Aminophenoxazinones are degradation products resulting from the metabolism of different plant species, which comprise a family of natural products well known for their pharmacological activities. This review provides an overview of the pharmacological properties and applications proved by these compounds and their structural derivatives during 2000–2021. The bibliography was selected according to our purpose from the references obtained in a SciFinder database search for the Phx-3 structure (the base molecule of the aminophenoxazinones). Compounds Phx-1 and Phx-3 are among the most studied, especially as anticancer drugs for the treatment of gastric and colon cancer, glioblastoma and melanoma, among others types of relevant cancers. The main information available in the literature about their mechanisms is also described. Similarly, antibacterial, antifungal, antiviral and antiparasitic activities are presented, including species related directly or indirectly to significant diseases. Therefore, we present diverse compounds based on aminophenoxazinones with high potential as drugs, considering their levels of activity and few adverse effects. Secondary metabolites are natural products produced by organisms to participate in their biological processes. As they are the result of natural selection through evolution, they are highly optimized structures. Thus, their affinity with the proteins to be synthesized and modified, or their influence on pathological processes , have made of these natural products an increasingly choice for the development of new drugs, either by themselves, or more frequently, through structural synthetic derivatives. These substances are excellent lead compounds in terms of selectivity, specificity, effectiveness and pharmacokinetics. Besides, their structural diversity allow one to secure suitable chemotheques .Aminophenoxazinones are degradation products resulting from the metabolism of different plant species. They comprise a family of natural products well known for their pharmacological activities. In fact, this particularly high interest on these compounds started more than a century ago, as their different industrial and medical applications as pigments, pesticides or drugs were being discovered. Aminophenoxazinones are tricyclic structures with double bonds in aromatic systems containing oxygen and nitrogen atom, which facilitates the development of synthetic derivatives to enhance the properties of these molecules.This review provides an overview of the research works that have focused on the pharmacological properties and applications of aminophenoxazinones and its structural derivatives. The most relevant previous review related to aminophenoxazinones was published in 2013 and was focused on Phx-3 [50) provided in the bibliography for the different assays have been included. The compounds named Phx-1, Phx-2 and Phx-3 have been the most evaluated in the literature, especially Phx-3, having shown a number of promising properties.First, we provide information about the biosynthesis of aminophenoxazinones in plants. Then, their anticancer activities will be presented, including the most relevant results that have allowed us to expand knowledge about their mechanism(s) of action. Antibacterial, antiviral and antiparasitic properties are subsequently detailed. To quantify the levels of activity and facilitate a comparison of the compounds, the values of the half maximal inhibitory concentrations  and 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) . Those, Because of the diversity of products that can be obtained from the degradation of hydroxylated benzoxazinones depending on their cultivation soil  or the eTriticum aestivum L., whereas DIBOA-Glc is mainly concentrated in S. cereale L. [H-phenoxazin-3-one (AMPO) through the action of edaphic microorganisms when submerged into aqueous solutions [H)-one (BOA) that turns into to Phx-3 . However, DIBOA has a high persistence and could have a more important role in chemical defence mechanisms than DIMBOA in plants that produce large amounts of hydroxamic acids [DIBOA and DIMBOA can be detected inside plants as their β-D-glucosides DIBOA-Glc and DIMBOA-Glc. β-D-Glucosides are transformed into DIBOA and DIMBOA when plants are damaged, by enzymatic mechanisms catalyzed by a β-glucosidase . The latreale L. . DIMBOA olutions . This faic acids .Arabidopsis thaliana) are related to the inhibition of histone deacetylases, which provokes locus-specific alterations in histone acetylation processes [In comparison to their precursors with benzoxazinone structures, aminophenoxazinones like Phx-3 are much stable and easier to extract. These advantages, which have enabled the development of numerous studies on their biological activities  and moderocesses . In certrocesses . Anotherrocesses .The degradation process of benzoxazinones functionalized with hydroxamic acid groups leads to benzoxazolinones . BOA andAminophenoxazines are an interesting family of tricyclic compounds related to the previously mentioned benzoxazinones and benzoxazolinones. They are originated as a result of the decomposition of benzoxazolinones into their corresponding 2-aminophenol and its subsequent dimerization.A wide variety of aminophenoxazines has been developed because of their pharmacological interest . Their antiviral , antitumo-aminophenol with catechol [N-heterocyclic (4-phenyl-piperazin-1-yl)methanones derivatives showed excellent inhibition growth activities against different tumor cell lines, being remarked the activity levels of compound 1  have been developed for in vivo tumor detection by formation of fluorescent polymer nanoparticles . SimilarAminophenoxazines  were firThe increasing number of the studies that demonstrated the pharmacological properties of aminophenoxazines made the main focus of attention to move from its use as a dye to the biological field. In fact, these tricyclic compounds are the structural base of several pharmacotherapeutic agents with a wide range of biological properties.Streptomyces antibioticus [A recent study on the biological activities of certain synthetic aminophenoxazinones rendered highly promising results regarding the development of new drugs with antibacterial, antifungal, anticancer, reversal resistance as well as anti-inflammatory, antiviral or antifilarials . Moreovebioticus . This anbioticus . Actinombioticus .50 = 8.87 ± 3.49 µM) [The study of structure-activity relationships (SAR) makes possible to define optimal structural characteristics to improve the properties of the molecules. That is the case of the natural product chandrananimycin A , whose h3.49 µM) . A secon3.49 µM) . More inIn general terms, cancer is an uncontrolled cellular division process that provokes the aggregation of cells and, subsequently, the formation of tumors. A number of additional factors such as genetic mutations, improper diet, smoking or the ingestion of heavy metals and other contaminants may also have some influence on this process by altering the signaling pathways ,40. The Gastric cancer is the third cause of cancer-related death worldwide and a common pathology with a high incidence rate , being n+/H+ exchanger called NHE-1 [The intracellular pH (pHi) varies in accordance with the acidification of the extracellular medium as homeostatic response. However, cancer cells do not decrease this pHi as expected, despite of tumors lead to the acidification of the extracellular pH. The regulatory system commonly concluded to be related is the membrane protein Naed NHE-1 . Severaled NHE-1 . Some exed NHE-1 ,48.2-Aminophenoxazin-3-one (Phx-3) in particular, stands out according to the numerous studies that have proven its effectiveness to change the pHi of different cancer cell lines . In the Kasuga et al.  determin50 value between 6–12 μM) and pro-apoptotic effects in these lines. Phx-3 also generates cytotoxic effects on HT-29 (IC50 = 16.7 μM) [50 = 20.03 ± 4.98 μM) [Colorectal cancer  is the third most frequent cancer and the second leading overall cause of cancer deaths . The in 16.7 μM)  and LoVo4.98 μM) . In the The review of Tomoda et al.  gatheredThus, studies like the ones detailed in this section support that Phx-3 would be an accurate agent itself to treat gastric and colon cancers. Regarding in vivo evaluations of adverse effects, experiments in which mice were treated with  Phx-3 showed that, in addition, this compound causes low adverse effects, since they did not show gastrointestinal lesions or diarrhea during 4 weeks of administration .N-(2-hydroxyphenyl)-2-phenazinamine, known as NHP (50 = 27.82 µg/mL). Moreover, NHP was active against other human lines: COC1 , HepG2  and A549 . At comparing the activity of NHP with that found for Phx-3 against two of these lines in other studies, it is worth highlighting that the corresponding IC50 value of NHP is significantly higher than that shown by Phx-3 against A549 (5.48 ± 0.38 µg/mL), whereas a value of 6.58 ± 0.61 µM was obtained for the HepG2 line. On the other hand, the value of NHP against A549 improves that of the aminophenoxazinone Phx-1 (78.29 ± 5.11 µg/mL) [Even though the Phx family (mainly Phx-3) has been the most studied, it is possible to find evaluations of other derivatives. This is the case of n as NHP , a secon1 µg/mL) ,61. The Glioblastoma is the most common type of aggressive brain tumor in adults, whose treatment is considered a high challenge for oncology since surgery, chemotherapy or radiotherapy are not enough to prevent the tumor progression. One of the main difficulties is achieving the cross of sufficient doses of the chemotherapeutic agents through the blood-brain barrier . The abiGlioblastoma is a complex disorder that appears as a response to a particular form of cellular damage by which cells’ signaling pathways get altered. Cell differentiation is then affected, and as different cell growth factors are over-expressed, the glioblastoma cells get activated and the oncogenesis process, the process by which a normal cell becomes a carcinogen, is promoted . Two of 50 values were 2.602 ± 0.087 µM (24 h) and 1.655 ± 0.093 µM (48 h), which confirms the potent inhibitory effect of Phx-3 at low concentrations against LN229 cell line, being one of the lowest values ever measured until 2013. As an example of the previous study on the effect of Phx-3 on glioblastoma cell lines, let us point out the remarkable cell growth inhibition confirmed for U251MG cell [Focusing on the aminophenoxazinone Phx-3, Che et al.  studied 1MG cell .Che et al. also clarified the molecular mechanisms of the apoptosis induced by Phx-3, by attending to the ERK and JNK signaling pathways. The levels of these phosphorylated pathways increased significantly (6 h) after treatment with Phx-3, being maintained for 20 h. The activation of JNK was suggested to be mediated by the generation of reactive oxygen species (ROS). On the contrary, the phosphorylation of AKT and mTOR  were suppressed. All these effects would contribute with the apoptosis induced by Phx-3, and the relevance of their roles was studied by the evaluation of the activity of Phx-3 in the presence or absence of specific inhibitors. The results showed as the phosphorylation of ERK would not represent a major pathway for the induction of apoptosis, whereas AKT would act under ERK signaling. In the case of JNK, its activation would play a key role in the induction of apoptosis. Long exposure to Phx-3 would activate alternative apoptotic mechanisms by mitochondrial demoralization, like the inhibition of mTOR activity.Previous studies confirmed that treatment of the lung adenocarcinoma cell line A549 with Phx-3 resulted in the depolarization of the mitochondria, causing a decrease of pHi and the generation of ROS . The supIn summary, all the information gathered indicates that Phx-3 depolarizes mitochondria and decreases pHi, which causes the generation of ROS in mitochondria. Then, ROS activates the JNK pathway, which is a signaling pathways highly related to glioblastoma, and also the main generator of apoptosis in cancer cells by caspase cascade. The long exposure to Phx-3 would activate alternative mechanisms.The incidence of melanoma has increased at a higher rate than other solid tumors, being currently the third most common skin cancer. When at advanced stages it represents a serious threat when it spreads to internal organs, which is the major cause of death in skin-cancer patients. This disease may be due to different causes, including both genetic and environmental factors. Several factors have already been identified as significant influencers on both the incidence and the clinical and oncogenic characteristics of this disease. These factors are UV exposure, use of tanning beds, a family history of melanoma, and phenotypic characteristics of fair skin and hair color ,74.Phx-3 has been considered as effective for inhibiting the proliferation of malignant B16 melanoma cells in mice in vitro and in vivo, causing few adverse effects . In the In a subsequent study , it was From the results mentioned in this section, Phx-3 could be proposed as suitable candidate drug for the treatment or prevention of melanoma, including its cosmetic use to treat hyperpigmentation and to prevent melanogenesis.50 values, the sensitivity of the mentioned cancer cell lines after 72 h of treatment with Phx-3 showed how almost all of them were vulnerable to Phx-3 at 10 µM (IC50 lower than 8 µM). KLM-1, Lovo-1 and Y79 lines were the exceptions (IC50 close to 20 µM). The normal cell lines HEL (embryonic pulmonary fibroblast) and HUVEC  were also tested, being obtained high IC50 values , which indicates that these cells are less sensitive to Phx-3 than cancer cells.First of all, we should highlight the study that Che et al. carried out to evaluate the cytotoxicity of Phx-3 on different types of cancer cells, such as MCF-7, A431, KCP-4, A549, KLM-1, MIA PaCa-2, ACHN, LoVo-1, U251MG and Y-79 lines . In the Regarding the pHi decrease, significant results were obtained for all cancer cell lines , being comparable to those of the normal lines. The decrease was proved dose-dependent for MCF-7 (breast cancer) and A431 (skin cancer) cell lines for 30 min and ranging between 0 and 100 µM . TherefoMCF-7 and A431 lines were further evaluated to provide conclusions of the mechanism of action of Phx-3, attending at the reduction of the mitochondrial membrane potential (first and irreversible step towards apoptosis). In other study, the cytotoxic and pro-apoptotic effects of Phx-3 on hepatocellular carcinoma dRLh-84 (rat) and HepG2 (human) cell lines, and the normal hepatocellular RLN-10 (rat) cell line have been studied . Phx-3 r50 = 12.09 ± 3.29 µM) [The list of cancer lines with cell whose growth can be inhibited by Phx-3 may be completed with HeLa  , U266 (m3.29 µM)  lines. TPhx-1  is an amRegarding the pHi reduction in MCF-7 and A431 cancer cell lines, similar or rather close results as those from Phx-3 were observed at the lowest concentrations (5–10 µM) were applied. However, Phx-1 proved to be significantly less active than Phx-3 at 50 µM, and concentration had to be increased up to 100 µM for any major differences to appear in the A431 line.The study of Phx-1 on diverse cell lines . These results are in agreement with the pHi changes undergone by the cells treated either Phx-1 or Phx-3 that have been summarized in the previous paragraph. This could represent a certain advantage regarding cancer treatment, since healthy cells would suffer a lesser damage.50 value of Phx-1 for Y-79 cells  is the highest, previous studies [Although the IC studies  showed iIt is worth highlighting that Phx-1 inhibited the proliferation and induced the apoptosis of diverse human leukemia cell lines  in a dose-dependent manner. This study also proved that Phx-1 reduced in vivo the tumor growth rate in mice, whereas just few adverse effects were found on weight loss and white blood cell .Once confirmed the ability of Phx-1 and Phx-3 to induce the apoptosis of diverse cancer cell lines, Tabuchi et al. studied the effects of these compounds to induce apoptotic cell death in human neutrophils, as this kind of cells are related with the mitochondrial depolarization and reduction of pHi. Both aminophenoxazines caused apoptosis or the loss of the morphology of neutrophils, while lymphocytes and monocytes did not undergo this process. These results would suggest that Phx-1 and Phx-3 are specific drugs to induce apoptotic cell death of neutrophils, being potential preventive anti-inflammatory drugs .50 value of Phx-3 much lower (0.5 µM vs. 20 µM) [The antitumor activity of Phx-1 and Phx-3 on NB-1 (neuroblastoma cell line) was demonstrated by another study. It was thereby confirmed that both aminophenoxazinones induced apoptosis and necrosis, being the IC. 20 µM) .From the structural point of view, the differences in the bioactivity exhibited by Phx-1 and Phx-3 would be associated to the methyl group in Phx-1, even this premise is still to be confirmed. Molecular dynamics simulations could cast some light on the solubility of these compounds in cell membranes, which would allow to determine how much their structural differences affect cross-membrane processes and, thereby, explain the differences in their activity levels. In this line, certain recent studies have proven that the replacement of hydroxyl groups by fluorinated esters (in the eudesmanolide structure), improves the cytotoxicity of these compounds to cancer cells  .Recent studies have tested the activity of different derivatives with an indole group on different cancer cell lines. Indole groups are heterocyclic compounds formed by a benzene atom linked to a pyrrole compound, that have a pair of free electrons in the nitrogen atom of their aromatic ring. They are fairly common components in perfumes, drug candidates and hormones (like melatonin) . Some naTaking into account the potential pharmacological effects of indoles, as well as those already recognized of phenoxazines and the synergistic effects exhibited by some pharmacophoric hybrids , Nunewar50 values. The most inhibited cell line was A549, by the derivative 2 (IC50 = 3.71 ± 0.57 µM), characterized by two isopropyl substituents (3 (IC50 = 4.43 ± 0.64 µM). In addition, 2 was the only derivative with the previously explained DNA intercalation capacity. These results confirm that hybrid compounds are a promising alternative in the search for new anticancer drugs.Therefore, the cytotoxicity of certain indol derivatives was tested by Nunewar et al. on A549 (lung), MG-63 (bone), BT-474 (breast), Hep G2 (liver) and HCT116 (colon) human cancer cell lines, along with a normal line of lung epithelial tissue (L-132). All the derivatives tested showed remarkable ICtituents , and folN-terminal group of L-lysine was linked to the chromophore through an amide bond  conjugated with the amino acid L-lysine, which were designed and synthesized with the aim of developing novel drug compounds with anticancer potential . Similaride bond .4–7 and 4′–7′ were tested on cancer cell lines of leukemia , colon (HT-29), breast cancer (MCF-7), cervical cancer (HeLa), papillary renal cell carcinoma (ACHN), and melanoma (SKMEL-28 and G-361). All the products inhibited the proliferation of a panel of human liquid and solid neoplastic cell lines, the latter being more sensitive to antiproliferative effects (only compound 7 showed similar activity in both states). The IC50 values of the derivatives with the L-lysine side chain attached to C-10 were higher than their corresponding derivative functionalized at C-9. The IC50 values of 4–7 were comparable to that of AMD and even lower than those of Doxo and VP-16 . It is worth highlighting the lowest values of IC50, all in the range of 0.001–0.007 µM, achieved by 4 and 7 for HT-29, SKMEL-28, MCF-7 and G-361 lines. The authors proved that both compounds are strong DNA intercalators, and possess the capacity to selectively target the topoisomerase Topo IIα.Products The difference in activity between the two series of derivatives was related to structure-activity correlations. Thus, the position of the nitrogen atom in D-ring would play an important role in the antiproliferative activity, whereas the position of the L-lysine side chain and the type of amine groups affect the cytotoxic activity both between the two series and within each series.4 and 7 in particular, provide really interesting opportunities for the development of new DNA-targeting anticancer drugs.All the mentioned properties indicate that this new series of pyridophenoxazinones conjugated to L-lysine have a great antitumor therapeutic potential. Products The emergence of resistant bacteria and its impact on health, together with the inappropriate use of antibiotics, has become a pressing problem for patient safety. Infectious diseases are one of the main causes of global morbidity and mortality, especially in developing countries.Mycobacterium scrofulaceum attending to minimum inhibitory concentration (MIC) values obtained (1.4–2.8 µg/mL). Phx-3 also inhibited Mycobacterium marinum and Mycobacterium intracellulare, with slightly higher MIC values, whereas Mycobacterium kansasii showed the best inhibition levels for Phx-1 and Phx-2 . Phx-3 was suggested for the treatment of T cell-mediated autoimmune diseases, and for those chronic-inflammatory diseases induced by bacteria.In the case of Phx-3, high antituberculosis activity levels have been also reported (MIC = 3.91 µM) , as wellHelicobacter pylori, which affects approximately 50% of the world population, is a causative agent of chronic gastroduodenal ulcer and, possibly, gastric cancer. The triple therapy that comprises amoxicillin, clarithromycin (CAM) and a proton pump inhibitor, has proven to be effective to eradicate this bacterium. However, resistance is a common issue that appears when applied to the control of bacteria like H. pylori. As an example, the recovery rate when this triple therapy treatment is applied in Japan is as low as 20% [w as 20% . This low as 20% .H. pylori  and against three clinical strains . According to the results regarding cell-mediated immunity (CMI), none of the strains, including TK 1029, which has proven to be resistant to the triple therapy, grow in the presence of Phx-3 at concentrations of 1 µg/mL or higher. After 48 h, the bacterial populations were below their detection limit. Phx-3 has also been tested against H. musterae (ATCC 43772), and 0.5 µg/mL CMI value was reported.This resistance issue has brought about the development of alternative drugs over the last decade, being Phx-3 one of the drugs of interest for this purpose . Phx-3 wH. pylori have the capacity to implement certain morphological changes [H. pylori and H. mustelae strains.Inhibitors of  changes . Such chH. pylori infections to gastric lymphomas and adenocarcinomas (currently known as type 1 carcinogens) [Information provided herein would be quite relevant for the development of new drugs for the control of H. pylori based on Phx-3. Some reports associate inogens) . TherefoHelicobacter, the appearance of certain antimicrobial resistance represents a menace to the prevention and treatment of infections caused by bacteria, parasites, viruses and fungi. The diseases associated to these organisms are the third leading cause of death in developed countries [Fusarium spp. (fungi) in plants is conditioned by the resistance generated by the detoxification of certain drugs, like benzoxazolinones BOA and MBOA (plant defense compounds). This process was found to be catalyzed by the fungal γ-lactamase enzyme encoded by FDB1 locus [Similarly to the genera ountries . For insB1 locus . TherefoFusarium verticilloides [Bacillus subtilis and Staphylococcus aureus, as well as on the fungus Candida albicans, whereas no activity against Escherichia coli was observed. The derivative hydroxylated at C-6 was active for the two first species, with slightly better results [S. aureus the article by Tse et al. [S. aureus, that was attributed to the release of silver ions into the cells after getting dissociated from the complex. Like Phx-3, this complex was inactive against E. coli, which was probably explained by the obstacle posed by the double layer cell wall that characterizes gram-negative species [Phx-3 has shown high levels of toxicity to the species illoides . Tested  results . We woule et al. . Later o species .Mucor miehei and, in the case of the first, also on B. subtilis [S. aureus, C. albicans, Chlorella vulgaris, Chlorella sorokiniana and Scenedesmus suspicatus. The better activity of chandrananimycin C in the concentration tested (20 µM) would be related to its structure, which presents the scaffold of chandrananimycin B with an additional fourth ring (cyclic amine). None of the three compounds were active on E. coli.Chandrananimycin A  and B arsubtilis . In the C. albicans (MIC = 64 µg/mL) [The natural product NHP , in addi4 µg/mL) .8–15) of phenoxazines against the bacteria Listeria ivanovii, E. coli, S. aureus and Klebsiellapneumoniae spp., and against the fungi C. albicans and Aspergillusniger spp. For the synthesis of the compounds, in a first stage, 5 was synthesized, then, the rest of compounds were obtained by arylation (compounds 9–12) or amination (compounds 13–15) reactions of 8  exhibited activity against E. coli, which was more selective. In the case of S. aureus, K. pneumoniae, C. albicans and A. niger, it should be highlighted that only compound 9 presented some activity against all these species. It should also be noted that compound 8 (scaffold of 9–15) showed sensitivity to all the organisms except S. aureus and L. ivanovii, both of which are gram-positive bacteria. Therefore, it could be concluded, with regard to molecular structures, that the compounds obtained either through the arylation or amination of compound 8 causes the inactivation of the molecule on fungi, but exhibits an increased activity when applied to gram-positive bacteria.The agar well diffusion method was employed for the sensitivity study of 11 and 15 on L. ivanonii (14.42 µg/mL), being a significantly lower value than that of the reference drug (21.30 µg/mL). This higher activity than currently commercialized drugs, let us think that new derivatives could be developed to a more efficient control of microorganisms.The data obtained from the experiments show how most of the compounds are active at low concentrations. The best minimum active concentration observed was for derivatives Klebsialla sp., Bacillus sp., Proteus vulgaris, Salmonella sp., Shigella flexneri, S. aureus, Streptococcus pneumoniae and Vibrio cholerae, with compound 16 being remarkable  by detecting β-alanyl aminopeptidase activity in clinical samples [The final study presented in this section will be the finding of new chromogen aminophenoxazinone derivatives with applicability for the detection of  samples . This faIn vivo antiviral studies of aminophenoxazinones Phx-1, Phx-2 and Phx-3 in mice allowed to propose the three compounds as potential drugs for preventing the replication of HSV and the aggravation of lesions that these viruses cause. Their activity on herpes simplex virus type-1 (HSV-1) was highlighted, whereas Phx-2 was the only that showed inhibitory effects against HSV-2 (moderately). Another point of interest regarding HSV-2 was the improvement of the survival rates after the treatment with Phx-1, Phx-2 or Phx-3  . Phx-2 aThe antiviral potential of the three compounds is also promising for the human T-lymphotropic virus type-1 (HTLV-1), which causes adult T-cell leukemia (ATL). Phx-1, Phx-2 and Phx-3 prevent the cell-to-cell transmission of this virus in model cell lines, as well as the induction of apoptosis in MT-1 cells in doses of 10 μg/mL . The samRecently, Phx-3 was computationally studied by docking to evaluate its pharmacokinetic related with the inhibition against MMP9 enzymes, whose overexpression is caused by the infection of hepatitis B virus . As resuAnopheles mosquitoes [Malaria is a parasitic disease that causes high fevers, chills, flu-like symptoms and anemia. It is caused by a parasite and is transmitted to humans through the bite of infected squitoes .17, 18 should also be highlighted. However, this compound presented the disadvantage of its shorter life; probably due to the shorter chains of the tertiary amine.Ge et al.  evaluate17 was considered a suitable candidate for the development of antimalarial drugs. In the first place, it exhibited an IC50 of 7.6 nM it against Plasmodium falciparum as well as a noticeable higher selectivity when compared to the other synthesized derivatives. Moreover, the in vivo experiments that were carried out on mice infected with Plasmodium berghei  achieved the cure after three daily doses for three consecutive days.Compound Leishmania major is a parasitic species that causes cutaneous leishmaniasis, a tropical disease that generates a large number of adverse effects on human health, mainly affecting the skin and mucous membranes. Human infections occur after the bite of sand fly (Phlebotomus). A number of haloacetamines derivated from N-substituted phenoxazines, as well as other related structures such as phenothiazines, have been studied through in vitro essays [L. major. The lowest IC50 values were obtained by the products shown in 19), 9.5 µM (20) and 9.6 µM (21).o essays . The halH. pylori or F. verticilloides are examples of high sensitive species to these aminophenoxazinones. Moreover, it should be remarked their activity against herpes simplex virus type-1 (HSV-1) and human T-lymphotropic virus type-1 (HTLV-1). Remarkable antiproliferative capacity have been proven for some structural derivatives, such as those conjugated to indole groups 2 and 3 , the pyridophenoxazinones 4 and 7 , compounds 8, 9, 11, 15 and 16 , 17  and 19–21 (against Leishmania major).Our compendium of relevant studies from the literature leads us to conclude that aminophenoxazinones possess a number of promising properties like anticarcinogenic, antifungal, antiparasitic, antibacterial or antimicrobial activities. Aminophenoxazinones are degradation products that present really interesting pharmacological properties, selectivity and minimal toxicity that make them suitable for the treatment of certain current and relevant diseases as explained in this review. Phx-1, Phx-2 and Phx-3 are the most studied aminophenoxazinones in the literature, especially Phx-3, whose activities and modes of action have been detailed herein. In the case of their cytotoxicity against cancer cell lines, Phx-3 stands out as promising anticancer drug, due to the capacity to decrease the pHi in cancer cells and inhibit the proliferation of cell lines like the related to gastric and colon cancer, glioblastoma or melanoma, among others. The activity of Phx-3 has been compared to that of anticancer drugs like camptothecin, known for its side effects. In this context, in vivo studies have shown that Phx-1 and Phx-3 cause low or null adverse effects, which indicates that they would be less aggressive drugs. Regarding their antibacterial and antimicrobial activities, Phx-1, Phx-2 and Phx-3 have proven to have the desirable characteristics that would allow certain drug resistance issues to be overcome. Some non-tuberculous mycobacteria, Therefore, the active compounds mentioned herein could be considered for the development or improvement of therapeutic treatments, from the perspective of the activity levels. This conclusion supports the research studies that are currently being conducted on this family of compounds. In the authors’ opinion, it is necessary the seek and evaluate new derivatives that could allow one to define the structural features that lead to the effects observed in the different types of assays. Even though different in vivo studies have been presented in this review, we consider that the proportion of this type of assays should increase over the next years, which would bring firm conclusions for the future use of aminophenoxazinones and related compounds as drugs.


The need to produce immortal, food-relevant cell lines is one of the most pressing challenges of cellular agriculture, the field which seeks to produce meat and other animal products via tissue engineering and synthetic biology. Immortal cell lines have a long and complicated story, from the first recognized immortal human cell lines taken from Henrietta Lacks, to today, where they are used to assay toxicity and produce therapeutics, to the future, where they could be used to create meat without harming an animal. Although work in immortal cell lines began more than 50 years ago, there are few existing cell lines made of species and cell types appropriate for cultured meat. Cells in cultured meat will be eaten by consumers; therefore, cultured meat cell lines will also require unique attributes not selected for in other cell line applications. Specifically, cultured meat cell lines will need to be approved as safe for consumption as food, proliferate and differentiate efficiently at industrial scales, and have desirable taste, texture, and nutrition characteristics for consumers. This paper defines what cell lines are needed, the existing methods to produce new cell lines and their limitations, and the unique considerations of cell lines used in cultured meat. Cellular agriculture is an emerging field that aims to produce animal products from cell cultures rather than whole animals. One of the anticipated products of cellular agriculture is cultured meat made in vitro from animal cell cultures, also known as cell-based meat, cultivated meat, in vitro meat, clean meat, artificial meat, or lab-grown meat. Multiple names exist due to a lack of consensus on a term that is both informative to consumers and accurate by technical and regulatory standards ,2,3,4. GAlthough primary cell cultures may be used to study the mechanisms of cultured meat production over short time scales, primary cells can only undergo a finite number of cell divisions before they lapse into senescence or arrest of the cell cycle. This makes longer-term studies and commercial-scale production difficult. Using primary cells for cultured meat production requires that donor herds of animals be kept to provide biopsies, and that these biopsies are regularly acquired and approved for use in food production. Unlike primary cell cultures, immortal cell lines do not undergo senescence, and can exhibit infinite divisions. They could, therefore, be easier to study, and allow for safer and more consistent cultured meat without ongoing need for animal biopsies . Currently, however, there are no cultured-meat-appropriate cell lines available to researchers and developers. The culinarily appropriate components of animal meat are primarily skeletal muscle and adipose tissue ; relevanEfforts have only recently begun in developing repositories of cell lines specifically appropriate for cultured meat in order to facilitate the research and development of novel food. One example is the partnership between the Good Food Institute (GFI) and Kerafast that aims to curate a repository of standardized terrestrial and aquatic cell lines appropriate for cultured meat research ,13. ThusAs no commercially available, agriculturally relevant, immortal cell lines have been confirmed food-safe and initial repositories are seeking deposits, this paper defines the existing methods to produce new cell lines and their limitations, and the unique considerations of cell lines for use in cellular agriculture.Cell lines become immortal when they lose their cell cycle checkpoint pathways and circumvent the process of senescence. There are currently three methods to establish immortal cell lines: the discovery of spontaneously immortalized cell lines, expression of the catalytic subunit of telomerase (TERT), or induction by viral genes that inactivate p53/p14/Rb. Each method utilizes either telomerase expression or inactivation/bypassing of the cell cycle, or both, as summarized in In rare events, most often in cancer, cells will spontaneously immortalize. These cells can then be isolated. Tissue must be sampled by biopsy and then dissociated, and the cell types must be isolated and tested for proliferative capacity and identity. This was the original method to obtain cell lines beginning with the first immortal cell line derived in the 1940s from mouse fibroblasts, as well as the HeLa cell line isolated from the cervical cancer of Henrietta Lacks. Immortality of the HeLa cells could be attributed to the cells’ infection with human papillomavirus 18, which may have either degraded the tumor-suppressor protein p53 or caused chromothripsis, a chromosome-shattering and rearrangement associated with 2–3% of all cancers and which changes the expressions of thousands of protein-coding genes ,15. In addition to being discovered in samples, spontaneous immortalization can be coordinated by scientists. Cancer can be induced by radiation or chemical carcinogens. A cell line can also be put under serial passaging to select for clones with immortalization markers, high TERT expression or low p15/p16/Rb expression. One example of spontaneously immortalized cell lines in cultured meat is the chicken fibroblast line used by the company Future Meat. The Future Meat cell line was created by culturing fibroblasts isolated from a chick embryo, and isolating, concentrating, and expanding the foci of more rapidly growing cells until there was a culture of uniform morphology that was able to survive past the 20–30 divisions undergone by an unmodified somatic cell [Spontaneous immortalization has its limitations, and may be more suitable for some situations and less suitable for others. For example, spontaneously immortalized cells would likely not be considered genetically modified (GM), which could allow them access to European markets that currently have strict regulations on GM foods . Under oIn a normal cell, chromosome telomeres shorten with every replication of DNA. Repeated shortening eventually exposes the chromosome ends to damage, which leads to cell senescence. The upper limit of divisions that a cell can reach before it enters senescence is known as the Hayflick Limit, and is typically between 20 and 30 divisions . Germ liThe introduction of telomerase has been successful in immortalizing cell lines, because telomere elongation helps cells escape senescence triggered by telomere shortening. This has been performed by ectopic expression of the catalytically active subunit of telomerase (TERT), or by the overexpression of TERT. Despite the checkpoints controlling human cell immortalization , human fTelomerase expression has not been as commonly successful in immortalization as methods that inactivate or bypass the p53/p16/Rb stress response  large T-antigen (TAg), which binds to and inactivates p53/Rb as well as other tumor suppressor factors in a number of species and organ types ,29,30. IIn many cases, TERT expression or inactivation of p15/p16/Rb alone are not sufficient to immortalize a cell line, suggesting that both telomere shortening and the p53/p16/Rb stress response must be bypassed. Myoblasts have previously been shown to require the bypass of both senescence triggers to become immortal . In 2016Cellular agriculture requires cell lines made from organisms that are agriculturally relevant rather than well-characterized; therefore, some cell lines have been difficult to establish. In particular, no permanent cell line is available for marine invertebrates, such as mollusks or snails or crustaceans, perhaps due to a lack of information regarding their physiology and biochemistry and understanding of the variables required for their immortalization. To create these cell lines, scientists must first characterize their stem cell expression markers and ideal culture conditions, and must also develop methods for inducing myogenesis in the created cell lines . CurrentOne of the most common concerns about cultured meat from consumers is the safety of ingesting cell lines. Currently, cultured animal cell lines are not commonly eaten by consumers. An immortal cultured meat cell line may contain expressed oncogenes, and if so, food products produced with these cells will need to be confirmed not to have tumorigenicity ,44. AlthIn the case that a cell line is confirmed safe for consumption, it will still need to be regularly monitored for contamination and genetic drift. Contamination of cell lines with other cell lines and adventitious agents can be common in cell culture ,46,47. ARegulatory agencies are beginning to adapt pathways for cultured meat products to be examined regarding safety for consumption. To some degree, this will involve updating the existing regulations on cell-derived products for relevance to consumption as food ,52,53. ARegulatory bodies in a few other regions have begun to adapt their regulations to new cultured meat products .The immortalization method itself may be an important factor in the decision of consumers to accept cultured meat. To the best of the authors’ knowledge, no study has yet compared the consumer acceptance of cultured meat made of primary cells and cultured meat made of immortalized cells. There have, however, been initial studies analyzing the acceptance of genetically modified and non-genetically modified cultured meat. These studies found that consumers are more willing to purchase non-genetically modified cultured meat compared to genetically modified cultured meat ,60. ProdThere are, however, some differences across applications and among consumers in how genetic engineering is evaluated that may inform decisions on the immortalization of cell lines for cultured meat. Consumers appear to be more concerned when genes are exchanged between different species, and see gene insertion as more unnatural than gene deletion ,65,66. UAs mentioned in Furthermore, some cell lines may be prohibited from consumption under religious food restrictions. Jewish kosher laws and Islamic halal laws do not allow the consumption of conventional pork and other meats , and it Cell lines for cultured meat production will not only be from animals and cell types not previously established, but will also require different attributes from those previously typical for immortal cell lines. Specifically, they should be food-safe, able to proliferate stably and efficiently in a large-scale production environment with minimum costs, and have desirable taste, texture, and nutrition. Multiple companies have filed patents on ways to overcome the unique challenges of growing food-relevant cells at scale. The cultured meat company Wild Type has filed one patent on a footprint-free method of genetic modifications to control cell differentiation and proliferation with a small genetic footprint . AnotherThe sensory experience of cultured meat may be affected by the cell types used. A scientific paper on the sensorial qualities of cultured meat compared to animal-sourced meat has not been published thus far . AlthougThe sensory experience of cultured meat may additionally be affected by certain attributes to be selected for in cell lines. Consumer surveys have found that many consumers expect cultured meat to be less tasty than conventional meat, and this perception contributes to their unwillingness to consume cultured meat ,12. The Cultured meat should be designed to provide similar, if not enhanced, available levels of nutrients compared to conventional meat. Meat provides 26% of the global protein supply, 24% of the global fat supply, and 9% of the global calorie supply per capita per day, as well as essential micronutrients such as iron, zinc, and vitamins A and B12 . For culOther cultured meats could be engineered for desired differences from the nutritional profile of conventional meat. As an initial exploration of nutritional engineering for cultured meat, Stout et al. have incorporated a biosynthetic pathway for carotenoids, a class of antioxidants native to some plants but not to animals, into primary bovine satellite cells (BSCs) . They deAlthough there are several techniques used to establish cell lines appropriate for bio-logical or medical research, there are still several unanswered questions about how to establish immortalized cell lines suitable for human consumption as cultured meat and which of these existing techniques are appropriate or would need to be modified for use in cultured meat. The lack of readily available cell lines is a current barrier to conducting research in cultured meat. Most academic cultured meat research, from media composition to bioscaffolding to bioreactor projects, must currently begin by creating cell lines for study. Cultured meat will require unique cell lines that are safe, appropriate for industrial-scale production, and desirable to consumers. Although development of these cell lines can be based on established immortalization methods, they will need to be applied to cell types appropriate to meat. In addition, the properties of food safety, industrial production, and consumer desirability have only begun to be explored in cell lines for cultured meat. There is still much work to be done before a collection of cell lines appropriate to cultured meat can be established and readily available for the use of researchers and developers.


CDKN2A, CDKN2B), expression of fusion genes  or of genes targeted by point mutations . MEF2D-BCL9 and PAX R38H mutations in cell lines have hitherto been undescribed, suggesting that YCUB-4 (MEF2D-BCL9), PC-53 (PAX R38H) and their sister cell lines will be useful models to elucidate the function of these genes. All aberrations mentioned above occur in both sister cell lines, demonstrating that the sisters derive from a common ancestor. However, we also found mutations that are specific for one sister cell line only, pointing to individual subclones of the primary tumor as originating cells. Our data show that sequential sister cell lines can be used to study the clonal development of tumors and to elucidate the function of common and clone-specific mutations.For many years, immortalized tumor cell lines have been used as reliable tools to understand the function of oncogenes and tumor suppressor genes. Today, we know that tumors can comprise subclones with common and with subclone-specific genetic alterations. We sequenced DNA and RNA of sequential sister cell lines obtained from patients with pre-B acute lymphoblastic leukemia at different phases of the disease. All five pairs of cell lines carry alterations that are typical for this disease: loss of tumor suppressors ( For many decades, cell lines have enabled the modeling of human disease in cell culture for a better understanding of a plethora of pathophysiological processes and also, most importantly, for drug screening. Clearly, cell lines represent a useful in vitro tool owing to their easy manipulability and reduced culture costs; furthermore, they constitute a renewable resource, and, provided they are grown under recommended conditions, they retain their characteristic features and the results are reproducible. Nevertheless, a detailed and fully annotated characterization is fundamental before their use. In fact, in many cancer research fields, cell lines offer the most comprehensively characterized platform ,2.Additionally, in the domain of hematopoietic tumors, cell lines represent vital and powerful models in experimental systems to unravel the pro-leukemogenic roles of specific genetic mutations. As all leukemia cell lines inevitably carry molecular alterations, it is also essential here for the utility of these models to appropriately detail these genomic changes .Despite the achievement of a complete remission in the vast majority of patients with acute lymphoblastic leukemia (ALL), ALL relapse remains a leading cause of childhood cancer-related death . RelapseTo obtain further insight into the relative impact of founding genomic alterations and acquired genetic alterations, we set out to examine the gene mutation status and gene expression of paired cell lines procured from ALL patients at diagnosis and at subsequent relapse or at other consecutive stages of their disease, aiming at a better understanding of the ALL relapse processes.Cell lines NALM-20 and NALM-21 were taken from the stock of the cell lines bank . All other cell lines were supplied for research purposes. Cell lines were authenticated by DNA profiling. Detailed references and cultivation protocols for NALM-20, NALM-27 and PC-53 have been described previously . www.bioinformatics.babraham.ac.uk/projects/fastqc, accessed on 28 May 2019 and aligned by STAR (2.5.3a) . NA. NABCR apositive  24]. NA. NABCR aMEF2D translocations were described in pre B-ALL, with BCL9 being the most common fusion partner of MEF2D [MEF2D exon 5/BCL9 exon 9 fusion transcript, resulting from t  . These c of PAX5 . PAX5 deLL cases .PAX5 is also a partner of translocations, and it is the target of point mutations in this disease [PAX5 R38H mutation  along with their sister cell lines (IKZF1 deletions and deletions of CDKN2A and CDKN2B are markers of disease recurrence in adolescent and adult Philadelphia chromosome-negative pre-B ALL [BCR-ABL1-positive cases and a lower proportion (<5–25% depending on the subtype) of BCL-ABL1-negative cases carry the IKZF1 deletion [IKZF1 deletion are from adults and carry the BCR-ABL1 fusion —NUCKS1 (ex 2) fusion is expressed in the cell line PC-53 but not in the sister cell line PC-53A  analysis showed that in PC-53 both genes, NSMAF and NUCKS1, were located in the middle of the transition between two copy numbers ) fusion transcript in PC-53 and not in PC-53A.The e PC-53A . This fu numbers . These d numbers . Thus, tBecause the CGH analysis was suitable for detecting molecular differences between the sister cell lines PC-53 and PC-53A, we performed copy number analyses to elucidate whether other sister cell lines carried clone-specific copy number differences as well. Most aberrations detected by the CGH analysis were found in both of the sister cell lines (KRAS A146T (Gca/Aca) or with NRAS G12C (Ggt/Tgt) mutations   were mutant. In AT-2, 3% were mutant  . HoweverGca/Aca) . Furthere mutant . KRAS homozygously wild-type (0/0)/NRAS heterozygously mutant (0/1) and “clone B” being KRAS (0/1)/NRAS (0/0). The easiest explanation for these results is that both sister cell lines comprise two subclones, “clone A” being BCL2) and clone-specific (MYC) alterations [BCL6 [We have previously shown that B-lymphoma cell lines can comprise subclones . Twelve erations . The difns [BCL6 . The immns [BCL6 . CDKN2A, CDKN2B, ETV6, IKZF1 and PAX5, all of them recurrent deletions in pre-B ALL —BCL9 (ex 9) in YCUB-4 and YCUB-4R was especially noteworthy. BCL9 is the most frequent fusion partner of MEF2D in pre-B ALL [MEF2D-BCL9 fusion transcript. What is also novel is the PAX5 R38H mutation (COSM5986423) in cell lines PC-53 and PC-53A  and NRAS G12C , were detected in each of the sister cell lines, albeit at different percentages and not with the 50% wt vs. 50% mu read proportion that would be expected if a cell clone carried a wild-type and mutant version of a gene (KRAS A146T and the other with the NRAS G12C mutation, had already existed in the patient. AT-1 and AT-2, established at first and second relapse, comprise both subclones, albeit at different percentages (Without DNA from the primary tumor, we could not formally exclude that the sister cell specific aberrations had developed in vitro. However, the WES analysis of f a gene  . It is centages .MEF2D-BCL9 fusion in YCUB-4 and YCUB-4R and the PAX5 R38H mutation in PC-53 and PC-53A are noteworthy. Mutations specifically occurring in one of the sisters suggest a derivation from individual clones of the primary tumor, whereas common mutations point to the common ancestor. The study shows that sequential sister cell lines allow one to study common and clone-specific mutations.In sum, the results of the RNA-seq, WES and CGH analyses of five pairs of sequential pre-B ALL cell lines show aberrations characteristic for the disease. Because they are unreported in cell lines so far, the


H-pyrazolopyridine and pyrazolopyridine embelin derivatives was synthesized through a multicomponent reaction from natural embelin, 3-substituted-5-aminopyrazoles and aldehydes. The synthesized compounds were evaluated against three hematologic tumor cell lines, HEL (acute erythroid leukemia), K-562 (chronic myeloid leukemia) and HL-60 (acute myeloid leukemia), and five breast cancer cell lines . The primate non-malignant kidney Vero cell line was used as the control of cytotoxicity. From the obtained results, some structure–activity relationships were outlined. Furthermore, in silico prediction of physicochemical properties and ADME parameters were determined for the derivatives with the best antiproliferative values.A set of new dihydro-1 H-pyrazolopyridines and pyrazolopyridines have raised great interest due to the biological activities that they exhibit. A) with cytotoxic activity against triple-negative breast cancer MDA-MB-231 cell line  pyridines and pyrazolopyridines are found in the literature. Of special interest are those based on multicomponent reactions (MCRs), such as those involving the condensation of 1,3-dicarbonyl compounds with aldehydes and aminopyrazole derivatives [b]pyridines] from isatins, pyrazol-5-amines and β-ketonitriles pyridine and pyrazolopyridine embelin analogues and evaluate their potential as antiproliferative agents against eight different human hematological , and breast cancer cell lines .With these antecedents and because of our interest in the preparation of bioactive derivatives from the natural benzoquinone embelin ,16,17,181) has a masked 1,3-dicarbonyl moiety, the condensation of (1) with 4-nitrobenzaldehyde (2a) and 3-phenyl-5-aminopyrazole (3a) was carried out in order to obtain the desired dihydro-1H-pyrazolopyridine derivatives, using EtOH as solvent at room temperature. Ethylendiamine diacetate  was also used as an effective organocatalyst for the initial Knoevenagel condensation. Under these reaction conditions the product 4a was obtained in 33% yield. The structure of 4a was determined and corroborated by 1D and 2D NMR experiments  and electron-donating groups (4l–4o) good yields (73–98%) were obtained. The use of heteroaromatic aldehydes led to the corresponding products (4p–4s) in moderate yields (42–63%). This methodology is also tolerant to aliphatic aldehydes since the corresponding dihydro-1H-pyrazolopyridine adducts (4t–4w) were obtained.As we can observe, different yields were obtained depending on the type of aldehyde used in the Knoevenagel condensation. Thus, with aromatic aldehydes substituted with both electron-withdrawing (4) is shown in 2) and embelin (1) results in the formation of a methylene quinone reactive intermediate (A), which is trapped by the 3-phenyl-5-aminopyrazole (3). The reaction takes place via Michael addition to the methylene quinone system by the nucleophilic carbon in the α position to the amino group of the 3-phenyl-5-aminopyrazole to give the intermediate (B) which after a proton transfer gives the dihydropyridine system by intramolecular cyclization and dehydration.A plausible mechanism for the formation of these embelin derivatives , K-562 (chronic myeloid leukemia) and HL-60 (acute myeloid leukemia), and five breast cancer cell lines . The primate non-malignant kidney Vero cell line was used as control of cytotoxicity. The results obtained are shown in 50 values between 0.7 and 7.5 μM.None of the compounds evaluated showed significant cytotoxic activity in the triple negative breast cancer cell lines MDA-MB-231 and HS-578T. Several of the compounds showed good cytotoxic activity against the rest of cell lines evaluated with IC4a, with a 4-NO2 group, exhibited the best cytotoxic activity in HL60 with an IC50 of 0.70 ± 0.14 μM. This compound also showed good activity values in HEL (1.05 ± 0.35 μM) and K562 (1.25 ± 0.35 μM). Derivatives 4c, 4d, 4e and 4g, with substituents 4-Cl, 4-Br, 4-F and 4-CF3, respectively, also presented good values of cytotoxic activity in the three leukemia cell lines with IC50 values between 0.90 and 3.30 μM. The best IC50 value in acute erythroid leukemia (HEL) was achieved with compound 4g (1.00 ± 0.42 μM), whereas in chronic myeloid leukemia cell line (K-562), the derivative 4k (4-CN-Ph) showed the best IC50 value (0.92 ± 0.32 μM).In general terms, the best results were obtained in the leukemia cell lines compared to the breast cancer cell lines. Concerning the leukemia cell lines, compound para position of the phenyl ring at the 1,4-dihydropyridine ring, is highlighted. For the derivatives 4f, and 4h, having a -F and a -NO2 group, at the meta position a loss of cytotoxic activity was detected compared to the corresponding para-substituted derivatives (4e and 4a). Interestingly, the replacement of these electron-withdrawing groups at the para position of the phenyl ring by others such as the -COOH (4i), or -CO2CH3 (4j) also leads to a loss of activity.According to these first results, the importance of substituents in 2, -3F-4MeO, 3-4-MeO, 3,4-methylenedioxy  leads to worse values of IC50 in the leukemia cell lines, compared with those of 4b (-Ph). A clear loss of activity when heteroaromatic rings were attached at the dihydropyridin nucleus (4p–4s) was also observed, while aliphatic substituents  leads to higher values of IC50 with the exception of the derivative 4t, with a cyclohexyl group, which keeps a good cytotoxic activity (1.30 ± 0.28 μM) against HEL.On the other hand, the presence of electron-donor groups at the phenyl ring such as -NMe4a (4-NO2) showed the best cytotoxic activity against SKBR3 (1.30 ± 0.28 μM). In the MCF7 cell line, only the derivatives 4a (4-NO2) and 4u (-CH2CH3) were active, with IC50 values of 3.25 ± 1.91 and 4.75 ± 1.20 μM, respectively. Finally, several of the derivatives evaluated in BT549 cell line  presented IC50 values around 3.00 μM.With respect to the breast cancer cell lines , derivative 4c, 4d, 4g, 4t) and breast cancer  cell lines. Taking advantage of the versatility of this multicomponent reaction, and given the good results of cytotoxic activity obtained for some of the dihydro-1H-pyrazolopyridine derivatives previously synthesized, we decided to carry out modifications in the quinone and aminopyrazole components to evaluate the effect of these structural variations on the cytotoxic activity.All previously synthesized nitrogenated embelin derivatives were also evaluated in the Vero cell line of primate kidney, to determine its cytotoxicity in a non-tumor cell line and to study its viability for carrying out future in vivo tests. Interestingly, inhibition of cell viability induced by several compounds were 8- to 20-fold lower in Vero cells compared to hematological  and the presence of fluorinated groups in biologically active molecules, such as the trifluoromethyl group, is of great interest pyridine derivatives through corresponding MCRs using embelin (1) and 4-(trifluoromethyl)benzaldehyde (2g). The results obtained are shown in The aminopyrazoles (14a–14f). Furthermore, as we can see in 14g (82%) having a 2-furyl group, or aliphatic groups such as 5-methyl-3-aminopyrazole .The corresponding derivatives were obtained in good yields, from 76 to 94%, regardless of the nature of the substituent at the pyrazole ring , 14d (3-F-Ph), 14e (4-MeO-Ph) and 14g (2-furyl) presented a significant improvement in their cytotoxic activity with values of IC50 from 2.01 ± 1.12 µM to 2.40 ± 0.28 µM. All of them also showed an IC50 > 10 µM in MDA-MB-231 and HS-578BT cell lines.Some of the new derivatives showed good cytotoxic activities, especially in breast cancer cell lines. The derivative 14d (3-F-Ph) with an IC50 of 1.05 ± 0.64 μM in HL60 cell line and 0.95 ± 0.4 μM in K562 cell line. Derivatives 14e (4-MeO-Ph) and 14g (2-furyl), as in some of the breast cancer cell lines, also have good cytotoxicity values in HL60 with IC50 of 1.10 ± 0.14 and 1.10 ± 0.04 μM, respectively.Concerning the hematological tumor cell lines, we can observe an improvement in the cytotoxic activity for the derivative 4g was treated with DDQ, the corresponding pyrazolopyridin derivative (15) was obtained in 82% yield .Since other embelin derivatives have shown inhibitory activity against the human protein kinase CK2 ,18, we tUnderstanding the physicochemical properties and the pharmacokinetic profile  of molecules is an essential step to avoid failures in the selection of lead molecules during the different stages of development and discovery of new drugs. Therefore, it is essential to design chemical leads with acceptable ADME and good drug-like properties. However, about the drug-likeness of natural product derivatives, it is important to mention that many derivatives of them do not comply with drug-like rules during the lead optimization process. A lot of these compounds are found outside the drug-like space because they usually have a higher molecular weight and can be more complex than drug-like. Although the greatest value of the use of the privileged structures present in natural products is that they were optimized during the course of evolution and also, they can explore parts of chemical space that synthetic drug-like compounds do not essentially cover.H-pyrazolopyridine embelin derivatives with the best antiproliferative values . This study was performed using the Qikprop module of Schrödinger software pyridine embelin derivatives were efficiently synthesized from a modular approach that includes Knoevenagel condensation/Michael addition/ intramolecular cyclization and dehydratation. The influence on the antiproliferative activity of structural variations was investigated. Thus, for the eight tumoral cell lines tested  the presence of the free hydroxyl group at the benzoquinone nucleus, and the dihydropyridine ring were key. The best results were obtained for a C-11 long chain. Regarding the nature of the substituents at the dihydropyran ring, phenyl substituents with halogens, or -NO2 or -CF3 groups at position 4 yielded good IC50 values, while the modifications carried out in the pyrazol moiety revealed that 4-F-Ph, 3-F-Ph, 4-OCH3-Ph and 2-furyl resulted be the best substituents for the antiproliferative activity. Furthermore, the QikProp module of Schrödinger software was used as a computational method for analyzing the pharmacokinetic descriptors of the compounds with the best antiproliferative activities . The findings from this study are paving the way for further investigations concerning to obtain more selective and active compounds.A new family of dihydro-1


Allogeneic hematopoietic stem cell transplantation  is the most effective treatment and curative approach for patients with hematological malignancies. The life-saving benefits of all-HSCT include graft-versus-leukemia (GvL) effects and healthy immune reconstitution. However, the majority of patients develops graft-vs-host-disease (GvHD), leading to organ/tissue damages in the skin, liver, and GI tract. For that reason, extensive researches on GvHD have been made and identified IFNs as therapeutic targets. Nonetheless, the pleiotropic nature of IFNs has hindered our goals to optimally control GvHD.The goals of the articles collected in the Research Topic are to define the roles and mechanisms of IFNs in GvHD and to provide insights into novel therapeutic approaches to restrain GvHD while enhancing the benefits of allo-HSCT.Hess et al. highlighted the development of humanized mouse models and xenogeneic transplant model system, leading to investigation on human T cell biology. The models essentially benefit from the human T cell receptors (TCRs) having cross-reactivity to murine MHC in addition to cytokines and co-stimulatory proteins. Hess et al. further explained the importance of TCR, the ability to recognize non-self-antigens on MHC, and cellular therapy by suppressing or enhancing the secretion of cytokines and co-stimulatory/inhibitory signaling pathways.To expand the investigation on mechanisms of primary human T cell responses in GvHD, xenogeneic cell transplant models have been utilized. In depth, Zhao et al. the authors discussed the effects of IFNs on T cell and their function and epigenetic regulations during GvHD. They elaborated the paradoxical roles of IFNs in alloreactive T cell differentiation and function and the development of GvHD. The process of GvHD is also critically controlled by epigenetic regulators, such as DNA methylation, histone modification, and chromatin remodeling. In turn, these regulators modulate T cell differentiation and function by influencing IFN signaling and therapeutic potentials. Conclusively, the authors suggest future studies on epigenetic mechanisms of IFNs to better understand how IFNs regulate GvHD.In the review by Haring et al. explored the pathophysiology of GvHD caused by the imbalance of cytokine network in the GI tract. The authors summarize that similar to IBD, type I and type III IFNs are important for maintaining the intestinal epithelial barrier integrity and controlling immune responses in GvHD. However, the conditioning regimen prior to allo-HSCT damages the epithelial cells and results in the release of DAMPs and PAMPs, leading to local inflammation together with inflammatory cytokines and activation of APCs. This in turn results in the activation and expansion of the alloreactive T cells causing further tissue destruction and inflammation together with cytokines including type II IFN and chemokines in the GI tract. Overall, the review shows the highly complex and interconnected cytokines network and the effect of its imbalance on intestinal mucosal inflammation, suggesting that identifying targets downstream of IFNs and receptors might provide novel therapeutic strategies for GvHD and IBD.Not only essential for differentiation and expansion of immune cells, IFNs play an important role in the GI tract. Assal and Mapara comprehensively reviewed the role of the JAK/STAT pathway in T cell activation and expansion, APC function, and Tregs expansion. The knowledge led to the idea of disrupting the JAK/STAT pathway by using ruxolitinib or baricitinib to ameliorate GvHD. Clinical evidence has also shown that ruxolitinib administration to GvHD patients has been successful. The study by Wang et al. also showed similar results; in the retrospective study, 70 Chinese patients with steroid-refractory chronic GvHD (SR-cGvHD) received ruxolitinib, then the overall response, complete remission, and partial remission rates were examined. Twenty-four weeks after ruxolitinib treatment, those rates were significantly improved, compared to those of the patient group without ruxolitinib treatment. The authors also showed that CD4+ cells, total B cells, and IL-10 were significantly increased after the treatment, whereas NK cells, Tregs, and ST2 were significantly decreased. Collectively, these results suggest that ruxolitinib is a safe and effective treatment for SR-cGvHD.The use of JAK inhibitors is a promising therapeutic strategy for GvHD and cytokine release syndrome. Lutfi et al. authors illustrated the CD27-CD70 co-stimulatory pathway and its therapeutic potential in combination with immune checkpoint inhibitors and in GvHD. The CD27-CD70 pathway leads to survival and activation of T, B, and NK cells, and the activity is shown to be increased under pro-inflammatory conditions and IFN-γ secretion. With the understanding of its nature, studies to targeting the CD27-CD70 pathway to improve outcomes have been made. For instance, a CD27 agonizing monoclonal antibody, varlilumab, showed to improve antineoplastic response in combination with immune checkpoint inhibitors by enhancing CD8+ T cell expansion and proliferation. On the other hand, an administration of anti-CD70 monoclonal antibody and CD70 KO mice resulted in increased GvHD. Further studies explained that while APC-expressed CD70 provides a co-stimulatory signal, T-cell-expressed CD70 served an inhibitory role in T-cell response. These findings proposed that this complex mechanism may provide a potential therapeutic intervention as an oncologic therapy and to attenuate GvHD by modulating the pathway.Targeting TCR and co-stimulatory pathways also serves as a therapeutic strategy to control GvHD. In the review by + T cells, thereby inhibiting tumor growth. Liu et al. in turn hypothesized that LYG1 participated in the development of GvHD and explored the role and mechanisms of LYG1 during GvHD. As hypothesized, the study discovered that LYG1 deficiency in donor T cells reduced the severity of GvHD. In depth, LYG1 deficiency in donor T cells inhibited the activation of CD4+ T cells and expression of IFN-γ, while promoting the expression of FOXP3, thereby suppressing the expression of CXCL9 and CXCL10 and the infiltration of allogeneic CD4+ T cells into target organs. Despite of a modest improvement in the overall survival by LYG1 blockade, these findings demonstrate that targeting LYG1 may be as a potential therapeutic strategy to reduce GvHD.Previous studies suggest that recombinant human LYG1 protein (rhLYG1) can promote the activation and IFN-γ production of CD4Huisman et al. provide significant insight on the risk of off-target toxicity via allo-HLA across-reactivity. Using third-party donor-derived virus-specific T cells, Huisman et al. investigated whether HLA-restriction, HLA background, and/or virus-specificity could predict the risk of allo-HLA cross-reactivity. The results demonstrated that HLA-B*08:01-restricted T cells, had the highest allo-HLA cross-reactivity regardless of virus-specificity, suggesting a potential strategy to reduce the risk of off-target toxicity by selecting T cells with a specific HLA restriction and background.Overall, the articles in the Research Topic provided sufficient reviews on the recent advances in the roles of IFNs in GvHD. They also highlighted promising therapeutic strategies, such as CD27 agonizing monoclonal antibodies, JAK inhibitors, and targeting LYG1, to modulate IFN signaling to restrain GvHD while preserving the benefits of all-HSCT. Indeed, further researches should be followed to reveal the exact mechanisms of IFNs in the context of transplantation to provide promising transplantation therapies.BK and JC wrote the manuscript. JC is a guest associate editor of the Research Topic. All authors contributed to the article and approved the submitted version.JC is supported by Emerson Collective, NIH/NIAID (R21 AI155990), the Alvin J. Siteman Cancer Center through The Foundation for Barnes-Jewish Hospital, and the Alvin J. Siteman Cancer Center through The Foundation for Barnes-Jewish Hospital and the National Cancer Institute (P30 CA091842).JC received an honorarium from Incyte Corporation and research funding from Mallinckrodt Pharmaceuticals.The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Despite continuous improvements in multimodal therapeutic strategies, esophageal carcinoma maintains a high mortality rate. Metastases are a major life-limiting component; however, very little is known about why some tumors have high metastatic potential and others not. In this study, we investigated thermogenic activity and adhesion strength of primary tumor cells and corresponding metastatic cell lines derived from two patients with metastatic adenocarcinoma of the esophagus. We hypothesized that the increased metastatic potential of the metastatic cell lines correlates with higher thermogenic activity and decreased adhesion strength. Our data show that patient-derived metastatic esophageal tumor cells have a higher thermogenic profile as well as a decreased adhesion strength compared to their corresponding primary tumor cells. Using two paired esophageal carcinoma cell lines of primary tumor and lymph nodes makes the data unique. Both higher specific thermogenesis profile and decreased adhesion strength are associated with a higher metastatic potential. They are in congruence with the clinical patient presentation. Understanding these functional, biophysical properties of patient derived esophageal carcinoma cell lines will enable us to gain further insight into the mechanisms of metastatic potential of primary tumors and metastases. Microcalorimetric evaluation will furthermore allow for rapid assessment of new treatment options for primary tumor and metastases aimed at decreasing the metastatic potential. Many solid tumors are characterized by a good prognosis in early stages if a complete resection can be achieved but by a tremendously bad prognosis in metastatic stages. Metastases usually present the limitation of therapy and are associated with a high mortality rate. One of the cancer types in which this is highly pronounced is esophageal carcinoma, which shows an increasing incidence of adenocarcinoma in the western population over the past decades . MortaliAn emerging alternative is the analysis of biophysical markers of tumor cells. The focus here lies on physical aspects of the cells themselves, such as cell deformability, cell membrane water transport, or adherence of cells to, for example, the extracellular matrix (ECM) ,4,5,6. MA further biophysical approach of tumor cell analysis is the investigation of the bioenergetic capability of tumor cells. This can, for example, be determined by measuring tumor thermogenesis, which in turn is linked with almost all cellular events in the cells’ energy turnover . In thisIn the current study, we used two paired stable esophageal adenocarcinoma cell lines generated from primary tumors and lymph nodes of the same patients. Comparing cell lines derived from the same patient makes our data unique even though the number of patients is low, as matched primary tumor and lymph node cell lines are rare. Furthermore, we investigated the thermogenic activity as well as the adhesiveness of primary cells and their corresponding metastatic cell lines in combination the first time. We evaluated any correlations between metastatic potential of the metastatic cell lines and higher thermogenic activity or decreased cell adhesion strength, respectively.2.Cell lines PT1590, LN1590, PT6216, LN6216c and LN6216gc were acquired from primary tumor and lymph nodes of patients with esophageal adenocarcinoma at the University Medical Center Hamburg-Eppendorf as described previously ,14,15. Ct-test or analysis of variance with a post-hoc test was used. p values less than 0.05 were defined as significant. The error bars in all bar plots represent one standard deviation.For microcalorimetric measurements, a 48-channel isothermal microcalorimeter  was used as previously described . Tumor c2SO4 (concentrated): H2O2 (30%) (v/v)) for 30 min. Cover glasses were washed in ultrapure water and incubated for 1 h with shaking in 2% v/v (3-Glycidoxypropyl) trimethoxysilane  in a solution of 88% v/v ethanol and 10% v/v water. Subsequently, the surfaces were washed in ethanol and incubated at 80 °C for 30 min. Silanized cover glasses were then functionalized with 1 mg/mL Collagen type I  in 100 mM HEPES pH 8 for 1 h, washed with ultrapure water and dried with nitrogen. Then, the surface was blocked with 5% heat-inactivated BSA in PBS for 30 min and washed with ultrapure water. Esophageal carcinoma cells were allowed to attach for 4 h at 37 °C. Before spinning, the cell suspension was removed and replaced with PBS; then, the cover glasses were mounted on the spinning disk device and immersed in PBS at room temperature. The spinning routine consisted of a 20 s acceleration ramp, 5 min steady spinning at the indicated rotations per minute value, and 20 s deceleration. The hydrodynamic forces at any point on the surface of the cover glass vary linearly with radial distance and can be described by the following equation:The spinning disk apparatus was built in-house based on previously described designs ,18. The 50) [τ is the applied shear stress, r is the radial position relative to the center of the cover glass, ρ is the spinning buffer density, µ is the fluid dynamic viscosity, and ω is the rotational speed. After spinning, the cover glasses were imaged at 10× magnification on an Olympus IX81 microscope ). A custom Python-based image analysis script was used to segment the cells from the background by applying a gray-value threshold. The script returns the radius of the cover glass and a list containing the area, XY coordinates and distance to the center of the cover glass, for each cell. The fraction of adherent cells (f), at different positions on the disk, was calculated by normalizing the density of cells at each section of the disk with the density of cells at the center of the disk, where the shear forces are close to zero. Detachment profiles (f vs. τ) for three cell replicates per line were plotted and fitted with a global sigmoid probabilistic model to extract the value of the shear stress at which 50% of the cells remain adherent (τ50) : (2)f=1/50 is used as a measure of mean adhesion strength for comparison of the different cell lines and is reported with 95% confidence interval determined by the fit.The τ1, M0, G3 in 1997 [PT1590 cell line was established from a patient with an apparently localized adenocarcinoma of the esophagus pT1, pN in 1997 . The pat in 1997 . After c in 1997 ,20. ThesThe human cell lines PT6216, LN6216c and LN6216gc were isolated from primary tumor and lymph node metastases of a patient with adenocarcinoma of the esophagus . After iWith respect to the location of the primary tumor at the distal esophagus, the closest lymph node from the tumor was LN6216c, and LN6216gc was further away.Isothermal microcalorimetry was performed to compare the heat production of cells from the primary tumor to that of tumor cells generated from lymph nodes from both patients separately. Cell line LN1590 showed an increased heat flow over 24 h as well as a much higher overall heat production compared with cell line PT1590 B,C. The 50 parameter is the shear stress at which 50% of the cells are detached from the disk. τ50 for PT6216 was 143.28 ± 6.01 dyn/cm2, while it was 87.72 ± 15.04 dyn/cm2 for LN6216gc and 57.72 ± 4.26 dyn/cm2 for LN6216c. The assay revealed that, after 4 h of incubation, both LN6216gc and LN6216c cells are less adhesive compared with PT6216 cells, which results in a greater readiness to detach . These same unfavorable conditions are also known to promote metastasis.The factors contributing to tumor cell adhesion are heterogeneous and complex. Several adhesion factors have been proposed to play a role in metastatic processes. For example, it has been shown that with a decrease of adhesion strength tumor cells display a decreased focal adhesion turnover . MoreoveOur own investigations in migrating neuroblastoma cells have revealed an increased expression of the water channel AQP1 that furthers migration by promoting water in- and efflux through the tumor cell membrane and facilitating restructuring of the cytoskeleton . On the Our future investigation will focus on the role of AQP1-facilitated membrane permeability and plasticity and possible role in tumor cell thermogenesis for the migratory process.In health, cell migration is a highly orchestrated process that is required for embryonic development. It is vital for tissue repair and regeneration ,39. In c


The ToGA trial has demonstrated, in HER2-expressing patients, that unresectable and advanced gastric cancer, chemotherapy and trastuzumab in combination increase overall survival, even if it is still unclear why after one year the same patients are non-responsive to trastuzumab treatment. Here, we have demonstrated that in HER2-positive gastric cancer cell lines, the addition of duligotuzumab, targeting HER3 receptor, or ipatasertib, targeting AKT protein, enhances the antitumor effect of trastuzumab in vitro through a full inhibition of the membrane signals, on HER2 and HER3, and of downstream signaling, including AKT, and MAPK pathways. Hence, this study suggests a novel and biomarker-driven therapeutic strategy supporting further evaluation of the anti-tumor efficacy of these combinations in HER2 human gastric cancer patients.The anti-HER2 monoclonal antibody trastuzumab is a key drug for the treatment of HER2-positive gastric cancer (GC); however, its activity is often limited by the onset of resistance and mechanisms of resistance are still poorly understood. Several targeted agents showed synergistic activity by concomitant use with trastuzumab in vitro and are under clinical investigation. The aim of this study was to assess the antitumor activity of duligotuzumab, an anti HER3/EGFR antibody or ipatasertib, an AKT inhibitor, combined with trastuzumab in a panel of HER2-positive human gastric cancer cells (GCC), and the efficacy of such combinations in HER2-resistant cells. We have assessed the efficacy of duligotuzumab or ipatasertib and trastuzumab in combination, analyzing proliferation, migration and apoptosis and downstream intracellular signaling in vitro on human HER2-positive GCC  and in negative HER2 GCC (MKN28). We observed a reduction of proliferation, migration and apoptotic rate in HER2-positive OE33, OE19 and N87 cell lines with the combination of duligotuzumab or ipatasertib plus trastuzumab. In particular, in OE33 and OE19 cell lines, the same combined treatment inhibited the activation of proteins downstream of HER2, HER3, AKT and MAPK pathways. Targeting both HER2 and HER3, or HER2 and AKT, results in an improved antitumor effect on HER2-positive GCC. Gastric cancer is the fourth most common malignant neoplasm and the second leading cause of cancer-related death in Western countries . AlthougSignificant progresses in the comprehension of pathogenesis of gastric cancer and a better knowledge of its biological heterogeneity have led to the identification of new targets and to the development of new molecular drugs .Trastuzumab, a recombinant humanized monoclonal antibody that targets the extracellular domain IV of HER2, is the first and only molecular target drug approved for the treatment of advanced HER2-positive gastric cancer ,6,7.The ToGA trial has shown that trastuzumab plus conventional chemotherapy has demonstrated a significant clinical benefit compared to chemotherapy alone in terms of response rate, progression-free survival (PFS) and overall survival (OS), and has therefore become the new standard of treatment for metastatic HER2-positive gastric cancer .However, some evidence indicates that the efficacy of trastuzumab plus chemotherapy may actually be less than expected on the basis of the results of the ToGA trial in actual clinical practice .Further clinical trials conducted with other anti-HER2 agents such as lapatinib, TDM-1 and pertuzumab, failed to show a survival advantage, compared to standard first- or second-line treatment ,11,12.Several preclinical studies have investigated the mechanisms of resistance to trastuzumab, in order to identify new molecular targets in HER2-positive gastric cancer patients .In particular, activation of the PI3K/AKT signaling pathway and enhanced signaling from HER family receptors, including overexpression of HER3, are involved in mediating trastuzumab resistance in HER2-positive GC cells ,17,18,19Thus, selective PI3K- or AKT-inhibitors and anti-HER3 antibodies represent a novel option to overcome resistance to anti-HER2 therapy and improve gastric cancer prognosis. To date, a number of drug candidates targeting HER3 and PI3K/AKT pathways are under clinical evaluation for the treatment of gastric cancer ,21,22.Ipatasertib (GDC-0068) is an oral, investigational small molecule ATP-competitive with the potential to inhibit of all three isoforms of AKT .In preclinical models of gastric cancer cells, ipatasertib acts synergistically with fluorouracil (5-FU) and platinum chemotherapy. A clinical trial aiming to evaluate the efficacy of ipatasertib in combination with FOLfox6 in (NCT01896531) HER2-negative metastatic gastric cancer patients is ongoing .Duligotuzumab  is a novel humanized IgG1 antibody that is directed at HER3 and EGFR, which is under investigation in phase I and II trials for metastatic colon rectal cancer and squamous cell carcinoma of the head and neck ,26.The aim of our study is to investigate the efficacy of duligotuzumab or ipatasertib in combination with trastuzumab by evaluating in vitro the antiproliferative, pro-apoptotic and anti-migration effects in HER2-positive gastric cancer cells.The antiproliferative effects of duligotuzumab or ipatasertib as single agents or in combination with trastuzumab was evaluated in a panel of Her2 positive or negative human gastric cancer cell lines with different biologic profiles.MKN28 gastric cells are human gastric adenocarcinoma-derived cell lines and were selected as a HER2-negative model. Among HER2-positive human gastric cancer cell lines, we chose three cancer cell lines: NCI–N87, 0E19 and OE33. Cell proliferation was measured with the 3--2,5 diphenyltetrazolium bromide (MTT) assay. As anti-HER2 agent, we selected trastuzumab, currently used for the treatment of metastatic gastric HER2-positive cancer patients. Different doses of ipatasertib, duligotuzumab, trastuzumab alone and the combination of trastuzumab with duligotuzumab or ipatasertib were tested.From the dose response curve results, using doses from 0.01 to 25 µM for each drug, IC50 values ranged from ≈0.1 to ≈0.5 µM for duligotuzumab and ipatasertib, whereas the IC50 values for trastuzumab ranged from ≈0.5 to ≈1 µM  The sameCombined treatment of duligotuzumab or ipatasertib and trastuzumab resulted in an important antiproliferative effect as compared to single agents all of HER2 gastric cancer cells A–F.The synergistic effect of the two drugs is evident with dose of 0.5 µM and therefore this dose was chosen for the subsequent experiments. The experiments were conducted on the two cell lines that showed higher response: OE19 and OE33 cells. For this reason, cells were treated with duligotuzumab, ipatasertib alone or in combination with trastuzumab observing that cells that manage to retain the ability to migrate to and form colonies with the single agent reach 70–80%, while the combination of two drugs, duligotuzumab plus trastuzumab and trastuzumab plus ipatasertib, drastically reduces these cell abilities under 40% of the initial population .Apoptosis analysis was performed after 72 h treatment with duligotuzumab or ipatasertib and trastuzumab combination in NCI–N87, 0E19, OE33 human gastric cancer cell lines. As shown in Western blot analyses were performed to evaluate the effect on intracellular signaling pathways on protein extracts from OE33 and OE19 cell lines after 48 h treatment with duligotuzumab or ipatasertib and trastuzumab, at IC50 doses from cell growth inhibition tests, as single agents or in combination .First, the expression of specific proteins in extracts of each cell line was verified, in particular the presence of all the markers involved in the signaling mechanism of PI3K/AKT and of HER2 and HER3 receptors, in both total and phosphorylated isoform. Then, Western blot analysis was performed following treatments to investigate the variation of these markers with single agents and combinations A.We found that single agents, duligotuzumab, ipatasertib and trastuzumab, have no effect on the expression of AKT and MAPK proteins, while the combination of two drugs, duligotuzumab plus trastuzumab and trastuzumab plus ipatasertib, resulted in a strong reduction in phosphor-AKT and phosphor-MAPK, leaving the expression of the total forms of AKT and MAPK unchanged both in KATOIII and in OE19 cell lines, thus demonstrating a downstream effect on the PI3K/AKT pathway. A similar “switch-off” effect was also observed in the expression of HER2 and HER3 receptors, which were reduced by the combination of the two drugs in both cell lines, though most significantly in the OE19 cell line B,C. The The phase III ToGA trial proved the superiority of trastuzumab, plus standard chemotherapy in patients with HER2-positive metastatic gastric cancer compared to chemotherapy alone in terms of OS, PFS and response rates. However, the objective response rate to trastuzumab combined with chemotherapy failed to reach 50% in a real-practice setting, with a median PFS of 6.7 months .The subsequent randomized phase III trials evaluating other anti-HER2 agents such as lapatinib or TDM-1 in this setting of patients failed to show any clinical benefit compared to chemotherapy alone ,11,12.Finally, the addition of pertuzumab to chemotherapy plus trastuzumab was also not associated with a survival advantage compared to trastuzumab plus chemotherapy .Based on these data, there is a compelling need to explore resistance mechanisms associated with anti-HER2 therapy and to establish rational strategies to improve trastuzumab efficacy in HER2-positive GC patients ,26,27,28In a study designed to explore potential causes of failure of anti-HER2 agents in HER2-positive GE adenocarcinomas, Kim et al. observed that the majority of HER2-amplified tumors harbored secondary oncogenic alterations involving genes related to cell-cycle regulation such as PI3K pathway mutations and receptor tyrosine kinase  amplifications. Several studies have demonstrated that somatic mutations in PI3KCA/AKT and in HER3 are present respectively in 4–25% and 16–59% of patients with gastric carcinoma ,30.This evidence suggests that additional oncogenic alterations may represent the key molecular features responsible for the lack of benefit derived from HER2 inhibition in patients with HER2-positive GC .In the present work, we developed a preclinical model using human HER2-positive gastric cancer cells in order to assess the activity of trastuzumab in combination with the AKT-inhibitor ipatasertib or the anti-HER3 antibody duligotuzumab. We reported evidence supporting the improved anti-proliferative in vitro effect of the combination vs. single agent treatment in multiple human gastric cancer cell lines. In particular, we demonstrated that the combination of duligotuzumab or ipatasertib, plus trastuzumab, induced a stronger inhibition of cell proliferation compared to single treatment alone by using the 3--2,5 diphenyltetrazolium bromide (MTT) assay in HER-2 positive, but not in HER2-negative cell lines.The synergistic effect of trastuzumab combined with different anti-HER3 antibodies, such as 1A 5 -3D4, and AKT inhibitors such as MK-2206, has been confirmed in several HER2-positive GC cell lines ,33,34.Our work showed that combination treatments resulted in reduced migration and invasion of gastric cancer cells in vitro, with an increased percentage of apoptotic cells in all cell lines tested by flow cytometric analysis. For instance, in OE33 and OE19 cells, the apoptotic rate increased from 35% to 60% when duligotuzumab or ipatasertib were combined with trastuzumab.Additionally, immunoblotting analysis showed that while the expression of the total forms of AKT and MAPK were unchanged in OE33 and OE19 cell lines, a strong reduction in phosphor-AKT and phosphor-MAPK levels was observed only with the combination of duligotuzumab or ipatasertib plus trastuzumab, thus demonstrating an improved inhibitory effect on the PI3K/AKT pathway. A similar “switch-off” effect was also observed by assessing expression levels of HER2 and HER3 receptors. A more marked effect associated with combination treatment was obtained in the OE19 cell line, probably because of the higher HER2 expression level of these cells, which translates in a stronger HER2 activity of this model. Taken together, these results suggest that a complete inhibition of HER3, AKT and MAPK signaling pathways is needed for an optimal antitumor effect of trastuzumab.This study suffers from multiple limitations, including the limited number of HER2-amplified gastric cancer cell lines, which makes our finding difficult for the generalization of our findings. Furthermore, our study did not evaluate simultaneously the effect of these combinations on trastuzumab-resistant HER2-positive gastric cancer cell lines, although experiments in this setting are ongoing.Finally, our findings demonstrate that inhibition of other pathways may enhance trastuzumab sensitivity in HER2-amplified gastric cancer cells, suggesting the potential for combinational strategies in this setting.2 and cultured with Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 20% fetal bovine serum  and 1% antibiotics/antimycotics  and Roswell Park Memorial Institute  medium added with 10% FBS and 1% antibiotics/antimycotics, OE33/OE19 and NCI-N87/MNK28, respectively.The human gastric cancer OE33, NCI-N87 and MNK28 cell lines were provided by American Type Culture Collection  and OE19 by Sigma Aldrich . Cells were maintained in stable humid conditions at 37 °C with 5% COSTR profiling (Promega) was used to confirm the identity of all cell lines prior to performing experiments, and was repeated after the majority of the experiments were performed.Each cell line contains genomic mutations in one or more of the following genes according to the Sanger COSMIC database: CDKN2A, TP53, SMAD4, CDH1, CTNNB1, KRAS, MLH1 and PIK3CA/AKT (8–11%). The OE19 cell line contains a 100-fold amplification of the ERBB2 gene, and highly expresses its messenger ribonucleic acid. OE33, has an ERBB2 amplification of a lesser copy number. The NCI -87 cell line expresses levels of c-myc and c-erb-B 2 RNA that is comparable to other cell lines. Trastuzumab, duligotuzumab and ipatasertib, provided by Roche Genentech, were dissolved in sterile dimethylsulfoxide (DMSO) while working concentrations were prepared in culture medium just before each experiment.50.Cancer cells were seeded in 96-multiwell plates  and treated for 72 h with an increasing range of concentrations of each drug to determine the ICThe proliferation rate was measured with the MTT assay, as previously described. The results showed the median of three separate experiments, each performed in quadruplicate. Synergism was calculated with ComboSyn software, ComboSyn Inc., Paramus, NK 07652, USA.The assay was performed in 6-well tissue culture dishes where cells were distributed at 300 cells per well and treated with duligotuzumab, trastuzumab and ipatasertib. All conditions were performed in triplicate using untreated cells as reference control. Cells were cultivated for 7 days, at which point they were fixed with 4% paraphormaldeid, stained with crystal violet and colonies counted using the GelCount  .The in vitro invasive ability of cancer cells was analyzed by using 24-well Transwell chambers  according to the manufacturer’s protocol . BrieflyApoptosis assay was performed by flow cytometry according to the manufacturer’s instruction . Early apoptotic cells , necrotic/late apoptotic cells (double positive), as well as living cells (double negative) were detected by FACS Calibur flow cytometer and then analyzed by Cell Quest software . Argon laser excitation wavelength was 488 nm, whereas emission data were acquired at wavelength 530 nm (FL-1 channel) for FITC and 670 nm (FL-3 channel) for propidium iodide.Following treatment, cancer cells were lysed with RIPA buffer (0.1% sodium dodecylsulfate (SDS), 0.5% deoxycholate, 1% Nonidet, 100 mmol/L NaCl, 10 mmol/L Tris–HCl (pH 7.4), 0.5 mmol/L dithiotritol, and 0.5% phenylmethyl sulfonyl fluoride, protease inhibitor cocktail  and isolated by centrifugation at 14,000 rpm for 10 min at 4 °C. After Bradford assay quantification , protein lysates were subjected to SDS-PAGE and Western blot analysis.Primary antibodies for western blot analysis against p-EGFR (Tyr1068), EGFR, p-MAPK44/42 (Thr202/Tyr204), MAPK44/42, p-AKT (Ser473), AKT, pHER2 (Tyr1221/1222), HER2, pHER3 (Tyr1289) and HER3 were obtained from Cell Signaling Technology; monoclonal anti-α-tubulin antibody (T8203) from Sigma Chemical Co. St. Louis, MO, USA. The following secondary antibodies from Bio-Rad were used: goat anti-rabbit IgG and rabbit anti-mouse IgG. Immunocomplexes were detected with the enhanced chemiluminescence kit ECL plus  using the ChemiDoc (Bio-Rad). Each experiment was done in triplicate.P values represent 2-sided tests of statistical significance effects.Results are expressed as means ± s.d. from three independent experiments. Differences between groups were assessed by one-way analysis of variance (ANOVA). For all analyses To date, trastuzumab remains the only biologic target agent approved for the treatment of HER2-positive metastatic gastric cancer. In the current study we have demonstrated that in HER2-positive gastric cancer cell lines the addition of duligotuzumab, targeting HER3 receptor, or ipatasertib, targeting AKT protein, enhances the antitumor effect of trastuzumab in vitro through a fully inhibition of the membrane signals, on HER2 and HER3, and of downstream signaling, including AKT, and MAPK pathways. These results suggest that targeting both HER2 and HER3 or HER2 and AKT provide a biological rationale for further evaluation of the anti-tumor efficacy of these combinations in HER2 human gastric cancer patients.