One of the factors that has increased the incidence and worse prognosis of breast cancer is obesity. In this condition, high amounts of leptin are secreted, which have proliferative, mitogenic, antiapoptotic, and proinflammatory activity that may be antagonistic to treatment with tamoxifen, considered the first choice. The modulation evaluation of leptin receptor expression in the presence of leptin and tamoxifen stimuli was performed in breast cancer cell lines MCF 7, MDA MB 231 and HCC 1937 as a model of initial approach for the study of breast cancer subtypes and their behavior to the action response of adipokines and their possible relationship with the mechanism of resistance to chemotherapeutics such as tamoxifen in ER positive cell lines and triple negative marker. It was determined that leptin increases the proliferation of the three breast cancer cell lines and tamoxifen is able to exert an antiproliferative effect on them, however, it was identified that the ability of tamoxifen to decrease the proliferation of cancer cells is diminished in the presence of leptin, in addition to changes in the modulation of the expression of its receptor. It was determined that tamoxifen induces a greater modulation of the expression of ObRb in cell lines, which may be related to the decrease of its antiproliferative activity, while leptin generates a proliferative effect in the three cell lines and could participate in the tamoxifen treatment resistance mechanism. This classification is very useful to identify the type of chemotherapeutic treatment that should be administered, as well as the degree of risk and prognosis of the condition4.Obesity is defined as the abnormal or excessive accumulation of fatty tissue in the body, is a health problem that has received worldwide attention in recent years as its incidence has increased and it is associated as a risk factor for the development of chronic degenerative diseases that include cardiovascular diseases, diabetes mellitus type 2, hypertension and some types of cancer, including breast cancer5.There are different mechanisms by which obesity has been linked to cancer, among which are the promotion of inflammation and angiogenesis through the release of some molecules by adipocytes such as fibroblast growth factor (FGF2), angiopoietin-2, angiostatin, endostatin, thrombospondin (TSP-1), resistin, and elevated levels of adipokines such as leptin, among others6. Under normal physiological conditions, leptin maintains energy homeostasis by influencing the central anorectic pathway, where it acts to reduce food intake and increase metabolism7.Leptin is a polypeptide hormone that is produced in adipose tissue, whose plasma levels are increased in patients who are obese, generating a condition known as hyperleptinemia9.On the other hand, it also participates in the regulation of pathological processes such as breast cancer by presenting mitogenic, antiapoptotic and proinflammatory activity, and it is only capable of inducing the proliferation of cancer cells and not of normal breast cells, which is indicative that leptin participates directly in the development of this type of cancer10.To exert its biological function, leptin has a specific receptor (ObR), which has six different isoforms, which vary in the amount of intracytoplasmic residues they contain. Among them, ObRb, also known as the long isoform of the leptin receptor, is considered to be the one with the greatest capacity to activate signaling pathways11.Within the signaling pathways activated by leptin are: JAK2-STAT3, mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT), which generates the activation of some genes such as: C-MYC, CYCLIN D1, P21 WAF1, C-JUN, JUNB, ERG-1 and BCL-2, which are strongly involved in cell growth and proliferation7.The relationship between obesity, leptin and breast cancer has taken a further slope, that of chemotherapy resistance mediated by adipokines, proteins released by adipose tissue and can be involved in the action of chemotherapeutic drugs, since the higher risk of recurrence and mortality in patients with obesity and breast cancer could be related to a lower efficacy of cancer treatments, this is probably due to the plasma variations of adipokine levels, since women with breast cancer and obesity are less sensitive to chemotherapy and have higher mortality rates12.In relation to the above, it has been determined that leptin can influence the development of resistance to treatment with tamoxifen, an antiestrogenic drug commonly administered to breast cancer patients with the presence of ER. It was observed that combination therapy involving the blockade of the leptin receptor and treatment with tamoxifen could inhibit proliferation and promote apoptosis in breast cancer cells resistant to tamoxifen, suggesting that the suppression of leptin could be a new means to circumvent resistance to antiestrogenic treatment in breast cancer, because the signaling pathways activated by leptin could directly participate in the cellular response to the chemotherapeutic treatmentTherefore, the objective of this work is to evaluate the modulation of gene expression of leptin receptors in three different breast cancer cell lines as an initial approach model for the study of the behavior of subtypes of this disease before its exposure to leptin and tamoxifen.The cell lines MCF-7, MDA-MB 231 and HCC1937 were obtained from the American Type Culture Collection (ATCC). The cells were cultured at 37 °C in a 5% CO2 atmosphere, with RPMI 1640 culture medium  supplemented with 10% FBS  and 1% penicillin-streptomycin. Additionally, for the MCF-7 and MDA-MB 231 cell lines, the culture medium was enriched with a 2 mM pyruvate concentration and for the HCC 1937 line, a 2 mM concentration of glutamine was used. The culture medium was changed every third day.Proliferation of 5,000 cells of the cell lines MCF-7, MDA-MB 231 and HCC1937 (n = 3) was carried out in 96-well plates for 48 hours in RPMI-1640 medium (10% SFB). Subsequently they were stimulated for 48 hours with the following concentrations of tamoxifen: 0.002 μM, 0.02 μM, 0.2 μM, 2 μM, 20 μM and 200 μM, changing the stimulus every 24 hours.At the end of the 48 hours of stimulation, the cells were fixed with 4% formaldehyde for 15 minutes. The fixed cells were washed with PBS and stained with crystal violet (0.02%) for 15 minutes, to remove the residual dye, 3 washes of 250 μl of water were carried out and finally 250 μl of 10% acetic acid was added to each well. The plate was read at an excitation wavelength of 490 nm and an emission length of 630 nm in a microplate reader .13.Proliferation of 5,000 MCF-7 cells, MDA-MB 231 and HCC1937 (n = 3) was carried out in 96-well plates for 48 hours. The cells were then washed with PBS and fresh culture medium (supplemented to 5% of SFB) was added with leptin stimulation  in concentrations of 0 ng/mL, 10 ng/mL, 100 ng/mL and 200 ng/mL for 72 hours, changing the stimulus every 24 hours. Additionally, another test was carried out using these concentrations of leptin plus a corresponding standard with the IC50 of tamoxifen  obtained experimentally for each of the cell lines  and another only with the tamoxifen standard. The decrease in SFB was performed to decrease growth factors that could interfere with the action of leptin and tamoxifenEvery 24 hours after the stimulation the cells were fixed with 4% formaldehyde for 15 minutes. At the end of the 72 hours of stimulation, the fixed cells were stained with crystal violet (0.02%) for 15 minutes, to remove the residual dye, 3 washes of 250 μl of water were carried out and finally 250 μl of 10% acetic acid was added to each well. The plate was read at an excitation wavelength of 490 nm and an emission length of 630 nm in a microplate reader .et al.14, with some modifications.The regulation of gene expression of leptin receptors was evaluated by adding leptin and tamoxifen in cell lines, using the following methodology proposed by Jardé 20,000 cells of MCF-7, MDA-MB 231 and HCC1937 (n = 3) were plated in 24-well plates for 48 hours. The stimulation was carried out with leptin  and tamoxifen  in culture medium RPMI-1640 (5% SFB) for 72 hours with stimulus change every 24 hours. At the end of the stimulation, the culture medium was removed and 750 μL of TRIzol Reagent was added to carry out the RNA extraction according to the protocol of use (Ambion).Once the RNA was obtained, complementary DNA (cDNA) was obtained from 2 μg of mRNA by means of the reverse transcriptase reaction according to the protocol of use  obtaining 20 μL of cDNA. The same was used for the amplification of ObRb, ObRt, ER, aromatase and 18S control genes under initial amplification conditions at 94 °C for 5 minutes, 35 cycles  and a final extension at 72 °C for 10 minutes, using the HotStarTaq DNA Polymerase,  and the following pairs of primers: ObRb , ObRt, , ER  and aromatase , gene 18S .15.Likewise, the cDNA obtained was used to perform the determination of gene expression of leptin receptors by real-time PCR  using TaqMan probes  for ObR (Hs00174497_m1) and as control gene on 18S (Hs99999901_s1) following the instructions of the LightCycler 2.0 thermocycler (Roche Diagnostics) for each sample in duplicate. The program used to perform the RT-PCR was 50 °C for 2 minutes, 95° for 10 minutes and 40 cycles at 95 °C for 15 seconds and 60 °C for 1 minute. For the determination of the degree of genetic expression of the leptin receptors, the relative quantification method of Pfaffl was used, based on the comparison of the CT of a constitutive gene (18S) against the problem gene taking the value of its differences against the control line for each sampleIt was performed using the supernatants of the stimulated cell cultures and under the protocol of use of the ELISA kit for human leptin receptor (Human Leptin receptor ELISA Kit. DEIA8050 Creative Diagnostics). The detection range of the test ranges from 156 to 10,000 pg/mL. The plate was read with the microplate reader  at a wavelength of 450 nm.The statistical analysis was performed using ANOVA for the growth curves of each cell line. The value of P < 0.05 will be considered as statistically significant.For tests of gene expression, the normality test was used using the Kolmogorov-Smirnov test. Subsequently, Student’s t test for parametric variables and the Mann-Whitney U test for nonparametric variables with a p value > 0.05 for each one were performed.This article does not contain any studies with human participants or animals performed by any of the authors.The determination of the constitutive expression of mRNA was carried out by RT-PCR of the long isoform of the leptin receptor (ObRb), homologous region for any of the six different isoforms of the leptin receptor (ObRt), estrogen receptor (ER) and aromatase CYP19A1) in the three breast cancer cell lines prior to stimulation with leptin and tamoxifen, as shown in Fig. 9A1 in thIn the MCF7 cell line, the presence of mRNA of the four genes of interest was found, in the MDA MB 231 cell line only mRNA of the general homologous form of the leptin receptor was found and in the HCC 1937 cell line the expression of the leptin receptor was detected in its two variants.From the proliferation assay a dose response curve was obtained for each cell line Fig.  from whiIn general, in the cultures of the three different breast cancer cell lines, a positive proliferative effect was observed by stimulation with leptin, the opposite effect was observed when stimulating with tamoxifen Fig. .Figure 3The cells were cultured for 48 hours and subsequently stimulated with leptin and tamoxifen, observing the following results: in the MDA MB 231 cell line stimulation of leptin in any concentrations causes a proliferative effect from 24 hours of stimulation  was determined in the different cell lines as shown in Fig. In the MCF-7 cell line, the expression of the ObR had an increase of 71.86% (10 ng/mL) in the cells treated with leptin with respect to the control, however, at concentrations of 100 and 200 ng/mL of leptin, a decrease in ObR expression is observed, for the stimulation of tamoxifen a 37.2% increase in the expression of the gene was observed, with respect to the control, while for the combination stimuli of leptin with tamoxifen a decrease in the expression of the gene was observed.In the HCC 1937 cell line, a decrease in all cases of ObR expression was identified.In the MDA MB 231 cell line, no amplification was obtained with the Taqman probe for the leptin receptor.The determination and quantification of the leptin receptor protein in the supernatant of each of the cultures stimulated by the ELISA technique was carried out, obtaining the results shown in Fig. In the MDA MB 231 cell line, a higher concentration of the ObR protein was found in all the groups of stimulated cells compared to the control, reaching the highest concentration in stimuli that include 100 ng/mL of leptin and 100 ng/mL of leptin plus tamoxifen, with a concentration of 147.3 pg/mL.In the MCF 7 cell line, greater expression of the ObR protein was found in all cases where stimulus was added, however, it is of great interest that the stimulus that showed a higher concentration of the protein was in which only tamoxifen was added, obtaining a concentration of 407.7 pg/mL, while in the trials that include leptin, something similar to the MDA MB 231 line is found, since the stimuli that generated the highest expression of the protein were those of 100 ng/mL of leptin and 100 ng/mL of leptin plus tamoxifen, with concentrations of 183.1 pg/mL and 215.3 pg/mL respectively.In the HCC 1937 cell line, greater expression of the ObR protein was found in the stimuli corresponding to 200 ng/mL of leptin plus tamoxifen and only tamoxifen, where the concentration of 385.7 pg/mL and 397.5 pg/mL respectively was reached. A different behavior was identified in the stimulus corresponding to 200 ng/mL of leptin since a decrease in the expression of the ObR protein was observed, finding a concentration of 110.1 pg/mL which is lower than the concentration identified in the control which was 225.3 pg/mL.2.The determination of mRNA expression by RT-PCR was determined for the long isoform of the leptin receptor (ObRb), homologous region for any of the six different isoforms of the leptin receptor (ObRt), estrogen receptor (ER), aromatase (CYP19A1) and the 18s gene in the three breast cancer cell lines, as part of the characterization prior to stimulation with leptin and tamoxifen, it was determined that in the MCF7 cell line the mRNA of the five genes of interest is expressed, which is consistent with the description of this cell line, since as an identification feature it expresses the estrogen receptor and therefore the presence of CYP19A1 is found, an enzyme that participates in the estrogen biosynthesis, likewise the presence of leptin receptor mRNA was found, both in the homologous form for any of the six different isoforms of the receptor, as for the long isoform (ObRb), which is of interest since it is considered as the isoform that has greater activity and capacity to activate signaling pathways such as JAK2/STAT3 due to the intracytoplasmic residues it presents16.In the MCF7 cell line, the maximum increase in the proliferation of cancer cells was identified when stimulated with leptin for 72 hours, after a growth prior to stimulation of 48 hours, reaching an increase in proliferation of 232.22% for 10 ng/mL, 315% for 100 ng/mL and 310% for 200 ng/mL, which indicates that leptin generates an increase in the proliferation of this cell line and that after 100 ng/mL a significant increase is no longer found, the increase in cell proliferation corresponds to the reported characteristics of the activation of leptin signaling pathways, which contribute to the progression of cancer through multiple mechanisms, since it is known that leptin activates several signaling cascades, such as the JAK/STAT, MAPK and PI3 kinase/AKT pathways, most of which are involved in the differentiation and proliferation of cancer cells. In addition to that it has been shown that treatment with leptin suppresses apoptosis and accelerates the progression of the cell cycle through the induction of genes related to it17.In this cell line a greater anti-proliferative effect of tamoxifen is observed in comparison with the other cell lines (MDA MB 231 and HCC 1937), reaching a 76.12% decrease in cell proliferation, this is because tamoxifen is an antiestrogenic drug that acts by binding with the estrogen receptor competing with estrogen and nullifying the biological effects of these molecules, the tamoxifen-estrogen receptor complex interacts with nuclear DNA and blocks the action of estrogen by inhibiting the production of growth factors, which negatively affects cell proliferationin vitro behavior of triple negative cell lines is crucial, in this study the MDA MB 231 and HCC 1937 lines were used.There are other types of cell lines that belong to the type of triple negative breast cancer, which is an aggressive form that is characterized by the absence of some membrane receptors such as ER, PR and Her-2, which leads to limited treatment options. Therefore, in order to understand the molecular basis of this type of cancer, the analysis of the In the MDA MB 231 cell line, neither the presence of estrogen receptor mRNA nor CYP19A1 was found, this is consistent with the characteristic of the cell line belonging to the triple negative cancer type, for which it lacks the expression of the estrogen, progesterone and Her-2 receptor. Regarding the presence of leptin receptor mRNA, it was found only for the general homologous form of the receptor, so it can not be established which of the six different isoforms of the receptor is the one that is being expressed in this cell line.In the HCC 1937 cell line, which possesses a mutation in the BRCA1 gene, the presence of mRNA expression of the leptin receptor was found in the two isoforms analyzed (long and homologous isoform), as in the MDA MB 231 cell line, no expression of the estrogen receptor or CYP19A1 was found, as it was also a negative triple-negative breast cancer cell line.et al.; they identified results similar to those obtained in this study when they found an increase in cell proliferation in lines MCF 7 and MDA MB 231 when stimulated with leptin and an increase in β-catenin levels was also identified18.In the two lines of cancer triple negative marker an increase in cell proliferation was identified when stimulated with leptin, which suggests that as in the cell line MCF 7, leptin participates activating signaling pathways that give this cell type greater proliferative capacity, one of these pathways is related to Wnt/β-catenin, which is key in the development of several types of cancer, since Wnt/β-catenin signal transduction fulfills fundamental functions in the regulation of the proliferation and differentiation of cancer cells. Activation of the Wnt/β-catenin pathway is enriched in different types of breast cancer. In a recent study Liang 19.In both negative triple marker cell lines it was identified that tamoxifen generates a cytostatic effect on cell proliferation, which is interesting since tamoxifen is an antiestrogenic drug and its molecular target is the estrogen receptor, which is absent in these cell lines, however recent studies have identified that tamoxifen shows a response rate of 10% to 15% in tumors without ER expression, these findings suggest that tamoxifen has certain anticancer properties independent of ER. One of these mechanisms is the one that involves the decrease of the regulation of the cancerous inhibitor of protein phosphatase 2A (CYP2A) and p-Akt, which correlates with tamoxifen-induced apoptosis in ER-negative breast cancer cells, the possible mechanism that contemplates the interaction between leptin and tamoxifen in these cell lines is shown in Fig. 20.Regarding the relative expression of the ObR in the cell lines determined by qPCR, it was found that in the cell line MCF 7 there is an increase in the expression of the receptor in a 71.86% compared to the control for the concentration of 10 ng/mL of leptin, for the concentrations of 100 and 200 ng/mL the expression is surely diminished by a blockade of the expression due to the saturation of the receptor by the action of leptin, however it is important to note that when the cell line is stimulated with tamoxifen there is also an increase in the expression of the receptor, this is possibly due to the fact that the cancer cells induce a greater expression of the leptin receptor to maintain its viability and proliferation, since it has been determined that breast cancer cells use the signaling pathways of leptin as a fundamental part of maintaining their proliferationIn the HCC 1937 cell line, a decrease in ObR expression was found in all stimulation cases compared to the control, this is due to the fact that upon finding a higher expression of the ObR protein in this cell line, mRNA expression is diminished, whereas for the MDA MB 231 cell line the ObR amplification was not identified by qPCR, this is because the probe used does not amplify the desired sequence in this cell line, since it is focused on the detection of ObRb, which by RT-PCR was determined in MCF 7 and HCC1937 cell lines, but was not detected in MDA MB 231.21.The ObR protein levels obtained by the ELISA technique show that for MCF 7 and MDA MB 231 cell lines, higher concentration was obtained compared to the control in all the stimuli, whereas in the HCC 1937 cell line an increase in the concentration of the protein was obtained when stimulated with leptin (10 ng/mL) and with leptin plus tamoxifen in its different concentrations, in the case of the stimulation of leptin at 100 and 200 ng/mL, a lower concentration of the protein was obtained compared to the control, this makes it possible to define that the expression of the ObR protein is expressed under normal physiological conditions and not in a marked increase in the concentration of leptin. It is important to note that in this study it was determined that in the three cell lines a higher concentration of ObR protein expression is observed when stimulated with tamoxifen, this probably corresponds to the ability of ER positive (MCF 7) and triple negative marker cells (HCC 1937 and MDA MB 231) to increase the expression of ObR as a survival mechanism against the damage caused by the chemotherapeutic agent21, however, it is of great interest the evidence found that when the tamoxifen stimulus is placed in combination with leptin in the three cell lines, greater cell proliferation is observed than when only tamoxifen is added, so it can be defined that probably the plasma variations of leptin levels in women with breast cancer and obesity participate in generating lower sensitivity to chemotherapy and this leads to higher mortality rates.The presence of the ObR in the three cell lines corresponds to that reported in the literature where it is mentioned that the ObR is necessary for the maintenance and renewal of cancer cellsIn this study it was determined that leptin favors the proliferation of cell lines MCF 7, HCC 1937 and MDA MB 231, noticing a time-dose dependent effect; it was also determined that tamoxifen is able to generate its antiproliferative effect in the three cell lines, being the most noticeable effect in the cell line MCF7, which is ER positive.It is observed that the combination of the leptin-tamoxifen stimulus generates a decrease in the antiproliferative capacity of tamoxifen, which leads to propose that leptin participates in the mechanism of resistance to tamoxifen treatment, commonly observed in patients with breast cancer and obesity.Similarly, in this study it can be observed that the administration of tamoxifen in the three cell lines favors a higher expression of the ObR at the protein level, however, at the level of mRNA, the same can not be confirmed, since a greater expression is observed in the MCF7 cell line, while in the HCC1937 cell line, a decrease in the expression of the receptor is observed and in the MDA MB 231 cell line could not be determined because in this cell line the amplified one was not obtained by the Taqman probe.Supplementary InformationSupplementary Information 3Supplementary Information 4


This review provides an overview on the active phytochemical constituents of medicinal plants that are traditionally used to manage cancer in Ethiopia. A total of 119 articles published between 1968 and 2020 have been reviewed, using scientific search engines such as ScienceDirect, PubMed, and Google Scholar. Twenty-seven medicinal plant species that belong to eighteen families are documented along with their botanical sources, potential active constituents, and in vitro and in vivo activities against various cancer cells. The review is compiled and discusses the potential anticancer, antiproliferative, and cytotoxic agents based on the types of secondary metabolites, such as terpenoids, phenolic compounds, alkaloids, steroids, and lignans. Among the anticancer secondary metabolites reported in this review, only few have been isolated from plants that are originated and collected in Ethiopia, and the majority of compounds are reported from plants belonging to different areas of the world. Thus, based on the available bioactivity reports, extensive and more elaborate ethnopharmacology-based bioassay-guided studies have to be conducted on selected traditionally claimed Ethiopian anticancer plants, which inherited from a unique and diverse landscape, with the aim of opening a way forward to conduct anticancer drug discovery program. Cancer is a major global health challenge that affects millions of people annually across the world. Recent estimates showed about 18.1 million new cases of cancer and 9.6 million cancer-related deaths worldwide [Medicinal plants have been a rich source of clinically effective anticancer agents for the past few decades. Over 60% of the currently used anticancer drugs are either directly derived from plants or inspired by their novel phytochemicals  and/or uCatha edulis Forsk [Artemisia annua L., Rumex abyssinicus Jacq. [Carissa spinarum L., Dodonaea angustifolia L.f., Jasminum abyssinicum Hochst. ex DC., Rumex nepalensis Spreng., Rubus steudneri Schweinf. and Verbascum sinaiticum Benth. [Viola abyssinica Steud. ex Oliv. [Xanthium strumarium L. [Senna singueana (Del). Lock [Glinus lotoides L. [Kniphofia foliosa Hochst [Sideroxylon oxyacanthum Baill., Clematis simensis Fresen, and Dovyalis abyssinica (A. Rich) Warburg [Ethiopia inherited a unique array of fascinating flora from its diverse landscape. Due to the geographical location and diversity, which favors the existence of different habitat and vegetation zones, Ethiopia is home to a variety of plant species. The Ethiopian flora is estimated to contain 6027 species of higher plants of which more than 10% are estimated to be endemic . Differeis Forsk ,9, Artemex Oliv. , Xanthiuarium L. , Senna sl). Lock , Glinus oides L. , Kniphofa Hochst , Siderox Warburg . Thus, f Warburg , in whicA total of 27 anticancer traditional medicinal plants that belong to 18 botanical families and 27 genera are identified in this review. The botanical families Euphorbiaceae and Cucurbitaceae were the most dominant, represented with 15% and 11% of the selected plant species, respectively . All of 50 (Concentration that inhibited cell proliferation by 50%)/ED50 (Effective dose for 50% of the population) > 50 µg/mL or > 100 µM, in most cases, except few where compounds tested against a panel of cell lines) or plants from which no anticancer compounds were isolated/reported. This review compiled and discussed the potential anticancer/antiproliferative agents based on the types of secondary metabolites, such as terpenoids, phenolic compounds, alkaloids, steroids, and lignans.The present review reports secondary metabolites isolated from 27 plants that are traditionally used to treat different types of cancer in Ethiopia. Phytochemical investigations of traditionally used Ethiopian anticancer plants have led to the isolation of compounds that belong to different classes of natural products ,57. In tTerpenoids are classified according to the number of their isoprene unit as hemi-, mono-, di-, tri-, tetra-, and polyterpenes . VariousPodocarpus falcatus (Thunb.) collected from Berga forest, Addis Alem, central Ethiopia. P. falcatusis traditionally used to treat jaundice, gastritis, and amoeba [1), 2β,16-dihydroxynagilactone F (2), 7β-hydroxymacrophyllic acid, nagilactone D (3), 15-hydroxynagilactone (4), and nagilactone I (5) (50 < 10 µM)  (6), demonstrated a moderate cytotoxic activity (IC50 = 50 µg/mL). The compound was shown to trigger caspase mediated apoptotic cell death. 3β-Hydroxylup-20(29)-ene-27,28-dioic acid dimethyl ester (7), isolated from root of Plumbago zeylanica collected from India, also exhibited anti-proliferative and anti-migration activity against triple-negative breast cancer cell lines at IC50 value of 5 µg/mL [Among the different types of triterpenoids, pentacyclic triterpnoids display the most potent anti-inflammatory and anticancer activity . Addo etd amoeba . Among tne I (5)  showed p< 10 µM) . Premna ectively . The met 5 µg/mL .8) from Zehneria scabra, collected from Cameroon, demonstrated potent cytotoxicity against brine shrimp assay [9), isolated from Euphorbia tirucalli from Taiwan against human gastric cancer cells. Euphol selectively promotes apoptosis by mitochondrial-dependent caspase-3 activation and growth arrest through induction of p27kip1 and inhibition of cyclin B1 in human gastric CS12 cancer cells. It also showed a selective and strong cytotoxicity against other groups of human cancer cell lines such as glioblastoma (the most frequent and aggressive type of brain tumor) [10), isolated from the leaves of Olea europaea has been studied, which induced apoptosis in HT29 human colon cancer cells by directly inhibiting the expression of Bcl-2, increasing that of Bax, releasing cytochrome-C from the mitochondria and activating caspase-9 and then caspase-3 [Ricinus communis collected from Malta was also reported for its cytotoxicity against several human tumor cells and induction of apoptosis against human breast tumors, SK-MEL-28. The monoterpenoids 1,8-cineole, camphor and α-pinene, and the sesquiterpenoid β-caryophyllene, isolated from R. communis, also showed cytotoxicity against similar cell lines in a dose-dependent manner [Several terpenoids have been isolated from Ethiopian plants that have claims of having anticancer activity, although these plants may have been collected from other sources. For example, sonhafouonic acid  ,68. The aspase-3 . Similart manner .Jatropha curcas is a medicinal plant traditionally used to treat a variety of ailments in different parts of the world including Ethiopia [J. curcas, collected from China, resulted in the isolation of twelve phorbol esters (diterpenoids) including jatrophalactone (11), curcusecon A–J, 4-epi-curcusecon E, curcusone E, 3-dehydroxy-2-epi-caniojane (12), curcusone A (13), curcusone B (14), curcusone C (15), curcusone D (16), jatrogrosidone, 2-epi-jatrogrossidone, and 4E-jatrogrossidentadion (17) [50 values ranging from 0.084 to 20.6 µM against HL-60, SMMC-7721, A-549, MCF-7, SW480, and HEPG2 cell lines [Ethiopia . Investiion (17) . Most ofll lines .18), isolated from Ekebergia capensis [50 values of 1.4 and 13.3 µM, respectively. Another pentacyclic triterpenoid, asiatic acid (19), isolated from Centella asiatica, also showed 80% growth inhibition of human colorectal (SW480), human stomach (SNU668), and murine colorectal adenocarcinoma (CT26) cell lines with IC50 values of 20 µg/mL [Cucumis prophetarum from Saudi Arabia yielded a series of cucurbitacin and analogs (cucurbitacin E (20), cucurbitacin B (21), cucurbitacin D (22), cucurbitacin F 25-O-acetate, cucurbitacin E glucoside (23), dihydrocucurbitacin D, hexanor-cucurbitacin D, and isocucurbitacin D (24)), of which compounds 20–24 showed cytotoxic activity against MCF-7, MDA MB 231, A2780, A2780 CP, HepG2, and HCT-116 with IC50 values ranging from 1 to 27.3 µM [The pentacyclic triterpenoid oleanonic acid  isolated from Maytenus senegalensis has showed potent cytotoxic activity against mouse lymphoma cell line (L5178Y) [26); effususol A; effusol; dehydroeffusol; dehydroeffusal; 2,7-dihydroxy-1,8-dimethyl-5-vinyl-9,10-dihydrophenanthrene and juncusol; dehydrojuncusol and 1-methylpyrene-2,7-diol) from Juncus effuses inhibited the proliferation of five human cancer cell lines (26)  has also demonstrated potent cytotoxicity against HT-22 cell by inducing caspase-3-mediated apoptosis [28), a naphthoquinone isolated from Plumbago zeylanica also induced apoptosis in human non-small cell lung (IC50 = 6.1–10.3 µM) [50 = 2.1 µM) [29) isolated from Kniphofia foliosa Hochst collected from Ethiopia, induced necrotic death in mouse melanoma (B16), mouse macrophage tumor (RAW 264.7), human acute monocytic (THP-1), and promonocytic leukaemic (U937) cell lines with IC50 values that range from 0.5 ± 0.05 to 3.3 ± 0.39 µM [Phenolic compounds are biosynthesized by plants through shikimate, phenylpropanoid, and flavonoid pathways, and have an aromatic ring bearing one or more hydroxyl groups. These compounds have been reported for their antioxidant, antiproliferative, and cytotoxic properties . Many ph(L5178Y) . Likewisll lines . Among tnes (26)  was testpoptosis . Plumbag10.3 µM)  and huma 2.1 µM)  cancer c 0.39 µM .30) and vincristine (31) (Catharanthus roseus [C. roseus [32–34) along with other five known compounds from the whole plant of C. roseus collected from China (36) showed the most potent cytotoxic activity with IC50 value of 0.73 ± 0.06 µM. Furthermore, the isolated three new compounds (32–34) also showed potent cytotoxicity against triple-negative breast cancer (MDA-MB-231) cell line with IC50 values ranging from 0.97 ± 0.07 µM to 7.93 ± 0.42 µM. Another alkaloid, cathachunine (40), also showed a promising cytotoxic activity against HL-60 by inducing an intrinsic apoptotic pathway [V. roseus, showed weak cytotoxic activity against HCT 116 [41), isolated from the seeds of Gloriosa superba, demonstrated moderate activity against six human cancer cell lines  [Vinblastine (ine (31)  are one s roseus . This is. roseus ,86. Wang. roseus  isolatedom China . Among t pathway . On the  HCT 116 . Furthernd SiHa) .44) and 5β,6β,14α,15α-diepoxy-4β,27-dihydroxy-1-oxowitha-2,24-dienolide (45)  with IC50 values of 0.45 ± 0.00 and 8.3 ± 0.21 µg/mL, respectively [48); and hellebrigenin 3,5-diacetate (49) [Bersama abyssinica collected from Ethiopia, demonstrated cytotoxic activities. β-Sitosterol-3-O-glucoside, a phytosterol from Prunus Africana, exhibited poor anticancer activity against three cell lines  , isolateectively . Severalectively ; hellebrate (49)  isolatedll lines .50), (−)-carissanol (51), and (−)-nortrachelogenin, isolated from Carissa spinarum,were found to be cytotoxic against A549, MCF-7, and WI-38 cell lines. Among these, (−)-carinol  showed more potent cytotoxic activity against these three cell lines with IC50 value of 1 µg/mL, as compared to (−)-carissanol and (−)-nortrachelogenin [52) and matairesino (53), two lignans isolated from Linum usitatissimum, exhibited cytotoxicity against MCF-7 cells with IC50 values of 10 and 1 µM, respectively [Lignans and isoflavonoids are the major classes of phytoestrogens  which shelogenin . Secoisoectively .52), and colchicines (41), isolated from plants collected elsewhere, were considered further for clinical trial. Preclinical studies generate data on the efficacy, safety, and pharmacokinetic properties of lead compounds, which will later be used to select better molecules for clinical trials. Assessment of the findings of preclinical in vivo animal studies supports the traditional use of plants to manage cancer in Ethiopia . Despite50 and ED50 values) provided in this review will help to identify the most potent source(s) of these compounds, as bioactive marker(s), of local flora. Based on the higher frequency of citation Croton macrostachyus, Jatropha curcas, Plumbago zeylanica, and Vernonia hymenolepsis are potential candidates for follow-up bioassay guided investigations. Furthermore, plants with reported antiproliferative compounds such as Podocarpus falcatus, Linum usitatissimum, and Zehneria scabra should also be examined for additional cytotoxic compounds and evaluated against a battery of cancer cell lines.Despite the traditional use of various Ethiopian plants for the treatment of cancer by herbal medicine practitioners for many decades, only a few active anticancer crude extracts, herbal preparations, and pure compounds were tested and so far no clinical trial was conducted on them. In this review, an attempt has been made to document antiproliferative, antitumor, and cytotoxic natural products small molecules isolated from medicinal plants that are traditionally used to treat cancer in Ethiopia. However, among the reported active compounds, only few have been isolated from plants that are originated and collected from Ethiopian geographic location, despite their wider presence and traditional claim at home. The majority of compounds reported in this review are isolated from plants (corresponding to Ethiopian species) that were collected from different regions of the world. However, the comprehensive list of active compounds (ICGenerally, the ecological variation has a huge impact on the biosynthesis, yield of active constituent and biological potency of secondary metabolites produced by plants of similar species from different geographical regions. Thus, Ethiopian anticancer plants might have novel active constituents to fight cancer, based on traditional medical use, than those collected from other regions due to their unique geographical location and inherent climatic condition of the diverse landscape. Unfortunately, these valuable plant resources are disappearing rapidly due to climate change, rapid urbanization, agricultural land expansion, and artificial deforestation; therefore, Ethiopian flora is facing a great challenge, and thus it is high time to examine the anticancer plants systematically with the aim to carry out chemical and biological invesigations, as well as clinical trials on promising anticancer plant extracts based on ethnopharmacological knowledge.


XL, and MCL-1 in DH/DE-diffuse large B cell lymphoma (DLBCL) cell lines. Ultimately, CKD-581 also induced apoptosis via poly(ADP ribose) polymerase 1 (PARP1) cleavage. In a DLBCL SCID mouse xenograft model, CKD-581 exhibited anti-cancer effects comparable with those of rituximab (CD20 mAb). Our findings suggest that CKD-581 could be a good candidate for the treatment of DLBCL.Double-hit lymphoma (DHL) and double-expressor lymphoma (DEL) are aggressive forms of lymphoma that require better treatments to improve patient outcomes. CKD-581 is a new histone deacetylase (HDAC) inhibitor that exhibited a better safety profile in clinical trials compared to other HDAC inhibitors. Here, we demonstrate that CKD-581 inhibited the class I–II HDAC family via histone H3 and tubulin acetylation. CKD-581 treatment also up-regulated the phosphorylation of histone H2AX , and reduced levels of MYC and anti-apoptotic proteins such as BCL-2, BCL-6, BCL- Diffuse large B cell lymphoma (DLBCL) is the most common subtype of lymphoma, accounting for approximately 30% of all lymphoma . DLBCL cThe standard chemotherapeutic regimen for DLBCL is rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). About 60–70% of patients are cured by R-CHOP therapy; the remaining 30–40% of patients respond poorly to standard therapy .MYC is a representative proto-oncogene that is overexpressed in lymphomas . MYC reaHistone deacetylase (HDAC) inhibitors are epigenetic drugs that modify gene expression, restoring the normal differentiation and death program usually altered in cancer cells . HDAC inHowever, no HDAC inhibitor has been approved for treating DLBCL, despite the urgent need for an appropriate chemotherapeutic regimen. CKD-581  is a new HDAC inhibitor that targets class I–II HDACs. A phase I clinical study was conducted to evaluate the safety and tolerability of CKD-581 for patients with Hodgkin lymphoma, non-Hodgkin lymphoma , and refractory MM. CKD-581 was well tolerated, and 16 of 36 (44.4%) patients achieved a stable disease (SD) or better [In this study, the in vitro and in vivo anti-cancer activities of CKD-581 were assessed in four different DLBCL cell types. The pharmacological mechanism was investigated with a focus on the regulation of MYC and the anti-apoptotic BCL-2 family.XL were obtained from Santa Cruz Biotechnology . Antibody for phosphorylated H2AX (γH2AX) was purchased from Abcam . Antibodies for MYC, CD20, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were supplied from Thermo Fisher Scientific . CKD-581 was provided from CKD Pharmaceutical Corp.  and suberoylanilide hydroxamic acid  with a purity of >98% was purchased from Sigma-Aldrich . Rituximab was purchased from Roche . Compounds were dissolved in dimethyl sulfoxide (DMSO) and stored at −20 °C. The CellTiter-Glo luminescent cell viability assay kit was supplied from Promega . Antibodies recognizing histone H3, acetylated histone H3, tubulin, acetylated tubulin, myeloid cell leukemia-1 (MCL-1), and poly (ADP-ribose) polymerase 1 (PARP1) were purchased from Cell Signaling Technology . Antibodies for BCL-2, BCL-6, and BCL-2. OCI-LY1 cells were cultured in IMDM (Thermo Fisher Scientific), containing 20% fetal bovine serum. All cells were seeded in a 96-well plate and allowed to incubate overnight, then treated with serial dilutions of CKD-581 or SAHA. After a 72-h incubation, cell viability was assessed by the CellTiter Bright-Glo system (Promega). Relative viable cell percentages were quantified by measuring luminescence using a Glomax luminometer (Promega). Human DLBCL cell lines, SU-DHL-2 (ABC/DH), and SU-DHL-4 (GCB/DH) were purchased from American Type Culture Collection . OCI-LY1 (GCB/DH) and U2932 (ABC/DE) cell lines were kindly donated by Dr. Yu HY . SU-DHL-2, SU-DHL-4, and U2932 cells were cultured in RPMI1640 (Thermo Fisher Scientific) containing 10% fetal bovine serum (Thermo Fisher Scientific) at 37 °C in 5% CO2, and 8.6% sucrose) and centrifuged at 1500× g for 10 min at 4 °C. Pelleted nuclei were suspended with 50 μL 0.4 N H2SO4, and incubated on ice for 1 h, then centrifuged at 1500× g for 10 min at 4 °C. The supernatant proteins were precipitated with 500 μL ice-cold acetone overnight at −20 °C. The precipitated proteins were collected by centrifugation at 1000× g for 10 min at 4 °C, air dried, and suspended in 50 μL deionized water.SU-DHL-2 cells were incubated with CKD-581 or DMSO for 6 h, harvested, and washed twice with phosphate-buffered saline (PBS). The cells were suspended with 50 μL ice-cold lysis buffer (10 mM Tris-HCl (pH 6.5), 50 mM sodium bisulfate, 1% Triton X-100, 10 mM MgClCells were lysed with a RIPA lysis buffer (Sigma-Aldrich) supplemented with protease and phosphatase inhibitor cocktails (Roche). Total cell lysates were resolved on 4–12% gradient SDS-PAGE gel (Thermo Fisher Scientific) and transferred to a nitrocellulose membrane. After blocking with 5% skim milk in Tris-buffered saline containing 0.05% Tween 20 (TBS-T), the membrane was incubated with primary and HRP-conjugated secondary antibodies. Chemiluminescence signals were detected by Chemidoc .3, the animals were randomized into treatment groups, including vehicle , CKD-581 , and rituximab . All the mice were intraperitoneally injected with vehicle or treatment agents. Tumors and body weights were measured 2 times a week. Tumor volume was monitored using external measurements with a caliper and tumor volumes were calculated using the formula (width2 × length)/2.Male NOD.CB17 SCID mice were subcutaneously implanted with various human B cell lymphoma cell lines. After tumor volumes reached 100–200 mmp < 0.05, ** p < 0.01, and *** p < 0.001. All data were analyzed using GraphPad Prism software .One-way ANOVA was used to determine the differences in all experiments except the tumor xenograft experiment. For the tumor xenograft experiment, two-way ANOVA was used; significance was expressed as * Histone H3 and tubulin are representative target proteins of HDACs. The acetylation status of either protein can be affected by HDAC inhibitors. It is well established that acetylated histone proteins are regulated by HDAC class I in the nucleus, and that acetylated tubulin is a target of HDAC class II in cytoplasm . The ace50) values of CKD-581 in SU-DHL-4, OCI-LY1, SU-DHL-2, and U2932 cells were 1.31 ± 0.47, 36.91 ± 2.41, 1.18 ± 0.29, and 31.99 ± 1.06 nM, respectively. Based on the IC50 values of SAHA, CKD-581 was more potent in all DLBCL cell lines  and one DE-DLBCL cell line U2932)  were tes32  were ll lines b. 50 values of CKD-581 in each cell type, the inhibitory effects of CKD-581 on the expression of oncoproteins seem to be related to its anti-cancer effects. To investigate the effects of CKD-581 on the expression of poor prognostic factors, several molecules were identified by immunoblotting a–d. CKD-The status of poly(ADP ribose) polymerase 1 (PARP1) and γH2AX (phosphorylated histone H2AX) was also assessed by immunoblotting. PARP1 cleavage can be triggered by DNA strand breaks (DSBs) and caspase-3-dependent cleavage. PARP1 is cleaved into 89 and 24 kDa fragments, which are representative markers of apoptosis . γH2AX hXL and MCL-1 are anti-apoptotic members of the BCL-2 family which are amplified in diverse cancer types [XL or MCL-1 delayed MYC-driven B cell lymphomagenesis [XL and MYC caused B-cell lymphoma, and overexpressed MCL-1 accelerated MYC-driven B-cell lymphoma development [XL and MCL-1 are known to be related to resistance to rituximab and other chemotherapies [XL and MCL-1 could be beneficial for treating B-cell lymphoma, including DLBCL. BCL-er types . Previouagenesis ,22. On telopment ,24. In aherapies ,26. HencXL  and MCL-1  have been developed for DLBCL and are undergoing clinical trials [XL expression was high in all four tested cell lines, as was the expression of MCL-1 in all cell lines, with the exception of SU-DHL-2 cells  twice a week. As shown in XL-expressing DLBCL [Rituximab (CD20 mAb) is an important therapeutic agent for NHL. Rituximab was effective as a single agent  and the ng DLBCL . TherefoXL expression, and would be a candidate for clinical treatment of lymphoma, especially refractory/relapsed lymphoma after rituximab-based therapy. As shown in 50 value of cancer cell death was similar to those of other cell lines  and Grade 4 thrombocytopenia (2 patients at 210 mg/m2) were reported [As a new HDAC inhibitor, the safety, pharmacokinetics, and pharmacodynamics of CKD-581 were assessed in a Phase 1 clinical study . Althougreported .max values, which were distinct from the non-linear PK profiles of other HDAC inhibitors [Cardiac toxicity is another important safety concern for HDAC inhibitors ,35. Howehibitors ,37. ConsTo improve the clinical outcomes of cancer, combination therapy is widely applied because single agents have insufficient efficacy, where resistance to therapy is likely to emerge ,39. AlthFor instance, bortezomib could be suitable for combination with CKD-581. It has also been reported that HDAC inhibitors enhance the accumulation of misfolded proteins induced by proteasome inhibitors ,41. BrutXL, and MCL-1, were all reduced. CKD-581 also induced apoptosis in aggressive DLBCL cell lines and showed outstanding in vivo anti-cancer activity in aggressive DLBCL. Despite many efforts to develop new regimens for DLBCL, there are currently no HDAC inhibitors approved for this indication. Because CKD-581 is an efficacious HDAC inhibitor that maintains a proper safety profile, CKD-581 could be a promising drug candidate for the treatment of DLBCL.CKD-581 is a new broad-spectrum HDAC inhibitor that targets type I and II HDACs. This investigation demonstrated for the first time that CKD-581 reduced the expression of MYC and anti-apoptotic BCL-2 family members in a concentration-dependent manner. Moreover, the expression levels of important prognostic markers for DLBCL, including BCL-2, BCL-6, BCL-


There is a need for novel treatment options for patients with testicular germ cell tumors, especially for those that are resistant to standard chemotherapy, who show poor prognosis. In this work, we test two compounds that inhibit epigenetic enzymes called histone deacetylases—belinostat and panobinostat. We show that these enzymes are expressed at different levels in different germ cell tumor subtypes (seminomas and non-seminomas) and that both drugs are effective in reducing tumor cell viability, by decreasing cell proliferation and increasing cell death. These results are promising and should prompt further works with these compounds, envisioning the improvement of care of germ cell tumor patients.Novel treatment options are needed for testicular germ cell tumor (TGCT) patients, particularly important for those showing or developing cisplatin resistance, the major cause of cancer-related deaths. As TGCTs pathobiology is highly related to epigenetic (de)regulation, epidrugs are potentially effective therapies. Hence, we sought to explore, for the first time, the effect of the two most recently FDA-approved HDAC inhibitors (HDACis), belinostat and panobinostat, in (T)GCT cell lines including those resistant to cisplatin. In silico results were validated in 261 patient samples and differential expression of HDACs was also observed across cell lines. Belinostat and panobinostat reduced cell viability in both cisplatin-sensitive cells  and, importantly, also in matched cisplatin-resistant subclones , with IC50s in the low nanomolar range for all cell lines. Treatment of NCCIT-R with both drugs increased acetylation, induced cell cycle arrest, reduced proliferation, decreased Ki67 index, and increased p21, while increasing cell death by apoptosis, with upregulation of cleaved caspase 3. These findings support the effectiveness of HDACis for treating TGCT patients in general, including those developing cisplatin resistance. Future studies should explore them as single or combination agents. Testicular germ cell tumors (TGCTs) are among the most common solid cancers in young-adult Caucasian men, and the incidence is rising due to several environmental factors. These tumors are closely related to developmental biology, which constitutes the basis of their classification ,2. AmongOverall, the prognosis of the disease is good, with five-year overall-survival rates over 95%, and most patients are effectively treated even in the event of metastasized disease, which is in great part due to the high sensitivity of these tumors to platin-based chemotherapy . HoweverDespite intense research efforts directed towards a better understanding of cisplatin sensitivity and resistance in this tumor model, a major unifying mechanism is still lacking which may be therapeutically targeted ,11,12,13Herein, we aimed to assess the therapeutic potential of the two most recently Food and Drug Administration (FDA)-approved HDACis, belinostat and panobinostat, in (T)GCT cell lines including those resistant to cisplatin. We show that both agents, at low nanomolar concentrations, effectively reduce cell viability, and reduce the proliferation of cell lines, whereas apoptosis was increased.2+ -dependent HDACs , disregarding the sirtuins’ family (class III), which is NAD+ dependent. We found that class I HDAC1 and HDAC2 were expressed at higher levels when compared to the remaining players. Also, most HDACs  were upregulated in NSs compared to SEs. HDACs class II , on the other hand, were upregulated in SEs compared to NSs   that were expressed at high levels overall or, importantly, we focused on those in which differential expression between SEs and NSs was significant . Using real-time quantitative polymerase chain reaction (RT-qPCR), HDAC2 disclosed the highest expression levels. We also verified that HDAC1, 2, 7, 8, and 11 were significantly upregulated in NS tumor samples compared to SEs B–G. When 0.0072) .p < 0.0001) and HDAC2 (p = 0.0268) GCT cell lines for deacetylation ) and HDA 0.0268) A–D. The Our data demonstrate that HDACs are differentially expressed among TGCT subtypes, being upregulated in NSs and show high protein expression in those exposed to cisplatin.Next, we explored the expression of the previously selected HDACs in four (T)GCT cell lines. HDAC1 and HDAC2, followed by HDAC7′s transcript levels were the highest and significantly differed among the various cell lines. Overall, the SE-like TCam-2 cell line showed the lowest expression levels for most HDACs, except for HDAC7, where it was upregulated compared to other cell lines. The highest expression of HDAC1, 2, 8, 9, and 11 was observed in NCCIT or 2102Ep cell lines . At the We also explored the expression of selected HDACs on the cell line showing the highest resistance to cisplatin  or panobinostat (1 to 25 nM) for 72 h. A dose-dependent and time-dependent effect on cell viability (both cisplatin-sensitive and -resistant clones) was observed for both drugs and for all cell lines . RegardiOur data show that both HDACis are effective in reducing cell viability at low nanomolar concentrations, in three cisplatin-sensitive cell lines and, more importantly, also in the three matched cell line clones made resistant to cisplatin.Subsequently, we aimed to assess the impact on cell proliferation and apoptosis. For this, we chose the NCCIT-R cell line as surrogate, for being the most resistant (the highest IC50) to cisplatin.p < 0.0001) decrease in cell proliferation was found comparing to the vehicle, assessed by BrDU assay (When treating NCCIT-R with different concentrations of belinostat (50 and 100 nM) and panobinostat (5 and 10 nM), corresponding approximately to the IC50 and 2 × IC50 for this cell line, a significant  and panobinostat (p = 0.0084) when using a specific apoptosis assay  proteins, usually associated with transcription repression, and are grouped into four classes. Changes in the expression of these enzymes have been reported in several cancers and, importantly, inhibitors have been used effectively as cancer treatment , includiPRAME in cell lines representative of NS [Few works have focused on HDACis for treating (T)GCT cell lines. Nettersheim et al.  first shve of NS . More reve of NS ,50, whicve of NS . These sve of NS . For allve of NS ), reportMoreover, we showed a significant increase in apoptosis and a decrease in cell proliferation when treating the cell line most resistant to cisplatin (NCCIT-R) with belinostat and panobinostat . We furtFinally, following the work of Steele et al. , who shoIn future works, we intend to pursue in vivo studies, to assess more closely the effectiveness of these agents and, importantly, their toxicity profile on control subjects. This will be fundamental to assess the practicability of implementing these drugs in the clinical setting. Moreover, we intend to validate these findings and to explore in more detail if and how HDACs may be involved in cisplatin-resistance, through the knock-down of specific HDAC isoforms using CRISPR-Cas9 technology. Of particular interest are the findings of Cipro et al. in neuroblastoma cell lines, who indicate that HDACis overcome hypoxia-induced cisplatin resistance by interfering with acetylation of HIF-1α ,56, suggTo explore the expression of the several HDACs among TGCT subtypes and select the most promising candidates for validation in our cohort, we explored The Cancer Genome Atlas (TCGA) database using the publicly available cBioPortal tool . RNA seqPatient tumor samples were retrospectively selected from type II TGCT patients undergoing radical inguinal orchiectomy between 2005 and 2017 at the Portuguese Oncology Institute of Porto (IPO Porto), Portugal. A total of 161 TGCT patients were included. Additionally, 14 metastatic samples exposed to cisplatin  were included. All patients were treated at IPO Porto by the same multidisciplinary team. Specimens were routinely fixed in formalin and embedded in paraffin (FFPE) for subsequent staining with Hematoxylin and Eosin (H&E) and histological examination. All histological material was reviewed by the same TGCT-dedicated pathologist according to the most recent 2016 World Health Organization classification (cohort full characteristics reported in ). ClinicFrom each patient, a representative tumor block , with >70% tumor cellularity and low necrosis content, was selected. Importantly, distinct tumor components within the 62 mixed tumors were individually dissected and independently considered for downstream analyses, allowing to conclude on expression patterns among histological subtypes (as previously reported by us ). Thus, A summary of the study cohort is depicted in Antigenic recovery was performed for 20 min with citrate buffer in a microwave. The immunohistochemistry protocol used is described in full in . Slides The immunoexpression of each target was assessed separately for each TGCT component . As the staining was overall diffuse in all cases, but with great variation in staining intensity, we focused on the latter. The intensity of staining was considered as “weak”, “moderate”, and “strong”, as previously defined .n = 4) TCam-2 (a SE-like cell line) and NCCIT, 2102Ep, and NT2 (representative of NS) were kindly provided by Prof. Leendert Looijenga. These cell lines have been previously characterized, including copy number alterations, and were cultured as described [n = 6) were kindly provided by Prof. Daniel Nettersheim and generated by Dr. Christoph Oing and Prof. Friedemann Honecker. The resistant clones  were obtained from the parental matched clone  upon culturing with increasing doses of cisplatin, as reported before [Mycoplasma spp. . Cisplatin was kindly provided by IPO Porto’s Department of Pharmacy. Belinostat and panobinostat were purchased from Selleckchem, Houston, TX, USA .The (T)GCT cell lines . cDNA synthesis (1000 ng) was performed as described elsewhere [Total RNA was extracted using FFPE RNA/DNA Purification Plus Kit  for FFPE tumor samples and TRIzol  for cell lines. RNA quantification and purity were assessed in NanoDroplsewhere .® 480 multiwell plate system  using the following gene expression assays: HDAC1, HDAC2, HDAC7, HDAC8, HDAC9, and HDAC11 . For normalization purposes, the 18S rRNA was used as housekeeping . Results were plotted as relative expression of targets (target gene mean quantity/18S mean quantity), multiplied by 1000 for easier tabulation. Serial dilutions of cDNA obtained from Human Reference Total RNA  were used to compute standard curves for each plate. All experiments were run in triplicates and two negative controls were used in each plate. For cell lines, data was plotted using the 2−ΔΔCt method. Five biological replicates were used, and reactions were also run in triplicates.RT-qPCR was run in the LightCyclerTotal protein was extracted from cells, in biological triplicates, using the radioimmunoprecipitation assay buffer (RIPA)  complemented with 10% of protein inhibitor cocktail (PIC). After 15 min on ice, samples were centrifuged at 13,000 rpm for 30 min at 4 °C and the supernatant was collected. Protein was quantified using the Pierce BCA Protein Assay Kit , according to manufacturer’s instructions.Aliquots of 30 µg total protein from each cell line were resuspended in loading buffer, denatured at 95 °C for 5 min, and loaded in 8% or 12.5% polyacrylamide gels (as appropriate), where they were separated by size through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at 120 V. Then, proteins were transferred to 0.2 μm polyvinylidene fluoride (PVDF) membranes  using 25 mM Tris-base/glycine buffer and a Trans-Blot Turbo Transfer System (Bio-Rad) at 25 V and 1.3 mA for 10 to 15 min, as appropriate. Membranes were blocked with 5% bovine serum albumin  or 5% dry milk in TBS with 0.1% Tween  as appropriate, and then incubated with primary antibody . Lastly,2 overnight. On the next day, cells were given the respective treatments. At 72 h of exposure to the drugs, cells were fixed with 4% paraformaldehyde (PFA) for 10 min and permeabilized with 0.25% Triton X-100 solution in phosphate-buffered saline (PBS) for 15 min. Cells were then blocked with 5% BSA for 30 min, followed by incubation with Ki67 primary antibody (TM) for 1 h, at room temperature. Nuclear staining was performed with 4′,6- diamidino-2-phenylindole  in mounting medium. The overall proportion of stained cells was ascertained in a fluorescence microscope. Pictures were taken by the fluorescence microscope Olympus IX51 with a digital camera Olympus XM10 using CellSens software .Immunofluorescence for Ki67 was performed at 72 h of treatment with belinostat and panobinostat. Cells were seeded in 96-well plates at 6000 cells/well (seeding determined after optimization) and allowed to adhere at 37 °C, 5% COantibody , overnig2. In each experiment, the vehicle alone was included .Briefly, the viability assay was performed at 24, 48, and 72 h of treatment with belinostat and panobinostat. Cells pre-treated with non-toxic concentrations of belinostat for 72 h were subsequently exposed to cisplatin for an additional 72 h . Cells were plated into 96-well plates in medium at a density of 6000 cells/well (seeding density previously optimized) and incubated overnight, at 37 °C in 5% COFor the viability assay, resazurin  was used. The culture medium was removed, and cells were incubated for 3 h at 37 °C with 100 μL of 1:10 resazurin solution in culture medium. The solution was then removed, and spectrophotometric measurement was done at 560 nm (reference wavelength: 600 nm) in a microplate reader . Wells with the resazurin solution were used as blank to correct the OD values. ODs obtained for each time point were all normalized for the 0-htime point. At each time-point, the HDACi and vehicle were freshly added to the wells and the procedure was repeated the next day. All experiments were performed with biological triplicates, each with experimental triplicates. IC50 values were extrapolated from the sigmoidal dose-response (four-parametric logistic equation) with a variable slope, as calculated on GraphPad Prism 6.2. At each time point, cells were previously incubated with 20 μM BrdU labeling solution for 12 h. After removing labeling medium, cells were fixed for 30 min at room temperature with FixDenat solution, after which anti-BrdU-POD antibody (1:100) was added. After 90 min the antibody was removed, and cells were rinsed three times with 1 × PBS. The immune complex formed was detected by adding 100 μL/well of substrate solution and incubated for 5 to 10 min, until color development. Then, the reaction was stopped with 1M H2SO4 added to each well, and the reaction product was quantified in a microplate reader by measuring absorbance at 450 nm (reference wavelength: 690 nm). ODs obtained for each time point were normalized for the 0-h time point. In each experiment, the vehicle alone was included. All experiments were performed with biological triplicates, each with experimental triplicates.The BrdU assay was performed at 24, 48, and 72 h of treatment with belinostat and panobinostat. Cells were plated into 96-well plates in medium at a density of 6000 cells/well (seeding density previously optimized) and incubated overnight at 37 °C in 5% CO2. After 24 h, the Cell-APOPercentageTM apoptosis assay kit  was followed according to the manufacturer’s instructions. Next, cells were incubated with 300 μL/well of APOPercentageTM dye solution at a ratio of 1:20, for 30 min at 37 °C. Cells were washed with PBS 1× and detached from the wells with TrypLeTM Express  at 37 °C. Then, APOPercentageTM dye release reagent was added and the plate was vigorously agitated for 15 min, followed by colorimetric measurement at 550 nm with 620 nm reference filter . H2O2 was used as a positive control. ODs obtained were normalized for the 0-h time point. In each experiment, the vehicle alone was included (DMSO).The apoptosis assay was performed at 24 h into treatment with belinostat and panobinostat. Cells were seeded in a 24-well plate at a density of 45,000 cells/well (seeding density determined after optimization) and incubated for 24 h at 37 °C and 5% COp-values were adjusted for multiple comparisons . Chi-square and Fisher exact tests were used as necessary for establishing associations between categorical variables. Statistical significance was set at p < 0.05 and is reported in graphs as following: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.Data were tabulated using Microsoft Excel 2016 and analyzed and plotted using GraphPad Prism 6. Percentages were calculated based on the number of cases with available data. Non-parametric  tests were used for comparing mRNA/protein expression levels among all samples (patients and cell lines), as necessary. All To conclude, we demonstrated, for the first time, the effectiveness of two recently FDA-approved agents, belinostat and panobinostat, for treating cisplatin-sensitive and, importantly, cisplatin-resistant (T)GCT cell lines, reducing cell viability, inducing cell cycle arrest and apoptosis. HDACis show promise as single or combination agents, and our work builds up evidence of their effectiveness for treating TGCT patients developing cisplatin resistance, which, in our view, should motivate clinical trials for assessing the clinical utility of these epidrugs.


The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.Medulloblastoma (MB) is the most common malignant brain tumour in children and is subdivided into four subgroups: WNT, SHH, Group 3, and Group 4. These molecular subgroups differ in their metastasis patterns and related prognosis rates. Conventional 2D cell culture methods fail to recapitulate these clinical differences. Realistic 3D models of the cerebellum are therefore necessary to investigate subgroup‐specific functional differences and their role in metastasis and chemoresistance. A major component of the brain extracellular matrix (ECM) is the glycosaminoglycan hyaluronan. MB cell lines encapsulated in hyaluronan hydrogels grew as tumour nodules, with Group 3 and Group 4 cell lines displaying clinically characteristic laminar metastatic patterns and levels of chemoresistance. The glycoproteins, laminin and vitronectin, were identified as subgroup‐specific, tumour‐secreted ECM factors. Gels of higher complexity, formed by incorporation of laminin or vitronectin, revealed subgroup‐specific adhesion and growth patterns closely mimicking clinical phenotypes. ECM subtypes, defined by relative levels of laminin and vitronectin expression in patient tissue microarrays and gene expression data sets, were able to identify novel high‐risk MB patient subgroups and predict overall survival. Our hyaluronan model system has therefore allowed us to functionally characterize the interaction between different MB subtypes and their environment. It highlights the prognostic and pathological role of specific ECM factors and enables preclinical development of subgroup‐specific therapies. © 2020 The Authors.  Medulloblastoma (MB) is the most common malignant brain tumour in children and is subdivided into four subgroups: WNT, sonic hedgehog (SHH), Group 3, and Group 4. These molecular subgroups are characterized by different metastasis patterns and related prognosis rates , 2, 3, 4et al observed and quantified characteristic tumour phenotypes according to the MB subgroups as radiological differences in primary tumour sizes and metastatic phenotypes [Metastatic dissemination is the most powerful predictor of poor outcome across all MB subgroups , 10, 11.enotypes , 13. In enotypes .in vitro model system that allows long‐term growth and remodelling of the local extracellular matrix by encapsulated tumour cells. The brain microenvironment is unique in its composition and characteristics [in vitro system currently exists that addresses the unique composition and characteristics of the brain microenvironment.Based on the above observations, we hypothesized that interaction with the surrounding extracellular matrix (ECM) might differ between the MB subgroups, thus explaining subgroup‐specific adhesion, invasion, and growth patterns. The radiological findings suggest that the most significant differences in ECM interaction and remodelling might be expected between Group 3 and SHH tumours, while Group 4 tumours would be characterized by an intermediate phenotype. Investigation of subgroup‐specific ECM interactions required a modifiable but realistic eristics , 16, 17.eristics . No in vHere, we developed a 3D cell culture model that allows investigation of the different metastatic tumour phenotypes observed in medulloblastoma subtypes. We used a 3D HA hydrogel model to show that the specific ECM composition as well as active ECM remodelling by MB tumour cells alters the adhesion, invasion, and growth characteristics of the tumour. Our model retains cell–HA interactions, which are potentially a crucial part of the brain tumour–matrix interface , 20. BasHyaluronan cross‐linked hydrogels were prepared according to the manufacturer's recommendations  using particular hyaluronan (1%) and Extralink (2% PEGDA) concentrations. Further details can be found in For cell viability assays, Prestoblue reagent  was used according to the manufacturer's instructions and fluorescence intensity was measured after 40 min incubation time using a microplate reader . For 3D hydrogel viability assays, all gels were washed four times with HBSS buffer (Thermo Fisher) before fresh medium was added to each well. Cell viability within each gel was measured weekly after drug or vehicle treatment.50 of a single VCR dosage (5 nm) as well as the two‐fold (10 nm) and ten‐fold IC50 (50 nm) as defined in spheroid cultures. DMSO was used as a vehicle control. For the 2D experiment, VCR was renewed 24 h after the first treatment. For the 3D gel experiment, 3‐week‐old hydrogels were treated with VCR and VCR renewal was performed 24, 72, and 144 h after the first dose. In order to assess the cell's recovery potential, all gels were washed and covered with fresh, drug‐free medium 1 week after the final VCR treatment and monitored for a further 4 weeks .Vincristine (VCR)  was added to the cell culture medium at three different concentration of VCR dissolved in DMSO . The VCR concentrations chosen were the ICpost hoc test to correct for multiple comparisons as indicated. Normal distribution and homogeneity of variance were tested using the Kolmogorov–Smirnov test and the F‐test. For nonparametric group results, the Kruskal–Wallis test with Dunn's post hoc test was performed. Significance levels are indicated as *p < 0.05, **p < 0.01, and ***p < 0.001. All statistical analyses and plots were carried out using GraphPad Prism 7.05  unless stated otherwise. The number of biological samples, corresponding statistical test, and significance levels are indicated in each figure legend. Kaplan–Meier survival data analysis was performed using the R2: Genomics Analysis and Visualization Platform (http://r2.amc.nl) and statistical significance was tested using the log‐rank test as described by Bewick et al [Results are shown as mean ± SEM of the indicated number of independent experiments. The statistical significance of differences of group results was compared using one‐ or two‐way analysis of variance (ANOVA) with a Dunnett's ck et al .Detailed methods for Medulloblastoma cell lines, culturing, and reagents; HA hydrogel preparation, long‐term cell culture, and quantification of invasion; Etoposide drug treatment in 3D; Western blotting; RNA isolation and RNA sequencing; RNA sequence data analysis; Microarray data analysis; Combined data analysis; Immunohistochemistry of HA hydrogels and tissue microarrays; and ECM protein enrichment analysis, ECM subtype definition, and overall survival analysis are presented in in vitro  in HA hydrogels and their growth was monitored for up to 60 days  is commonly used for MB treatment, for example as part of the Packer protocol [50 VCR concentration did not significantly reduce cell viability of the SHH cell line but the two‐fold and ten‐fold VCR IC50 were effective  and monitored nodular growth for several weeks  was the most highly expressed gene in Group 3 tumours relative to SHH and showed intermediate expression in Group 4 , ITGA6, and ITGA7  Figure –S10. TheSince we already observed MB subgroup‐specific invasion patterns and chemotherapy responses after long‐term growth in HA hydrogels, we hypothesized that these differences might also reflect a cell line‐specific ECM deposition. To test for the presence of secreted laminin and vitronectin, we processed pure HA hydrogels after 3 weeks of cell encapsulation for immunohistochemistry and stained the nodules for CD44, laminin, and vitronectin Figure . The HA To summarize, ECM factors that have been identified from MB patient's RNA and protein data were expressed and secreted by MB cell lines growing as nodules inside our HA hydrogel model, confirming the biological relevance of our realistic 3D models. Therefore, differences in ECM factor expression level and distribution throughout the nodule might contribute to the observed functional differences in invasion and chemoresistance of the MB subgroups Figures  and 2.et al's data set [high/VTNlow’ subtype correlated with a very good prognosis, while patients with low expression of laminin and vitronectin had a significantly worse survival prognosis . In contrast, low laminin expression but high levels of vitronectin were associated with the worst overall survival in Group 3 patients . Interestingly, the five Group 3 patients with high laminin and low vitronectin expression had the best overall survival. While a high laminin and low vitronectin expression defined patients with good overall survival rates in the SHH and Group 3 cohorts, the opposite was true for Group 4, indicating differences in the response to laminin between the MB subgroups . In Group 4 patients, the ‘LAMhigh/VTNlow’ subtype correlated with a very poor prognosis, thus identifying a new high‐risk Group 4 subtype. ECM subtypes can therefore be used to identify subgroup‐specific high‐risk patients.Having provided evidence that laminin and vitronectin can alter the growth and adhesion patterns of MB subgroups and that both are actively secreted by the tumour cells, survival analysis based on ECM subtypes was then performed using Cavalli data set   treatment kills the SHH MB cell line ONS76 effectively, while Group 3 nodules display strong resistance comparable to clinical observationsFigure S4. Differential gene expression analysis of the data set from Cavalli et al [li et al  reveals Figure S5. MB patients express subgroup‐specific levels of laminin (LAM) and vitronectin (VTN) genesFigure S6. Protein expression of laminin and vitronectin in TMAsFigure S7. Analysis of the data set from Cavalli et al [li et al  reveals Figure S8. Cell growth on pure HA hydrogels reveals remarkable differences between SHH cell lines and Group 3 and Group 4 cell linesFigure S9. Cell growth on laminin‐supplemented HA hydrogels revealed remarkable differences between SHH cell lines and Group 3 and Group 4 cell linesFigure S10. Cell growth on vitronectin‐supplemented HA hydrogels revealed remarkable differences between SHH cell lines and Group 3 and Group 4 cell linesFigure S11. ECM subtypes are composed of different MB subgroup proportionsTable S1. Clinicopathological characteristics of MB patients included in the TMAsClick here for additional data file.


In several fish species homozygous and heterozygous clonal lines have been produced using gynogenetic and androgenetic techniques. These lines are standardized and can be reproduced over generations. In rainbow trout such lines have existed for decades and has become important research tools in genome studies as well as in studies of commercially important traits. The Atlantic salmon is one of the best studied fish species globally, but all experiments are done on fish of wild or domesticated origin and access to standardized immortal fish lines would be of great benefit. Here, we describe the protocols developed to produce mitotic gynogenes, and from these the first clonal lines in Atlantic salmon.Atlantic salmon eggs fertilized with UV irradiated sperm combined with a pressure shock applied at 4700–4800 minC at 8 °C gave all homozygous (doubled haploid) gynogenetic progeny with high survival. From the six first maturing females, five all homozygous clonal lines were produced by meiotic gynogenesis and were verified as clonal and identical to their mother with microsatellite markers.We have now produced the first documented cloned Atlantic salmon lines. This work demonstrates the potential for production of further Atlantic salmon clonal lines, potentially with distinct characteristics. Such lines will provide an important resource for further elucidation of phenotypic and genetic traits in this globally important species. Brachydanio rerio  (t2-t1)− 1 )SSsp2216 , Ssa197,, Ssa202 , SsaD157 SsaD144 , Ssa289,SsaOsl85 , MHC I [5 [MHC I  and MHC  [MHC II . Polymerp ≤ 0.05. Length, weight, and K factor data were first checked for normality within group using the Shapiro-Wilk test. Subsequently, parametric data were analysed using one-way ANOVA whereas non-parametric data were analysed using the Kruskal-Wallis test with treatment (3–6 levels depending on the timepoint) as a categorical variable. When main effects were significant, we used Tukey’s or Dunn’s multiple comparisons tests for parametric and non-parametric data, respectively. Each time point was analysed separately.Data were analysed in GraphPad Prism, version 6.0. Significance was assigned at Additional file 1 Table S1. Mean ± SD (N) weight, length and condition factor (K) and sexual maturation  of control fish and fish from the different treatment protocols.Additional file 2 Table S2. Microsatellite analysis of 2 parents and 362 offspring from Exp2011 (row 1–366). The first 45 individuals (controls) were fertilized with diluted, but unirradiated milt. The others were fertilized with UV-irradiated milt and pressure shocked. Alleles that are exclusive for the Sire (sperm donor) are in green and blue and for the female in red and yellow. Only two individuals from the putative mitotic gynogenetic groups showed any paternal contribution (rows 55 and 216). The females that matured in 2014 and were used to produce the isogenic lines are shown in col. A with the number indicating which line they originated from. Rows 369 to 424 compares the microsatellites of the clonal founders with their progeny. Microsatellite data from Exp2012 is shown in lines 430 to 702 with the parents in lines 430 and 431, the doubled haploid progeny in lines 433 to 672 and the controls in lines 673 to 702.Additional file 3. Details on condition of PCR reactions


Cancer cell lines are widely used in basic research to study cancer development, growth, invasion, or metastasis. They are also used for the development and screening of anticancer drugs. However, there are no clear criteria for choosing the most suitable cell lines among the wide variety of cancer cell lines commercially available for research, and the choice is often based on previously published reports. Here, we investigated the characteristics of liver cancer cell lines by analyzing the gene expression data available in the Cancer Cell Line Encyclopedia. Unsupervised clustering analysis of 28 liver cancer cell lines yielded two main clusters. One cluster showed a gene expression pattern similar to that of hepatocytes, and the other showed a pattern similar to that of fibroblasts. Analysis of hepatocellular carcinoma gene expression profiles available in The Cancer Genome Atlas showed that the gene expression patterns in most hepatoma tissues were similar to those in the hepatocyte-like cluster. With respect to liver cancer research, our findings may be useful for selecting an appropriate cell line for a specific study objective. Furthermore, our approach of utilizing a public database for comparing the properties of cell lines could be an attractive cell line selection strategy that can be applied to other fields of research. Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world, accounting for more than 700,000 new cases and 600,000 deaths globally per year . Direct-One of the main issues with using cancer cell lines for research and development is their limited predictive value regarding the efficacy of novel treatments, and they are not always appropriate as cancer models . In prechttps://www.cancer.gov/about-nci/organization/ccg/research/structural-genomics/tcga). Given the benefits of current technologies, we were able to take full advantage of these detailed datasets. To determine unknown biological characteristics, unsupervised clustering based on transcriptome similarity has emerged as a powerful application of these datasets. In some cases, cancer cell lines are used as a surrogate for epithelial cells. Moreover, the dataset of the atlas of human primary cells can be used for comparing gene expression profiles between cancerous and non-cancerous cells . For clinical samples from TCGA, the fold change between the two clusters was calculated. Genes were then sorted according to fold change after subtracting the average expression level between two clusters.We analyzed the clinical data of patients classified into two clusters defined based on cell line analysis. The weight of the surgical specimen, a continuous variable, was assessed using the Brunner-Munzel test. The operating procedure, a categorical variable, was assessed using Fisher’s exact test. Cox proportional-hazards model and log-rank test were used for survival analysis. A p-value of less than 0.05 was considered statistically significant.https://david.ncifcrf.gov/) via the Bioconductor “RDAVIDWebService” package, with a Benjamini-Hochberg-adjusted p-value < 0.2. We used “GOTERM_BP_FAT” level annotation. The top ten GO terms were visualized using bar plots, with the q-value and the number of enriched genes.We performed GO enrichment analysis of the A and B genes through DAVID  using several R  and Bioconductor (3.5 and 3.6) packages (https://github.com/fk506cni/cell_line_comparison). Our protocols were deposited in protocols.io (dx.doi.org/10.17504/protocols.io.bq5vmy66). Comparison of a continuous variable in 2 groups was assessed using the Brunner-Munzel test. Qualitative data were compared using the Fisher’s exact test to compare categorical variables. A p-value of less than 0.05 was considered statistically significant.Statistical analysis and data visualization of the expression array and RNA-seq were performed on the R studio (version 1.2.5042) server and R studio (1.3.959) , whereas the B-dominant group did not include T1 .We performed GO enrichment analysis for the A and B genes using DAVID. Enriched terms in the A genes were associated with lipid metabolism, and those in the B genes were associated with extracellular matrix organization and cell adhesion . AlthougWe integrated the expression data of liver cancer cell lines and primary cells to analyze the DEGs that allowed us to investigate similar cellular properties. We used datasets from the CCLE and the atlas of human primary cells . Cell liWe constructed a representative vector from several classes of primary cells and clusters of cell lines to investigate which cells categorized as stromal cells resemble the cluster B gene expression pattern. This vector is presented as the log2 median signal intensity of the expression values of each probe set ID. The correlation analysis was performed among the representative vectors of cells, and primary fibroblasts were found to be similar to the cluster B cell lines. However, in this analysis, group C was the most similar to cluster B, followed by cluster A. Therefore, high similarity was confirmed among the hepatoma cell lines .The results of GO enrichment analysis, DEG analysis, and correlation analysis revealed that the gene expression pattern of cluster A is similar to that of hepatocytes, and the gene expression pattern of cluster B is similar to that of fibroblasts.We analyzed 947 cancer cell lines in the CCLE to identify fibroblast-like cell lines by correlation analysis with the representative vector. Setting the value of primary fibroblasts to 1, only 23 cell lines with values >0.88 in cluster B were identified . From thHCC is one of the most lethal cancers worldwide, and advanced-stage patients or those with recurrent disease have poor prognosis owing to a lack of effective therapies. A wide gap remains between the demand for effective drugs for treating HCC and the successful development of such drugs . TherefoTranscriptomes enable us to compare the gene expression pattern between cancer cell lines and clinical human cancer tissues. For instance, Chen et al.  comparedRecently, Caruso et al.  performeIt has been hypothesized that cancer cells may acquire fibroblast-like properties via epithelial to mesenchymal transition (EMT), which plays a role in cancer progression. However, it is difficult to recognize EMT processes in clinical practice, as EMT-related biomarkers are yet to be identified . MoreoveHeterogeneous cell populations are observed in the tumor stroma of HCC and may contain fibroblasts named cancer-associated fibroblasts (CAFs). Our results did not provide evidence of the origin of the cluster B cell line, which was established from human HCC. However, the possibility that its origin was CAFs rather than liver cancer cells cannot be excluded.In summary, liver cancer cell lines were classified into two main clusters by unsupervised clustering gene expression analysis. One cluster had a gene expression pattern similar to those of primary hepatocytes and most clinical HCC specimens. The other had a gene expression profile resembling that of the fibroblasts, and a few clinical HCC specimens exist with a similar profile. This demonstrates the usefulness of referring to datasets in public databases to select cell lines according to their gene expression profile and could potentially improve future studies in the field of cancer and anticancer drug development. The limitation of our study is that the expression patterns of cell lines are not completely consistent with those of clinical specimens. A possible cause of this discrepancy is that the bulk of tumor cells, including stromal cells, was analyzed for the clinical study. This is in contrast to the cell line analysis of almost monoclonal cells. Single-cell analysis, which has advanced considerably in recent years, can facilitate comparing the expression patterns of cell lines and clinical cancer cells.This study, comparing the properties of various cell lines using the public database, provides a promising strategy for selecting the appropriate cell line.S1 FigThe liver cancer cell lines were clearly clustered into two groups. Clusters A (red plots) and B (blue plots) show negative and positive PC1 values, respectively. The loading plots of A genes (orange vector) and B genes (green vectors) also showed negative and positive PC1 values, respectively. Thus, these genes contributed to the separation of cluster A and B cell lines.(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S1 Document(DOCX)Click here for additional data file.