The presence of hypoxia is a poor prognostic factor in prostate cancer and the hypoxic tumor microenvironment promotes radioresistance. There is potential for drug radiotherapy combinations to improve the therapeutic ratio. We aimed to investigate whether hypoxia-associated genes could be used to identify FDA approved drugs for repurposing for the treatment of hypoxic prostate cancer.Hypoxia associated genes were identified and used in the connectivity mapping software QUADrATIC to identify FDA approved drugs as candidates for repurposing. Drugs identified were tested in vitro in prostate cancer cell lines . Cytotoxicity was investigated using the sulforhodamine B assay and radiosensitization using a clonogenic assay in normoxia and hypoxia.Menadione and gemcitabine had similar cytotoxicity in normoxia and hypoxia in all three cell lines. In DU145 cells, the radiation sensitizer enhancement ratio (SER) of menadione was 1.02 in normoxia and 1.15 in hypoxia. The SER of gemcitabine was 1.27 in normoxia and 1.09 in hypoxia. No radiosensitization was seen in PC3 cells.Connectivity mapping can identify FDA approved drugs for potential repurposing that are linked to a radiobiologically relevant phenotype. Gemcitabine and menadione could be further investigated as potential radiosensitizers in prostate cancer.The online version contains supplementary material available at 10.1186/s12894-021-00856-x. The goal of drug repurposing is to find new clinical indications for existing pharmaceuticals that are currently on the market or failed in phase II/III trials. Repurposing is feasible because disease mechanisms are multifactorial and small drug molecules have multiple targets. Drug repurposing is both time and cost effective since the pharmacology and toxicity profile of approved drugs are already established. Approximately 30% of food and drug authority (FDA) applications for repurposed drugs are approved compared with 10% for new drugs . There iDrug and transcriptomic connectivity mapping can identify drug candidates for repurposing. The most widely used method is CMap (connectivity map project) which connects gene expression profiles to drugs based on data obtained from human cell lines treated with FDA approved drugs . CMap cuComputation-based approaches to drug repurposing provide an opportunity to identify novel agents to combine with radiotherapy . ProstatGenes significantly differentially expressed after 24 h exposure to 1% oxygen in more than two cell lines  were identified previously as seed genes. For each cell line, genes differentially expressed between normoxia and hypoxia conditions across triplicates were selected using a rank product probability of false positive rate < 0.05 . The 848The STRING database is a free online tool for exploring protein–protein interactions . The useQUADrATiC uses the LINCS database to identify connections between gene expression profiles and FDA-approved drugs . The dat2 in air), screened for mycoplasma and authenticated using short tandem repeat profiling. DU145 and LNCaP cells were cultured in RPMI 1640  with 10% fetal bovine serum (FBS) and 2 mM L-glutamine . PC3 cells were cultured in Ham's F12  with 10% FBS and 2 mM L-glutamine .Three prostate cancer cell lines were used to test drugs in vitro: DU145 (HTB-81), PC3 (CRL-1435) and LNCaP (CRL-1740). The cell lines were obtained from the American Type Culture Collection and authenticated by short tandem repeat profiling using the Promega Powerplex 21 system. Cell lines were cultured under normal conditions , gemcitabine  and tirapazamine  were purchased in lyophilized form. Drugs were reconstituted to a concentration of 50 mM in dimethyl sulfoxide (DMSO) as recommended by the manufacturer. Stock solutions were aliquoted and stored at -80 °C; aliquots were not repeatedly freeze thawed.2 hypoxia chamber  or kept in a normoxia incubator for 24 h. The assay endpoint was either 24 h or 4 days post-treatment. For the 24 h time point, the cells were fixed and stained after 24 h incubation with the drug. For the 4-day time point, the drug was removed after 24 h and fresh media added to the cells. Plates were incubated for a further 4 days in normoxia. Cells were fixed and then stained with sulforhodamine B according to the published method  and 22 µM (48 h at 3% O2) [As expected, we have shown the hypoxia selective toxicity of tirapazamine. Although previously studied in PC3 cells, this is the first study of tirapazamine in DU145 and LNCaP cells. The ICt 3% O2) , 33. Tirt 3% O2) . Furthert 3% O2) . Tirapazt 3% O2) .2 because the HIF-1 transcription factor is stabilised and changes in gene expression occurs. However, the level of O2 at which significant resistance to radiation is observed is < 0.13% hence the in vitro experiments were performed at 0.1% O2 [2 which is supraphysiological compared to the level of oxygen in the prostate gland. However, in vitro cell lines are routinely established and cultured in the laboratory under these conditions and have adapted to grow at 21% O2 and therefore the difference in gene expression may not reflect in vivo changes.To identify gene expression changes in response to hypoxia the cells were exposed to 1% O 0.1% O2 . The phy 0.1% O2 . Normoxi2 for 24 h were low over the dose ranges studied but similar to those previously reported for DU145 and PC3 with the exception of PC3 transfected with mir210 inhibitors giving an OER of ~ 2 [The OERs for the DU145 and PC3 cell lines exposed to 0.1% OR of ~ 2 –42. FurtR of ~ 2 .The guidelines for preclinical and early phase assessment of radiosensitizers state that relatively low SER values in the range 1.2–1.5 may indicate a useful effect, particularly if sensitization occurs at clinically relevant doses of radiation . In the 3, is a quinone and synthetic vitamin that can be converted into active vitamin K2 in the body. Menadione induces the production of reactive oxygen species (ROS) through redox cycling and disrupts the interaction between HIF-1a and its coactivator p300 thus inhibiting HIF-1a transcriptional activity [Menadione, also known as vitamin Kactivity –49. Apatactivity , 51. PSAGemcitabine is a chemotherapeutic agent used to treat several cancers, it is a nucleoside analogue that is incorporated into the DNA and inhibits DNA synthesis resulting in cell death. Nucleoside analogues such as gemcitabine are considered as potential radiosensitizers because they inhibit the repair of radiation induced DNA damage . In musc2.A limitation of our study is that radiosensitizing effects were weak and only observed in DU145 cells. This cell line is derived from a central nervous system metastasis of primary prostate adenocarcinoma . In compTwo approaches have been employed to identify FDA-approved drug for repurposing: in silico analytics and experimental screening studies . In a hiIn summary, this study highlights how connectivity mapping can be used to identify FDA approved drugs linked to biological phenotypes for potential repurposing. Our work shows the importance of downstream validation and proof-of-principle studies when identifying drugs for repurposing using in silico analytical approaches. Tirapazamine is an effective hypoxia-selective agent in prostate cancer cell lines and could be tested alongside ADT in future studies. In the DU145 cell line, menadione was a hypoxic radiosentiziter and gemcitabine was a normoxic radiosensitizer. Gemcitabine could be further investigated given that the guidelines for preclinical and early phase assessment of radiosensitizers report an SER in the range of 1.2–1.5 could be clinically useful.Additional file 1.Supplementary Table 1. GSE21032 hypoxia associated genes n = 103. Supplementary Table 2. TCGA hypoxia associated genes n = 66. Supplementary Table 3. GSE21032 hypoxia associated genes Affymetrix probe IDs. Supplementary Table 4. TCGA hypoxia associated genes Affymetrix probe IDs. Supplementary Table 5. The Z-scores for menadione and gemcitabine in the prostate cancer cell lines.Additional file 2.Supplementary Figure 1. Bubble plots representing the strength of the top negative connections identified by QUADrATiC. The larger the bubble the stronger the connection between the identified drug and the input genes. The drug and the cell line, that the connection was derived from, are shown in the bubbles. (A) GSE21032 (B) TCGA.Additional file 3.Supplementary Figure 2. Top drug and cell line connections identified by QUADrATiC. The plots represent the top ranked drugs and the cell line that the connection was derived from in the LINCs database. (A) GSE21032 (B) TCGA.Additional file 4.Supplementary Figure 3. No loss of cytotoxicity of menadione and gemcitabine in hypoxia versus normoxia after 24 h. DU145 (A–C), PC3 (D–F) and LNCaP (G–I) cell lines were exposed to menadione, gemcitabine or tirapazamine under normoxia or 0.1% O2 hypoxia. Three independent experiments were carried out, with six intra-assay replicates per experiment. Data points represent the mean ± SEM, statistical analysis was performed using a t-test with Holm-Sidak correction; **p < 0.01).Additional file 5.Supplementary Figure 4. (A) Under normoxia DMSO at a concentration of 0.02% (v/v) did not alter the surviving fraction of DU145 cells that were mockirradiated or irradiated with 4 Gy. (B) Under hypoxia DMSO at a concentration of 0.02% (v/v) did not alter the surviving fraction of DU145 cells that were mock irradiated or irradiated with 4 Gy. Data points represent the mean ± SEM of 3 biological repeats.


NKILA, localized to 20q13.31, is a negative regulator of NF-κB signaling implicated in carcinogenesis. As a CpG island is embedded in the promoter region of NKILA, it is hypothesized as a tumor suppressor lncRNA silenced by promoter DNA methylation in non-Hodgkin’s lymphoma (NHL). By pyrosequencing-verified methylation-specific PCR, NKILA methylation was detected in 1/10 (10%) NHL cell lines, but not in normal peripheral blood buffy coats or tonsils. NKILA methylation correlated with the repression of NKILA in cell lines. Hypomethylation treatment with 5-Aza-2′-deoxycytidine resulted in promoter demethylation and the re-expression of NKILA. In 102 NHL primary samples, NKILA was methylated in 29 (51.79%) diffuse large B-cell lymphoma (DLBCL) and 4 (20%) peripheral T-cell lymphoma cases, but unmethylated in all 26 mantle cell lymphoma cases. Mechanistically, the knockdown of NKILA resulted in promoting IkBα phosphorylation, associated with nucleus translocation of total p65 and phosphorylated p65 in SU-DHL-1 cells, hence constitutive NF-κB activation. Functionally, the knockdown of NKILA in SU-DHL-1 cells led to decreased cell death and increased cellular proliferation. Collectively, NKILA was a tumor suppressor lncRNA frequently hypermethylated in DLBCL. Promoter DNA methylation-mediated NKILA silencing resulted in increased cellular proliferation and decreased cell death via the repression of NF-κB signaling in NHL.The long non-coding RNA (lncRNA)  Non-Hodgkin’s lymphoma (NHL) encompasses a heterogeneous group of diseases, including B-cell, T-cell and natural killer (NK)-cell lymphoma based on the origin and lineage of tumor cells . B-cell 3) to C5 position of cytosine in a CpG dinucleotide via DNA methyltransferases [PTPL1 [SHP1 [miR-155-3p [miR-146a [DNA methylation refers to the addition of methyl group , localized to 20q13.31, was firstly found to be downregulated in breast cancer. Overexpression of NKILA resulted in the inhibition of metastasis and the increase in apoptosis by repression of NF-κB signaling activity in breast cancer cells, indicating the tumor suppressor property of NKILA [NKILA in lymphoma remains unknown.Long non-coding RNAs (lncRNAs) are broadly defined as a class of non-coding RNAs measuring >200 nucleotides ,15. lncRof NKILA . MoreoveNKILA, we postulated that NKILA is a tumor suppressor lncRNA reversibly silenced by promoter DNA methylation in NHL. Herein, the methylation of NKILA will be studied in NHL, and the role of NKILA in lymphomagenesis will also be investigated.As a CpG island is present at the promoter region of A total of 102 formalin fixed, paraffin-embedded (FFPE) or fresh frozen diagnostic lymph node biopsy tissues, including 56 DLBCL, and 26 mantle cell lymphoma (MCL) and 20 peripheral T-cell lymphoma (PTCL) cases, were acquired from 5 hospitals in Hong Kong, China . The diagnosis of lymphoma was based on the WHO  classification [2 at 37 °C.Five MCL cell lines , two DLBCL cell lines (SU-DHL-6 and SU-DHL-16), two ALK (+) anaplastic large cell lymphoma (ALCL) cell lines (KARPAS-299 and SU-DHL-1) and one T-cell lymphoblastic lymphoma cell line (SUP-T1) were used in this study. SP53 and REC-1 were kindly provided by Professor Raymond Lai . Other cell lines were purchased from Deutsche Sammlung von Mikroogranismen und Zellkulturen (DSMZ) . Cell lines were maintained in RPMI-1640 (DMEM for GRANTA-519) supplemented with 10–15% fetal bovine serum, 50 U/mL of penicillin and 50 ug/mL streptomycin in a humidified atmosphere of 5% CO6 cells/mL) were seeded in 25 cm2 flasks and cultured with 0.5–1.5 μM of 5-aza-2′-deoxycytidine (5-azadC) (Sigma-Aldrich) for 7 days. The 5-azadC was replaced every 24 h. Afterwards, the cells were harvested for DNA and RNA extraction.SU-DHL-6 cells . DNA extraction from frozen patient biopsies was conducted with an automated DNA extraction system (DNA Tissue Kit from Qiagen). DNA extraction from FFPE tissues were performed by using QIAamp DNA FFPE Tissue Kit (Qiagen).NKILA gene by the online tool Methprimer (http://www.urogene.org/methprimer/ (accessed on 31 May 2019). Details of the primer sequence and PCR condition for MSP are listed in Sodium bisulfite conversion was conducted with an EpiTect Bisulfite Kit (Qiagen). Afterwards, MSP was performed in bisulfite-treated DNA with two sets of primers, which were specific to unmethylated (U-MSP) or methylated (M-MSP) DNA sequences. MSP primers were designed at the CpG island upstream to the ® III . The qRT-PCR was performed with a SYBR® Select Master Mix , and the human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the endogenous control. The relative quantity of NKILA expression was calculated by the method of 2−ΔΔCt and normalized against the endogenous control. The primer sequences for NKILA and GAPDH are listed in Total RNA was isolated with a Direct-zol™ RNA MiniPrep kit (Zymo Research), followed by reverse transcription with SuperScriptNKILA overlapped with the amplicon of MSP was amplified in the bisulfite converted DNA with methylation-unbiased primers. Primer sequences and condition for PCR were listed as follows: forward primer, 5′-GTT GGG GAG AGG GTA TAG-3′; reverse primer, 5′-Biotin-CTC CTC CTC CTC ATT CAA ATC-3′ and condition, 56 °C/45 x/1.5 mM MgCl2. A Qiagen PyroMark PCR Kit was employed to perform PCR amplification. Afterwards, a stretch of DNA containing 10 consecutive CpG dinucleotides were pyrosequenced with a sequencing primer, 5′-GTT AGG GGA GGG GGT G-3′, on a PSQ 96MA system and analyzed using PyroQ-CpG 1.0.9. software.The promoter region of NKILA. Briefly, SU-DHL-1 cells were seeded at a density of 0.5 × 106/mL in a six-well plate and transfected with NKILA siRNA  or Silencer Negative Control (Ambion) at a final concentration of 150 nM with an RNAiMAX transfection reagent . The transfected cells were cultured for 24 h or 48 h.RNA interference by small interfering RNA (siRNA) was used to knockdown the expression of After 24 h and 48 h transfection, viable cells were analyzed by trypan blue exclusion and the cell number was measured by the Countess II (Invitrogen). Cell proliferation was calculated by normalization with the negative control at 48 h post-transfection. Cell death was analyzed by trypan blue exclusion at 24 h post-transfection. All experiments were repeated at least three times.NKILA siRNA and Silencer Negative Control were treated with 200 ng/mL TNFα for 1 hour before being lysed in RIPA buffer . Totally, 15 µg protein of each sample was loaded and separated in Mini-PROTEAN TGXTM Precast gel , followed by transference onto a 0.45 µm PVDF membrane . The membrane was blocked in a super blocking buffer and incubated in primary antibodies, including p65 ; p-p65 ser536 ; IkBα ; p-IkBα ser32 ; β-tubulin  and nucleolin  at 4 °C overnight with a gentle rotation. The membrane was then washed and incubated with HRP-linked secondary antibodies, anti-mouse  and anti-rabbit  for one hour, followed by TBST washing. The ECL HARP substrate was used on the membrane before being developed in X-ray films.SU-DHL-1 cells transfected with Nuclear and cytosol fractionations of SU-DHL-1 cells were prepared by the Nuclear/Cytosol Fractionation Kit , according to the manufacturers’ instructions. Western blot was then performed in these samples.NKILA siRNA and Silencer Negative Control were compared by Student’s t-test. The difference of the NKILA methylation frequency in different subtypes of NHL primary samples was analyzed by the χ2 test. All p-values were 2-sided; p < 0.05 was considered as the significant difference.The difference of cell proliferation and cell death between SU-DHL-1 cells transfected with NKILA was reported to repress the NF-κB signaling pathway [NKILA. Hence, the methylation status of NKILA was investigated by MSP in the bisulfite-converted DNA of normal healthy controls, including 10 peripheral blood buffy coats and 11 normal tonsil tissues, in addition to 10 NHL cell lines. Direct sequencing of M-MSP products from methylated positive control DNA demonstrated that all unmethylated cytosines were converted into thymidines after PCR, whereas all methylated cytosines remained unchanged, indicating the complete bisulfite conversion and specificity of MSP  in SU-DHL-6 and completely unmethylated (UU) in GRANTA-519, JEKO-1, MINO, REC-1, SP-53, KARPAS-299 and SU-DHL-1. However, neither U-MSP nor M-MSP signals were observed in SUP-T1 cells  DLBCL and 4 (20%) PTCL cases (p < 0.0001) and PTCL (p = 0.007).To investigate the methylation of  samples A. HoweveCL cases B,C, hencNKILA has been reported to suppress NF-κB signaling pathways by blocking IkBα phosphorylation in breast cancer, non-small cell lung cancer and nasopharyngeal carcinoma [NKILA function in lymphoma, NKILA-targeted siRNA was used to knockdown NKILA and non-targeting siRNA was used as a control in SU-DHL-1 cells. The qRT-PCR result confirmed that NKILA was knocked down at 24 h and 48 h post-transfection  at the promoter region of NKILA, suggesting the expression of NKILA regulated by histone modification. In addition, in breast cancer cells, some oncogenic microRNAs, such as miR-103 or miR-107, could directly target and downregulate the expression of NKILA, which was confirmed by the luciferase reporter assay [Thirdly, to our knowledge, this is the first study that reported that promoter DNA methylation mediated the reversible silencing of g et al.  showed ter assay .NKILA was first shown to suppress NF-κB signaling in breast cancer. It can directly bind to the NF-κB/IκB complex, and potentially mask the phosphorylation site of IκB, thereby suppressing IKK-induced IκB phosphorylation and, hence, NF-κB activity [NKILA and its interaction with NF-κB in lymphomas. To the best of our knowledge, this is the first study showing the role of NKILA in lymphomas. Previous studies reported that phosphorylation p65 at ser536 led to the enhanced transcriptional activity of NF-κB [NKILA led to the upregulation of NF-κB signaling pathways by promoting IκBα phosphorylation, and the consequent nuclear translocation of total p65 and phosphorylated p65 in lymphoma cells. Furthermore, as a transcription factor, NF-κB regulates the expression of multiple downstream effectors, enhancing cell proliferation, inhibiting cell apoptosis or promoting cell migration and invasion [NKILA resulted in an increase in cellular proliferation and decrease in cell death, consistent with the tumor suppressor role of NKILA in multiple tumors, including melanoma, lung cancer, rectal cancer, laryngeal cancer and breast cancer [NKILA has also been shown to inhibit tumor invasion and migration in epithelial cancers, such as breast cancer, hepatocellular carcinoma and tongue squamous cell carcinoma [NKILA can inhibit cellular proliferation and induce cell death by suppressing the NF-κB signaling pathway in NHL cells.Fourthly, activity . As the activity , and henof NF-κB ,33,34. Winvasion . Herein,t cancer ,38,39,40arcinoma ,39,40. CNKILA was mediated by promoter DNA methylation in a tumor-specific manner in NHL. Frequent hypermethylation of NKILA was preferentially detected in DLBCL patients. NKILA exerted its tumor suppressive property by the inhibition of cellular proliferation and increase of cell death, in association with suppression of the NF-κB signaling pathway via inducing IκBα phosphorylation, and the consequent nucleus translocation of total p65 and phosphorylated p65 in NHL cells.Taken together, the epigenetic silencing of lncRNA


Breast cancer (BC) is one of the most common types of cancer and the second leading cause of death in women. Local anaesthetics (LAs) and opioids have been shown to influence cancer progression and metastasis formation in several pre-clinical studies. However, their effects do not seem to promote consensus. A systematic review was conducted using the databases Medline (via PubMed), Scopus, and Web of Science (2010 to December 2021). Search terms included “lidocaine”, “ropivacaine”, “levobupivacaine”, “morphine”, “methadone”, “breast cancer”, “breast carcinoma” and “breast neoplasms” in diverse combinations. The search yielded a total of 784 abstracts for initial review, 23 of which met the inclusion criteria. Here we summarise recent studies on the effect of analgesics and LAs on BC cell lines and animal models and in combination with other treatment regimens. The results suggest that local anaesthetics have anti-tumorigenic properties, hence their clinical application holds therapeutic potential. Regarding morphine, the findings are conflicting, but this opioid appears to be a tumour-promoting agent. Methadone-related results are scarce. Additional research is clearly required to further study the mechanisms underlying the controversial effects of each analgesic or LA to establish the implications upon the outcome and prognosis of BC patients’ treatment. In 2020, 355,000 women were diagnosed with breast carcinoma (BC) in Europe, constituting 13.3% of all cancer cases detected that year [Early diagnosis and adequate treatment provide patients with a greater probability of cure and a better prognosis, since intervention is possible in the earliest stages of the disease and when treatment is, in most cases, more effective. The early diagnostic tests commonly used include clinical breast examination, imaging techniques  and in suspected cases, biopsy under local anaesthesia .Currently, surgery, radiotherapy, and systemic treatment, such as chemotherapy, are the three main treatments used in the therapeutic scheme for BC , with suThe perioperative period has been one of the targets of increased attention among the scientific community, due to its potential influence on the development of metastases and tumour recurrence. Perioperative factors such as pain, stress and anaesthetic techniques are increasingly recognized for affecting the necessary balance for the development of metastases, which combines metastatic immune activity and the ability of cancer cells to metastasize .V1.5 channel function on circulating colon cancer cells, an action that attenuates invasion.Despite the treatment advances achieved in the oncology field, during surgery there is immunosuppressive stress induction and cancer cells can be inadvertently disseminated into circulation, increasing the risk of metastases ,8. In clCancer-related pain is a problem that plagues most patients with BC. The incidence of this pain increases from diagnosis to more advanced stages, and in some cases the pain can be severe and debilitating . The ineMoreover, it is crucial to understand whether the concentration in which a drug is applied may influence the effect it exerts on a patient and its implication on the mode-of-action of other drugs administered to them. Clinically recommended concentrations of lidocaine, ropivacaine, levobupivacaine and morphine, as well as their maximum single doses are listed in Thus far, there is no literature gathering all the existing information focused on the influence of opioids and LAs on the behaviour of BC tumours and therapeutic response, as well as the molecular mechanisms underlying theses effects, particularly for the LAs ropivacaine, lidocaine and levobupivacaine and the opioids morphine and methadone. In this regard, this review emerges and aims to map and analyse the preclinical studies existing in the literature to assess the applicability of these drugs in BC treatment and its mechanism of action.The initial search identified 1027 references, of which 321 were from Medline (Pubmed), 315 from Scopus and 391 from Web of Science. After removing the duplicates, 784 references were reviewed by title and abstracts, resulting in 62 selected articles for full reading. After full-text analysis, 23 references were eligible for analysis and data extraction according to the purposes of this systematic review, as shown in The data collected from each selected article were gathered in Among all the studies analysed, 22 assessed the effect of these drugs on the behaviour of tumour cell lines. Three of these studies, namely Lirk et al. , Ge et aA multidisciplinary approach for the treatment and diagnosis of BC might increase the patients’ survival and quality of life . DespiteLidocaine is an amide-linked LA associated with reduced pain scores and thus reduced need to resort on analgesics . NeverthOverall effects of lidocaine on BC cell lines, at concentrations clinically relevant, seem to be promoted in a dose-dependent manner. In doses ranging from 0.3 to 10 mM, it promoted inhibition of viability, invasion, migration, and proliferation selectively depending on the sensitivity of each cell line to this LA ,29,30,31−1·h−1, during sevoflurane anaesthesia [The role of lidocaine in supressing migration was reported for several cell lines, however, they remain controversial for MCF-7 cells at ranges of 0.1–10 mM. Lidocaine used at an anti-arrhythmic concentration (10 μM) does not appear to have any major effect on BC cell lines . Neverthesthesia . Howeveresthesia .The cytotoxic effects observed in vitro were associated with cell death by apoptosis for MDA-MB-231  cells atTRPM7 gene did not lead to a significant suppression of migration and viability probably because there might be more mechanisms involved in the response to lidocaine when a chronic lack of TRPM7 function is present. This effect did not apply to MCF-7 cells [Several molecular mechanisms have been proposed for the cytotoxic effects of lidocaine in cancer cells. It was reported that lidocaine effects are mediated by membrane ionic channels, such as melastatin-like transient receptor potential 7 (TRPM7) ion channels. Liu et al.  used sev-7 cells . Neverth-7 cells  showed t2+ increase and impairs the cascade of cytoskeleton remodelling, essential to promote polarisation and cell motility during cell migration [There was also evidence that transient receptor potential cation channel subfamily V member 6 (TRPV6) has a major impact on cellular calcium influx, essential for the migration of tumour cells. Still, the exact role of TRPV6 in tumour progression and development remains unclear. In a TRPV6-expressing cancer cell line, MDA-MB-231, Jiang et al.  demonstrigration .The modification of epigenetic information is recognized as a major hallmark of cancer. Still, the magnitude of lidocaine’s effect on BC cell lines may be dependent upon epigenetic features of the tumour type. In oestrogen receptor (ER)-negative, BT-20 cell line, and (ER)-positive MCF-7 cell line, 0.01 to 1 mM of lidocaine demethylated DNA in a time- and dose-dependent manner, suggesting that this LA could regulate epigenetic events ,35. In fTaken together, these results are clinically encouraging and strongly suggest lidocaine as an extremely useful adjunct to BC treatment, not only to reduce tumour growth and metastasis but also to promote apoptosis of existing tumour cells.Ropivacaine is a long-acting amide-linked LA, voltage-sensitive sodium channel inhibitor, commonly used in BC surgery as a perioperative neuraxial anaesthetic and to reduce postoperative pain . The overall effects of ropivacaine on BC cells seem to be promoted in a concentration-dependent manner after 48 h of treatment ,31,37. IThe cytotoxic effects observed in vitro were associated with cell death by apoptosis for MDA-MB-231 cells with a cell cycle arrest in S phase, after a 24 h treatment with plasma and 10× plasma concentrations of ropivacaine [Several molecular mechanisms have been proposed for the cytotoxic effects of ropivacaine in cancer cells. It was reported that ropivacaine effects are supported by the inactivation of the PI3K/Akt/GS3K/β-catenin survival pathway and Wnt/GSK3β/β-catenin cell proliferation pathway, paralleled with an increase of P53 protein and interaction with the inhibitor of cyclin-dependent kinases P21, which leads to a well-established cell cycle arrest after a 24 h treatment with 50 μM of this LA in MDA-MB-231 cells, as shown by Castelli et al. . 2+ cell lines to evaluate the effects of ropivacaine on mitochondrial function and its possible role mediating cell apoptosis and proliferation. In fact, a 72 h treatment with 0.5 to 1 mM ropivacaine led to inhibition of cell proliferation and induction of apoptosis of either cell lines in a concentration-dependent manner [2+ cell line seemed to be more sensitive to ropivacaine than MDA-MB-438 cells. Furthermore, at this range of concentrations and timeline, this LA supressed the Akt/mTOR signalling pathway, which leads to impaired mitochondrial function and subsequently induced oxidative stress on BC cell lines [There was also evidence of an association between the Akt/mTOR signalling pathway and mitochondrial function in BC. Gong et al.  used MDAt manner . Still, ll lines . Hence, Alternatively, it was suggested by Zhao et al.  that 1 mRopivacaine’s effect on BC cell lines may be dependent on epigenetic characteristics of the tumour. In fact, Lirk et al.  showed tChemotherapy still represents one of the main treatments used in the therapeutic scheme for BC . NeverthLevobupivacaine is the long-lasting S-enantiomer of amide-linked bupivacaine, presenting additional advantages, namely its lower toxicity, which makes it safer to use than bupivacaine  in nerveThe results of the evaluation of levobupivacaine use in BC are consistent. Levobupivacaine at 2.5 μM had no major effect on BC cells lines, while in doses from 0.3 to 10 mM it was the most effective LA, comparative to lidocaine, mepivacaine, ropivacaine, bupivacaine, and chloroprocaine, to promote cell death and selectively inhibit cell migration in a concentration-relevant manner ,38. In nNumerous mechanisms have been associated with the decrease of cell proliferation, induced by this LA . AccordiThe role of levobupivacaine in inducing cell death by apoptosis have been reported for MDA-MB-231 and MCF-7 cell lines. According to Castelli et al.  treatmenTaken together, this complex crosstalk between the PI3K/Akt/mTOR signalling pathway and PI3K/Akt/GS3K/β-catenin survival pathway might constitute a potential target for levobupivacaine applicability as a cytotoxic agent.However, despite consistent searching, few in vitro studies were found, and no in vivo studies were retrieved from search. Thus, further studies are recommended on the effect of this LA on the therapeutic response of BC and its adjacent mechanism.There are indications in the literature that morphine can modulate cell migration and adhesion in BC, influencing metastatic potential, however its effect remains controversial , possiblIn fact, the majority of in vitro studies showed that morphine might increase cell proliferation and migration and inhibit apoptosis at ranges from 10–100 ng/mL and 1 to 100 μM ,43,45. N3 cells/well) it is possible to see that antiproliferative effects of morphine on BC cell lines seem to be associated with a reduced exposure time (up to 3 days) [3 cells/well), a less marked proliferative effect was observed [4 cells/well).Comparing studies using a similar density per well (from 2 to 5 × 10 3 days) ,39, whil 3 days) ,43. Furtobserved ,41,43 inobserved  where thAlternatively, at experimental concentrations far higher (10 μmol/mL), Cheng et al.  proposedMorphine is extensively used for anaesthetic pre-medication and management of cancer pain in patients undergoing cancer treatment, such as chemotherapy. Thus, understanding how morphine interacts with chemotherapeutic agents may be relevant to improve therapeutic management. Ge et al.  argued tIn fact, at clinically recommended doses (0.714 mg/kg), Ustun et al.  demonstrTaken together, these results suggest that morphine’s effects may alter tumour response in accordance with the duration of opioid exposure and the dosage used, which could influence the treatment of BC patients.There are several limitations in this review that preclude drawing definitive conclusions regarding the influence of analgesics and LAs on cancer progression and metastasis formation. This systematic review summarizes recent studies on the effect of lidocaine, ropivacaine, levobupivacaine and morphine on BC cell lines and animal models and in combination with other treatment regimens.Firstly, heterogeneity between experimental designs was noted in the identified studies. Different cancer cell types may function distinctly, and as the overall understanding of malignancy evolves, it becomes important that the biochemical similarities and differences between cancer cell types are elucidated. Furthermore, different mechanisms of action were proposed for the same effect of LAs in similar BC cells, and as a result, no clear consensus was established. Secondly, the animal models included in this review reflect different aspects of the BC, and no animal model is a perfect match to represent the full clinical situation. Thus, the significance of these conclusions needs to be further examined to translate these findings in terms of clinical significance and establish the implications upon the outcome and prognosis of BC patients’ treatment.Thirdly, in general, the concentration and duration of LAs’ isolated application required to exert a significant anti-tumour effect are not achievable through local infiltration. However, despite LAs bearing potential as synergistic agents of conventional therapies, there are few results regarding the effect of LAs and opioids in combination with different therapeutic approaches and there is a high variation of results between studies. In fact, studies that concern the effects that LAs may have in combination with chemotherapeutic agents, as well as their specificity to tumour cells are scarce.In addition, concerning ropivacaine and levobupivacaine few in vitro studies were found and only one in vitro and in vivo study was found for ropivacaine. No in vivo studies were found for levobupivacaine. This influences the reliability of the conclusions drawn from this systematic review and arises the great need for more consistent reporting of essential details regarding experimental design for future animal experiments and studies of combination with other therapies.The methods of this systematic review were performed following the PRISMA  guidelines .This study is a systematic review to identify eligible preclinical studies concerning the use of pain medications and its influence in BC treatment response. A search on the literature was performed in Medline retrieved from PubMed, Scopus and Web of Science databases, using the searching formulas presented in In order to be included in the analysis, the articles need to meet the following inclusion criteria: (i) preclinical studies, i.e., in vitro and/or in vivo; (ii) studies devoted to the study of ropivacaine, levobupivacaine, lidocaine, morphine or methadone; (iii) studies on BC field. Clinical studies, all types of reviews, editorials, opinion articles, preclinical studies non-related to the topic, articles written in other languages than English, written before 2010 and unavailable as complete articles were excluded. The search on the literature was undertaken in December 2021.The articles found with the search were downloaded into the reference manager software (Mendeley Desktop v1.19.4) and duplicates were eliminated. Then, a first screening was performed based on the title and abstract by two independent reviewers. The selected articles were read in full for evaluating whether they meet all the inclusion and exclusion criteria. In case of non-consensus, a third reviewer was included in the selection process. For each eligible reference, descriptive and quantitative data were collected by the reviewers. In particular, the reviewers extracted data regarding to: (i) the authors and year of the publication; (ii) the type of study, i.e., in vitro or in vivo; (iii) the specific study characteristics as population, i.e., the cell line/animal model used, and the anaesthetic/analgesic used; (iv) the main outcomes, such as the range of concentration and the effects observed; (v) the limitations of the studies. The study research methodology is illustrated in Local anaesthetics lidocaine, ropivacaine, and levobupivacaine present promising and consistent results regarding the anticancer influence of LA on BC cell lines, when administered alone or in combination with other treatment regimens. Thereby, this may positively affect patient outcomes and global survival. Although scarcely studied, levobupivacaine appears to be the most effective LA against BC studied in vitro. Nonetheless, overall results suggest that the LAs’ effects seem to be promoted in a selectively and dose-dependent manner, at concentrations clinically relevant (0.3 to 10 mM). Still, at lower concentrations, these LAs exhibit some effects on epigenetic information and cell cycle. In contrast, the use of morphine, while effective in the management of cancer-related pain, mostly seems to act as a tumour-promoting agent, despite showing anti-proliferative properties in combination with chemotherapeutic agents. However, overall results propose that morphine’s effects might change tumour response according to the dose and duration of opioid exposure, which could influence the treatment and clinical outcome of BC patients. In turn, there is a paucity of data regarding the influence of methadone on the therapeutic response to BC thus raising the need for future pre-clinical studies.This review additionally highlighted the relevance of deepening the study of the mechanisms behind these controversial effects, in order to evaluate the benefits and disadvantages of using each LA or analgesics in specific clinical settings and diseases, allowing the optimization of its application for BC patients improving their cancer treatment outcome and prognosis.


Aberrant PI3K signalling is implicated in trastuzumab resistance in HER2-positive gastric cancer (GC). The role of PI3K or MEK inhibitors in sensitising HER2-positive GCs to trastuzumab or in overcoming trastuzumab resistance is unclear.PIK3CA and ERBB-family  genes in gastric tumour samples from 69 patients. A panel of gastric cell lines  were profiled for anti-proliferative response to the PI3K inhibitor copanlisib and the MEK1/2 inhibitor refametinib alone and in combination with anti-HER2 therapies.Using mass spectrometry-based genotyping we analysed 105 hotspot, non-synonymous somatic mutations in PIK3CA were only identified in HER2-negative tumours, while ERBB-family mutations were identified in HER2-positive and HER2-negative tumours. Copanlisib had anti-proliferative effects in 4/5 cell lines, with IC50s ranging from 23.4 (N87) to 93.8 nM (SNU16). All HER2-positive cell lines except SNU16 were sensitive to lapatinib (IC50s 0.04 µM–1.5 µM). OE19 cells were resistant to trastuzumab. The combination of lapatinib and copanlisib was synergistic in ESO-26 and OE-19 cells  and additive in NCI-N87 cells (ED50:1.01 ± 0.55). The combination of copanlisib and trastuzumab significantly improved growth inhibition compared to either therapy alone in NCI-N87, ESO26 and OE19 cells (p < 0.05).Patients with HER2-positive GC had significantly poorer overall survival compared to HER2-negative patients (15.9 months vs. 35.7 months). Mutations in PI3K or MEK inhibition alone or in combination with anti-HER2 therapy may represent an improved treatment strategy for some patients with HER2-positive GC, and warrants further investigation in a clinical trial setting.The online version contains supplementary material available at 10.1186/s12967-021-02842-1. Gastric cancer (GC) is one of the most common cancers with over 1 million new cases each year, and despite a decrease in specific mortality, remains the second leading cause of cancer related death worldwide with over 780,000 deaths from the disease occurring annually . 5-year Approximately 6–30% of GCs overexpress human epidermal growth factor receptor 2 (HER2), and expression is dependent on the location of the primary tumour . HER2 plPIK3CA, which encodes the p110a subunit of PI3K, occur in approximately 20% of GCs [PIK3CA mutations are less frequent in HER2-positive GC, occurring in only 2% of tumours [Intracellular pathways, including the PI3K/AKT and RAS/RAF/MEK pathways are activated by ligand binding to ERBB-family members . Anti-HE tumours .EGFR, ERBB2, ERBB3 and ERBB4 (known as ERBB-family mutations herein) occur in 12% of GCs (n = 434) [PIK3CA and/or ERBB-family mutations in gastric adenocarcinoma. We also investigated whether targeting the PI3K or MAPK pathway in vitro using copanlisib, a p110α/δ inhibitor, or refametinib, a MEK1/2 inhibitor, could potentially improve responses to anti-HER therapies in HER2-positive GCs and provide evidence to support a future Phase Ib clinical trial. These inhibitors were chosen as we have previously shown that copanlisib and refametinib can restore sensitivity to anti-HER2 therapies in vitro in HER2-positive breast cancer cell lines with acquired resistance to trastuzaumab and lapatinib, therefore we hypothesised that we would observe a similar effect in HER2-positive GC cell lines [Mutations in n = 434) , 23. As ll lines , 25.Archival formalin fixed paraffin embedded (FFPE) tumour samples were obtained from tumour banks under the auspices of Institutional Review Board approved protocols. Tumour selection was based on a diagnosis of GC, the availability of sufficient tissue from the tumour, and the availability of relevant clinical data. All patients were diagnosed between January 2006 and December 2012. Samples were assessed for HER2-positivity using immunohistochemistry (IHC) , Ventana, Tuscon, AZ, USA). Samples with an IHC score of 2 + were assessed subsequently with fluorescence in situ hybridisation (FISH). Clinical characteristics of the patients included in the study are described in Table From each tumour sample 6 × 10 µM sections were cut from the paraffin block. Hematoxylin and eosin staining was performed on the first and last sections and checked by an experienced Pathologist to ensure that there was tumour present. If the tumour content was lower than 30%, tumour area was macrodissected. DNA was extracted using an All Prep DNA FFPE kit (Qiagen), as per manufacturer’s instructions. DNA concentration was calculated using the Qubit dsDNA kit.PIK3CA, EGFR, ERBB2, ERBB3 and ERBB4. Mutations in ERBB-family genes were identified using publically available data from the TCGA database and a literature search [Mass Spectrometry single nucleotide polymorphism genotyping technology  was applied to DNA extracted from the FFPE samples to detect a total of 108 nonsynonymous somatic mutations in e search . AVSIFT From each tumour 5 × 10 µM sections were cut from the paraffin block. Protein extraction was carried out as previously described  using a Reverse phase protein array (RPPA) analysis was carried out as previously described , 27. The2. Cell line identity was confirmed by DNA fingerprinting (Source Biosciences). Cell lines were mycoplasma tested before and after in vitro experiments. Trastuzumab (21 mg/ml) was obtained from Beaumont Hospital pharmacy and was prepared in sterile water. Lapatinib (10.8 mM) was purchased from Selleckchem and stock solutions were prepared in dimethylsulfoxide (DMSO). The PI3K inhibitor copanlisib and the MEK1/2 inhibitor refametinib were obtained from Selleckchem and stocks  were prepared in 100% DMSO with 10 mM TFA and 100% DMSO, respectively.The GC cell lines NCI-N81, SNU16 and KATOIII were obtained from ATCC and ESO26 and OE19 were obtained from ECACC. All cell lines were cultured in RPM1-1640 media (Sigma) supplemented with 10% FBS and 1% Penicillin/Streptomycin, and maintained at 37 °C with 5% COCells were made into cell blocks using 1% low melting point agar before formalin-fixation and paraffin embedding. 3 μM sections for Her2 (4B5) IHC  were stained using the Ventana BenchMark XT stainer.5 cells were seeded in 6-well plates and grown until confluent. Total protein was extracted using 100 µl lysis buffer  Protein was quantified by the bicinchoninic acid (BCA) assay and stored at – 80 °C until analysis.4 × 1030–40 μg protein was subjected to electrophoresis through 4–12% Bis–Tris precast gels . After transfer, the membranes were incubated with corresponding primary antibodies at 4 °C overnight and secondary antibodies at room temperature for 1 h. Primary antibodies and dilutions were as follows: 1:1000 rabbit S6K1 ); 1:1000 p-S6K1  ; 1:3000 Akt ; 1:1000 p-Akt (Thr308) ; 1:3000 MAPK Erk1/2 ; 1:3000 p-MAPK Erk1/2  ; 1:1000 PI3K p110α . All membranes were additionally probed for ß-actin . Antibody binding was visualised by electrochemiluminescence reagents  for 5 min or 1 min for ß-actin and images were developed digitally .4 cells were seeded in 96 well plates. Plates were incubated overnight at 37 °C to allow cells to adhere. Drugs were added to the plates and incubated at 37 °C. Copanlisib and refametinib were combined with lapatinib in cell lines at ratios determined from the IC50 values of the single agent drugs. Following 5-day incubation, all media was removed from the plates and the plates were washed once with PBS. Proliferation was measured in KATO III, ESO26, NCI-N87 and OE19 cells using the acid phosphatase assay, as previously described [3 × 10escribed  and in St-test. IC50 and combination index (CI) values at effective dose 50 (ED50) were calculated using CalcuSyn software (Biosoft). A CI value of < 0.9 is considered synergistic, 0.9–1.1 is considered additive and > 1.1 is considered antagonistic. A Kruskal–Wallis non-parametric test was performed to compare trastuzumab alone, copanlisib or refametinb alone or the combination of trastuzumab and copanlisib or trastuzumab and refametinib, p < 0.05 was considered statistically significant. Statistical analysis was carried out using GraphPad Prism.Kaplan–Meier curves for overall survival (OS) and progression free survival (PFS) were statistically analyzed by the log rank test. Differences in mutation frequencies between HER2-positive and HER2-negative tumours and differences in protein expression between wildtype and mutated tumours were determined using students Sixty-nine GC patients were included. Forty (58%) patients had HER2-negative disease and 29 (42%) patients had HER2-positive disease. Of the HER2-positive patients, 13 (44.8%) had gastric adenocarcinoma and 16 (55.2) had gastro-oesophageal junction adenocarcinoma. Of the HER2-negative patients, 21 (52.5%) had gastric adenocarcinoma and 19 (47.5%) had gastro-oesophageal junction adenocarcinoma. The median age at diagnosis was 64.5 years (range 32–79) in the HER2-positive cohort and 78 years (range 40–87) in the HER2-negative cohort. 20/29 (69%) and 22/40 (55%) of patients were male in the HER2-positive and HER2-negative cohorts, respectively. Stage at diagnosis was similar in both cohorts with the majority of patients diagnosed with stage 3 or 4 disease. Clinical characteristics are described in Table Of the HER2-positive patients 17/29 (58.6%) were treated with a trastuzumab containing regimen. Twelve patients (41.4%) did not receive trastuzumab as part of their treatment. Among the 17 patients who received trastuzumab, the drug was given in combination with oxaliplatin and capecitabine or fluorouracil in 14 patients, and with fluorouracil alone in three patients. In the HER2-negative patient cohort only 12/40 (30%) patients received systemic therapy. Seven patients received fluorouracil and leucovorin, one patient received docetaxol and carboplatin, one patient received docetaxol, carboplatin and fluorouracil, one patient received oxaliplatin and fluorouracil and two patients received capecitabine alone , however median OS was significantly shorter in the HER2-positive patient cohort compared to the HER2-negative patient cohort   HER2-negative tumours and 2/29 (6.9%) HER2-positive tumours. ERBB3 mutations were identified in 1/40 (2.5%) HER2 negative tumours and 1/29 (3.4%) of HER2-positive tumours, while mutations in ERBB4 were identified in 2/40 (5%) HER2-negative tumours and 2/29 (6.9%) HER2-positive tumours. PIK3CA mutations were identified in 5/40 (12.5%) HER2-negative tumours. No PIK3CA mutations were identified in HER2-positive tumours  . In the HER2-negative group expression of MEK1 was significantly higher in the mutated tumours compared to the wildtype tumours (p = 0.016)  and PIK3CA (Q546H). NCI-N87 cells harboured two ERBB2 mutations (F425L and L436V). SNU16 cells had a KRAS G12D mutation, while KATOIII cells had a PIK3CA L387L mutation. OE19 cells were wildtype for all mutations tested. No mutations in EGFR, ERBB3 or BRAF were identified in any of the cell lines.We used publically available data from the cancer cell line encyclopaedia (CCLE) database to search for mutations in PIK3CA mutation. Expression of p-AKT was also high in the ERBB2 mutated NCI-N87 cell line and the wildtype OE19 cell line compared to the KATOIII and SNU-16 cell lines. p-P70S6K and PI3K P110α expression was highest in the KRAS mutated SNU16 cell line. ERK 1/2 (T202/Y204) expression was higher in the ERBB4/PIK3CA mutated ESO26 cell line than any of the other cell lines. Expression of all proteins was lowest in the wildtype HER2-negative KATOIII cell line  than KRAS wildtype cell lines. We also investigated the effect of refametinib, a MEK1/2 inhibitor, in our panel of cell lines. IC50s for refametinib ranged from 2.75 ± 0.37 nM in OE19 cells to 6.05 ± 0.84 nM in KATOIII cells. ESO26 and NCI-N87 cells did not achieve an IC50 for refametinib at the concentrations of drug used in our study.We investigated response to treatment with the anti-HER2 therapies trastuzumab and lapatinib in the cell lines. Lapatinib IC50s ranged from 39.8 ± 13.3 nM in NCI-N87 cells to 1660 ± 151.7 nM in OE19 cells. SNU16 and KATOIII cells, which have low and negative HER2 expression, respectively, are resistant to lapatinib  and an additive effect in OE19 cells (CI@ED50 1.09 ± 0.45). The combination of lapatinib and copanlisib was antagonistic in NCI-N87, SNU16 and KATOIII cells . The combination of lapatinib and refametinib was antagonistic in OE19 cells and the HER2-negative KATOIII cell line  p < 0.05) , AKT (T308) and MEK1/2 (S217/221) were significantly upregulated in NCI-N87 cells. MAPK (T202/Y204) was significantly upregulated in ESO26. After co-treatment with copanlisib and lapatinib, expression of mTOR (S2481) was upregulated in ESO26. PI3-Kinase p110alpha was downregulated in NCI-N87 cells compared with the other cell lines tested. Following co-treatment with copanlisib and trastuzumab, expression of AKT (T308), PDK1, PDK1 (S241) and RAF were upregulated in NCI-N87 cells, while MAPK (T202/Y204), and mTOR (S2481) were upregulated and MEK1/2 (S217/221) was downregulated in ESO26 cells compared with the other cell lines tested. After co-treatment with refametinib and lapatinib, expression of PI3-Kinase p110alpha and VEGF Receptor 2 were upregulated in ESO26 and NCI-N87 cells, respectively, while co-treatment with refametinib and trastuzumab resulted in upregulated expression of MAPK (T202/Y204) and mTOR (S2481) in ESO26 cells. Co-treatment with refametinib and trastuzumab also resulted in decreased expression of p38 MAP Kinase (T180/Y182) in NCI-N87 cells compared with the other cell lines tested.Trastuzumab is currently the only first-line targeted therapy approved for the treatment of advanced GC . HoweverPI3K pathway activation is common in many cancers, including GC , 31. WhiPrevious studies have suggested that continuously active HER2 downstream signalling pathways, including the PI3K and MAPK pathways, might result in the relatively lower sensitivity of trastuzumab in HER2-positive GC than in HER2-positive breast cancer , 36. We Several studies have investigated targeting the PI3K pathway in GC. The dual PI3K/mTOR inhibitor BEZ235 demonstrates anti-tumour activity in vitro and in vivo in GC models . However50: 39.8 ± 13.3 nM; copanlisib IC50: 26.8 ± 9.7 nM). When we combined trastuzumab with copanlisib, we again observed greater proliferation inhibition relative to either drug alone in two out of the four HER2-positive cell lines tested. The only cell line where we did not observe this was the SNU16 cell line, which is only very weakly HER2-positive. Interestingly, the combination of trastuzumab and copanlisib was effective in the OE19 cell line, which was resistant to either drug as a single agent.In this study we have used copanlisib, a selective alpha/delta isoform dominant PI3K inhibitor with in vivo and in vitro activity , 45. We 50: 2.75 ± 0.37 µM). The combination of refametinib and trastuzumab did not result in greater proliferation inhibition relative to testing either drug alone in any of the cell lines we tested. However, the HER2-positive OE19 and SNU16 cell lines, as well as the HER2-negative KATOIII cell line were sensitive to single agent refametinib, with IC50s ranging from 2.75 ± 0.37 µM in OE19 cells to 6.05 ± 0.84 µM in KATOIII cells.The role of MEK inhibition in the treatment of HER2-positive GC is less clear. Refametinib is a potent allosteric MEK1/2 inhibitor that we, and others, have shown to have anti-proliferative effects, including in HER2-positive breast cancer cells , 49. We In summary, ERBB-family mutations in HER2-positive GC may result in increased signalling through the PI3K and MAPK signalling pathways. PI3K and MAPK may therefore represent a rational strategy to improve responses to anti-HER2 therapy in GC. In vitro we have shown that the PI3K inhibitor copanlisib and the MEK inhibitor refametinib have anti-proliferative effects as monotherapy in some HER2-positive GC cell lines, including those that are intrinsically resistant to trastuzumab. Interestingly, combinations of copanlisib or refametinib and lapatinib also induce additive or synergistic anti-proliferative effects in some cell lines. The combination of copanlisib and trastuzumab also offers greater benefit than using either drug alone. Our data suggest that the addition of copanlisib to HER2-targeted therapy should be considered for clinical trial evaluation in patients with HER2-positive GC, particularly in those patients with PI3K pathway activated tumours.Additional file 1: Table S1. Nonsynonymous somatic mutations in PIK3CA and ERBB family genes 108 Non-synonymous mutations in EGFR, ERBB2, ERBB3, ERBB4 and PIK3CA analysed by Agena MassArray in this study. Table S2. The full list of antibodies used in the RPPA assay.Additional file 2: Table S3. Raw data from RPPA experiments.


S-Nitrosoglutathione (GSNO)), termed Group 1, and a combination therapeutic, inhibitor-4 (SMYD-3 inhibitor) plus NO (from GSNO), termed Group 2, were evaluated for their efficacy on breast carcinoma cell lines MCF7 and MDA-MB-231, and the normal MCF10A breast cell line, using cellular viability, colony formation capacity, cytotoxicity, and cellular apoptosis analysis. These results indicated that, in Group 1, breast carcinoma lines MCF7 and MDA-MB-231, cells experienced a moderate reduction in cellular viability (~20–25%), a large reduction in colony formation capacity (~80–90%), a moderate increase in the relative number of dead cells, and a moderate increase in cellular apoptosis. Group 2 was significantly more impactful, with a ~50% knockdown in cellular viability, a 100% reduction in colony formation capacity, a large increase in the relative number of dead cells, and a large increase in cellular apoptosis. Additionally, Group 2 induced a very small impact on the normal MCF10A cell line. Cumulatively, this work revealed the exciting impact of this combination therapeutic, indicating its potential for clinical application and further research.Despite enormous advances in the detection and treatment of breast cancer, it still remains the leading cancer diagnosis and has the second highest mortality rate. Thus, breast cancer research is a high priority for academics and clinicians alike. Based on previous research indicating the potential of nitric oxide (NO) and SMYD-3 inhibition, this work sought to expand upon these concepts and combine the two approaches. Both NO (from  Female breast cancer cases in the United States have been continually increasing over the past 20 years . FortunaN-diazeniumdiolates, and S-Nitrosothiols (RSNOs) . Mo. Mo29]. The next method employed to determine therapeutic potential of Group 1 and Group 2 was LIVE/DEAD cytotoxic analysis using fluorescence microscopy. Both breast carcinoma lines, MCF7 and MDA-MB-231 cells, as well as the normal breast cell line, MCF10A, were evaluated and exposed to both treatment groups. In both of the breast carcinoma lines, MCF7 and MDA-MB-231 cells, both treatment groups induced cell death, as shown by the green (dead)-stained cells below. These images also revealed that cell death did occur in MCF10A cells treated by both treatment groups; however, this occurred to a lesser extent than in the breast carcinoma cell lines. Collectively, these images further support the cellular viability and colony formation data analyzed above, highlighting the potential potency and discriminatory capability of the combination treatment approach (Group 2) on breast carcinomas.Group 1Optimistically, the LIVE/DEAD fluorescence microscopic analysis revealed that both breast carcinoma cell lines, MCF 7 and MDA-MB-231 cells, were significantly more impacted by Group 1 than normal MCF10A breast cells. This can be seen in the middle images in , where tGroup 2Encouragingly, the LIVE/DEAD fluorescence microscopic analysis of Group 2 MCF7, MDA-MB-231, and MCF10A cells also reiterated the cell viability and colony formation results from above . SignifiFinally, the last major indicator of therapeutic efficacy assessed in this work was the evaluation of cell apoptosis. The impact of both treatment groups, Group 1 and Group 2, was assessed for the induction of cell apoptosis of MCF7 and MDA-MB-231 breast carcinoma cell lines, as well as normal breast cell line, MCF10A. Two different methods were employed, including a Caspase-Glo 3/7 Assay, to probe for caspase 3/7-mediated induction of apoptosis and Annexin V/FITC and PI staining to display early/late apoptosis and necrosis. Overall, the results showed that Group 2 showed a significantly higher amount of cellular apoptosis in the breast carcinoma cell lines MCF7 (seen via Annexin V/FITC and PI staining) and MDA-MB-231 (seen via increased Caspase 3/7 activity and Annexin V/FITC and PI staining). Annexin V/FITC and PI staining revealed only a very slight increase in apoptosis in the normal MCF10A line. These results support and further reinforce the data collected above, emphasizing the promising potential for this combination therapeutic approach (Group 2) in breast carcinomas.Group 1Caspase 3/7 activation was probed for all three cell lines, MCF7, MDA-MB-231, and MCF10A, using the Caspase-Glo 3/7 assay with luminescence detection for Group 1 . CaspaseGroup 2Caspase 3/7 activation was also evaluated for all three cell lines, MCF7, MDA-MB-231, and MCF10A, using the Caspase-Glo 3/7 assay with luminescence detection for Group 2 . Again, The second method employed to evaluate apoptosis and the final method to determine overall efficacy in NO (Group 1) and the combination therapeutic (Group 2) was Annexin V-FITC and PI staining with fluorescence microscopy. These data showed the occurrence of apoptosis (early or late stage) as well as necrosis in each sample group  for all three cell lines, MCF7, MDA-MB-231, and MCF10A. Untreated PC cells and GSH-treated cells for all three did not show anything of note, so only the images of S cells are included below . OverallGroup 1Apoptosis of NO (Group 1)-treated MCF7, MDA-MB-231, and MCF10A cells was analyzed using Annexin V-FITC and PI staining to observe early  or late-stage apoptosis  as well as necrosis  . ObviousGroup 2Similarly, MCF7, MDA-MB-231, and MCF10A cells consecutively exposed to the combination therapeutic (Group 2), were analyzed for apoptosis using Annexin V-FITC and PI staining . Again, Ultimately, these results highlight the Group 2 combination therapeutic, NO (from GSNO) + SMYD-3 inhibition (from inhibitor-4) as an extremely promising treatment for continued application in breast carcinoma treatment. In particular, this treatment shows discriminatory potential to breast carcinomas while imparting very few negative effects on normal breast cells. Future studies of both NO and NO combinations with SMYD-3 inhibition on breast cancers are necessary to determine their efficacy in clinical applications, such as 3D cell culture platforms and animal models.


Neuroblastoma is a type of cancer intimately related with early development and differentiation of neuroendocrine cells, and constitutes one of the pediatric cancers with higher incidence and mortality. Protein tyrosine phosphatases (PTPs) are key regulators of cell growth and differentiation by their direct effect on tyrosine dephosphorylation of specific protein substrates, exerting major functions in the modulation of intracellular signaling during neuron development in response to external cues driving cell proliferation, survival, and differentiation. We review here the current knowledge on the role of PTPs in neuroblastoma cell growth, survival, and differentiation. The potential of PTPs as biomarkers and molecular targets for inhibition in neuroblastoma therapies is discussed. Neuroblastoma is the most common extracranial solid tumor diagnosed in infants and the paediatric cancer with higher risk of death, with high-risk neuroblastoma (about 50% of neuroblastoma cases) showing a survival rate of about 50% . NeuroblProtein tyrosine phosphatases (PTPs) dephosphorylate key homeostatic phospho-proteins and are major regulators of developmental processes, including neuronal survival and differentiation . AccordiMYCN gene is the most frequent genomic alteration in neuroblastoma, in association with poor cell differentiation and bad prognosis, which occurs in about 25% of primary neuroblastoma tumors and constitutes the major hallmark of high-risk neuroblastoma (MYCN gene encodes the MYCN (N-Myc) transcription factor, whose expression and functional activity is highly regulated in the nervous system during embryonic and early-life development, playing an important role in the maintenance of neural stem cell pluripotency and in the differentiation of neural progenitors . TrkA and TrkC high expression are favorable prognostic markers in neuroblastoma, in association with a differentiated phenotype and absence of MYCN amplification, whereas high TrkB expression associates with aggressive neuroblastomas and chemotherapy resistance (Trks constitute a family of neurotrophin receptors with multiple and complementary roles in the nervous system. Trk family members include TrkA (NTRK1), TrkB (NTRK2), and TrkC (NTRK3), whose neurotrophin binding preference is nerve growth factor (NGF), brain-derived neurotrophic factor and neurotrophin-4/5 , and neurotrophin-3 (NT3), respectively . No relesistance . This resistance . In thissistance , althougsistance . A complALK is the gene more frequently mutated in sporadic neuroblastoma, as well as in the germline of familial neuroblastoma patients. Most of these mutations are gain-of-function missense mutations which target the intracellular catalytic domain of ALK and generate a constitutively active RTK (ALK gene is amplified in a subgroup of MYCN-amplified neuroblastoma tumors due to the proximity of the two loci (2p23-24) at the 2p-gain region associated with high-risk neuroblastoma . As illustrated, high mRNA expression of a group of PTPs associates with low patient survival, suggesting pro-oncogenic roles for the product of these genes. Conversely, several PTPs display low mRNA expression in association with low patient survival, suggesting tumor suppressive roles for them in neuroblastoma. Protein expression analysis of the distinct PTPs in neuroblastoma tumor samples is required for further validation of these observations.Classical PTPs are defined by the presence of a classical PTP catalytic domain, which displays the conserved HCxxGxxR signature motif containing the catalytic cysteine residue, and they can be broadly classified into two major groups: receptor-like  and non-receptor enzymes . Receptor-like PTPs (20 genes in human genome) harbor a transmembrane and an extracellular region capable of ligand binding, and possess in most of the cases two classic PTP domains located in the cytoplasm and positioned in tandem, one with catalytic activity (D1) and one with regulatory activity (D2). Non-receptor PTPs (17 genes in human genome) are non-transmembrane proteins harboring a single classic PTP cytoplasmic domain, as well as additional regulatory domains important for function and subcellular location . The mRNPTPRA (RPTPα) has been documented to be involved in neuronal differentiation, as well as in neuroblastoma cell motility, in association with dephosphorylation and activation of Src  (PTPRD (RPTPδ) and PTPRS (RPTPσ), together with PTPRF (LAR) belong to a subfamily of classical receptor-like PTPs which display large extracellular regions susceptible to proteolytic shedding and containing tandem repeats of fibronectin type III- and immunoglobulin-like domains  Chagnon. A majoractivity . A tumore.cloud) . Low expe.cloud) . Howevere.cloud) . It shouPTPRS enzyme is highly expressed in neuroblastoma cells  belongs to a subfamily of receptor like-PTPs with a large extracellular region enriched in fibronectin type III-like domains and a single intracellular catalytic classical PTP domain , although the functional consequences of these mutations need to be evaluated.P domain . Other mP domain . PTPRH pP domain   was expressed in PC12 cells, and cytosolic PTPRR expression down-regulated ERK1/2 phosphorylation , PTPN5 (STEP), and PTPN7 , belong to the subgroup of kinase interaction motif (KIM)-containing classical PTPs, whose major substrates are the MAPK ERK1/2 and p38s . A majorrane PTP . PTPN7 irane PTP . PTPRR mrylation . In SH-Sand PP2A . Interesand PP2A . These oand PP2A . Since aand PP2A , therapeand PP2A . WhetherPTPRZ1  is a receptor-like PTP predominantly expressed in the central nervous system and in endothelial tumor cells. Its extracellular domains include a carbonic anhydrase-like domain, a fibronectin type III-like domain, and a carbohydrate-rich region , the founding member of the PTP gene family  is highly related to PTPN1. It also displays a wide tissue distribution and exists in humans as two isoforms, one nuclear and one associated to the endoplasmic reticulum  and PTPN11 (SHP2) are structurally related PTPs which harbour two tandem regulatory Src homology 2 (SH2) phospho-tyrosine binding domains preceding the catalytic PTP domain . Intramo process . This is process . In this process .PTPN11 acts as an oncogenic phosphatase in several cancer types by dephosphorylation of a wide variety of effector and regulatory signaling proteins, mainly acting downstream of RTKs to promote activation of the RAS/MAPK pathway. PTPN11 direct substrates include RTKs, RAS proteins and RAS negative regulators, as well as SFKs and SFK regulators, among others . In addition, PTPN11 is mutated in the germline of patients with several developmental disorders  and hematological malignancies  . In addition, high expression of PTPN11 mRNA associates with poorer survival of high-risk neuroblastoma patients with MYCN amplification  . PTPN11  cancers  (https:/fication . This suhibitors . This hihibitors , making PTPN14 has been shown to be involved in migration, invasion, and proliferation of neuroblastoma cells, as well as in the regulation of the nuclear translocation of the transcription coactivator YAP (PTPN14 gene have been found specifically in relapsed neuroblastoma tumors, suggesting the existence of an operative functional axis PTPN14-YAP in neuroblastoma relapse (PTPN21 is structurally related to PTPN14, and it has been shown to be involved in negative regulation of apoptosis in PC12 cells in association with ERK1/2 activation (ator YAP . Mutatio relapse . YAP int relapse . PTPN21 tivation . The idePTPN4 possesses a FERM and a PDZ protein domain specialized in protein-protein interactions, and the binding of PTPN4 PDZ domain with protein partners has been postulated to positively regulate cell survival in cancer cells, including neuroblastoma cells (PTPN9 harbors a lipid-binding Sec14 domain that target the enzyme to secretory vesicles, regulating vesicle size and fusion, and PTPN9 has been shown to directly dephosphorylate TrkA in P19 neuroblastoma cells, which would regulate its transport to the plasma membrane (PTPN12 is highly expressed in SH-SY5Y cells (Other non-receptor PTPs related with neuroblastoma include PTPN4, PTPN9, and PTPN12. ma cells . PTPN9 hmembrane . Finally5Y cells , where i5Y cells .The involvement of PTPs in the regulation of neuroblastoma cell signaling and development mediated by the distinct RTK/MYCN axes argues for PTPs as relevant biomarkers and potential therapeutic targets in this type of cancer. This is reinforced by the differential association found between neuroblastoma patient outcome and expression of specific PTP genes in neuroblastoma tumor samples, as illustrated along this review. A prominent example of pro-oncogenic PTP in neuroblastoma is PTPN11, whose gene is mutated with relative frequency in high-risk neuroblastoma tumors and whose inhibition by both allosteric and catalytic inhibitors is under intense scrutiny in neuroblastoma experimental models. PTPs are difficult to target specifically using small molecule inhibitors. In this regard, the diversity of regulatory and protein interaction domains present in PTPs offers a wide variety of potential intervention points in addition to direct modulation of catalysis by compounds targeting the enzyme active site . Since d


LHX6, a gene required for, and a marker of medial ganglionic eminence (MGE)-derived cortical interneurons. Upon differentiation of the LHX6-mEmerald and LHX6-mCherry hPSCs towards the MGE fate, both reporters exhibited restricted expression in LHX6+ MGE derivatives of hPSCs. Moreover, the reporter expression responded to changes of interneuron inductive cues. Thus, the LHX6-reporter lines represent a valuable tool to identify molecules controlling human interneuron development and design better interneuron differentiation protocols as well as for studying risk genes associated with interneuronopathies.GABAergic interneurons control the neural circuitry and network activity in the brain. The dysfunction of cortical interneurons, especially those derived from the medial ganglionic eminence, contributes to neurological disease states. Pluripotent stem cell-derived interneurons provide a powerful tool for understanding the etiology of neuropsychiatric disorders, as well as having the potential to be used as medicine in cell therapy for neurological conditions such as epilepsy. Although large numbers of interneuron progenitors can be readily induced in vitro, the generation of defined interneuron subtypes remains inefficient. Using CRISPR/Cas9-assisted homologous recombination in hPSCs, we inserted the coding sequence of mEmerald and mCherry fluorescence protein, respectively, downstream that of the  Cortical interneurons are gamma-aminobutyric acid-containing (GABAergic) inhibitory neurons that connect locally in the neocortex. They provide inhibitory inputs that shape the responses of pyramidal cells and prevent runaway excitation ,2,3,4. C+ neural progenitors of the medial ganglionic eminence (MGE), while the 5HT3aR interneurons are derived from the dorsal caudal ganglionic eminence (CGE). CGE neural progenitors express Coup tfii and Prox1, but not Nkx2.1. The PV and SST neurons are the most numerous subtypes in the cortex [+) can be abundantly produced, the generation of the LHX6 lineage interneurons, i.e., those that express PV or SST, remains inefficient. The current state-of-the-art reflects our limited knowledge about transcription factor regulation and signaling pathways that control MGE development and interneuron fate specifications despite recent significant advances [Cortical interneurons are a heterogeneous cell population consisting of three major subtypes based on their expression of parvalbumin (PV), somatostatin (SST) and ionotropic serotonin receptor 5HT3a (5HT3aR), respectively ,13,14,15e cortex . They are cortex ,18. Apare cortex ,20,21. Ae cortex , providie cortex , thus pre cortex ,27,28,29advances ,30,31.+ neurons of hPSC-derived MGE progeny.PSCs engineered with a lineage or cell type specific reporters have proven to be valuable tools for identifying novel determinants of a defined cell fate, or purifying cell types of interest for a number of downstream applications ,32,33,34https://zlab.bio/guide-design-resources, accessed on 22 May 2016) to target the 3′ UTR of the human LHX6 gene. Using a multiplex CRISPR/Cas9 assembly system [https://hpscreg.eu/cell-line, accessed on 22 May 2016) at a ratio of 1 Cas9/3xsgRNAS: 3 targeting vector. Individual G418-resistant colonies were picked, expanded and genotyped by PCR. Targeted clones identified by PCR were further verified by Sanger sequencing. Selected targeted clones were then transiently transfected with a Cre-expressing plasmid and individual clones isolated again. The loss of the drug selection cassette was confirmed by PCR and G418 resistance test. The cell lines used in this study were two homozygous mCherry lines and one genotype each of the mEmerald lines. All lines were genotype verified by microarray-based CNV analysis .Three gRNAs were designed using the online tool  H7 and KOLF2 lines were used as negative control where relevant. The two clones of mCherry and mEmerald lines behaved indistinguishably in terms of respective fluorescence intensity and pattern and were therefore referred to in short as LHM for the mCherry and LHE for the mEmerald lines. Routine hPSC culture and MGE differentiation followed procedures described previously . All hPSimagej.net; accessed on 22 May 2016).The cultures were fixed with 3.7% PFA for 15–20 min at 4 °C. For nuclear antigen detection, an additional fixation with methanol gradient was performed, which included 5 min each in 33% and 66% methanol at room temperature, followed by 100% methanol for 20 min at −20 °C. The cultures were then returned to PBS-T (0.3% Triton-X-100 solution in PBS) via inverse gradient and were then permeabilized during three 10-minute washes and then blocked in PBS-T containing 1% BSA and 3% donkey serum. The cells were incubated with primary antibodies in blocking solution overnight at 4 °C. Following three PBS-T washes, Alexa-Fluor secondary antibodies (Thermo Fisher Scientific) were added at 1:1000 in blocking solution for 1 h at ambient temperature in the dark. Three PBS-T washes were then performed that included once with DAPI at 1:1000 (Thermo Fisher Scientific). Images were taken on a Zeiss LSM710 confocal microscope from at least 5 randomly selected fields/samples and staining quantification was acquired manually in ImageJ1.51 . The data for mCherry+ cells were acquired using a Fortessa analyzer. Parental H7 cells at the same stage of differentiation were used at each time-point as negative controls. DAPI staining was used to discard dead cells from the analysis. t-test or non-parametric alternatives, as stated in the figure legends where relevant. All statistical tests were performed in SPSS .All data were collected from at least three independent experiments and are presented as mean ± SEM. The data were tested for normality with the Shapiro–Wilk test and for equal variance with the Levene test before performing statistical analyses by unpaired LHX6 locus  neural progenitors  and examined its effect on MGE induction and reporter expression using the LHX6-mEmerald line . Compare- neural progenitors, leading to a ‘dilution’ of LHX6-mCherry+ cells in the cultures. To test this hypothesis, we treated day 35 cultures for 2 h with demecolcine, a drug that inhibits mitosis by inhibiting spindle formation during cell division  remains very low [Currently, the robust generation of cortical interneurons from hPSCs remains a challenge. Although the induction of NKX2.1iciently ,27,28,29very low ,27,28,29very low . This isvery low ,44,45. TIn summary, this work established a valuable tool to study human cortical interneuron development. It is anticipated that these reporter cells could also be used for investigations into the etiology of neuropsychiatric diseases in combination with CRISPR/CAS9 genome editing of associated risk genes.


The aim of this in vitro study was to reveal the pharmacological interactions between meloxicam and risedronate sodium, used jointly to induce a cytotoxic effect in canine (D-17) and human (U-2 OS) osteosarcoma cell lines. Meloxicam, a non-steroidal anti-inflammatory drug, is capable of intensifying the cytotoxic activity of risedronate sodium routinely used in bone tissue metabolic diseases. The cell cultures were incubated, tested, and evaluated according to standard protocols. The study demonstrated a greater susceptibility of canine osteosarcoma cells in vitro to the investigated drug combination than the human. In both cases, meloxicam alone showed low cytotoxic activity against the tested cell lines, but the two compounds combined were synergic.50 values, computed for risedronate sodium and meloxicam cytotoxicity, showed comparable effects against the canine OS cell line in similar concentration of both drugs. In case of human OS, the stronger cytotoxic effect of risedronate sodium was proved. The EC50 values for meloxicam in both cell lines were, statistically, significantly different (* p < 0.05). Moreover, the cytotoxic effect of a combined administration of meloxicam and risedronate sodium in doses 100 µg/mL, compared with the negative control showed statistically significant differences. The human OS cell line was more resistant to both compounds than the canine OS cell line. The apoptotic effect in canine and human osteosarcoma triggered by risedronate sodium and meloxicam was statistically significant (p < 0.05). The cytotoxic effect induced with 100 µg/mL of risedronate sodium proved statistically significant differences between both tested cell lines compared to negative control. The results obtained with 10 and 100 µg/mL of meloxicam were not statistically significant. The study showed the synergic mechanism of action of risedronate sodium and meloxicam, but the concentrations used in vitro will not be possible to achieve in in vivo. Therefore, our results serve as basis only to design future studies on the tissue level.The study describes the cytotoxic effect against human and canine osteosarcoma (U-2 OS and D-17) cell lines induced by risedronate sodium and meloxicam per se and in combination. Both cell lines were prepared according to standard procedures for cell cultures studies. The cell viability was estimated in both cell lines treated with chosen concentrations of risedronate sodium and meloxicam. The apoptosis assessment was carried out using TUNEL  assay. EC Osteosarcoma (OS), a malignant primary bone tumor of mesenchymal origin with a highly diverse histopathological structure, constitutes a treatment challenge in veterinary and human medicine. OS is diagnosed in approximately 80–85% of dogs with bone tumors, which makes it the most common type of bone neoplasm . HoweverIn dogs, OS affects most frequently the appendicular skeleton. It must be stressed, the genetic factor plays an important role in OS etiology . Other pConsidering histopathological structure, location, age of occurrence, and also predisposing factors, human and canine OS exhibit similar features ,11,12,132); prostaglandin H (PGH2); and finally, prostaglandin D, E, and F, prostacyclin (PGI2), and thromboxane A2 (TRA2). COX-1 (constitutive form) is present in organs, in which prostnoids play the physiological function, i.e., decrease the secretion of hydrochloric acid (HCL) in the glandular stomach mucosa or cause luteolysis in the ovary. Moreover, prostaglandins influence the circulatory system, i.e., blood vessel dilatation and thrombocytes aggregation modulation. They also stimulate the mobility of spermiums and modulate the smooth muscles’ contraction. The synthesis of the second isoform of cyclooxygenase (COX-2), known as inductive form, is stimulated with the inflammatory process mediators  from monocytes, macrophages, fibroblasts, osteocytes, and chondrocytes. The additional variant of COX-1 is COX-3, isolated from the central nervous system and its role is not fully known. Routinely used in therapy, non-steroidal anti-inflammatory drugs (NSAIDs) are capable of non-selective inhibition of both cyclooxygenase isoforms, preferential inhibition of COX-2, or even selective inhibition of COX-2 [The pharmacological effect of meloxicam administration consists of the subsequent mechanism of action, in which three isoform cyclooxygenases play a crucial role: COX-1, COX-2, and COX-3 as a biochemical pathway of the arachidonic acid. The aforementioned acid is subsequently transformed into prostaglandin G (PGGof COX-2 .Meloxicam is an enolic acid derivative (oxicam) with strong anti-inflammatory, analgesic, and anti-pyretic effect, used both in humans and dogs. The mechanism of action is based on the preferential inhibition of COX-2 in dogs and moderate-selective inhibition of COX-2 in humans ,15. The The risedronate sodium belongs to the third generation of bisphosphonates. It shows very strong affinity to the bone tissue. Although risedronate molecules do not penetrate the cell cytoplasm, they accumulate in the bone matrix, causing the inhibition of hydroxyapatite degradation and the decrease in osteoclasts activity  .50 to be above 100 µM. These results encouraged us to design our experiment with the doses of the aforementioned substance in the range of 0.15 and 300 µg/mL. Moreover, Murayama et al. [Risedronate sodium is routinely used in Paget’s disease and osteoporosis. The literature lacks wider information about the risedronate sodium in vitro cytotoxicity. The cytostatic activity of risedronate sodium has been evaluated rarely and only clinically in OS therapy. Murayama et al.  studied a et al.  stated ta et al.  showed tBisphosphonates deeply influence the bone tissue metabolism. This caused its use not only in osteoporosis therapy, but also in oncology as osteoclasts-mediated bone diseases treatment. It is known that this group of drugs is also able to inhibit the growth of soft tissue tumors, like breast cancer, renal cell carcinoma, and prostate cancer . The theThe treatment of human and canine OS follows a similar regimen. The routine treatment involves a surgery (amputation/resection of tumor tissues or limb-sparing surgery) combined with a post-surgical chemotherapy protocol; radiotherapy can also be part of the treatment . RegardlIn summary, the combined actions and interactions between bisphosphonates and NSAIDs in anti-cancer therapy seems to be a new research challenge. Before introducing any compound for in vivo studies in vitro investigations, such as those chosen by us in form of cell cultures, studies are needed. Therefore, the aim of this study was to evaluate the potential cytotoxic activity of risedronate sodium alone and in combination with meloxicam in human and canine OS cell lines. Moreover, the comparison of results achieved for the use of risedronate sodium/meloxicam, both combined and separately, may help to answer whether the compounds have any synergic or additive effect.2 were incubated under a constant 5% flow of CO2 at 37 °C. Eagle’s Minimum Essential Medium (EMEM) and McCoy’s 5A culture medium  were supplemented with 10% fetal bovine serum (FBS) , 4 nM L-glutamine , 100 U/mL penicillin, and 100 µg/mL streptomycin .Canine (D-17) and human (U-2 OS) osteosarcoma  cell lines in culture flasks with a bottom area of 25 cmMeloxicam and risedronate sodium  were diluted in double-distilled water. The culture media were used to obtain the desired concentrations of the tested compounds, for meloxicam (mel) 100 and 10, for risedronate sodium (rd) 100, 10 and 1 µg/mL. The concentration ranges of the tested substances were based on their chemical characteristic of solubility in water, on the literature data, and their maximal concentration in serum. Higher doses of meloxicam and risedronate (above the routinely used therapeutic doses), were used to observe their potential synergistic activity in vitro. The negative control was non-treated cells, and the positive control was cells treated with doxorubicin at 0.5 µg/mL.2.Cells from D-17 and U-2 OS lines were placed onto sterile, 96-well cell culture plates . After 24 h incubation, the culture medium was replaced. Then, the cells were incubated with the tested compounds alone or in combinations , at 37 °50) was made. Cell viability was measured with MTT assay .Further to assessing cellular viability, an attempt at determining the half maximal effective concentration  for a 24 h incubation. After that, the culture medium was replaced, and the cells were incubated with selected concentrations of the tested compounds and their combinations  was performed. The nuclei were stained with 1% hematoxylin solution . In the last step of the assay, the slides were immersed in 70% ethanol  for 30 s, then in xylene  for 30 s, and covered with a cover slip and slide adhesive .The cells from the canine and human OS lines were concentrated to 2 × 10inations  for 24 hThe percentage of apoptotic cells was determined in five fields of vision at a magnification of 40× under an optical microscope, Olympus BX53 . In the first step all cells visible in the field of vision were counted (100%). Next, the number of apoptotic cells was estimated, and their percentage computed. The procedure was repeated in five fields of vision for every well .The observation results were expressed as a mean of five evaluated fields of vision. This assessment was performed by two independent researchers with considerable experience in evaluation of immunohistochemical reactions.p < 0.05.The statistical analysis of the data was performed with StatisticaPL 10.0 software . The normality of results was verified with a Shapiro–Wilk W test. Subsequently, the values estimated for particular compounds and their combinations were compared with a Kruskal–Wallis test. A Mann–Whitney U test was used to compare the results for canine and human cell lines. The correlation analysis was performed with Spearman’s correlation test. The significance level was established at 50 value was computed for both compounds’ activity .The non-steroidal, anti-inflammatory drug meloxicam showed higher cytotoxicity against the canine OS cell line than the human. At its highest concentration (300 µg/mL), it narrowed the viability of the cells to 1.92 ± 4.89% for D-17 and 22.37 ± 4.63% for human OS (U-2 OS). The concentration of 100 µg/mL of the aforementioned compound caused low cytotoxicity against the canine OS and did not influence the variability in case of the human OS cell line . The comp < 0.05). Simultaneously, the comparison of cytotoxic effect between concentrations of 30 and 300 µg/mL proved statistically significant differences in both cases (human and canine OS) (p < 0.05). The administration of risedronate sodium in doses of 100 µg/mL caused a moderate cytotoxic effect in both investigated cell lines , around 50% in comparison to the negative control  narrowed the cell variability of human and canine OS to 14.84 ± 2.36% and 29.1 ± 2.17%, respectively. The cytotoxic effect of risedronate sodium was stronger against human OS. Risedronate sodium cytotoxicity compared in two concentrations (100 and 300 µg/mL) showed the statistically significant differences only in the case of human OS .The combination of risedronate sodium and meloxicam in doses of 100 µg/mL (100 mel/100 rd) compared with negative control showed statistically significant differences in the viability of human and canine OS cell lines, U-2 OS: 20.14 ± 3.27% and D-17: 10.01 ± 3.13% (negative control 98.51 ± 0.55%), p < 0.05. Finally, 10 µg/mL of meloxicam and 100 µg/mL risedronate sodium combination compared to the negative control proved the statistically significant differences in the human OS cell line viability, U-2 OS: 29.62 ± 6.32% and 98.51 ± 0.55%, p < 0.05. The positive control (doxorubicin 0.5 µg/mL) proved cell viability at 20.89 ± 5.95% for canine OS and 9.14 ± 0.98% for human OS, respectively. The use of a ten-times lower dose of meloxicam, together with the same concentration of risedronate sodium, enhanced the cytotoxic effect against the investigated cell lines, and the viability of human (U-2 OS) was lower than canine (D-17) OS .Moreover, the combination of meloxicam, in doses of 100 µg/mL, and risedronate sodium, in doses of 10 µg/mL, showed lesser viability of canine OS than the negative control , D-17: 34.18 ± 3.75% and 98.58 ± 0.91%, p < 0.05) against human OS , which was estimated at 49.06 ± 3.77%. Meloxicam alone did not exert significant proapoptotic activity either in canine or human OS  used in veterinary medicine, while in humans its application is less popular. It is often assumed that this substance exhibits anticancer activity against different types of tumors, both in humans and animals, as confirmed for hepatocellular carcinoma (HCC), non-small cell lung cancer, bladder cancer, colon cancer, or OS ,28,29,3050 of human than canine OS cells (p < 0.05) than humans OS cell lines (Studies assessing the use of meloxicam against cells from the established canine (D-17) and human (U-2 OS) OS lines demonstrated significantly higher viability and ECOS cells . This fill lines . This php < 0.05, r = 42) was observed. In a study on another line of canine OS cells (D-17), Wolfesberger et al. [50 values of 215.9 µM/mL, which were lower than our findings. The most probable explanation is the different types of active substances and diluents used in the two studies. That is, Wolfesberger et al. [Additionally, the concentration-dependent cytotoxic activity of meloxicam could be observed in both canine and human OS cell lines. In this study, the highest cytotoxic activity was found for the highest concentrations, i.e., 100–300 µg/mL, while the lowest tested concentrations induced only a slight stimulation of cellular proliferation. In both tested cell lines, a negative correlation between viability and meloxicam concentration . For example, in D-17 and U-2 OS treated with 100, 10, and 1 µg/mL risedronate sodium solution, the viability equaled 53.38 ± 1.46 and 49.56 ± 0.7%; 97.08 ± 3.32 and 74.92 ± 4.01%; and 102.67 ± 3.56 and 94.56 ± 3.52%, respectively. The negative correlation between the concentration on the tested substance and cell viability was proved in both canine  and human OS . The analysis of alive cell percentage and EC50  indicated a higher susceptibility of human than canine OS cells to the cytotoxicity of risedronate sodium (p < 0.05) (p < 0.05). In summary, among all tested combinations of meloxicam and risedronate sodium, the highest cytotoxic activity was detected for 100 µg/mL risedronate sodium + 100µg/mL meloxicam (p < 0.05) and, in comparison with negative control (non-treated cells), the statistically significant differences in the viability of human and canine OS cell line were proved  combined with meloxicam. Even though the literature contains some reports on cytotoxic activity of older bisphosphonates , risedro < 0.05) . Similar < 0.05) . While c < 0.05) . Moreovee proved . The cytp < 0.05) and it is possible to conclude that combined use of meloxicam and risedronate sodium in comparison to both negative control and to separate administration can be classified as synergy. In the case of apoptosis, it was clearly visible that the combination of risedronate sodium and meloxicam induced apoptosis in human and canine OS cell lines .The study indicated a synergetic effect of risedronate sodium and meloxicam used in combination. Despite the fact that meloxicam alone did not exhibit important pro-apoptotic effects at the tested concentrations, its combinations with the second investigated compound enhanced the capability of risedronate sodium to induce apoptosis in the investigated neoplasm cell lines.