 cell line, caco2 cell line, PC3 cell line, and A-375 cell line. Glucosinolates were further screened at different concentrations to calculate their IC50. All extracts were assayed over normal human cells (BJ-1 cell line) to study the safety of glucosinolates. In this study, we examined the anticancer effect of all samples using the MTT assay, which is a reliable assay for studying cellular metabolic activity and mitochondrial dysfunction. As a result, when comparing glucosinolates from different extracts, cell suspension derived from leaf (GL extract) was most effective on HepG2 cancer cells in a dose-dependent manner, so it was subjected to study their possible apoptotic mechanism through their effect on BCL2, BAX, and caspase 3.Apoptosis is one of the most effective ways to eliminate cancer cells. The search for novel natural compounds that can induce apoptosis is attracting much interest in cancer research. In our study, glucosinolates from leaf and root cell suspension cultures of Our results revealed that GL extract downregulates the anti-apoptotic protein BCL2 and upregulates the pro-apoptotic protein BAX, a key pro-apoptotic molecule in the mitochondria-dependent apoptotic pathway, and disrupt the BAX/BCL2 ratio, which signals an apoptotic cascade inside HepG2 cancer cells. HepG2 cancer cells treated with GL extract upregulate capasase 3 protein compared to the untreated HepG2 cancer cells.50 concentrations of GL (47.5 ug/ml) for 24 h, the cells were labeled with the two dyes. The corresponding red (PI) and green (FITC) fluorescence were detected with the flow cytometry. The findings revealed that GL extract cytotoxic activity against HepG2 cancer cells was related to S-phase cycle arrest.As cancer is thought to be a cell cycle disease, cell cycle blockage is considered an attractive strategy to fight cancer , so we sLepidium sativum. Since cancer cannot be cured, additional study is needed to find compounds that can fight it without harming human health, in contrast to the petroleum ether fractions from two suspension cultures, which had a lethal effect on the normal cells. In our study, all glucosinolates extracted from cell suspensions influenced cancer cells while not impacting healthy cells. Understanding cancer biology has become essential for obtaining compounds that fight cancer cells, and it has become the focus of recent studies. Apoptosis is one of the crucial control mechanisms used to combat cancer cells. The glucosinolates extract from our study’s L. sativum cell suspension demonstrated its anticancer efficacy, promoted apoptosis, and reduced hepatocellular growth by inducing S phase arrest.Utilizing components from cell suspension culture combined with precursor feeding has proven to be a successful method for obtaining special compounds with critical industrial uses. L-Tyrosine and L-methionine additions to the cell suspension culture improved the production of glucosinolates in


This finding was classified as moderate resistance to TRAIL. The Caco-2 cell line showed resistance to oxaliplatin and irinotecan. MSC successfully overexpressed soluble TRAIL and induced cancer cell death at a 1:6 ratio in co-culture. Oxaliplatin pre-treatment in the Caco-2 cell line increased the cell death percentage (50%) and apoptosis by sTRAIL. This finding was statistically different from the negative control (p < 0.05), and activity was even higher with the oxaliplatin–flTRAIL combination. Thus, oxaliplatin increases apoptotic activity induced by soluble TRAIL in a chemoresistant CRC cell line. A key problem in colorectal cancer (CRC) is the development of resistance to current therapies due to the presence of cancer stem cells (CSC), which leads to poor prognosis. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a protein that activates apoptosis in cancer cells through union with TRAIL death receptors. Cell therapies as delivery systems can produce soluble TRAIL (sTRAIL) and full-length TRAIL (flTRAIL), showing a high capacity to produce apoptosis in vitro and in vivo assays. However, the apoptotic activity of TRAIL as monotherapy had limitations, so it is important to explore other ways to enhance susceptibility to TRAIL. This study evaluated the cytotoxic and proapoptotic activity of soluble TRAIL overexpressed by mesenchymal stem cells (MSC) in an oxaliplatin-resistant CRC cell line. Bone marrow-MSC were lentiviral transduced for soluble TRAIL expression. DR5 death receptor expression was determined in Caco-2 and CMT-93 CRC cell lines. Sensitivity to first-line chemotherapies and recombinant TRAIL was evaluated by half-maximal inhibitory concentrations. Cytotoxic and proapoptotic activity of soluble TRAIL-MSC alone and combined with chemotherapy pre-treatment was evaluated using co-cultures. Caco-2 and CMT-93 cell lines expressed 59.08 ± 5.071 and 51.65 ± 11.99 of DR5 receptor and had IC Colorectal cancer (CRC) is the second cause of death-related cancer. It is associated with a late diagnosis and metastasis . ChemoreTNF-related apoptosis-inducing ligand (TRAIL) has been used as an alternative treatment for cancer resistant to current chemotherapies. TRAIL activates extrinsic apoptosis by trimerization and activation of TRAIL death receptors DR4 and DR5, which leads to a Fas-associated death domain (FADD), which recruits caspase 8/10 to form the death-inducing signaling complex (DISC), leading to activation of a caspase cascade which triggers apoptotic changes and eventual cell death ,8,9. A lMesenchymal stem cells (MSC) are adult multipotent cells that have been developed as delivery vehicles for proapoptotic proteins, such as TRAIL. They can be found in different sources, such as adipose tissue, the umbilical cord, the placenta, and bone marrow . This hep > 0.05, t-test)  C. Thus, 50 in cell lines. We then compared IC50 with a previously determined maximum peak plasma concentration of each drug and established the resistance sensitivity. Based on maximum peak plasma concentrations, CMT-93 was sensitive to the three chemotherapies, but Caco-2 cancer cells were resistant to oxaliplatin and irinotecan  in the Caco-2 and CMT-93 cell lines (CDP 50 to 70) . In addition, there was no significant difference between sTRAIL treatments and flTRAIL and rmTRAIL controls. This finding implies that our sTRAIL delivery system can induce at least 50 of CDP in colorectal cancer cell lines. Once TRAIL sensitivity of colorectal cancer cell lines was confirmed, we tested our expression system of sTRAIL by MSC. We included rmTRAIL and flTRAIL-MSC as positive controls. A cytotoxicity increase directly related to the number of MSCs added was found for both colorectal cancer cell lines. This finding shown in 0 to 70) A,B. Howep < 0.01, Kruskal–Wallis, Dunn’s multiple comparisons); however, it was not statistically different from individual treatments for this cell line . In addition, an increase of sTRAIL-MSC activity when oxaliplatin pretreatment was applied was observed (p < 0.05 One-way ANOVA with Tukey’s multiple comparisons). This finding implies increased sensitivity of cells to TRAIL, and this activity was even higher than the combination of oxaliplatin with flTRAIL   C,D. ThusColorectal cancer is still the second cause of cancer-related death, mainly because of the development of resistance to current therapies and metastasis ,26. In t50. TRAIL-resistant pancreatic cancer cell lines require concentrations above 500 ng/mL of recombinant TRAIL to reach IC50  × 100. IC50 was calculated with linear regression.We determined the IC3 cells per well and incubated for 24 h with or without oxaliplatin pretreatment at a maximum peak plasma concentration (2.9 µg/mL)  × 100. Evaluation of activated caspases luminescence signal was associated with apoptosis. The colorectal cancer cell line Caco-2 was cultured in 96-well plates at a density of 2 × 103 cells per well and incubated 24 h with oxaliplatin (2.9 µg/mL) . Co-culture with 12 × 103 MSC expressing sTRAIL or flTRAIL was incubated for another 24 h. Apoptosis was analyzed with a Caspase-Glo 3/7®  kit. The 100 µL reagent was added to each well, agitated for 30 s, and incubated for 2 h at room temperature. Luminescence was quantified in a Cytation 3 plate reader (BioTek). Colorectal cancer cell lines were cultured in 96-well plates at a density of 2 × 10v/v) and washed three times with Tris-Buffered saline Tween 20 (TBST). Slides were then incubated with the primary antibodies, anti-CD44 conjugated with PE/Cy7 (1:100), and anti-CD24 conjugated with FITC (1:100). All antibodies were diluted in PBS and incubated at 4 °C for 12 h. The slides were washed again three times with TBST. The secondary antibody Alexa Fluor® 488 (1:1000 in PBS) was added and incubated for 1 h at 25 °C. As a mounting medium, 25 µL of VECTASHIELD® mixed with DAPI  was added. Slides were observed with a ZEISS Imager.A2 epifluorescence microscope (Carls ZEISS), taking five pictures per slide with an AxioCam MRc coupled to the microscope. Images were analyzed with ZEISS ZEN Blue and Image J software. The expression percentage was calculated with relative luminescence units as:  × 100. We evaluated cell death by fluorescence. The Caco-2 cell line was cultured in 6-well plates at a concentration of 5 × 104 cells per well above a coverslip. After cell attachment to the coverslip, we applied pretreatment with oxaliplatin (2.9 µg/mL)  and incubated for 24 h. Using 0.4 µm Transwell® , we co-culture 3 × 105 MSC expressing sTRAIL and incubate for 24 h. After that, we removed the coverslips with cells and added 25 µL of VECTASHIELD® mixed DAPI . Slides were observed with a ZEISS Imager.A2 epifluorescence microscope (Carls ZEISS), taking five pictures per slide with an AxioCam MRc coupled to the microscope. Images were analyzed with ZEISS ZEN Blue and Image J software.The CSC markers CD44 and CD24 were analyzed by immunofluorescence. Cell lines were cultured in Lab-Tek® microchambers . After 24 h, they were fixed with methanol-acetone , GraphPad Prisma (significance p < 0.05). All experiments were performed twice in triplicate.The According to the results, MSCs are a promising strategy as a delivery system for antiapoptotic proteins such as sTRAIL and flTRAIL, which can induce cytotoxicity and apoptosis in CRC cell lines. We found a ratio of 1:3 and 1:6 with naïve-MSC, sTRAIL-MSC, and flTRAIL-MSC-induced cell death and apoptosis against tumor cells (Caco-2 and CMT-93) that had moderate resistance to recombinant TRAIL which was statistically different from the negative control. We also demonstrated increases in the antitumoral activity of MSC expressing sTRAIL at a ratio of 1:6 with oxaliplatin pretreatment. Our treatment with sTRAIL-MSC as monotherapy induces apoptosis in colorectal cancer cells expressing cancer stem cell markers. This treatment is effective even in a chemoresistant CRC cell line.


Depression is a serious neuropsychiatric disease affecting an increasing number of people worldwide. Cognitive deficits  are common in the clinical picture of depression. Cognitive impairment has been hypothesized to be one of the most important components of major depressive disorder , although typical cognitive symptoms are less frequent in people with depression than in people with schizophrenia or bipolar disorder . The importance of α-Klotho in the aging process has been well-documented. Growing evidence points to the role of α-Klotho in regulating other biological functions, including responses to oxidative stress and the modulation of synaptic plasticity. It has been proven that a Klotho deficit may contribute to the development of various nervous system pathologies, such as behavioral disorders or neurodegeneration. Given the growing evidence of the role of α-Klotho in depression and cognitive impairment, it is assumed that this protein may be a molecular link between them. Here, we provide a research review of the role of α-Klotho in depression and cognitive impairment. Furthermore, we propose potential mechanisms (related to oxidative stress and glutamatergic transmission) that may be important in α-Klotho-mediated regulation of mental and cognitive function. Depression is recurrent, and the most common mental disease manifests as depressed mood and the loss of pleasure or interest in activities for a long time. According to the WHO, approximately 280 million people worldwide suffer from depression, i.e., 3.8% of the general population, including 5% of the adult population. Depression is the leading cause of suicide. Approximately 700,000 suicides are committed per year. Importantly, suicide is the fourth leading cause of death among people aged 15–29 ,5. NeverDepression is most often accompanied by cognitive impairment. Sometimes, the loss of higher mental functions may dominate the clinical picture and significantly impact patient functioning . For manKlotho is a relatively recently described transmembrane protein related to β-glucuronidase, which regulates aging . Klotho-Because considerable evidence has confirmed the importance of Klotho in both depression and cognitive impairments, which often coexist, it is suggested that Klotho may be a molecular factor linking these disorders .This review aimed to discuss the role of the Klotho protein in the pathogenesis of depression and cognitive impairments. Eligible studies were identified by searching web-based databases  using the following search terms: Klotho; depress*; bipolar*; cognitive funct*; cognitive impair*; Nrf2 depress*; glutamate; NMDA receptor; MDD; BD. In addition, reference lists from the retrieved articles were reviewed.We hypothesize a strong connection between Klotho with glutamatergic neurotransmission and oxidative stress. Moreover, Klotho is a promising molecular target for future pharmacotherapy of depression, especially in patients with severe cognitive impairment.KL gene resulted in increased myelin formation [KL gene expression is associated with an improvement in cognitive processes and a reduction in OS markers [Klotho is primarily produced in the kidney . In addition, it is present in the brain parenchyma, reproductive organs, pancreas, blood vessels , parathyroid glands, bladder, intestines, muscles, and inner ear ,23,24,25ormation . Klotho ormation ,33, accoormation . On the  markers ,36.KL gene located on the long arm of chromosome 13 (13q12) in humans [KL gene in humans, mice (on chromosome 5), and rats (on chromosome 12), it shows high cross-species similarity  but lacks the TATA box, which is typical for eukaryotic genes [KL expression [KL gene is also tumor necrosis factor-alpha (TNF-α), which inhibits its transcription by producing the nuclear factor κ-light chain enhancer of activated B-cell (NF-kB). TNF-α induces the translocation of IKB-α  from NFkB-β, which allows NFkB-β to enter the nucleus [KL gene regulation has been widely discussed [Klotho belongs to the type I transmembrane proteins encoded by the n humans ,39,40,41pression ,43. An i nucleus ,45; thisiscussed ,53,54,55iscussed ,57.Human Klotho is a single-pass transmembrane protein (1012 aa) consisting of a large extracellular domain (residues 34–981), a helical transmembrane region (residues 982–1002), and a short intracellular domain (residues 1003–1012). In the extracellular domain, the protein contains two regions of glycosyl hydrolase 1: KL1 (residues 57–506) and KL2 (residues 515–953), which may have weak glycosidase activity ,58,59,60The best-known physiological function of alpha Klotho is the interaction with fibroblast growth factor receptors , significantly increasing their affinity to FGF23 (fibroblast growth factor 23). This indicates that both membrane-bound and soluble Klotho forms may act as co-receptors for FGF23 . GeneralResearch on the significance of Klotho in depression is relatively rare , which is surprising given that depression is associated with accelerated cellular aging and aging-related phenotypes and comorbidities seen in Klotho deficiency. In addition, Klotho is present in brain structures closely related to the pathophysiology of depression  and is essential in the regulation of oxidative and inflammatory processes  ,27,28,29KL gene in the NAc resulted in depression-like changes in naïve mice. In contrast, its overexpression induced an antidepressant-like effect in normal mice and improved behavioral responses in CSDS-susceptible mice, which was associated with the modulation of the GluN2B-containing NMDA receptor. KL gene knockout resulted in a selective reduction in GluN2B expression comparable to that observed in CSDS-susceptible mice. In addition, overexpression of Klotho in the NAc reversed the reduction in GluN2B expression, accompanied by a change in synaptic and structural plasticity in the NAc of CSDS-susceptible mice. The use of a specific GluN2B antagonist  abrogated the beneficial effects of increasing Klotho level in CSDS-susceptible mice [KL gene expression in hippocampal neurons is regulated by estrogen, which translates into the resistance of female rats to 3-week chronic unpredictable mild stress. CUMS induced depression-like behavioral changes  only in male rats , and these changes were correlated with decreased Klotho protein levels in the hippocampus. AAV-mediated downregulation of Klotho expression in the hippocampus of rats (both sexes) decreased stress resilience, which was observed in both females and males, being particularly pronounced in females. These results confirm that Klotho is an important factor in sex differences in stress resistance. The same study also showed that Klotho is a significant factor in the estrogen-dependent increase in the number of presynaptic vesicular glutamate transporter 1 (Vglut1)-positive clusters on the dendrites of hippocampal neurons [Depression is multifacial and conditioned by many factors, among which stress is considered one of the most important predisposing factors . The firble mice . Moreove neurons ,75  and peroxiredoxin 1 (Prx-1) and increases Nrf2 expression and the levels of reduced glutathione (GSH) in human aortic smooth muscle cells (HASMCs) [2 (Prx-2) [Confirmation of the hypothesis that Klotho may be related to the pathophysiology of depression comes from an increasing number of human studies that indicate changes in Klotho protein in the cerebrospinal fluid and blood. Prather et al. (2015) showed for the first time a relationship between severe chronic stress and reduced serum Klotho levels in women . Similarll types ,91 (see (HASMCs) . On the (HASMCs) . In diab (Prx-2) ,93. More (Prx-2) . This al (Prx-2) . Immune  (Prx-2) . Therefo (Prx-2) . Moreno  (Prx-2) . Regardi (Prx-2) . Additio (Prx-2) ,98. Thes (Prx-2) . An incr (Prx-2) . A betteGao et al., 2021 suggest that peripheral changes in Klotho protein concentration are more important for elderly MDD patients. While young MDD patients  showed no changes in plasma α-Klotho levels, a significant decrease was observed in elderly MDD patients, associated with increased disease severity and the number of T rs9315202 alleles. Moreover, young MDD patients manifested a significantly earlier age of onset, higher α-Klotho levels, and lower HAMD  scores compared with elderly MDD patients . SimilarIn patients with bipolar disorder (BD), higher serum Klotho levels have been reported than those in the MDD group . SimilarKL gene in response to selective serotonin reuptake inhibitor (SSRI) treatment in patients (>65 years) diagnosed with late-life MDD [Growing evidence indicates that Klotho may be related to the effectiveness of antidepressant therapy, both pharmacologically and non-pharmacologically. Although Sartorius et al. (2019) showed no effect of 4-week administrations of escitalopram, venlafaxine, and buprorion on Klotho levels, another research team found a strong association between two single-nucleotide polymorphisms (SNPs) of the life MDD ,110. Siglife MDD . The implife MDD . These olife MDD . CSF Klolife MDD . Furtherlife MDD . Similarlife MDD . Howeverlife MDD ,112,113 atients >5 years dlife MDD ,74. It should be noted that the positive effects of Klotho on cognition and synaptic plasticity have been documented in young and old animals and a model of AD . It is aAs for human studies, the same authors conducting a meta-analysis found a correlation between the KL-VS Klotho genetic variant and increased cognition in 718 mostly Caucasian individuals . The obsIf the mechanisms of enhanced cognitive performance are deliberate, several scenarios should be considered. It should be noted that Klotho does not cross the blood–brain barrier (BBB) when delivered peripherally. At the same time, preclinical studies have observed improvements in cognitive performance . Moreove2+ homeostasis [L in the hippocampus [Several other scenarios related to cognitive improvement and Klotho can be considered: mitochondrial mechanisms with oxidative stress, myelin metabolism, and changes in Caeostasis . In a moeostasis . Subsequeostasis . Klotho eostasis . In addipocampus . α-Tocoppocampus . This stpocampus . When hupocampus , indicatpocampus . These mpocampus .According to Shafie et al. (2020), mechanisms associated with calorie restriction  establish a vital role of Klotho in neurodegeneration. Klotho levels and its co-receptor—FGF23—were elevated in the hippocampus and prefrontal cortex of rats fed high-protein, low-calorie, and low-calorie–high-protein diets . In the 2 promoter [Gao et al. 2021) proposed an interesting concept: Klotho may represent a neurobiological link between depression and dementia. According to this, depression may be a risk factor for AD. The reduction in Klotho during depression through oxidative stress and inflammatory mechanisms paves the way for the development of cognitive impairment observed in AD [ proposedGlutamate (Glu) is a primary excitatory neurotransmitter in the nervous system. In synaptic transmission, Glu is released by the presynaptic terminal and binds to specific ionotropic  and metabotropic (mGlu) receptors located in the postsynaptic membrane, producing various effects in neuronal networks . A growiThe first target, linked to Klotho function and glutamatergic activity, is related to excitatory amino acid transporters (EAATs). EAATs comprise a class of five transporter isoforms (EAAT1–5) that are mainly expressed in neurons (EAAT3–4) and glial cells (EAAT1–2) of the central nervous system. The primary function of EAATs is glutamate transport (and uptake) across the plasma membrane . AbnormaThe ability to regulate the function of AMPA and NMDA receptors is another molecular pathway through which Klotho may be involved. Key evidence  have demonstrated the lack of described molecular mechanisms that regulate Klotho in the central nervous system. Most human studies have focused on peripheral (serum) changes in Klotho levels in depression. The size of the group, study design, medical history, and parameters of post-mortem tissue (such as pH) can limit the relevance of funding. Importantly, defining the baseline level of Klotho in depression (independent of age) is needed.


Crinum jagus (48.06 ± 3.35%) and Eucharis bonplandii (45.79 ± 3.05%) at 30 µg/mL. The research focused on evaluating the identified alkaloids on the Bcl-2 protein family (Mcl-1 and Bcl-xL) and HK2, where the in vitro, in silico and statistical results suggest that powelline and buphanidrine alkaloids could present cytotoxic activity. Finally, combining experimental and theoretical assays allowed us to identify and characterize potentially useful alkaloids for cancer treatment.Cancer is a major cause of death and an impediment to increasing life expectancy worldwide. With the aim of finding new molecules for chemotherapeutic treatment of epidemiological relevance, ten alkaloid fractions from Amaryllidaceae species were tested against six cancer cell lines  with HaCat as a control cell line. Some species determined as critically endangered with minimal availability were propagated using in vitro plant tissue culture techniques. Molecular docking studies were carried out to illustrate binding orientations of the 30 Amaryllidaceae alkaloids identified in the active site of some molecular targets involved with anti-cancer activity for potential anti-cancer drugs. In gastric cancer cell line AGS, the best results (lower cell viability percentages) were obtained for  Cancer is a major cause of death and a severe impediment to increasing life expectancy worldwide . Breast,Nowadays, chemotherapy, immunotherapy, radiotherapy, and surgery are traditional methods that are still considered effective ,10. SomeCatharanthus roseus) are used for myeloid leukemia treatment [Camptoteca accuminata) are used to treat lung, colon, and breast cancer, and homoharringtonine  to treat chronic myeloid leukemia [Narcissus poeticus L., a species of Amaryllidoideae useful to treat uterine tumors [In the last 40 years, cancer drug development has led to the discovery of many bioactive compounds from natural resources, of which 46% of anticancer-approved drugs have been provided ,20. In areatment . In the leukemia . Naturalleukemia ,24. Seveleukemia . These aleukemia . These pleukemia . Early he tumors . Recentle tumors .Crinum jagus (J. Thomps.) Dandy, Caliphruria subedentata Baker, Caliphruria tenera Baker, Eucharis bonplandii (Kunth) Traub, Eucharis caucana Meerow, Eucharis formosa Meerow, Phaedranassa lehmannii Regel, Phaedranassa ventricosa Baker, Zephyranthes carinata Herb., and Zephyranthes puertoricensis Traub. in AGS, BT-549, MCF7, PC3, MDA-MB 231, and HEC-1B tumor cell lines and human keratinocytes, HaCat, as a control. Several of these species are native/endemic to Colombia. Due to the low availability of some plant material, some of these plants were collected and subsequently micropropagated through a biotechnological strategy. Finally, the compounds identified in the most active fractions have been docked to target the Bcl-2 family  and HK2 enzymes to predict the affinity of small compounds to potential molecular targets.With the aim of finding new molecules for chemotherapeutic treatment of epidemiological relevance, we characterized the cytotoxic activities of chemically characterized alkaloid fractions from The specimens were explored, collected, and herborized. Subsequently, the identification of the material was made using specialized bibliography and comparison with the reference collection deposited in the herbarium of the University of Antioquia (HUA). C. subedentata, C. tenera, E. caucana, and E. formosa were collected in the field and a bulb of each was selected for the micropropagation process. E. caucana showed the lowest multiplication rate corresponding to 1.5 shoots/explant, while the other three species did not show significant differences with a multiplication rate of 2.8 shoots/explant. In the rooting phase, two to three roots were obtained per shoot.The species 50 less than 30 μg/mL is a candidate for chemical characterization [E. formosa, the alkaloidal fractions did not affect the viability in the control cell line HaCat, as the percent cell viability is greater than 90%, showing their low toxicity to the control line. A percentage equal to or below 50% of cell viability was taken as a control point.The cytotoxic effects of the ten alkaloid fractions of Amaryllidaceae species  were assrization ,30. As iE. caucana was the only fraction that showed significant results (<50% cell viability) for the MFC-7 cell line. For the rest of the breast cancer cell lines, Z. puertoricensis fraction showed the best activity for BT-594 (51.40 ± 1.42%) and E. formosa for MDA-MB-231 (54.81 ± 4.61%). It is important to clarify that although E. formosa presented anti-proliferative activity against AGS and MDA-MB 231, it was the cell line that presented the lowest viability for the HaCat control line (<90%). However, the best results (lower cell viability percentages) were obtained for C. jagus (48.06 ± 3.35%), C. subedentata (52.15 ± 2.10%), and E. bonplandii (45.79 ± 3.05%) in the AGS cell line. Therefore, using this cell line, IC50 values were determined through the MTT assay for some Amaryllidaceae alkaloids such as haemanthamine (13.18 ± 0.47 μg/mL or 43.74 ± 1.56 μM) and lycorine (4.17 ± 0.18 μg/mL or 14.51 ± 0.62 μM). In addition, doxorubicin (5.73 ± 0.80 μg/mL) was used as a positive control.In the case of the PC3 and HEC-1B cell lines, none of the alkaloidal fractions showed significant cell death results, with survival percentages above 70%. These results suggest that under the conditions in our laboratory, and with an acute treatment for 24 h at a concentration of 30 µg/mL, some alkaloidal fractions are selective and present cytotoxic potential against certain types of cancer cell lines, without compromising the safety in this case, of immortal keratinocytes (HaCat cell line). Interestingly, 80% of the alkaloidal fractions that were active are native/endemic species from Colombia, showing the chemotherapeutic potential of the chemistry of Colombian Amaryllidaceae in molecular targets of pharmacological interest.C. subedentata  and Z. carinata , with 8-O-demethylmaritidine, haemantamine, hamayne, galanthamine, and lycorine alkaloids being the major contributors in C. subedentata.All examined alkaloid fractions were chemically characterized by the presence of their different profiling of Amaryllidaceae alkaloids using GC/MS. The analysis of alkaloid identification was performed by comparing the fragmentation pattern reported in the literature and database of Amaryllidaceae alkaloids. C. jagus is characterized by the production of an exclusive group of crinane-type alkaloids corresponding to crinine acetate, buphanidrine, crinamine, and powelline, which were identified only in this species. According to the Eucharis genus, it was found that E. caucana produces a total of 14 alkaloids followed by E. bonplandii with 12 and E. formosa with ten. This last species is the major producer of crinane, galanthamine, and lycorine-type alkaloids with values of 0.649, 0.933, and 2.400 mg/g DW, respectively. E. caucana produces the greatest diversity of alkaloids with the exclusive presence of 6-O-methylpretazettine, narwedine, and N-formylnorgalanthamine. A total of nine alkaloids were identified in Phaedranassa plants, the species with the lowest diversity and production of alkaloids being crinane, galanthamine, and lycorine-type with values of 0.015, 0.019, and 0.119 mg/g DW for P. lehmannii and 0.054, 0.015, 0.013 for P. ventricosa, respectively. In the alkaloid analysis of Zephyranthes species, fourteen alkaloids were identified in Z. carinata and eight in Z. puertoricensis, with the exclusive presence of galanthine, lycoramine, aulicine, norlycoramine, and assoanine (identified only in Zephyranthes species).The species C. subedentata, C. tenera, E. caucana, P. ventricosa, and Z. carinata correspond to the crinane-type alkaloids: crinine (X1), haemanthamine (X6), hamayne (X10), and deacetylcantabracine (X11), followed by type-lycorine alkaloids: 11,12-dehydroanhydrolycorine (X19), 5,6-dihydrobicolorine (X13) and galanthine (X30). Similarly, galantamine-type alkaloids such as sanguinine (X14), lycoramine (X27), and galanthamine (X12) are differentiating molecules for these species. These results show that multivariate statistical analysis allows the grouping of Amaryllidaceae species concerning their alkaloid content.The PCA was performed using information from the Amaryllidaceae species and their alkaloidal composition . The PCAC. Jagus, C. subedentata, E. bonplandii, and E. formosa in gastric cancer cell line AGS and breast cancer cell line MDA-MB-231, with higher cytotoxic potential in AGS cells. Therefore, the resulting cytotoxic data expressed as percentage values of the cell growth relative to the control were used to develop multivariate statistical analyses; in particular, the supervised partial least square discriminant analysis (PLS-DA) was utilized to establish a correlation between the alkaloid profile and cell viability in gastric cancer cell line AGS  and 8-O-demethylmaritidine (X2) present in C. subedentata and E. bonplandii. Finally, 8-O-demethylmaritidine (X2) was identified in E. formosa. Thus, crinane-type alkaloids could be related to possible cytotoxic effects on gastric cancer cells.The screening of alkaloid fractions in cancer cell lines showed that the most promising species were line AGS . The maxMolecular docking analysis is frequently a useful tool for predicting the affinity of small compounds toward potential molecular targets through the study of binding orientation. E. grandiflora by direct organogenesis showed that from a mother bulb, it is possible to obtain 3.8 shoots/explant and 133 plants in 5 months [C. subedentata, C. tenera, E. caucana, and E. formosa, which allows obtaining more than 1000 plants in 6 months, allowing the initiation of habitat enrichment programs with these species. Additionally, it is necessary to develop protocols to stimulate the production of alkaloids with biological potential, which are necessary for many species of Amaryllidaceae.Amaryllidaceae is one of Colombia’s rarest and most striking plant families and is endangered due to habitat destruction . In vitrC. jagus shows that this species produces an exclusive group of crinane-type alkaloids in combination with lycorine-type alkaloids, which agrees with previous phytochemical reports [E. bonplandii, E. caucana, P. lehmannii, and Z. carinata were shown to produce several crinane, galanthamine, and lycorine-type alkaloids [C. tenera, E. formosa, P. ventricosa, and Z. puertoricensis. C. jagus and Z. puertoricensis. In general, all these species were characterized by presenting a notable diversity of alkaloids, the vast majority with all types of alkaloids identified, including miscellaneous-type alkaloids. Therefore, the wide diversity of Amaryllidaceae alkaloids  , anti-apSome alkaloids presented low interaction energy with the Mcl-1 protein ; Table 4O-methylpretazettine is stabilized by six hydrogen bridge-type interactions with Gln608, Val655, Phe602, Phe604, Pro513 and Ser603  for 2 min and then with 1% sodium hypochlorite  for 20 min. The modified “twin-scales” technique [®, Lenexa, KS, USA) [®, Lenexa, KS, USA) and naphthaleneacetic acid  was used, and rooting was carried out in MS medium with activated charcoal . Plantlets were acclimatized under nursery conditions.The species corresponding to echnique  was usedKS, USA) , supplem2SO4 . Neutral compounds were removed with hexane , and the accusation phase was based on NH4+OH− . The alkaloids were extracted from the aqueous phase with chloroform , and the fraction of alkaloids obtained was stored at 4 °C until the time of analysis.Bulbs of wild and micropropagated plants were collected, washed, cut into small portions, and dried at 40 °C for 48 h. The dehydrated bulbs were extracted with methanol : water (1:1) by extraction assisted by bath ultrasound at a temperature of 40 °C for 30 min ,40. The Alkaloid analyses were carried out by GC/MS, according to the methods described by Cortes et al. . For theThe alkaloids were identified by comparison of fragments of the mass spectra of the Amaryllidaceae alkaloid spectrotheque of the AgroBio Institute  and information from scientific databases. The compounds were also identified by comparing the mass spectral fragmentation of the compounds with standard reference spectra from the NIST 17 database . The compounds’ Kovats Retention Index (RI) was recorded with a standard calibration mixture of n-hydrocarbons . The percentage of TIC  was estimated for each alkaloid. The abundance of each compound was calculated using codeine (50 μg/mL) as an internal standard. The area of GC/MS peaks depends on the concentration of the related compounds and the intensity of their mass spectral fragmentation.™), prostate cancer cell line PC3 (CRL-1435™), breast cancer cell lines BT-549 (HTB-122™), MCF7 (HTB-22™) and MDA-MB 231 (HTB-26™), uterine cancer cell line HEC-1B (HTB-113™) and human keratinocytes HaCat (PCS-200-011™) as a non-carcinogenic control cell line  [2 and 95% air at 37 °C. Cells were monitored on a Nikon Eclipse TS100  inverted phase contrast microscope. Cell viability experiments were performed when cells reached 75–80% confluence using 0.25% trypsin 1 mM EDTA .Cell viability screening of alkaloid fractions of the ten species of Amaryllidaceae was performed in gastric cancer cell line AGS (CRL-1739m ATCC®) ,59. The 4 cells/well for 24 h. After incubation, the medium was changed, and the cells were treated with the alkaloid fraction at a concentration of 30 µg/mL, according to the cytotoxic activity criteria for a crude extract as established by the American National Cancer Institute (NCI) in IC50 < 30 µg/mL. This criterion for a crude extract has recently been used in research as a screening parameter [® cell viability reagent  was added to each well. The plate was left agitating for 5 min to induce cell lysis, and the plate was incubated at room temperature for 15 min to stabilize the luminescent signal. Relative luminescence units were measured using the GloMax 96 microplate luminometer . Cell viability was expressed as a percentage of control values set at 100%.Cell viability was determined by bioluminescence assay, based on the quantification of ATP present, as an indicator of metabolically viable cells . The celarameter ,30, maki2 for 48 h. Doxorubicin  was used as a positive control . Subsequently, 50 μL of MTT  was added. After 4 h of incubation at 37 °C, the MTT culture medium was removed and replaced with 150 μL DMSO  to dissolve the formazan crystals. Plates were incubated in dark with agitation for 2 h. Optical density was determined at 540 nm using a microplate reader. All experiments were conducted with three independent trials, each with six replicates.Cells viability was also determined using 3--2,5-diphenyltetrazolium bromide  assay. Lycorine  and haemanthamine compounds were evaluated at different concentrations  in the cell line with the lowest percentage of cell viability (AGS) and incubated at 37 °C with 5% COhttps://www.schrodinger.com/citations#Maestro (accessed on 1 March 2023), belonging to the Schrodinger suite. In this process, the hydrogen atoms were added, and the protonation states were checked for a pH between 7.0 ± 2.0. Three-dimensional (3D) experimentally known structures of the anti-apoptotic Bcl-2, Bcl-xL, and Mcl-1 proteins were obtained from the Protein Data Bank (PDB), pdb codes: 2W3L [The molecular docking analysis of the Amaryllidaceae alkaloids has been carried out using the Autodock 4.2 program . The tries: 2W3L , 2YXJ [6es: 2W3L , and 3KZes: 2W3L , respectes: 2W3L . The reaes: 2W3L ,67,68,69es: 2W3L . For thip ≤ 0.05 and with respect to the control.The results of cell viability are shown as the mean ± SD. The statistical significance between the control group and the treatments with the alkaloid fraction were evaluated by analysis of variance (one-way ANOVA) followed by the Newman–Keuls multiple-comparison test, using GraphPad Prism version 5.00  data analysis system. In addition, multivariate principal component (PCA) and partial least squares (PLS-DA) statistical analyses were performed using the MUMA package at RStudio . The differences were considered significant for Crinum jagus and Eucharis bonplandii against the gastric cancer cell line AGS. Our results suggest that the cytotoxic activity might be associated with lycorine and crinine-type alkaloids, which is consistent with previous studies. In addition, this is the first report of phytochemical data for C. tenera, E. formosa, P. ventricosa, and Z. puertoricensis. The research focused on evaluating the identified alkaloids in the Bcl-2 protein family (Mcl-1 and Bcl-xL) and HK2, where the in vitro, in silico, and statistical results suggest that powelline and buphanidrine alkaloids could present cytotoxic activity. More tangible evidence is required based on testing the activity of isolated compounds. Finally, combining experimental and theoretical assays allow us to identify and characterize potentially useful alkaloids for cancer treatment.In summary, the present study explored the cytotoxic activity of ten Amaryllidaceae species, characterized by the content of their alkaloids, against six cancer cell lines. The best results (lower cell viability percentages) were obtained for


AHR and ELAVL1 genes were silenced by siRNA transfection. The RNA and protein were extracted for quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot (WB) analysis. Direct binding between the ELAVL1 protein and AHR mRNA was examined through immunoprecipitation (IP) assay. Cell viability, clonogenicity, and migration assays were performed. Our study revealed that both AHR and ELAVL1 inter-regulate each other, while also having a role in cell proliferation, migration, and chemoresistance in PDAC cell lines. Notably, both proteins function through distinct mechanisms. The silencing of ELAVL1 disrupts the stability of its target mRNAs, resulting in the decreased expression of numerous cytoprotective proteins. In contrast, the silencing of AHR diminishes cell migration and proliferation and enhances cell sensitivity to gemcitabine through the AHR-ELAVL1-deoxycytidine kinase (DCK) molecular pathway. In conclusion, AHR and ELAVL1 interaction can form a negative feedback loop. By inhibiting AHR expression, PDAC cells become more susceptible to gemcitabine through the ELAVL1-DCK pathway.The aryl hydrocarbon receptor (AHR) is a transcription factor that is commonly upregulated in pancreatic ductal adenocarcinoma (PDAC). AHR hinders the shuttling of human antigen R (ELAVL1) from the nucleus to the cytoplasm, where it stabilises its target messenger RNAs (mRNAs) and enhances protein expression. Among these target mRNAs are those induced by gemcitabine. Increased AHR expression leads to the sequestration of ELAVL1 in the nucleus, resulting in chemoresistance. This study aimed to investigate the interaction between AHR and ELAVL1 in the pathogenesis of PDAC in vitro.  Pancreatic cancer (PC) is a devastating disease that has a less than 10% five-year survival rate , with paOne of the biggest problems in PC treatment is its heterogeneity and resistance to first-line drugs. Resistance to chemotherapy can be innate  or acquired  , with thTherefore, we hypothesise that the modulation of AHR and ELAVL1 expression can decrease the chemoresistance of PDAC cells in vitro.AHR and ELAVL1 genes were silenced almost completely after 24 h in both lines. In BxPC-3, siAHR decreased AHR mRNA to 20% and the protein was not detected; in Su.86.86, AHR mRNA decreased to 29% and the protein decreased to 2% compared with nontreated control cells.Human PDAC cell lines  were transfected with small interfering RNAs (siRNAs): siAHR or siELAVL1 for 24 h. Western blot analysis revealed that the ELAVL1 in BxPC-3 cells, the ELAVL1 mRNA levels decreased to 31%, and the ELAVL1 protein levels decreased to 38% compared with nontreated control cells. Similarly, in Su.86.86 cells, the ELAVL1 mRNA levels decreased to 9%, and the ELAVL1 protein levels decreased to 15% compared with nontreated control cells. These results indicate successful silencing of both the target mRNA and the subsequent protein expression for ELAVL1   . The samELAVL1 silencing caused a decrease in AHR mRNA (in BxPC-3 AHR mRNA decreased significantly to 76% and in Su.86.86 to 77%) as well as in protein (in BxPC-3 AHR protein was absent and in Su.86.86 decreased to 73%).These findings suggest a mutual influence and potential regulatory relationship between AHR and ELAVL1. As ELAVL1 primarily exerts its activity in the cytoplasm, it was important to investigate the mRNA and protein expression levels of its downstream targets, specifically DCK and HMOX1.AHR increased Heme oxygenase 1 (HMOX1) mRNA (4.45-fold) and protein (1.4-fold) in BxPC-3 cells; however, Su.86.86 showed no changes in either mRNA or protein of HMOX1 (DCK mRNA (1.6-fold) and protein slightly (1.1-fold) in the BxPC-3 cell line; however, it did not change DCK mRNA and even decreased protein levels (to 56%) in the Su.86.86 cell line.The silencing of of HMOX1 . SiAHR aELAVL1 decreased HMOX1 mRNA  and protein  in both cell lines. DCK mRNA did not change in either cell line; however, the DCK protein decreased in both .The silencing of These results show that ELAVL1 pathway genes and proteins somewhat correspond to changes in AHR and ELAVL1 expression levels; however, it is more noticeable for protein level and differs between cell lines.AHR mRNA and the ELAVL1 protein. ELAVL1 is known to bind to various mRNAs, including DCK [HMOX1 [AHR gene has nine adenylate uridylate (AU)-rich elements (AREs) (ATTTA), which is used as a binding motif for ELAVL1 protein [AHR mRNA .AHR mainly resides in the cytoplasm of both PDAC cell lines. The shift of AHR protein was not noticeable in either cell line when silencing ELAVL1 and/or treating with GEM (photos not included).AHR silencing and GEM treatment increased cytoplasmic ELAVL1 concentrations even more than separately, indicating that the silencing of AHR could be beneficial to ELAVL1 translocation in response to GEM treatment  but decreased protein levels in both cell lines   and protein .GEM treatment significantly increased  to 38%) . TreatmeAHR prior to GEM treatment, it increased BxPC-3 ELAVL1 mRNA (1.46-fold) and protein (2.6-fold); however, Su.86.86 ELAVL1 mRNA did not change, and protein decreased to 74%. Silencing ELAVL1 prior to GEM treatment increased AHR mRNA in both cell lines  but greatly decreased AHR protein .When silencing ELAVL1 pathway genes, specifically HMOX1 and DCK.These findings highlight the differences observed between mRNA levels and protein expression levels, as well as variations across different cell lines. These differences suggest the involvement of post-transcriptional regulatory mechanisms in the cellular response to gemcitabine. To gain a deeper understanding of these distinctions, we conducted an analysis of HMOX1 mRNA ; however, an increase in protein was much less noticeable , as well as DCK mRNA  and protein  (GEM greatly increased .3-fold) .AHR prior to GEM treatment, HMOX1 mRNA increased ; however, the HMOX1 protein increased in BxPC-3 (1.5-fold) and decreased in Su.86.86 (to 75%). A similar pattern was also seen with DCK mRNA and protein levels. DCK mRNA increased in both cell lines ; however, protein levels only increased in BxPC-3 (2.5-fold) and did not change in Su.86.86.When silencing ELAVL1 prior to GEM treatment increased HMOX1 mRNA  and protein . DCK mRNA was also increased , as well as protein levels .The silencing of HMOX1 and DCK, respond to gemcitabine (GEM) treatment. However, notable differences exist between mRNA and protein expression levels of AHR, suggesting the involvement of post-transcriptional regulation mechanisms that affect the translation of AHR mRNA into protein. Furthermore, significant differences were observed between the cell lines, indicating the presence of additional mechanisms that contribute to the observed variations.The observed changes indicate that both AHR and ELAVL1 genes and proteins, as well as the ELAVL1 pathway genes AHR or ELAVL1 gene silencing on cell chemoresistance, AHR or ELAVL1 genes were silenced for 24 h by transfection with siAHR or siELAVL1. After silencing, the cells were treated with an IC50 dose of GEM for 48 h. The metabolic activity of all groups was compared with the control group (100%) of nontreated cells for GEM alone or siControl for transfected cells. GEM alone significantly reduced cell viability to 57% for the BxPC-3 cell line and 48.4% for the Su.86.86 cell line. There was no significant effect on BxPC-3 or Su.86.86 cell viability after the silencing of AHR or ELAVL1. However, the silencing of AHR together with GEM significantly decreased the viability of both cell lines when compared with GEM alone (BxPC-3 to 25.8% and Su.86.86 to 23.1% cell viability). In BxPC-3, the silencing of ELAVL1 together with GEM did not have a significant effect on cell viability when compared with GEM alone; however, in Su.86.86, the combined effect of siELAVL1 and GEM when compared with GEM alone significantly decreased cell viability to 23.1% . The silencing of ELAVL1 had a significant effect on BxPC-3 colony formation (colony formation decreased to 74.7%) but had no effect on Su.86.86 cell line colony formation. The silencing of AHR significantly increased the effect of GEM (BxPC-3 to 3.7% and Su.86.86 to 11.3%); however, due to severe effects on both GEM and siAHR, it is unclear whether the effect is due to increased sensitivity to GEM or the additive effect of siAHR and GEM. The silencing of ELAVL1 did not significantly change the effects of GEM on either cell line.The ability of cell lines to form colonies was measured by clonogenicity assay . The effAHR significantly reduced BxPC-3 cell line migratory abilities, and 36.2% of the wound was closed after 24 h. On the other hand, the silencing of ELAVL1 had no significant effect on the migration of BxPC-3 cells. The silencing of AHR or ELAVL1 and treatment with GEM had a significant effect when compared with controls (siAHR+GEM—16.5% and siELAVL1+GEM—19.7% of the wound closed); however, neither siAHR nor siELAVL1 increased GEM effect on migratory abilities. Su.86.86 , and it did not increase the effects of GEM on cell migration.Migratory ability was measured by the wound healing (scratch) assay . After 2Su.86.86 b,d had aKRAS [MUC4 [TP53 [miR-106b [miR-181b [miR-21 [miR-29a [Chemoresistance of pancreatic cancer is a major problem limiting the success of chemotherapeutic treatments in patients. There are plenty of suggested mechanisms that cause PC resistance to chemotherapy, including common genetic mutations such as those in the KRAS ,25, MUC4AS [MUC4 , and TP5C4 [TP53  genes. MmiR-106b , miR-181miR-181b , miR-21  [miR-21 , and miR[miR-29a  have als[miR-29a  or heat [miR-29a  and many[miR-29a ,21,22, i[miR-29a  and in t[miR-29a ,35,36. T[miR-29a . TherefoAHR mRNA is a direct target for the ELAVL1 protein, and this interaction stabilises AHR mRNA, thus possibly increasing protein synthesis. AHR has been shown to sequester ELAVL1 in the nucleus [AHR. By silencing AHR mRNA and in turn protein synthesis, we were able to show an increase in ELAVL1 expression as well as its localisation shift towards cytoplasm, which agrees with previous studies. However, the results of the expression of ELAVL1 pathway genes and proteins are conflicting, and both cell lines had different changes in some cases, showing that the effect of this modulation is hard to predict and can be different depending on the cell characteristics. By silencing ELAVL1, we showed that AHR mRNA and protein levels decrease, proving again that AHR can be a post-transcriptional target of ELAVL1. This complex interaction between AHR and ELAVL1 shows that they can alter the expression of each other by forming a negative feedback loop, which has never been shown before.Our results show that  nucleus , which iAHR greatly reduces cell migration and colony formation and might ease ELAVL1 translocation from the nucleus to the cytoplasm. However, different responses of ELAVL1 pathway genes and differences between cell lines show that there are more mechanisms involved in this regulation, warranting further investigation into the relationship between AHR and ELAVL1. Increased AHR expression can be seen in many different malignancies, for example, lymphoma [Due to the fact that AHR is a transcription factor and ELAVL1 is a post-transcriptional gene expression regulator, this interaction and its changes involve many different cell mechanisms involved in cytoprotection, migration, overall viability of the cell, and most importantly to PC treatment chemoresistance. Our results show that by lowering AHR or ELAVL1 expression, the cells become more susceptible to gemcitabine; however, the mechanisms most likely differ. The silencing of lymphoma  and leuklymphoma , as welllymphoma , kidney lymphoma , gastroilymphoma , and panlymphoma ,21. Sinclymphoma ,40,43, aELAVL1 has a much lesser effect on cell migration or colony formation than AHR; however, our results show that it decreases HMOX1 mRNA levels and DCK and HMOX1 protein levels. This lowers the protective mechanisms of the cell but can also increase the chemoresistance of the cell. Lowering ELAVL1 was also shown to decrease the synthesis of other protective proteins, such as COX-2 [MAPK and JNK signalling pathways and cause breast cancer resistance to tamoxifen [ELAVL1 was also shown to increase the response to chemotherapy, although it was attributed to a decrease in cytoprotective proteins rather than a direct influence on gemcitabine activation [The silencing of as COX-2  or IFN-βas COX-2 , making as COX-2 , prostatas COX-2 , and othamoxifen ; its cytamoxifen . In pancamoxifen . It can amoxifen . The siltivation . Howevertivation ,13,14,51Since ELAVL1 can be both a good and bad marker for chemotherapy resistance, it is imperative to understand how it works and find ways to utilise it in a way that would not cause any harm. Our findings suggest a mechanism  by whichOverall, targeting AHR and decreasing its expression would ease the shuttling of ELAVL1 to the cytoplasm, which in turn would decrease cell resistance to gemcitabine and, at the same time, decrease cell colony formation and migration capabilities. Notably, the increase in ELAVL1 activity can increase cell cytoprotective mechanisms through the stabilisation of proteins such as HMOX1, although this effect depends on the cell characteristics according to our results, warranting further studies into the mechanisms involved.MAPK and JNK [Both AHR and ELAVL1 are involved in various molecular mechanisms. ELAVL1 is known to be involved in various cell signalling pathways, such as  and JNK , as well and JNK . It is a and JNK  and the  and JNK ,53. Simi and JNK  and xeno and JNK . It is a and JNK ,57. The Both AHR and ELAVL1 are involved in the immune system of the body. This poses a limitation to in vitro experiments due to absence of immune cells. Nonetheless, this novel mechanism provides a stepping stone for combating gemcitabine resistance in PC and should be investigated in in vivo setting. This would further elucidate the relationship of AHR and ELAVL1 in a setting with various other systems with which both AHR and ELAVL1 might interact.There are some limitations to our study. Only lipofectamine-mediated siRNA transfection was used, which would prove to be difficult to use for patients, and so further studies with protein inhibitors would broaden the knowledge of the AHR-ELAVL1-DCK pathway. Also, only two cell lines were used, which had some different responses to AHR and ELAVL1 modulation; so, further investigation into the mechanisms causing such differences and the stratification of cancer subtypes that would benefit most from such modifications are required. Lastly, only in vitro experiments with cell cultures were performed; so, further experiments in vivo would be necessary to prove this relationship.2-enriched environment.Two human pancreatic ductal adenocarcinoma cell lines, BxPc-3 and Su.86.86, were used for analysis. The BxPC-3 and Su.86.86 cell lines were a gift from the European Pancreas Centre . Both cell lines were grown in RPMI medium  with 10% FBS  and 1% penicillin/streptomycin solution (Gibco). Cells were grown in monolayers in sterile flasks/plates in an incubator, which maintains a moist temperature of 37 °C with a 5% CO3 cells/well; Su.86.86 1.2 × 103 cells/well) and treated with varying concentrations of gemcitabine (GEM)  ranging from 1–10,000 nM by logarithmic dilution to test the half-maximal inhibitory concentration (IC50) of GEM. Treated cells were maintained at 37 °C in a 5% CO2 incubator for 48 h. To measure the proportion of metabolic activity, the MTT -2,5 diphenyltetrazolium bromide) metabolism method was used (see ‘MTT metabolic activity assay’). No less than 4 replicates of the experiment were carried out.Cells were seeded in 96-well cell culture plates  assay. After treatment, MTT was added to a concentration of 0.5 mg/mL, and the cells were incubated for 4 h at 37 °C. After 4 h of incubation, the medium with MTT was removed and the remaining formazan product was dissolved with DMSO (dimethyl sulfoxide)  by agitation in the spectrophotometer for 60 s. The absorbance was measured with the spectrophotometer (TheSunrise (Software v7.1.), Tecan, Grodig, Austria) at a wavelength of 570 nm and reference of 620 nm.2 flasks. Lipofectamine 2000  was used according to the manufacturer’s instructions for all transfections with RPMI medium. All MTT experiments included two groups of control cells: untreated control and a control treated with an siRNA negative control. Four replicates of the experiment were carried out. Silencing efficiency after 24–72 h was evaluated by Western blot (WB) analysis.siELAVL1, siAHR, and negative control siRNA were purchased from Ambion . Transfection was performed in 96, 6-well plates or 25 cmELAVL1 (Hs00171039_m1), AHR (Hs00169233), HMOX1 (Hs01110250_m1), and housekeeping gene GAPDH (Hs02758991_g1) were from Applied Biosystems. Three replicates of the experiment were carried out.Total RNA extraction was performed from cultured cells using the RNA extraction kit  according to the manufacturer’s protocol. Purified RNA was quantified and assessed for purity by UV spectrophotometry (NanoDrop 2000 (Software v.1.4.2), ThermoFisher Scientific, Waltham, MA, USA). cDNA was generated from 2 μg of RNA with High-Capacity cDNA Reverse Transcription Kit . The amplification of specific RNA was performed in a 20 μL reaction mixture containing 2 μL of cDNA template, 1X PCR master mix, and the primers. The PCR primers used for detection of g for 10 min at 4 °C. The supernatants were assayed for protein concentration with BCA protein assay kit . Protein samples were heated at 97 °C for 5 min before loading, and 25–50 µg of the samples was subjected to 4–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), then transferred to poly-vinylidene fluoride (PVDF) membranes 40 min 30 V. Next, membranes were blocked with a 5% skimmed milk blocking buffer for 60 min at room temperature. Membranes were then incubated for 1.5 h at room temperature or overnight at 4°C with primary antibodies. The following primary antibodies were used: 1:100 mouse monoclonal anti-ELAVL1 ; 1:1000 mouse monoclonal anti-AHR ; 1:2000 rabbit monoclonal anti-HO-1 ; 1:3000 mouse monoclonal anti-GAPDH ; and 1:2000 mouse monoclonal anti-DCK . The membranes were washed with 1X Tris-Buffered Saline, 0.1% Tween 20 Detergent (TBST) antibody washing buffer or antibody wash buffer  and incubated in the appropriate peroxidase-conjugated secondary antibody solution  for 30 min or in horseradish-peroxidase-conjugated secondary antibody solution (LSbio) for 1 h. After that, membranes were washed again with TBST antibody washing buffer or antibody wash buffer  and incubated with chemiluminescence substrate  or West Pico Stable peroxidase buffer + luminol enhancer (Thermo Scientific) for 5 min. Results were analysed with a documenting system . Three replicates of the experiment were carried out; however, only one is being shown as a representative replicate.Whole cells were lysed using RIPA lysis buffer with protease inhibitors  and centrifuged at 10,000× Cells were cultivated on chamber slides for 96 h either with or without treatment. A mixture of 96% ethanol with 5% glacial acetic acid was used for fixation and 0.5% Triton X-100 in PBS- for permeabilisation. Cells were subsequently incubated with 1:250 primary mouse monoclonal ELAVL1 antibody (LSBio) or 1:500 primary mouse monoclonal AHR antibody  and 1:2000 secondary antibody-Alexa Fluor 488 Goat Antimouse IgG (H+L)  and washed with PBS followed by washing with nuclease-free water. Slides were then mounted with ProLong Diamond Antifade Mountant with DAPI  for cell nuclei staining and analysed with Olympus IX71 fluorescent microscope . Three replicates of the experiment were carried out; however, only one is being shown as a representative replicate.The colony formation of pancreatic cancer cells was evaluated using a crystal violet stain. The cells were cultivated for 96 h with or without treatment with siAHR/siELAVL1/GEM. After treatment, the cells were detached by trypsin/EDTA, counted, and seeded into 6-well culture plates at concentration of 600 cells/well. After 7 days of growth, formed colonies were fixed with 96% ethanol and stained with crystal violet stain. After staining, crystal violet was removed, and the wells were rinsed with water. Plates were dried at room temperature, the morphology of cells was observed, and colonies were counted under an Olympus IX71 phase-contrast microscope . No less than 4 replicates of the experiment were carried out.5 cells/well. After 24 h, a scratch was made with a 200 µL pipette tip, and the medium was changed into a fresh medium without FBS. The scratch was observed and photographed under an Olympus IX71 phase-contrast microscope at 0 and 24 h after making the scratch. No less than 3 replicates of the experiment were carried out.The cells were cultivated for 96 h with or without treatment with siAHR/siELAVL1/GEM. After treatment, the cells were detached by trypsin/EDTA, counted, and seeded into 24-well culture plates at concentration of 2 × 102 flasks until they reached a confluence of 80–90%. The cells were then lysed using Magna RIP  kit and the IP assay was carried out using ELAVL1 antibody  and GAPDH antibody  as a negative control. The samples for WB and qRT-PCR analysis were collected before and after immunoprecipitation. WB and qRT-PCR assays were performed as described above.Cells were cultivated in 150 cmp < 0.05.Statistical analysis was performed using GraphPad  software. The data are presented as mean ± SD of three or more independent experiments. A nonparametric Mann–Whitney test was used for comparison between groups. Statistical significance was defined as AHR silencing appears to be more pronounced than that of ELAVL1, and both proteins act through distinct mechanisms. The silencing of ELAVL1 disrupts the stability of its target mRNAs, resulting in the decreased expression of numerous cytoprotective proteins. In contrast, the silencing of AHR diminishes cell migration and proliferation and enhances cell sensitivity to gemcitabine through the AHR-ELAVL1-DCK molecular pathway.Our study revealed that both AHR and ELAVL1 inter-regulate each other, as well as having a role in cell proliferation, migration, and chemoresistance in PDAC cell lines. Notably, the effect of These findings underscore the complex interplay between AHR, ELAVL1, and important cellular processes in PDAC. The differential effects and distinct molecular pathways associated with AHR and ELAVL1 modulation highlight their potential as therapeutic targets for PDAC treatment, particularly in overcoming chemoresistance and inhibiting tumour progression. Further investigations are necessary to fully elucidate the underlying mechanisms and explore the clinical implications of targeting these pathways in PDAC management.


In the current study, to elucidate the clinicopathological significance of the appearance of in vitro three-dimensional (3D) spheroid models of oral malignant tumors, pathologically, three different squamous cell carcinoma (OSCC) cell lines and one oral adenosquamous carcinoma (OASC) cell line were included. Using these, the biological measurements of the two-dimensional (2D) cultured cells through the cellular metabolic analysis of a Seahorse bio-analyzer and the cytotoxicity of cisplatin (CDDP) resulted in the appearance of their 3D spheroids by phase contrast microscopy and others. In Seahorse real-time cellular metabolic analysis, ATP-linked respiration and proton leaking were significantly different among the four cell lines, and the malignant stages of these cultures were significantly associated with increased ATP-linked respiration and decreased proton leakage. Alternatively, the appearances of these 3D spheroids were also significantly diverse, and the increasing order of their differences was identical to the increased efficacies of CDDP-induced cytotoxicity. The current obtained findings indicate that cellular metabolic functions and 3D spheroid architectures may be valuable and useful indicators when estimating the pathological and drug-sensitive aspects of OSCC and OASC malignancies.The objective of the current study was to elucidate the clinicopathological significance and appearance of in vitro three-dimension (3D) spheroid models of oral malignant tumors that were prepared from four pathologically different squamous cell carcinoma  and oral adenosquamous carcinoma  obtained from patients with different malignant stages. To characterize the biological significance of these cell lines themselves, two-dimensional (2D) cultured cells were subjected to cellular metabolic analysis by a Seahorse bioanalyzer alongside the measurement of the cytotoxicity of cisplatin (CDDP). The appearance of their 3D spheroids was then observed by phase contrast microscopy, and both 2D and 3D cultured cells were subject to trypsin digestion and qPCR analysis of factors related to oncogenic signaling and other related analyses. ATP-linked respiration and proton leaking were significantly different among the four cell lines, and the malignant stages of these cultures were significantly associated with increased ATP-linked respiration and decreased proton leakage. Alternatively, the appearances of these 3D spheroids were also significantly diverse among them, and their differences increased in the order of LEM2, MO-1000, SSYP, and Mesimo. Interestingly, these orders were exactly the same in that the efficacies of CDDP-induced cytotoxicity increased in the same order. qPCR analysis indicated that the levels of expression of oncogenic signaling-related factors varied among these four cell lines, and the values for fibronectin and a key regulator of mitochondrial biogenesis, PGC-1α, were prominently elevated in cultures of the worst malignant Mesimo cells. In addition, although 0.25% trypsin-induced destruction was comparable among all four 2D cultured cells, the values for the 3D spheroids were also substantially varied among these cultures. The findings reported herein indicate that cellular metabolic functions and 3D spheroid architectures may be valuable and useful indicators for estimating the pathological and drug-sensitive aspects of OSCC and OASC malignancies. Oral squamous cell carcinoma (OSCC), the most common malignancy of the oral cavity, is recognized as one of the main causes of mortality worldwide, especially in developing countries ,2. DespiFor this purpose, initially, conventional two-dimensional (2D) planar cell culture methods have been used as in vitro disease models. Subsequently, however, it was recognized that the nature of these 2D cultured models was distinct from those of in vivo physiological states. This conclusion is based on several differences in changes of several gene expression profiles, intercellular binding, extracellular matrix protein interactions, and others between normal or cancerous tissues ,11. By cIn addition, it was also found that the shapes of 3D spheroids were dependent on the origin of the cells. Globe-shaped or non-globe-shaped 3D spheroids were obtained from non-cancerous cells ,20,21 orTherefore, in the current study, to elucidate the clinicopathological significance of the in vitro 3D spheroid model of OSCC, 3D spheroids were prepared using four OSCC or oral adenosquamous carcinoma (OASC) cell lines, LEM2, M0-1000, SSYP and Mesimo which were established from patients with clinically different malignant stages, and the morphological and functional aspects of these cultures were compared.2 at 37 °C. When the cells reached 80–90% confluence, they were washed with PBS and detached by treatment with a 0.05% EDTA/trypsin solution for 5–10 min at 37 °C. After centrifugation at 1500 rpm for 5 min at room temperature, the cells were resuspended in a medium and seeded in 100 mm dishes. The passage was serially performed until a cell line was established. The malignancy levels of these cell lines were estimated based on the Yamamoto–Kohama (YK) classification of invasion [Three oral squamous carcinoma cells termed SSPY, MO-1000, LEM2, and one type of oral adenosquamous carcinoma cells (OASC) termed MeSimo were uniquely established in our laboratory. In a typical experiment, tumor tissues were minced into 1–2 mm pieces with scissors and placed in a primary culture in Dulbecco’s modified Eagle’s medium/Ham’s F12 medium  supplemented with 10% fetal bovine serum  and 1% penicillin–streptomycin  in a humidified atmosphere containing 5% COinvasion  for theiinvasion ,24,27,282-free incubator at 37 °C for 1 h prior to the measurements. OCR and ECAR were simultaneously measured in an XFe96 extracellular flux analyzer at the baseline and under the following sequential injections of 2.0 μM oligomycin, 5.0 μM carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), a mixture of 1.0 μM rotenone and 1.0 μM antimycin A, and 10 mM 2-deoxyglucose. The values were normalized to the amount of protein, as determined by a BCA protein assay (TaKaRa BCA Protein Assay) in each well after the measurement was complete.The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of 2D-cultured OSCC and OASC cells, SSYP, MO-1000, LEM2, and Mesimo, were measured using a Seahorse XFe96 real-time metabolic analyzer  according to the manufacturer’s instructions. In a typical run, twenty thousand 2D-cultured cells, as described above, were placed in each well of an XFe96 Cell Culture Microplate  the day before the assay and were incubated at 37 °C. On the day of the assay, the culture medium was replaced with a Seahorse XF DMEM assay medium  containing 5.5 mM glucose, 2.0 mM glutamine, and 1.0 mM sodium pyruvate). The assay plates were then incubated in a CORegarding the morphological aspects of the above-prepared 3D spheroids, their downward and lateral views were observed by phase contrast microscopy, as reported in our previous study ,24,29. T−2, 1.0 × 10−1, 1.0 × 100, 1.0 × 101 or 1.0 × 102 μM concentrations for 72 h. To determine the survival rates, after incubation with 10 μL of a reactive solution of the commercially available kit  for 2 h, the absorbance at 450 nm was measured using a microplate reader .To estimate the cytotoxic effects against cisplatin (CDDP), four different 2D cultured OSCC or OASC cell lines; SSYP, MO-1000, LEM2, and Mesimo (5000 cells/well) were exposed to CDDP at 0, 1.0 × 10To compare the trypsin-induced destruction of the 2D and 3D cultured cells among 4 OSCC and OASC cells, the period required for them to disperse against 0.05% trypsin was determined using phase contrast microscopy.t-test for the two-group comparison or one-way ANOVA followed by Tukey’s multiple comparison tests was used.Quantitative PCR using specific primers  and statIn the current study, we established four OSCC or OASC cell lines that originated from patients with pathologically different malignant levels , proton leakage increased and an FCCP-induced increase in oxygen consumption was impaired, suggesting that these lesser malignant cells damaged the mitochondria. Glycolytic capacity was relatively low in low-grade SSYP cells but remained at high levels in all four OSCC cell lines. These associations between malignancy and cellular metabolism may reflect the fact that these cells have a high energy demand to continuously proliferate in squamous carcinoma cells. Interestingly, Mesimo, which had the highest mitochondrial respiratory capacity, was the most resistant to the anticancer agent CDDP. We (and others) have reported the possibility that enhanced mitochondrial respiration or mitochondrial adaptation can be associated with a variety of anticancer drug resistance in cancer cells ,33. ThusECM proteins are important molecular components that are involved in cellular architectures and have several signaling properties ,37,38. THowever, current conclusions remain speculative at present, and the following study limitations need to be investigated regarding unidentified issues, including the relationship between the appearance of the 3D spheroids and several additional clinical, physiological, and pharmacological aspects. Thus, to elucidate the additional significance of the in vitro 3D spheroid model regarding the pathogenesis of oral malignant tumors in more detail, additional investigations with the objective of identifying additional regulatory factors and mechanisms using approaches such as RNA sequencing and others will be our next project.In the current study, we described the clinicopathological significance of newly developed in vitro 3D spheroid models of oral malignant tumors using pathologically different OSCC and OASC cell lines. Several biological measurements, including a real-time cellular metabolic analysis, the cytotoxicity of cisplatin (CDDP), the appearance of their 3D spheroids, and others resulted in cellular metabolic functions and 3D spheroid appearance, which could become pivotal and useful bio-indicators for evaluating clinicopathological significance; that is, evaluations could be conducted on the pathological and drug-sensitive aspects of OSCC and OASC malignancies. Therefore, our currently developed in vitro 3D OSCC spheroid model may be a useful methodology for related research investigations.