STAT3 is a transcription factor which is activated via various signaling transduction pathways or Epstein-Barr virus (EBV) infection and plays an oncogenic role in lymphoid malignancies including Hodgkin lymphoma (HL). The tumor cells of HL are derived from germinal center B-cells and transformed by chromosomal rearrangements, aberrant signal transduction, deregulation of developmental transcription factors, and EBV activity. HL cell lines represent useful models to investigate molecular principles and deduced treatment options of this malignancy. Using cell line L-540, we have recently shown that constitutively activated STAT3 drives aberrant expression of hematopoietic NKL homeobox gene HLX. Here, we analyzed HL cell line AM-HLH which is EBV-positive but, nevertheless, HLX-negative. Consistently, AM-HLH expressed decreased levels of STAT3 proteins which were additionally inactivated and located in the cytoplasm. Combined genomic and expression profiling data revealed several amplified and overexpressed gene candidates involved in opposed regulation of STAT3 and EBV. Corresponding knockdown studies demonstrated that IRF4 and NFATC2 inhibited STAT3 expression. MIR155 (activated by STAT3) and SPIB (repressed by HLX) showed reduced and elevated expression levels in AM-HLH, respectively. However, treatment with IL6 or IL27 activated STAT3, elevated expression of HLX and MIR155, and inhibited IRF4. Taken together, this cell line deals with two conflicting oncogenic drivers, namely, JAK2-STAT3 signaling and EBV infection, but is sensitive to switch after cytokine stimulation. Thus, AM-HLH represents a unique cell line model to study the pathogenic roles of STAT3 and EBV and their therapeutic implications in HL. Hodgkin lymphoma (HL) is a germinal-center-derived B-cell malignancy, although rare cases with T-cell origin have been described . The typHRS and LP cells lack typical B-cell characteristics including expression of B-cell-associated developmental transcription factors (TFs). Even the absence of master TFs PAX5 and EBF1 have been reported; however, their reactivation was insufficient to reconstitute the B-cell phenotype ,15,16. THere, we analyzed the EBV-positive HL cell line AM-HLH . InteresCell lines were obtained from the DSMZ : AM-HLH (ACC 908), HDLM-2 (ACC 17), KM-H2 (ACC 8), L-428 (ACC 197), L-540 (ACC 72), L-1236 ACC 530), SUP-HD1 (ACC 574), U-HO1 (ACC 626). Cell culture conditions, culture media and other relevant information on each cell line are described elsewhere , SUP-HD1. Analyze6 cells by electroporation using the EPI-2500 impulse generator  at 350 V for 10 ms. Transfected cells were harvested after 20 h cultivation. Cell stimulations with 20 ng/mL interleukin (IL)6 or IL27  were performed for 4 h, and for 20 h with 10 µg/mL histone deacetylase inhibitor TSA . All knockdown and stimulation experiments were performed twice generating similar results.Gene activities were suppressed via siRNA-mediated knockdown. Gene-specific siRNA oligonucleotides and AllStars negative control siRNA (siCTR) were purchased from Qiagen . A green fluorescence protein (GFP)-labeled STAT3 expression construct was cloned into vector pCMV6-XL4 and obtained from Origene . SiRNAs (80 pmol) and plasmid DNA (2 µg) were transfected into 1 × 10For functional testing, treated cells were analyzed with the IncuCyte S3 Live-Cell Analysis System . Apoptotic cells were quantified using the IncuCyte Caspase-3/7 Green Apoptosis Assay diluted at 1:2000 . Live-cell imaging experiments were performed twice with four-fold parallel tests.Total RNA was extracted from cell line samples using TRIzol reagent . cDNA was synthesized by random priming from 5 µg RNA using Superscript II . Expression analysis of EBV-encoded genes LMP1, LMP2A, EBNA1, EBNA2, EBNA3A, EBNA3C and of control gene YY1 was performed as described previously . Reverset-test and the derived p-values are indicated by asterisks .Real-time quantitative (RQ)-PCR analysis was performed with the 7500 Real-time System using commercial buffer and primer sets . For normalization of expression levels, we analyzed the transcript of TATA box binding protein (TBP). We used the ddCT-method, and the obtained values are indicated as fold expression in relation to one selected sample which was set to 1. Quantitative PCR analyses were performed in triplicate. Standard deviations are calculated for each experiment and presented in the figures as error bars. Statistical significance was assessed by Western blots were generated using the semi-dry method. Protein lysates from cell lines were prepared using SIGMAFast protease inhibitor cocktail (Sigma). Proteins were transferred onto nitrocellulose membranes  and blocked with 5% dry milk powder dissolved in phosphate-buffered saline buffer (PBS). The following antibodies were used: alpha-Tubulin (Sigma), STAT3 , phospho-(P)-STAT3 , and IRF4 (Origene). For loading controls, blots were reversibly stained with Poinceau (Sigma), and the detection of alpha-Tubulin (TUBA) was performed thereafter. Secondary antibodies  were linked to peroxidase for detection by Western-Lightning-ECL . Documentation was recorded using the digital system ChemoStar Imager .Immuno-cytology was performed as follows: cells were spun onto slides, air-dried and fixed with methanol/acetic acid for 90 s. The STAT3 antibody (see above) was diluted 1:20 in PBS containing 5% BSA and incubated for 30 min. Washing was performed 3 times with PBS. Preparations were incubated with fluorescent secondary antibody (diluted 1:100) for 20 min. After final washing the cells were mounted in Vectashield , containing DAPI for nuclear staining. Images were captured with an Axion A1 microscope using Axiocam 208 color and software ZEN 3.3 blue edition .For genomic profiling by cytogenetic microarray, the genomic DNA of cell line AM-HLH was prepared by the Qiagen Gentra Puregene Kit (Qiagen). The labelling, hybridization and scanning of HD Cytoscan arrays was performed at the Genome Analytics Facility, Helmholtz Centre for Infection Research , using HD arrays according to the manufacturer’s protocols . Data were visualized and interpreted using the Chromosome Analysis Suite, software version 2.0.1.2 (Affymetrix). https://www.ncbi.nlm.nih.gov/gds/, accessed on 1 February 2022) [Gene expression profiling data from cell lines were generated at the Genome Analytics Facility using HG U133 Plus 2.0 gene chips , as reported previously . Expressry 2022) . Transcrry 2022) .In HL, DLBCL and ALCL, STAT3 aberrantly activates NKL homeobox gene HLX which plays an oncogenic role in these lymphoid malignancies ,19,20. AHowever, RQ-PCR analysis of NKL homeobox gene HLX in cell lines derived from HL, DLBCL and ALCL detected only weak expression levels in AM-HLH. This result contrasted to EBV-negative HL cell line L-540, EBV-positive DLBCL cell line DOHH-2 and all examined EBV-negative ALCL cell lines C. HLX isRQ-PCR analysis of STAT3 in eight HL cell lines showed low expression levels in AM-HLH while HDLM-2, L-428 and L-540 expressed the highest levels A. AccordHowever, the treatment of AM-HLH with IL6 for 4 h resulted in STAT3 phosphorylation and translocation from the cytoplasm into the nucleus C, demonsThe treatment of L-540 cells with the deacetylase inhibitor TSA resulted in the translocation of STAT3 from the nucleus into the cytoplasm D. AccordGenomic aberrations play a basic role in the pathogenesis of HL. Accordingly, the tumor cells contain several chromosomal rearrangements and copy number alterations as described in patients and cell lines ,11,12,13We focused on seven amplicons exhibiting at least a 10-fold rise in their copy numbers which were located at 6p25, 9p24, 9p13, 16p13, 16p12, 16p11 and 20q13 . Gene exIRF4 encodes a hematopoietic TF and was amplified at 6p25 and overexpressed in AM-HLH as confirmed by RQ-PCR and Western blot analysis A. IRF4 hBAG1 encodes an activator of survival factor BCL2 . BAG1 waThe amplifications at 16p harbor several overexpressed genes which, however, indicates no connection to STAT3 or EBV C. In conTo validate these findings and to identify additional genes differentially expressed in AM-HLH, we performed genome-wide comparative gene expression profiling analysis, analyzing the top-1000 up/downregulated genes . AccordiOur approach analyzing genomic and gene expression profiling data from AM-HLH and HL control cell lines revealed interesting candidates potentially involved in STAT3 regulation or lymphomagenesis. These genes and their downstream effects were analyzed by the modulation of their activity. SiRNA-mediated knockdown of IRF4 was confirmed by RQ-PCR and Western blot analysis A. The daBoth, IL6- and IL27-signalling activates STAT3 ,22. AccoKMT2A is a direct target of amplified Zinc-finger TF ZFP64 . KMT2A iTaken together, these data showed that overexpressed IRF4 and NFATC act as STAT3 inhibitors. Nevertheless, STAT3 was still ready for reactivation as demonstrated by treatment with IL6 or IL27. Furthermore, we detected downregulation of STAT3-activators NFIB and HIPK2 and of STAT3-target genes HLX, MIR155 and PAX5. CELF2 is a reported inhibitor of MIR155 and was activated by overexpressed NFATC and KMT2A.We identified IRF4 as an amplified and overexpressed STAT3-inhibitor in AM-HLH. To examine its potential oncogenic activity in AM-HLH we performed live-cell-imaging after siRNA-mediated knockdown of IRF4. The results indicated that IRF4 supported proliferation A and inhThe results of this study are summarized in IRF4 played a central role in STAT3 inhibition. Its gene was amplified at 6p25 and consequently overexpressed. IRF4 also inhibited the expression of NFIB which has been shown to activate STAT3 . FunctioJAK2 represents a major STAT3-activator and was downregulated in AM-HLH although located in an amplicon. This observation indicated that this cell line escapes STAT3-activation. As compared to HLX-positive cell line L-540, HLX target gene SPIB was elevated in AM-HLH. SPIB plays an important role for both, B-cell differentiation and EBV activity, thus, indicating a conflict of two pathognomonic mechanisms ,49,50. PAdditional deregulated players identified in AM-HLH included ZFP64, KMT2A, CELF2, and MIR155. ZFP64 was amplified at 20q13 and overexpressed. This TF has been described as potent activator of KMT2A transcription , which wUsing cell line AM-HLH as a model for EBV-positive HL, we recognized downregulated STAT3 activity. Amplified and overexpressed IRF4 played a major role in STAT3 inhibition. Genes located downstream of STAT3 have been described as both oncogenes and regulators of EBV. STAT3 was still ready for reactivation via IL6- and IL27-signalling, indicating a possible switch in EBV activity. Thus, our study revealed conflicting roles of STAT3 in HL and showed that AM-HLH represents a unique cell line model to gauge therapeutic options for EBV-positive HL patients.


TP53 (p53) is mutated in 80–90% of cases of triple-negative breast cancer (TNBC). Statins, which are widely used to treat elevated cholesterol, have recently been shown to degrade mutant p53 protein and exhibit anti-cancer activity. The aim of this work was to evaluate the potential of statins in the treatment of TNBC. The anti-proliferative effects of 2 widely used statins were investigated on a panel of 15 cell lines representing the different molecular subtypes of breast cancer. Significantly lower IC50 values were found in triple-negative (TN) than in non-TN cell lines  indicating greater sensitivity. Furthermore, cell lines containing mutant p53 were more responsive to both statins than cell lines expressing wild-type p53, suggesting that the mutational status of p53 is a potential predictive biomarker for statin response. In addition to inhibiting proliferation, simvastatin was also found to promote cell cycle arrest and induce apoptosis. Using an apoptosis array capable of detecting 43 apoptosis-associated proteins, a novel protein shown to be upregulated by simvastatin was the IGF-signalling modulator, IGBP4, a finding we confirmed by Western blotting. Finally, we found synergistic growth inhibition between simvastatin and the IGF-1R inhibitor, OSI-906 as well as between simvastatin and doxorubicin or docetaxel. Our work suggests repurposing of statins for clinical trials in patients with TNBC. Based on our findings, we suggest that these trials investigate statins in combination with either doxorubicin or docetaxel and include p53 mutational status as a potential predictive biomarker. Patients with triple-negative breast cancer (TNBC) lack estrogen receptors, progesterone receptors as well as HER2 gene amplification/overexpression. Consequently, these patients cannot be treated with 2 of the most effective therapies currently available for breast cancer, i.e. endocrine and anti-HER2 therapy. Indeed, until recently, the only form of systemic therapy available for patients with TNBC was cytotoxic chemotherapy . RecentlOne of the most successful forms of anti-cancer treatment introduced in recent years is the use of drugs for targeting cancer driver genes. The most frequently occurring driver gene in TNBC is mutations in p53 which are present in 80–90% of these patients , 5. ThusHistorically, however, mutant p53 has proved difficult to target and was thus frequently regarded as “undruggable” , 7. ThisWhile mutant p53-reactivating compounds have been widely investigated , less woIn addition to HSP inhibitors, a group of commonly used drugs known as statins, have also been shown to degrade mutant p53 but not WT p53 , 16. StaBased on the high prevalence of p53 mutations in TNBC, the ability of statins to degrade mutant p53, and thereby inhibit tumour cell proliferation, the aim of our study was to test the hypothesis that these drugs might provide a new treatment for patients with TNBC.All cell lines were originally sourced from the American Type Culture Collection (ATCC), apart from Hs578Ts(i8) which was supplied by Dr. Susan McDonnell, University College, Dublin. All cell lines were maintained in RPMI, supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin. All cell culture reagents were purchased from Biosciences  and Sigma-Aldrich . Cells were maintained at 37 °C in a 5% CO2 environment. The p53 mutation status of these cell lines is shown in Table Cell response to statins was assessed by seeding 96-well plates with 5,000 cells per well and allowing to adhere overnight. The following day, fresh media were added, containing statins at the indicated concentration or DMSO. Cells were incubated for 5 days, at which point viability was assessed by adding 3--2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich) to 10% of media volume and incubating for 2 h. Absorbance was measured at 570 nm. In some experiments, mevalonate was added at a concentration of 100 or 200 µM.Cells treated with statins for 24 h were harvested and fixed by resuspending in 500 µL PBS followed by slowly adding 4.5 mL ice-cold 70% ethanol. Cells were prepared for cell cycle analysis using FxCycle™ PI/RNase Staining Solution (Thermo-Fisher), following manufacturer’s protocol. Fluorescence was measured on a BD FACSCanto II, using a 488 nm excitation source and a 576 nm emission filter. Cell cycle percentages were calculated using Flowjo software.Cells treated with statins for 48 h were harvested and prepared for analysis using the FITC Annexin V Apoptosis Detection Kit with 7-AAD  following manufacturer’s guidelines. Fluorescence intensity of FITC and 7-AAD was measured on a BD FACSCanto II, using a 488 nm excitation source and a 525 nm or 695 nm emission filter, respectively.Expression of apoptotic proteins was analysed using the Raybiotech Human Apoptosis Array following manufacturer’s guidelines. Briefly, protein was isolated from SUM159 cells following treatment for 24 h with 10 µM simvastatin or vehicle control, using the provided lysis buffer. Membranes were blocked and incubated with protein samples overnight at 4 °C. After washing with the provided buffers, membranes were incubated overnight again at 4 °C with detection antibody. Membranes were incubated at room temperature with HRP-Streptavidin for 2 h. Following a final wash stage, membranes were incubated with detection buffer and imaged using a Licor C-DiGit Blot Scanner. ImageJ software was used to generate densitometry values which were then normalised to the internal controls on the membrane.Protein was isolated and prepared for analysis by Western blotting as previously described . PrimaryEfficacy of drug combinations was performed following the same protocol as proliferation assays. Drugs were tested as both single agents and in combination. Combination index (CI) values were calculated using Compusyn software . CI valuAll experiments were repeated at least 3 times. All statistical analysis was performed using GraphPad Prism 8 software. Significance was determined using Student t tests or ANOVA post-hoc analysis. A p value of less than 0.05 was deemed statistically significant.r = 0.94, p < 0.0001)   , HER2 (2 cell lines) and TNBC (9 cell lines). Both statins reduced cell viability in a dose-dependent and cell type-dependent manner , it also reduces levels of the intermediate product, MVA . To confAs simvastatin was the more potent of the two statins investigated, we focussed on it for the rest of our study. To investigate if simvastatin could induce cell cycle arrest in TNBC cells, we measured DNA content by propidium iodide staining and flow-cytometry in 4 TNBC cell lines following 24 h treatment. Representative graphs of simvastatin-treated MDA-MB-231 cells are shown below  and 46% (p < 0.001) cells staining positive for annexin-V, respectively. Similar levels of annexin-V positivity were seen in Hs578T  annexin-V staining . The combination was also effective in SUM159 cells .Finally, we tested the combination of simvastatin and OSI-906 on apoptosis. MDA-MB-231 and SUM159 cells were treated with a low dose of simvastatin (1 µM) that, by itself, did not significantly induce apoptosis in either cell line Fig. A, B. SimAs statins are unlikely to be used alone in cancer treatment, we also tested the anti-proliferative effect of combined treatment with a statin and standard chemotherapy drugs used for the treatment of breast cancer. As shown in Table Here, we show that although 2 widely used statins decreased the proliferation of breast cancer cell lines representing the main molecular subtypes of breast cancer, their inhibitory impact was significantly more potent in TNBC than in non-TNBC cell lines. A possible explanation for the enhanced sensitivity of the TNBC versus the non-TNBC cell lines may relate to the higher prevalence of p53 mutations in this breast cancer subtype. Indeed, as mentioned in the Introduction above, our findings are consistent with previous observations in which treatment with statins was reported to result in the degradation of mutant but not wild-type p53 , 16. PrePreviously, Parrales et al. reported that lovastatin degraded p53 with conformational mutations but had minimal effects on contact mutations . Based oAlthough our results show that two different statins reduced the proliferation of TNBC cell lines, these drugs are unlikely to be used alone for the treatment of this form of breast cancer. We therefore evaluated the effects of combining simvastatin with two frequently used drugs to treat TNBC, i.e. docetaxel and doxorubicin. Both these cytotoxic drugs in combination with simvastatin synergistically enhanced growth inhibition in the 2 TNBC cell lines investigated, suggesting that combined treatment with simvastatin and docetaxel or doxorubicin might be investigated in a clinical trial.A novel finding in this report was that simvastatin upregulated levels of IGBPF4. Although we did not investigate the mechanism of this upregulation or its possible implication for statin actions, previous studies showed that IGFBP4 binds and sequesters IGF-1 ligands, thus acting to dampen IGFR1 signalling . ConsistWe should state however, that there are several other mechanisms other than via upregulation of IGBP4 by which statins could decrease cell proliferation or promote apoptosis. These include a reduction in different cell signalling systems such as from RAS, RHO, Hedgehog, YAP or TAZ . Furthep = 0.01), longer breast cancer-free interval  and longer distant recurrence-free interval  [Consistent with our preclinical studies, several clinical studies have shown that the use of statins was associated with improved outcome in breast cancer patients. Thus following a systematic review of the literature and meta-analysis, Manthravadi et al.  identifi = 0.03) .p = 0.022) and overall survival  in patients with TNBC (n = 1534). In contrast, there was no association with breast cancer-specific survival  or overall survival  in those without TN disease . Since cell lines containing mutant p53 appear to be more sensitive to statins than p53 wild-type cells [Of particular relevance to our study was the recent report showing a significant benefit of statins in patients with TNBC but not in those with non-triple-negative disease . Thus, ipe cells  theoretiDespite the multiplicity of preclinical and epidemiological studies linking statin use with anti-cancer activity across different types of cancer, there are little data from randomized clinical trials that treatment with these drugs improves outcome for patients with cancer. Statins however, are one of the most commonly prescribed classes of drug worldwide, with well-established safety and dosage profiles . Their lIn conclusion, our data described in this article may be informative with respect to the design of clinical trials involving statins for the treatment of breast cancer. We show that the anti-proliferative effect of simvastatin is enhanced by combination with docetaxel or doxorubicin. Furthermore, our preliminary finding when combined with that of others  suggests


The attachment of sugar residues to proteins is known as glycosylation. Glycosylation is a stepwise process which is significantly altered during cancer and may thus serve as both a therapeutic and/or diagnostic target. However, our understanding of how and which glycan structures contribute to aggressive cancer behaviors is lacking. In this study, we examined how specific N-glycans contribute to aggressive neuroblastoma behavior. Here, we use genetic editing to direct N-glycan expression in neuroblastoma cells, thus enabling us to assign specific N-glycan structures to cellular properties of neuroblastoma. In addition, we also examine how altering the process of N-glycosylation influences the expression of endoplasmic reticulum (ER) folding and transport proteins. It is critical to understand how N-glycosylation processing may influence ER folding and transport protein expression. This study demonstrates that alterations of N-glycosylation influence neuroblastoma progression by diminishing or enhancing cellular properties associated with cancer progression, along with influencing the expression pattern of ER folding and transport proteins.Mgat1), NB_1(-Mgat2) and NB_1(-Mgat3), as well as the parental cell line NB_1. The two earlier mutant cells have compromised N-acetylglucosaminyltransferase-I (GnT-I) and GnT-II activities. Lectin blotting showed that NB_1(-Mgat3) cells had decreased activity of GnT-III compared to NB_1. ESI-MS profiles identified N-glycan structures in NB cells, supporting genetic edits. NB_1(-Mgat1) had the most oligomannose N-glycans and the greatest cell invasiveness, while NB_1(-Mgat2) had the fewest and least cell invasiveness. The proliferation rate of NB_1 was slightly slower than NB_1(-Mgat3), but faster than NB_1(-Mgat1) and NB_1(-Mgat2). Faster proliferation rates were due to the faster progression of those cells through the G1 phase of the cell cycle. Further higher levels of oligomannose with 6–9 Man residues indicated faster proliferating cells. Human NB cells with higher oligomannose N-glycans were more invasive and had slower proliferation rates. Both rat and human NB cells revealed modified levels of ER chaperones. Thus, our results support a role of oligomannose N-glycans in NB progression; furthermore, perturbations in the N-glycosylation pathway can impact chaperone systems.Enhanced N-glycan branching is associated with cancer, but recent investigations supported the involvement of less processed N-glycans. Herein, we investigated how changes in N-glycosylation influence cellular properties in neuroblastoma (NB) using rat N-glycan mutant cell lines, NB_1(- Neuroblastoma (NB), a pediatric cancer derived from the sympathetic nervous system, accounts for 15% of pediatric cancer-related deaths despite continued improvements in treatment . HoweverN-glycosylation is a common co- and post-translational modification that is known to be altered in the progression of cancer ,4,5,6. TThe chaperone systems and oxidative protein folding in the endoplasmic reticulum (ER) are crucial for the proper folding of secretory soluble and membrane proteins, including glycoproteins ,25,26,27Mgat1 or Mgat2 in the rat NB_1  cell line resulted in altered N-glycosylation pathways that produced higher levels of oligomannose and hybrid N-glycans, respectively [Mgat1) and NB_1(-Mgat2) cell lines, cell proliferation and invasiveness were altered compared to the NB_1 cell line. The loss of Mgat1 resulted in decreased proliferation, but increased invasiveness, while the loss of Mgat2 resulted in decreased proliferation and invasiveness. In this study, we engineered a cell line by editing the Mgat3 gene, called NB_1(-Mgat3). Lectin blotting showed that NB_1(-Mgat3) expressed lowered bisecting GlcNAc (N-acetylglucosamine) N-glycans relative to NB_1 cells, while cell invasion was similar to NB_1 cells, and cell proliferation was slightly faster. Cell–cell interactions of NB_1(-Mgat3) were stronger than NB_1, and furthermore, the cell adhesion properties of NB_1(-Mgat3) cells could be rescued. Next, we used electron spray ionization (ESI) mass spectroscopy to characterize the N-glycan structures in the parental NB_1 cells and the mutant cell lines: NB_1(-Mgat1), NB_1(-Mgat2) and NB_1(-Mgat3). The highest level of oligomannose N-glycans was present in NB_1(-Mgat1) cells, intermediate in NB_1(-Mgat3) and NB_1 cells and lowest in NB_1(-Mgat2). Higher levels of oligomannose N-glycans, along with lower levels of fucosylated and sialylated N-glycans, supported a more invasive phenotype, while increased proliferation rates were associated with those cell lines consisting of elevated oligomannose N-glycans with 6–9 Man residues and truncated N-glycans. Western blotting of ER chaperone proteins in the various rat NB cell lines revealed marked differences in protein levels, supporting perturbations in the N-glycosylation pathway impacting the ER chaperone systems, including oxidative protein folding. To expand the roles of N-glycans and chaperones, as well as Ero1 in cell growth and invasiveness, we examined clonal cell lines from human NB (HuNB) SK-N-BE(2): BE(2)-C and BE(2)-M17. Lectin binding studies revealed that higher amounts of oligomannose were associated with the more invasive and slower growing cell line BE(2)-M17. Further levels of chaperone proteins and Ero1-Lα were different between both cell lines. Overall, these results show that oligomannose type N-glycans impact NB progression, and that changes in the N-glycosylation pathway adjust chaperone systems and Ero1-Lα.Previously, our lab showed that the editing of ectively ,35,36. IMgat1) [Mgat2) [Mgat3). Gene editing of beta-1,4-mannosyl-glycoprotein 4-beta-N-acetylglucosaminyltransferase (Mgat3) was accomplished using CRISPR/Cas9 technology. The sgRNA oligonucleotide (ggtggacttcgtgttgccgg) was designed using chop-chop software [Mgat3 was the following: NM_019239. Recombinant DNA procedures were used to paste the Mgat3 gRNA into pSpCas9(BB)-2A-Puro vector (Addgene plasmid ID: 48139) [Mgat3 in NB_1 cells as previously described [Mgat3 gRNA sequence, were amplified and cloned into the pCR2.1TOPO vector for DNA sequencing. Sequencing of 9 separate fragments revealed indels in each of them that generated 4 different frameshift mutations: 1 nt insertion, 1 nt deletion, 5 nt deletion and 5 nt indel. Premature stop codons are at nucleotides 420–422 or 470–472 of the coding sequence (CDS) while the next in-frame start sequence occurs at nucleotide 370. This mutated cell colony was amplified and used for experiments. All cells were maintained in DMEM containing 10% FBS, 50 U/mL penicillin and 50 μg/mL streptomycin at 37 °C in a 5% CO2 atmosphere. The mouse Mgat3-pCDNA3.1-Hygromycin vector kindly provided by Dr. Pamela Stanly, College of Albert Einstein, was used to rescue the NB_1(-Mgat3) cell line by employment of the Lipofectamine® 2000  protocol [Human neuroblastoma BE(2)-C, BE(2)C-M17 and Rat B35 neuroblastoma cells were purchased from American Type Culture Collection . The rat NB_1 cell line is a clonal cell line derived from the B35 neuroblastoma (NB) cells  and was Mgat1)  and NB_1software  and the protocol .Total membranes of NB cells were isolated via ultracentrifugation as previously described . RIPA buTM gels . Separated proteins were transferred to PVDF membranes and were incubated in EveryBlot Blocking Buffer, primary and secondary antibodies, and then imaged using a ChemiDoc MP imaging system. Primary antibodies with rat and human reactivity include: BiP, PDI (PDIA1), calreticulin, Grp94, KIF5B and ERp57  and rhodamine conjugated actin . Primary antibodies with only human reactivity include: Ero1-L-α and calnexin . The secondary antibody  used was anti-rabbit IgG starBright Blue 700. Immunobands were quantified using image lab software  by comparing the ratio of the protein of interest to β-actin, housekeeping protein. In short, multichannel images of Western blots were used to detect protein of interest (starBright Blue 700), along with the housekeeping protein (rhodamine-conjugated β-actin) in each lane. Moreover, multiplexed Western blotting permitted correction for loading and transfer of separated proteins to membranes for each sample since β-actin was measured in the same lane as the protein of interest. The ratio of the immunoband intensities of the protein of interest to β-actin are reported as a mean from at least 3 separate lanes. To verify that proteins of interest and β-actin migrated as reported by the literature which accompanied product, Western blots with protein markers can be viewed in supplementary section  at 250 mA for lectin blotting, as previously described . Transfe section . AdjusteTo measure cell proliferation, a colorimetric 5-bromo-2-deoxyuridine (BrdU) assay was used  following the manufacturer’s protocol and as previously described . Cell pr4 cells were plated in 2 mL DMEM media and allowed to incubate for 2 h at 37 °C. After the incubation, the cells were labeled for 16 h in 800 μL labeling volume with Premo CDT1-RFP and Premo geminin-GFP viral particles (80 MOI) as outlined in the accompanying kit protocol. The media were replenished and labeled cells were allowed to incubate for another 8 h before imaging using an Olympus IX-71 microscope  with an ORCA R2 deep cooled mono CCD camera. Image J software was used to determine the percentage of cells in S/G2/M phases (geminin-GFP expression), G1 phase (CDT1-RFP expression) and G1-S transition (geminin-GFP and CDT1-RFP expression) per field.The Premo FUCCI (fluorescence ubiquitination cell cycle indicator) Cell Cycle Sensor Kit  was used to examine the cell cycle of the parental and mutant cell lines. In brief, 5 × 10Cells were grown to confluency on CellBind Culture Dishes . Cell monolayers were rinsed twice with DMEM and then cells were detached in DMEM using a cell scraper. Detached cells were dissociated by pipetting ten times with a 1 mL pipet tip. Images were obtained of 25–30 fields per dish using a 10× objective from a IX71 Olympus microscope. Areas of cell clusters with more than 10 cells were measured using Image J Software.Cells were plated on 60 mm cell bind dishes  and allowed to grow to confluency. Media were removed and wounds were generated using a beveled 200 µL pipette tip . Washing of cellular debris from the plate was carried out twice using 2 mL DMEM. Media were replenished using 3.5 mL of DMEM. Images were obtained at the 0 h and 19 h time points at 4× magnification using an Olympus IX73 Microscope . Wound size was measured using Photoshop  as previously described . Percent2O, Buffer B: 80% ACN, 0.1% formic acid, 1 mM NaOAc). Precursor ion scan was acquired at 120,000 resolution in the Orbitrap analyzer, and precursors at a time frame of 3 s were selected for subsequent MS/MS fragmentation in the Orbitrap analyzer at 15,000 resolution. MS/MS fragmentation was conducted with fixed CID (Collision Energy 40%) using a data-dependent scan (DDS) program, which performs an MS/MS acquisition for the most abundant ions in the MS1 spectrum. Precursors with an unknown charge state, or charge state of + 1 were excluded, and dynamic exclusion was enabled (30 s duration). LC-MS/MS data were analyzed using Thermo Xcalibur 4.2, Thermo FreeStyle 1.7 and GlycoWorkBench software. The area under the curve of different charge states of each N-glycan peak were extracted from the LC-MS chromatogram, added and the relative percentages of individual N-glycans were calculated. The data acquisition and analysis were conducted in duplicate. The total membrane protein fractions extracted from parental and mutant cells were diluted with 100 µL of 50 mM ammonium bicarbonate buffer, followed by the addition of 25 µL of 25 mM Dithiothreitol (DTT) and incubated at 50 °C for 30 min. The samples were subsequently desalted by centrifugal filtration using Amicon centrifuge filters  and then ultrasonicated to dissolve the proteins. The N-glycans were released from the samples by adding 5 µL of PNGaseF  and incubating at 37 °C for 48 h. The released N-glycans were permethylated by using methyl iodide in the presence of NaOH-DMSO base . The pert-test while one-way ANOVA with Bonferroni adjustments was used for more than two groups.The N-glycan structures were assigned with the aid of GlycoworkBench software based on precursor masses (sodium adduct) obtained by ESI-MS/MS and common mammalian biosynthetic pathway . Image JMgat genes) are responsible for adding branch points through the addition of GlcNAc residues to the conserved pentasaccharide of N-glycans . The parental NB_1 cell line is a clonal cell line isolated from rat B35 NB cells [Mgat3 from a selected cell colony revealed four different indels: a nucleotide insertion and deletion; and two different five nucleotide indels (Mgat3) cells, along with those of the parental cell line, were separated on a reducing SDS gel and probed with Phaseolus vulgaris Erythoagglutinin (E-PHA) (Mgat3) cell line was reduced compared to the parental cell line. The adjacent SDS gel stained with Coomassie blue verifies that the amount of protein loaded per well was similar. Thus, the lectin blot indicated that indels identified by DNA sequences reduced the activity of GnT-III in the NB_1(-Mgat3) cell line relative to the parental cell line.GnT enzymes  C. This lMgat1 [Mgat2 [Mgat3) and NB_1 cell lines by incorporation of BrdU into the DNA during its replication process (Mgat3) cells proliferated slightly faster than NB_1 cells. The NB_1(-Mgat1) [Mgat2) [Previously, it was shown that NB_1 cell lines with introduced indels in Mgat1  or Mgat21 [Mgat2  genes ha process A. NB_1(-(-Mgat1)  and NB_1 [Mgat2)  cell linMgat3) cell lines had the largest percentage of cells in the S/G2/M phases and G1-S transition, respectively, while NB_1(-Mgat1) and NB_1(-Mgat2) had lower levels of cells in these proliferating states (Mgat1) and NB_1(-Mgat2) had more cells in the nonproliferating phase, the G1 phase. Therefore, a lowered expression of either GnT-I or GnT-II in NB_1 cells reduced the proliferation rate, and the reduction in cell proliferation was due to impeding the progression of cells through the G1 phase of the cell cycle.To ascertain whether the different proliferation rates of the N-glycosylation mutant and parental cell lines correlated with changes in cell cycle progression, we performed fluorescent microscopy analysis of these cell lines. Unsynchronized cells were labeled with CDT1-RFP and geminin-GFP, and then micrographs were obtained after 24 h. The percentage of cells in S/G2/M phases (Green), G1 phase (red) and G1-S transition (yellow) of the cell cycle were determined by the expression of geminin-GFP, CDT1-RFP or both fluorescently labeled proteins, respectively, from each micrograph B. The NBg states C. MoreovMgat3) cells formed larger cell clusters than the parental cell line, NB_1. When NB_1(-Mgat3) cells were transiently transfected with a mammalian expression vector, containing Mgat3 cDNA, referred to as NB_1(−/+Mgat3) cells, the cell cluster size decreased relative to the NB_1(-Mgat3) cell line. Moreover, the cell cluster area of the rescued cell line was 86% of NB_1(-Mgat3) and 114% of NB_1 cells  and NB_1 cell lines (Mgat3) and NB_1 cell lines. Invading cell spheroids from the NB_1(-Mgat3) cell line was much like those of the NB_1 cell line, and in both cases, the invasion was more pronounced when cell spheroids were allowed to invade for a longer period. Since NB cell invasion was dependent on sphere area, cell invasiveness was quantified as the ratio of invasion area to sphere area [Mgat3) at 16 h and 20 h invasion periods, respectively. As expected, the cell invasiveness of cell spheroids from NB_1(-Mgat3) and NB_1 at the 16 h  (Mgat2) (Mgat3) (Mgat1) and NB_1(-Mgat2) were a Man5 oligomannose , and a hybrid containing three Man residues , respectively. Bisecting GlcNAc N-glycans  could be observed in the spectrum for NB_1, while only one of these N-glycans were present in the spectrum of NB_1(-Mgat3). Based on the relative abundancies of the N-glycans, both NB_1 and NB_1(-Mgat3) cell lines exhibited similar levels , 1.3% ± 0.03) of bisecting GlcNAc hybrid ; however, levels , 0.9% ± 0.02) of possible bisecting GlcNAc complex  N-glycans were different. Of note, the bisecting GlcNAc complex N-glycans could not be differentiated from triantennary N-glycans. Thus, these results are consistent with genetic edits and biochemical data according to which GnT-I, -II and -III activities are reduced in the NB_1(-Mgat1), NB_1(-Mgat2) and NB_1(-Mgat3) cell lines, respectively, compared to the parental cell line NB_1.In the current and past studies ,35,36, tuplicate . Averageuplicate A, NB_1(- (Mgat1) B, NB_1(- (Mgat2) C and NB_ (Mgat3) D cell liMgat1) cells expressed the most, NB_1(-Mgat2) cells expressed the least and both NB_1 and NB_1(-Mgat3) cells had intermediate levels of oligomannose N-glycans. However, NB_1(-Mgat3) cells had significantly higher levels than the parental cell line. Hybrid-type N-glycans were also different between the cell lines with the NB_1(-Mgat2) cell line expressing higher levels greater than 2-fold. Complex-type N-glycans were more highly observed in the NB_1 and NB_1(-Mgat3) cell lines than the other two cell lines with NB_1 cells expressing the highest level. The level of truncated N-glycans was highest in NB_1(-Mgat1) cells and lowest in NB_1 cells (Mgat2) and NB_1(-Mgat3) cells had intermediate levels with the earlier cell line expressing significantly higher levels. The full range of oligomannose N-glycans was observed  in the NB cell lines (m/z 1346). The overall trend of the various oligomannose structures from Man9 to Man6 displayed a higher expression for NB_1 and NB_1(-Mgat3) and a lower expression for the cell lines expressing more truncated N-glycans, NB_1(-Mgat1) and NB_1(-Mgat2). Moreover, NB_1(-Mgat1) had significantly lower levels of Man9, Man8, Man7 and Man6 than NB_1(-Mgat2). Although NB_1 had higher levels of Man9 than the NB_1 (-Mgat3) cell line, it had significantly lower levels of Man7 and Man6. The levels of Man5 or Man4 structures were similar in cell lines with an active GnT-I: NB_1; NB_1(-Mgat2); and NB_1(-Mgat3). The summation of percentages of the Man9 to Man6 structures revealed that the expression level was lowest for NB_1(-Mgat1) while the NB_1 cell line (-Mgat2) was second to lowest (Mgat3) cell lines had higher levels with NB_1(-Mgat3) expressing the highest level. Differences existed in the levels of fucosylated (Mgat1) cell line produced the least amount of fucosylated and sialylated N-glycans while the NB_1(-Mgat2) cell line had the highest levels. Both NB_1 and NB_1(-Mgat3) cell lines expressed intermediate levels but NB_1 had significantly more fucosylated and sialylated N-glycans than NB_1(-Mgat3). Hence, although all cell lines expressed high levels of oligomannose N-glycans, the N-glycan populations were significantly different. The levels of oligomannose structures detected in the various NB cell lines support that the release of the Man residues in the ER and cis-Golgi were slowed in the NB_1 and NB_1(-Mgat3) cell lines relative to the other two cell lines. Moreover, the stalling was more apparent in the ER than cis-Golgi for NB_1 than NB_1(-Mgat3).The quantification of the various N-glycan types showed that the major type of N-glycan was oligomannose but significant differences in the levels of this type of N-glycan were observed A. NB_1, NB_1(-Mgat2) and NB_1(-Mgat3) cells were loaded from left to right. The anti-BiP antibody detected similar levels of BiP in all cell lines (top left panel) while anti-Grp94 (middle left panel), anti-CRT (bottom left panel), anti-ERp57 (top right panel), anti-PDI (middle right panel) and anti-KIF5B (bottom right panel) antibodies revealed differences in their respective immunobands in the various NB cell lines (Mgat3) cells than in NB_1(-Mgat1) and NB_1(-Mgat2) cells. CRT levels were about 1.5-fold higher in NB_1 and NB_1(-Mgat1) cells than in NB_1(-Mgat2) and NB_1(-Mgat3) cells. The NB_1(-Mgat3) cell line expressed the highest level of ERp57 followed by NB_1 and then NB_1(-Mgat1), while NB_1(-Mgat2) expressed the lowest amounts. The expression of PDI was about 1.3-fold higher in NB_1 than NB_1(-Mgat1), NB_1(-Mgat2) and NB_1(-Mgat3) cell lines. KIF5B levels were rather similar in NB_1, NB_1(-Mgat1) and NB_1(-Mgat3) while those of NB_1(-Mgat2) cells were about 2-fold higher. KIF5B expressions of NB_1, NB_1(-Mgat1) and NB_1(-Mgat3) cell lines were rather similar while the NB_1(-Mgat2) cell line expressed about 2-fold higher levels. Thus, these results indicate the levels of proteins in the chaperone systems, including those of the oxidative protein folding process, could be altered due to disruptions in the N-glycosylation pathway.To determine whether changes in N-glycan structure expression impacted the ER protein folding process, the abundancies of various chaperones and a protein (KIF5B) assisting in ER exit of folded proteins were evaluated. Whole-cell lysates from confluent cell culture dishes of the various NB cells were employed for Western blotting. In all cases, samples of NB_1, NB_1(-ll lines A. Bar grll lines B. BiP leMgat1) cell line, which has an inactive GnT-I [Mgat1). Furthermore, the NB_1(-Mgat1) was much more invasive but had slower proliferation rates than NB_1 [Previously, our lectin blotting studies showed that glycosylated proteins from the NB_1 cell line bind less Galanthus Nivalis Lectin (GNL) and more L-PHA than the NB_1-C (lane 1) and BE(2)-M17 (lane 2) NB cell lines A were peMgat3), and characterized it alongside the parental (NB_1) and two previously engineered (NB_1(-Mgat1) [Mgat2) [Mgat3) cells, and was followed rather closely by NB_1, while the NB_1(-Mgat1) [Mgat2) [Mgat1) cells proliferating the slowest. Furthermore, the two slowest proliferating cell lines progressed slower through the G1 phase of the cell cycle. Cell invasiveness was highest in NB_1(-Mgat1) [Mgat3) and NB_1, and then NB_1(-Mgat2) [Mgat2, as the parental cell line was more invasive and proliferated faster [Mgat3), had less truncated N-glycans than the slower growing cell lines, NB_1(-Mgat1) and NB_1(-Mgat2), indicating that more processed N-glycans in NB cells had faster cell proliferation rates. Certain N-glycan structures could also be associated with cell invasiveness for the parental and N-glycosylated mutant NB_1 cell lines. Higher oligomannose-type N-glycans, as well as lower fucosylated and sialylated N-glycans, supported the more invasive cell lines, such as NB_1(-Mgat1). To expand our findings, we showed that the human BE(2)-C NB cell line which had more complex-type N-glycans was less invasive and had faster proliferation rates than the human BE(2)-M17 NB cell line. Taken together, these results from the parental and mutant NB_1 cell lines, along with those of the two human NB cell lines, contribute significantly to our knowledge of how certain N-glycan structures, such as oligomannose-type N-glycans, promote various phases of NB.Our current and past studies ,35,36 pr(-Mgat1)  and NB_1(-Mgat1)  and NB_1(-Mgat1) , followe(-Mgat2) ,35 cell Mgat2)) with the highest level was the least invasive and proliferated at a relatively low rate [High abundancy of oligomannose N-glycans was identified by ESI-MS spectra in the parental NB cell line. Further GNL interactions of BE(2)-C and BE(2)-M17 cell lines indicated high levels of oligomannose-type N-glycans. These high levels were also suggested in NLF (MYCN-amplified) and SY5Y (MYCN-nonamplified) NB cells with higher levels in NLF cells . It was low rate ,35. ThusMgat3) which had higher levels of ERp57. These results indicate that disruptions in the N-glycosylation pathway were connected to CNX/CRT and GRP78 (BiP)/GRP94 chaperone systems. Further oxidative protein folding was modified in the NB_1 cell line compared to the mutant NB_1 cell line. It should be mentioned that the interaction of ERp57 with CRT or CNX enhances its activity [Mgat3) cell line. Our examination of human NB cell lines showed that the BE(2)-C cell line which had more and less of the complex and truncated N-glycans , respectively, had modified ER chaperone levels compared to BE(2)-M17. BiP, CRT, PDI, ERp57 and Ero1-Lα levels were raised in BE(2)-C while CNX was lowered. Like NB_1, disulfide isomerase activity appeared to be significantly increased in BE(2)-C as both CRT and ERp57 levels increased relative to BE(2)-M17. Further increased levels of PDI, along with Ero1-Lα, supported higher disulfide isomerase activity as oxidative protein folding involves reoxidation of PDI by Ero1 [Since the processing of N-glycosylated proteins occurs in the secretory pathway, we evaluated ER chaperone proteins in the various NB cell lines. Overall, the N-glycosylation mutant NB_1 cell line had similar or lower levels of the various chaperone proteins to the parental cell line, except NB_1 cells relative to NB_1 (-Mgat2) and NB_1 (-Mgat3) cells, and in the case of NB_1 the higher calreticulin levels were associated with increased ERp57. Since the ER chaperone network utilizes a compensatory relationship between the multiple families of chaperones to ensure efficient and accurate protein folding [Mgat1, Mgat2 and Mgat3 caused a reduction in the expression of GnT-I, GnT-II and GnT-III in the NB_1(-Mgat1), NB_1(-Mgat2) and NB_1(-Mgat3) cell lines, respectively. NB_1(-Mgat1) cells expressed at least 98% of oligomannose N-glycans and the most prevalent oligomannose was the Man5 structure. Furthermore, the level of the Man4 structures was quite like the Man8 and Man7 structures, indicating that the glycoproteins with Man5 structures are moving through the secretory pathway and not retained. The low levels of hybrid and complex N-glycans are supported by previous work which showed that the deletion of 120 amino acids from the N-terminus of GnT-I causes a complete loss of activity [Mgat1) cell line does not have an in-frame start codon until amino acid 251. NB_1(-Mgat2) cells expressed the highest levels of hybrid N-glycans and the most abundant hybrid N-glycan structure had less than four Man residues; however, close to 10% of the N-glycans were of complex type. Since GnT-II uses hybrid N-glycans with the α1–3 mannose removed [Mgat2) supports that the indel introduced in the Mgat2 gene reduces GnT-II activity. The N-glycan profiles of NB_1(-Mgat3) cells supported changes in the N-glycosylation pathway as the bisecting GlcNAc complex N-glycans were reduced in NB_1(-Mgat3) compared to NB_1. As such, it appears the indel in Mgat1 almost completely silenced the gene, while indels in Mgat2 and Mgat3 caused some reduction in the activity of GnT-II and GnT-III. Alternative start codons are the likely reason for the remaining activities of GnT-II and GnT-III. In both the Mgat2 and 3 genes, there are start codons downstream of the sequenced indels that could serve as alternative start sites at nucleotide positions 292 and 370 of their coding sequences, respectively. N-Truncated proteins of 345 and 415 amino acid residues could be produced for GnT-II and GnT-III, instead of their full-length forms, 442 and 538 amino acid residues, respectively. However, these alternative start sites would be less favored for protein translation [Our current study showed varied expressions of ER chaperones in rat NB cell lines with disruptions in the N-glycosylation pathway due to genetic editing, and the human NB cell lines with different N-glycosylation populations acquired during tumor growth. Calreticulin was more abundant in BE(2)-C, while calnexin was more abundant in BE(2)-M17. Moreover, the enrichment of calreticulin in BE(2)-C was accompanied with more BiP and ERp57. This trend was like a previous study that reported cells depleted of calnexin was associated with increased levels of calreticulin, ERp57 and BiP . Calreti folding ,43, it mactivity . As mentnslation ,47. TakeMgat1) cells were the most invasive, and they had minimal fucosylation and sialyation while the least invasive cell line, NB_1 (-Mgat2), had the greatest levels of fucosylation and sialyation. NB_1 and NB_1 (-Mgat3) cells represented intermediate cell invasiveness and levels of fucosylation and sialyation. Since we suspected that the populations of complex glycans were underrepresented, fucosylation and sialyation were also accounted for on truncated N-glycans which gave a similar pattern. Overall, this highlights the complexities of N-glycans in cancer progression and indicates that high fucosylation and sialyation levels may suppress NB cell invasiveness.The fucosylation and sialyation of N-glycosylated proteins are reported to be increased in multiple cancers and are thought to contribute to cancer aggressiveness ,13,15,16Mgat3) proliferated faster. As such, when evaluating ER stress via chaperone systems, the significance of disruptions in the N-glycosylation pathway must not be neglected.Rapid proliferation is a common feature of cancerous cells which makes them prone to ER stress . Faster Mgat genes in a rat NB_1 cell line. Furthermore, two clonal cell lines of the SK-N-BE(2) human cell line (BE(2)-C and BE(2)-M17) were implemented to parallel the rat NB cell lines. Specifically, this study revealed a functional role of oligomannose glycans in cell invasiveness and proliferation, thus impacting the progression of NB. Additionally, our studies supported that lowered levels of fucosylated and sialylated N-glycans increased cell invasiveness. Moreover, a correlation between the N-glycosylation pathway and the ER protein folding process via altered ER chaperone expression, along with Ero1, was noted. Our systematic approach clearly showed that differences in the N-glycosylation pathway resulted in modified cell proliferation and invasion. We propose that increased levels of various oligomannose-type N-glycans promote NB aggression.In summary, we present a simplified approach correlating cell invasiveness and proliferation, along with the expression of ER chaperone proteins and Ero1, with changes in the N-glycosylation pathway by editing


Vitis vinifera L.) seeds are rich in polyphenols including proanthocyanidins, molecules with a variety of biological effects including anticancer action. We have previously reported that the grape seed semi-polar extract of Aglianico cultivar (AGS) was able to induce apoptosis and decrease cancer properties in different mesothelioma cell lines. Concomitantly, this extract resulted in enriched oligomeric proanthocyanidins which might be involved in determining the anticancer activity. Through transcriptomic and metabolomic analyses, we investigated in detail the anticancer pathway induced by AGS. Transcriptomics analysis and functional annotation allowed the identification of the relevant causative genes involved in the apoptotic induction following AGS treatment. Subsequent biological validation strengthened the hypothesis that MDM2 could be the molecular target of AGS and that it could act in both a p53-dependent and independent manner. Finally, AGS significantly inhibited tumor progression in a xenograft mouse model of mesothelioma, confirming also in vivo that MDM2 could act as molecular player responsible for the AGS antitumor effect. Our findings indicated that AGS, exerting a pro-apoptotic effect by hindering MDM2 pathway, could represent a novel source of anticancer molecules.Grapevine ( Vitis vinifera L.) is a plant rich in bioactive compounds that are principally distributed in berry skin, stem, leaves and seeds. Among the substances accumulated in grapes, the most biologically interesting ones are represented by polyphenols. They constitute the most important class of natural bioactive molecules. Therefore, there is an increasing interest in their use for the treatment and prevention of different chronic pathologies, including cancer. Grape seeds are a significant source of polyphenols, containing about 70% of these compounds accumulated in grapes , PC(O-14:0/2:0), PC(2:0/O-16:0)[U], PC(O-14:0/2:0), PE(18:0/0:0), PC(O-16:1(11Z)/2:0) and PC(0:0/18:1(9Z) were positively regulated in all AGS-treated cells. Notably, Alpha-CEHC, a tocopherol catabolite, was increased in all cell lines. Finally, for the additional 12 ions tentative chemical formulas were assigned, although it was not possible to retrieve an unequivocal identification. A targeted metabolomics approach allowed us to identify, in AGS-treated samples, 87 compounds involved in cell metabolism. Very interestingly, we found among them molecules belonging to primary pathways involved in cancer metabolism such as glycolysis and the acid citric cycle  .These compounds were used to perform a hierarchical clustering (HCL) analysis in order to evaluate the metabolite profile changes determined by AGS treatment . MetabolAs shown in A more detailed analysis of the metabolic changes occurring in each cell line provided evidence that AGS treatment induced a strong modulation in different metabolic classes. In particular, and in agreement with gene expression data, we found several compounds involved in cholesterol metabolism and catabolism, but the majority of them did not change in the AGS-treated samples compared to control. However, other compounds were found to be decreased after AGS treatment. They included molecules involved in cholesterol biosynthesis. For instance, 14-demethyllanosterol/ 20-alpha,22beta-dihydroxycholesterol decreased in MSTO and PC3, while 4alpha-methylzymosterol-4-carboxylate/Calcitetrol decreased in all cell lines . AdditioOf relevance, all the AGS-treated cell lines cells were characterized by further alterations in non-polar and semi-polar metabolic fractions; notable examples of the former are a group of fatty acids which were increased in PC3 and MDA AGS-treated cells  or in MSTO and PC3 . In addition, levels of 13-keto-9Z,11E-octadecadienoic acid and 14,15-dihydroxy-5,-8,11-eicosatrienoic acid were higher in treated cells . Additional changes were found in several compounds of interest: folic acid increased in all cell lines; glucose/fructose and their phosphate forms and two other intermediates in the glycolysis pathway, phosphoenolpyruvate (PEP) and 2,3-phosphoglyceric acid, showed the same positive sign in PC3 and MDA; glyceraldehyde-3-phosphate (GAP) decreased in all treated cell lines. The same trend was observed in both MSTO and MDA cells for 6-O-phosphono-D-gluconic acid and 6-phosphonoglucono-D-lactone, which belongs to the pentose phosphate pathway (PPP), as well as for citric acid and its isoform (isocitric acid) which represent fundamental intermediates of the TCA cycle. Finally, AGS treatment determined the differential accumulation of other groups of interest, such as molecules belonging to the N compound group. For example, AMP was reduced in PC3 and MDA, while thymidine was reduced in MSTO and PC3. Among organic acids, we found benzoic acid to be over-accumulated in all lines and 4-hydroxy and phosphoric acid over-accumulated in PC3 and MDA. On the contrary, lactic acid was reduced in all cells analyzed.Subsequently, a Venn diagram was used to determine the number of common and specific DAMs within the three cell lines analyzed . In agreHowever, the analysis revealed only a partial overlap among the different comparisons, with ten metabolites that changed in all cell lines after AGS treatment .Finally, to highlight the main metabolic pathways affected by AGS treatment, a functional enrichment analysis, performed using the Metaboanalyst tool, was carried out, considering the list of all differentially accumulated compounds. Interestingly, a strong impact on glucose/energy metabolism and more specifically on the Warburg effect, gluconeogenesis and pyruvate metabolism/glycolysis was observed following AGS treatment. Additionally, glutamate and glutathione metabolism as well as carnitine synthesis resulted as the top enriched pathways in AGS-treated cells .in vivo tumor growth. To this aim, we set up an experiment using an ectopic xenograft tumor in a mouse model using MSTO cells. The experimental design is reported in On the basis of results obtained by transcriptomics and metabolome analyses, we decided to assess the efficacy of AGS in reducing p-value < 0.0001). As reported in the p < 0.0001; The results showed that the administration of AGS (100 mg/kg) three times a week for four weeks was able to significantly delay the growth of the ectopic tumors as compared to the untreated control group (p < 0.0004). Accordingly, the apoptotic index was significantly higher in tumors of mice treated with AGS (p < 0.0004). Furthermore, the immunohistochemical analysis of MDM2 expression in the tumors revealed a strong decreased expression in AGS treated samples with respect to control (p < 0.0001)  . in vivo that MDM2 can be considered as one of the molecular players responsible for the antitumor effect of AGS.These results indicate that AGS treatment acts on both proliferation and apoptosis. Moreover, they confirmed Cancer represents the second leading cause of death all over the world and nowadays different therapeutic strategies are available to treat various cancers. However, the use of conventional drugs results in severe side effects due to their toxicity towards healthy cells. Another adverse aspect about the use of standard chemotherapeutics is that their efficacy is often reduced due to the development of cancer resistance—a multifaceted phenomenon related to cancer heterogeneity and occurring through DNA mutations, metabolic changes and drug inhibition or degradation . In the In this study, we further dissected, at the molecular level, by means of transcriptomic and metabolomic analyses, the anticancer activity of AGS to find key players responsible for its effect. Deregulated genes were functionally annotated in IPA and this analysis highlighted that, among “Disease and disorders” categories, “Cancer” was indicated as the class containing the majority of genes deregulated by AGS treatment, thus confirming our previous observations. Subsequently, we performed an IPA Bioprofiler analysis that allowed us to identify genes associated with a specific molecular profile of a biological process. This analysis detected MDM2, which was downregulated, as the relevant causative gene linked to the apoptosis induced by AGS treatment.The successive network analysis depicted an interaction between MDM2 and the other two genes involved in apoptosis: p53 and JUN, which were, respectively, upregulated and downregulated. In this context, the expression level of all these genes agrees well with the reduction in cancer cell proliferation.MDM2 is an oncogene that acts as a negative regulator of the tumor-suppressor p53  and it iThe interaction between p53 and JUN is crucial for cell cycle regulation. In the absence of JUN, the basal level of p53 is increased resulting in reduced cell proliferation. On the contrary, JUN overexpression negatively regulates the transcription of p53, blocking the p53-mediated growth suppression .The balance between MDM2 and p53 maintains a healthy cellular state. MDM2 is an E3 ubiquitin ligase that negatively regulates p53 , but it To better analyze if MDM2 could be the relevant causative gene linked to AGS anticancer effects, we verified the efficacy of AGS treatment and the molecular pathway activated in p53-defective cancer cell lines. To this aim, we chose to use two cell lines characterized by high aggressive behavior and invasiveness: MDA, a breast cancer cell line that lacks of functional p53 due to a missense mutation  and PC3,Our results confirmed that AGS treatment in both cell lines determined an increase in apoptosis, while no effect was evidenced in the corresponding normal non-tumor cell lines, MCF10A and PNT2. q-PCR analysis confirmed, in both cell lines, the expression levels of the genes in the network involved in apoptosis. Protein expression analysis clearly showed, in both cell lines, a decreased expression of JUN and MDM2, thus reinforcing the possibility that MDM2 could be the molecular target of AGS treatment. Finally, to understand the signaling pathway activated in the absence of p53 and involved in MDA and PC3 cell death, we analyzed the protein expression of p27 and p21, two CDK inhibitors whose upregulation has been reported to inhibit cell proliferation in both cancers. p21 and p27 have been described to bind MDM2 and activate apoptosis also in p53-defective contexts ,30,31. Our results confirmed that MDM2 downregulation is linked to upregulation of p21 and p27 expression in breast cancer cells. On the contrary, in PC3, decreased MDM2 appears to be linked to the exclusive upregulation of p21. These results suggest a p21 pro-apoptotic role in the absence of p53.In order to gain insights about the biochemical changes occurring in the cancer cells following AGS treatment, we carried out a metabolomics analysis either at “global” untargeted or targeted levels. Cancer cells are characterized by metabolic changes that are needed for their proliferation and different metabolic rearrangements which are involved in cancer growth . In factIn our study, metabolomic analysis of MSTO, PC3 and MDA cells following AGS treatment detected 115 DAMs. Interestingly, in all the cell lines analyzed, we observed a metabolic shift with a reduction in lactic acid, the glucose fermentation product, and an increase in the intermediates of the citric acid pathway, such as citric acid, isocitric acid and α-ketoglutaric acid. These data suggest an inhibition of the Warburg effect and an activation of the mitochondrial activity, resulting in apoptosis. Notably, PC3 and MDA cell lines displayed a more extreme attitude compared to MSTO, as revealed both by PCA and HCL analysis, as well as by the number of common and DAMs metabolites. This aspect could reflect the lack of functional p53.Using a metabolomics annotation pipeline and focusing on ions with a consistent trend in at least 2 out of 3 cell lines, we were able to assign 37 chemical formulas and 25 identities. In most of the cases, metabolic alterations agree well with the decreased tolerance to cancer induction and proliferation. A notable example is the increase in all cell lines of Alpha-CEHC, produced in the frame of tocopherol catabolism that has been described to have anticancer properties . We found changes in metabolites, belonging to a lipid class, that have been described to have anticancer properties (e.g. eicosatrienoic acids)  or to beWe also observed that AGS was able to modulate metabolites belonging to the amino acid class. It has been described that MDM2, independently of p53, is able to transcriptionally regulate amino acid metabolism and, in particular, act on serine/glycine metabolism contributing to cancer cells’ anabolic demand .Other metabolic changes consistent with AGS anti-cancer activity were found in folic acid  as well Our functional enrichment analysis highlighted the involvement of several metabolic pathways modulated by AGS treatment: Warburg effect, pyruvate/glycolysis and gluconeogenesis. All these pathways have been reported to play a key role in cancer .Overall, these results agree well with previous studies, indicating that activation of p53 affects cancer properties by modulating the glucose metabolism and the Warburg effect through inhibition of MDM2 . Furtherin vivo, the AGS anticancer effects, we generated a xenograft mesothelioma mouse model. The results indicated that administration of AGS three times/week for up to four weeks strongly delayed tumor growth, when compared to control, untreated tumors. According to this observation, immunohistochemical analysis indicated that tumor samples from AGS-treated mice showed a decrease in proliferation index and an increase in apoptosis. In addition, we also confirmed in vivo that AGS determined a reduction in MDM2 expression, thus suggesting that MDM2 could represent the causative gene of the apoptosis induced by AGS treatment. To validate, In vivo analysis confirmed the downregulation of MDM2 and the concomitant activation of the apoptotic pathway, suggesting that MDM2 can be viewed as a candidate target to induce an anticancer effect. Notably, MDM2 has been shown to act as oncogene in human cancers, and different strategies to inhibit MDM2 are under analysis for their treatment. The promising results of our study rely on the efficacy of AGS treatment also in p53-defective cancer cells and on the finding that MDM2 represents the key mediator of AGS activity. Taken together, our data suggest that AGS could be viewed as a natural source for selecting novel anticancer drug molecules exerting pro-apoptotic activity in different cancers. Combined transcriptomics and metabolomics analysis allowed us to unravel the unknown mechanism of action of AGS against mesothelioma, breast and prostate cancer. Overall, our results indicate that apoptosis induced by AGS-treatment occurred through MDM2 downregulation and concomitant modulation of glucose metabolism.


The emergence of resistance to pancreatic cancer (PC) current treatment requires the development of new therapeutic strategies. In this context, bioactive molecules from plant extracts have shown excellent properties to improve classical therapy against this type of tumor. This systematic review aims to collect all the in vitro studies related to the antiproliferative activity of isolated plant molecules that support their applicability in PC. A total of 620 articles published in the last 10 years were identified, although only 28 were finally included to meet the inclusion criteria. Our results reflect the most important biomolecules from natural compounds that induce cell death in PC and their essential mechanism of cell death, including apoptosis, pathways activated by the KRAS mutation and cycle cell arrest, among others. These in vitro studies provide an excellent molecule guide showing applications against PC and that should be tested in vivo and in clinical trials to determine their usefulness to reduce PC incidence and to improve the prognosis of these patients. However, natural compounds are isolated in small amounts, which prevents comprehensive drug screening, being necessary the role of organic synthesis for the total synthesis of natural compounds or for the synthesis of their simplified and bioactive analogs. Pancreatic cancer is the fourth leading cause of cancer death in the United States and Europe. One of the major problems with this type of tumor is the lack of effective therapy, with only 20% of tumors resectable by surgery since the mass is localized and in the early stages. However, most tumors are in more advanced stages when diagnosed, and these are often metastatic . This abIn recent years, the use of natural products in various areas related to cancer, such as preventive diets or complementary therapy, has gained great importance ,5. Due tIn this systematic review, different plant families will be analyzed from which functional extracts with antitumor activity against PC are obtained or from which compounds with antitumor activity can be isolated that can be used as a possible therapy and adjuvant in the current treatment of PC.The purpose of this systematic review was to assess the most recent and representative information on extracts and natural biomolecules with antitumor activity against PC. This systematic review was developed according to the PRISMA guide . For thi50 values and specify the mechanism of action by which the plant extracts or isolated compounds exert their antitumor power. All the research articles had been published in peer-reviewed journals, and those with fully accessible texts were selected. Articles found through bibliographic references could also be included, as long as they met the inclusion criteria.Research papers from March 2011 to March 2021, which included isolated biomolecules or plant extracts tested on PC cell lines, were incorporated in this systematic review. In addition, the studies had to include IC50 or only explained the anticancer mechanism of action, without providing IC50 values, were excluded. In turn, nonoriginal articles, such as reviews, meta-analyses, epidemiological studies, book chapters or conferences, were also excluded from the study.Studies that did not test the extracts or compounds isolated in PC cells, those in which the biomolecules or extracts were synthesized or purchased, that did not specify an extraction method, that the article was not freely accessible or that were written in Chinese or Japanese were excluded. Articles that studied extracts or compounds from other kingdoms than plantae were also discarded. In addition, articles that only included ICThis systematic review was carried out using the electronic databases: MedLars Online International Literature, through PubMed; SCOPUS; Web of Science; and Cochrane Library Plus. The medical subject headings (MeSH) were defined using “pancreatic neoplasm” and “plant extract” as descriptive terms. The final equation was ((“Pancreatic Neoplasms” [MeSH Terms] OR (“pancreatic” [Title/Abstract] AND “neoplasms” [Title/Abstract]) OR “Pancreatic Neoplasms” [Title/Abstract] OR “pancreatic cancer” [Title/Abstract]) AND (“Plant Extracts” [MeSH Terms] OR “Plant Extracts” [Title/Abstract])) AND (2011/3/1:2021/3/1 [pdat]). For the other databases, adaptations were made to carry out the search. In addition, the bibliography of the selected articles was reviewed to analyze if they met the inclusion criteria.Three of the authors  carried out the literature search, the review of the abstracts and the selection of the appropriate ones for further full-text examination. In turn, nonoriginal articles, such as reviews, meta-analyses, epidemiological studies, book chapters or conferences, were also discarded from the study. There were restrictions by language, excluding those who were in a language other than English or Spanish. After this first step, the author examined the full-text articles, considering the inclusion and exclusion criteria. Since the objective of this systematic review was to review in vitro studies, those where only in vivo studies were performed were excluded.To continue the study selection process, the same three authors independently reviewed the studies included and extracted data from them. There was good agreement between the researchers  accordinKRAS gene, 7 demonstrated cell death due to cell cycle arrest cell and 4 other articles describe other actions through important factors in PC. The most frequent part of the plant used to obtain the bioactive compounds were the roots, followed by the leaves, stems, fruits and seeds. The shoots and bark were less studied  Miq. was analyzed in two studies. In the first, ethanol and distilled water were used to obtain the plant stem extracts. Then, liquid chromatography (HPLC) was used to obtain four preparations of the active compound DCA. The IC50 for the different compounds ranged between 0.22 and 5.5 µg/mL on the PANC-1 pancreatic cell line. The value found for the distilled water extract was negligible. On the BxPC-3 cell line, the values varied between 0.82 and 34 µg/mL, without having tested the aqueous extract. In the different DCA preparations, only 1 and 3 showed activity. The DCA and ethanolic extracts showed more potency than the aqueous extract. It was observed that the products of O. horridus produced an increase in the number of nuclear apoptotic bodies, visualized with fluorescence. In turn, the effect of DCA on 35 genes associated with apoptosis was studied, suggesting cell death by apoptosis through the extrinsic and intrinsic pathways [50 of 5.8 and 21 nM in the PANC-1 and BxPC-3 pancreatic cell lines, respectively. The DCA, for its part, showed an IC50 of 0.73 and 2.71 µM in PANC-1 and BxPC-3 cells, respectively. Apoptosis mechanism was mediated by an increase in caspase-3 and cytochrome C in the cell cytoplasm and a decrease in bcl-2 and bax mRNA expression. It should be noted that the ethanol extract showed the ability to stop the cell cycle in G0/G1 and in phase S.pathways . In the pathways  used theEugenia involucrate DC fruits and seeds obtained using ethanol (99.4%) were tested for their antitumor activity. Only the seed extracts showed a cytotoxic effect, with an IC50 of 645 µg/mL in the PANC-1 pancreatic cell line, showing the ability to induce apoptosis through the alteration of the mitochondrial membrane (intrinsic pathway). Alteration in the structure of the cytoskeleton and an increase in the production of reactive oxygen species (ROS) were also observed in this cell line [Cleistocalyx operculatus (Roxb.) shoots were used to extract the biocomposite 2′,4′-dihydroxy-6′-methoxy-3′, 5′-dimethylchalcone (DMC) by using 70% ethanol. This compound showed an IC50 of 10.5 and 12.2 µM in PANC-1 and MIA PaCa-2 pancreatic cell lines, respectively. A significant increase in caspase-3 and -9 and a greater inhibition of bcl-2 suggested an apoptosis mediated by the mitochondrial pathway [E. robusta Sm, E. microcorys F. Muell and E. saligna Sm) were analyzed. Only the extracts of the last two species showed antitumor activity. E. microcorys leaf extract showed an IC50 between 68 µg/mL (aqueous extract) and 76 µg/mL (70% ethanol extract) in MIA PaCa-2 pancreatic cell line. Furthermore, distilled water extract stimulated the activity of caspase-3/7, suggesting induction of cell death by apoptosis. In E. saligna, only the ethanol extract showed activity (IC50 of 53 µg/mL) [Natural extracts of the ell line . Cleisto pathway . In addi3 µg/mL) .Gloriosa superba L. seeds, with high levels of colchicine (GS), were used to generate extracts of rich and poor GS. The first extract showed high cytotoxicity in PANC-1 and Panc02 cells . By contrast, the second extract (designated GS2B) showed an IC50 of 9.49 µg/mL in Panc02 cells (50 times higher). In vivo assays of both extracts using Panc02 tumors showed a reduction in tumor volume with a decrease in ki67 expression and an increase in apoptosis-mediated caspase-3 [Prunus armeniaca L. kernels which was tested in PANC-1 cells showing an IC50 of 704 µg/mL with an increase in bax/bcl-2 ratio and an overexpression of caspase-3 [On the other hand, aspase-3 . Moreoveaspase-3 .C. nutans stems and leaves were generated using methanol (polar compounds), hexane, and diethyl ether (nonpolar compounds). The results showed that only the nonpolar extracts had antitumor activity, with an IC50 ranging between 30.91 and 39.12 µg/mL in the three pancreatic tumor lines tested  This extract showed synergistic action in combination with gemcitabine, increasing the expression of proapoptotic proteins (BAX and BID) and decreasing the expression of antiapoptotic proteins  [C. cordifolioidea roots was used to isolate the cordifoliketonas A biomolecule. This compound showed an IC50 ranging from 4.18 to 5.56 µg/mL in pancreatic tumor cell lines. AsPC-1, BxPC-3 and PANC-1 produced apoptosis through overexpression of proapoptotic proteins  and decreased expression of antiapoptotic proteins (BCL-2 and BCL-XL). An in vivo assay showed similar activity, decreasing tumor growth compared to controls [S. oleracea. This molecule was tested in vitro , obtaining an IC50 between 18.5 and 26.9 µg/mL through apoptosis associated with the overexpression of PARP, caspase-3 and BAX (proapoptotic proteins). The in vivo assay performed on MIA PaCa-2 tumors showed that MGDG had synergy with irradiation [S. baicalensis obtained in ethyl acetate. The in vitro assay showed cytotoxic activity on pancreatic cell lines BxPC-3 and PANC-1, with IC50 ranging from 6.5 to 41.7 and 8.9 to 47.4 µg/mL, respectively. TFAE had no effect on the nontumor cell line HPDE6-C7. This extract produced cell toxicity through the induction of autophagy (via PI3K/AKT/mTOR). Its antitumor activity was also confirmed in an in vivo BxPC-3 model decreasing tumor growth [As shown in  CIAP-2) . In addicontrols . In addiadiation . Anotherr growth .T. officinale roots were used to generate a cytotoxic aqueous extract against BxPC-3 and PANC-1 pancreatic cell lines . The cytotoxic mechanism was mediated by autophagy with an increase in LC3-II levels. However, a high expression of caspase-8 was also detected [U. dac stem (methylene chloride extract), showed activity against the PANC-1 cell line (IC50 14.5 µM) through the inhibition of the AKT/mTOR pathway, key in the start of the autophagy process [detected . In addi process .V. anthelmintica dried fruits were extracted using 80% ethanol. Only isorhamnetin and luteolin showed antitumor activity against PANC-1 . Isorhamnetin induced inhibition of the RAS/MAPK pathway and cell cycle arrest in the S phase [C. hystix fruit extracts. Bergamottin was the most interesting molecule, showing an IC50 of 4.6, 2.2 and 9.4 µM in PANC1, MIA PaCa-2 and PSN-1, respectively. Its cytotoxic activity was due to the induction of cell death by autophagy [On the other hand, nine compounds from  S phase . In anotutophagy .I. helenium (trizoma and dried root) using 95% ethanol induced an IC50 of 4.3 µg/mL in CFPAC-1 pancreatic cells. This extract induced autophagy, apoptosis (with increased proapoptotic proteins and decreased antiapoptotic proteins) and cell cycle arrest in G0/G1 phase. Furthermore, EEIHL inhibited cell migration through increased e-cadherin expression [Finally, the EEIHL extract obtained from pression .H. hirsute were processed with 40% methanol to obtain a saponin-enriched extract. HPLC allowed separation of six fractions (F0–F5). Apart from F0, all fractions showed an IC50 between 3.11–7.76 μg/mL and 3.71–17.12 μg/mL for MIA PaCa-2 and BxPC-3, respectively. The mechanism of action of F2, F3, F4 and F5 was cell cycle arrest in the S phase [Ocimum sanctum L. dried leaves. EEOL was tested on AsPC-1 and MIA PaCa-2 cell lines, showing an IC50 of 46 and 69 µg/mL, respectively, and a significant arrest of the cell cycle in G2/S. AEOL was tested in an AsPC-1 in vivo model, showing a reduction in antiapoptotic protein expression and increasing expression of BAD (proapoptotic) and E-cadherin [P. acerifolium bark (PaEBE) was tested on PANC-1 cells, showing an IC50 of 74.22 µg/mL and intense cell cycle arrest in the sub-G1 phase accompanied by an increased ROS production and altered mitochondrial membrane potential [A relevant group of isolated compounds was able to induce cell death through arrest in some phases of the cell cycle, as shown in  S phase . On the cadherin . In anototential .Meliae fruit, Cinnamon bark and Sparganium rhizome (named H39) showed a significant antitumor effect on PC cells PANC-1 alone (IC50 0.07 mg/mL) or in combination with gemcitabine (3 nM) (IC50 of 0.05 mg/mL). H39 induced cell death by arresting the cell cycle at the G0/G1 phase. This extract was able to decrease cell migration and the expression of antiapoptotic genes such as JAK2 and XIAP [O. horridus (root bark), ethanolic (70%) extract was associated to gemcitabine, cisplatin and paclitaxel against the PANC-1 cell line in 2D and 3D conditions, showing that the extract significantly enhanced the antiproliferative activity of both gemcitabine and cisplatin at some concentrations. The cell death mechanism described was a cell cycle arrest in the S phase [The association of extracts has also been assayed to determine the antitumor potential in PC. A 40% ethanolic extract of the combination of and XIAP . In some S phase .E. microcorys and M. oleifera leaves. In the first case, five fractions (F1–F5) were isolated after HPLC, but only F1 showed antiproliferative potential in the MIA PaCa-2 cell line (IC50 93.11 μg/mL) through G2/M cell cycle arrest [50 between 1.1 and 1.8 mg/mL. The cytotoxicity was caused by the cell cycle arrest in the sub-G1 phase showing synergistic activity with cisplatin [Aqueous extraction (distilled water) has also been used as in the case of e arrest . In the isplatin .P. koreana (50% ethanol extraction), which demonstrated high antitumor activity in five different pancreatic cell lines . Furthermore, this molecule inhibited both HIF-1α and VEGF, two potent inducers of angiogenesis and was capable of inducing apoptosis by the intrinsic pathway [A. membranaceus, A. gigas and T. Kirilowii), which was tested on endothelial cells (HUVECs) observing a reduction in angiogenesis (inhibiting VEGF phosphorylation) and a delay in tumor growth derived from the Panc-28-LUC cell line [A. millefolium called Yarrow SFE, which showed an IC50 of 31.45 µg/mL on MIA PaCa-2 cells. Its activity was related to the alteration of lipid homeostasis, showing a decrease in the expression of SREBF1, FASN and SCD. These genes are overexpressed in PC and are key factors for fatty acid production. In vivo, this extract produced a significant tumor growth inhibition, a decrease in KI67-positive cells and a downregulation in SREBF1 [S. oleracea allowed the isolation of the monogalactosyl diacylglycerol (MGDG). This compound induced an IC50 of 22, 15.1 and 18.8 nM in PANC-1, BxPC-3 and MIA PaCa-2, respectively and exerted its antitumor activity through the selective inhibition of polymerases α, δ and ε and inhibition of DNA replication [Some plant species and isolated compounds induced cell death through the alteration of other important factors in the etiology of pancreas cancer . Tumor c pathway . SH003 in SREBF1 . Lastly,lication .O. horridus, it was illustrated how the extracts obtained from the stem of the plant using ethanol showed a higher IC50 than those obtained with distilled water [C. nutans showed the relevance of the extraction method and the part of the plant used [Both the extracts and the biomolecules analyzed in this review were characterized by showing significant antitumor activity in vitro against different PC cell lines through several molecular pathways.Most of the analyzed extracts were obtained using polar solvents, generally ethanol. Therefore, most of the extracted compounds with biological activity against PC were phenols and flavonoids with high affinity for these solvents . Other ped water . Likewisant used . Howeverant used .C. nutans, E. microcorys and S. oleracea was described. M. oleifera extract showed greater activity in combination with platins. It should be remembered that the FOLFIRINOX regimen contains oxaliplatin among its drugs [In addition, various studies used these plant products together with current antitumor drugs. Indeed, a synergistic effect between gemcitabine and extracts and/or molecules of ts drugs ,47,48,52ts drugs . Furtherts drugs . In addits drugs ,60,61,62ts drugs . Most ofts drugs ,64,65.S. baicalensis being worth noting since not only was a reduction in tumor size demonstrated but also an in vitro absence of effect on HPDE6-C7 nontumor cells [Of the 28 included studies, only eight analyzed in vivo effects of these drugs. The study on or cells . These eor cells . Meanwhior cells . On the or cells ,69. In tor cells .U. dac showed high antitumor activity against cells in nutrient-poor environments and had no effect on cells in more favorable environments and that some antitumor drugs lacked activity in this adverse environment. These results are consistent with other studies in which plant extracts generated high cytotoxicity under conditions of low nutrient content [Finally, it is worth noting the analysis that some researchers carried out on the hypoxic and nutrient-deficient microenvironment of PC, which could reduce the antitumor drug effects. Consequently, the effect of plant extracts and molecules on tumor cells cultured in nutrient-rich and nutrient-poor media was tested. In fact, Ueda et al.  showed h content ,72,73,74 content .In vitro studies have been a first step to determine biomolecules that guarantee an effect on these tumor cells. Although more studies will be necessary to corroborate its activity, the results obtained to date will make it possible to analyze other parts of the plant, to improve extraction methods and to develop more complex tests improving pancreatic cancer therapy. It will be necessary to increase the number of tests in nontumor cells, the in vivo assays and the toxicity studies to take the step of carrying out clinical trials that allow them to be used as adjuvant therapy in the future. The role of organic synthesis would provide improvements in these isolated compounds of plant origin.Plant extracts are presented as an opportunity to improve the treatment of PC and to obtain bioactive molecules that implement novel strategies to address this type of tumor. Various mechanisms of cell death related to apoptosis, KRAS mutation and cell cycle modulation have been described when extract or isolated biomolecules have been used in pancreatic tumor culture cells.


The potential anticarcinogenic ability of the compounds was studied via cytotoxicity assay in three different human tumor cell lines, i.e., epithelial cervix carcinoma (HeLa), promyelocytic leukemia (HL-60), and histiocytic lymphoma (U-937). Data obtained showed that complexes with methyl substitutions did not modify cell viability, while no-methyl substituted compounds had a moderate cytotoxic effect on all three cell lines. The complexes with phenyl substitutions displayed the lowest IC50 values, which ranged between 46.39 ± 3.99 μM and 62.74 ± 6.45 μM. Moreover, Pd accumulation inside the cell was observed after incubation with any of the four complexes mentioned, and the two complexes with phenyl rings were found to induce an increase in the percentage of apoptotic cells. These results suggested that the presence of bulky substitutions on the ligands such as phenyl groups may influence the cytotoxicity of the chemotherapeutic agents synthesized.The synthesis of analogs of cisplatin, which is a widely used chemotherapeutic agent, using other metal centers could be an alternative for cancer treatment. Pd(II) could be a substitute for Pt(II) due to its coordination chemistry similarity. For that reason, six squared-planar Pd(II) complexes with thiazine and thiazoline ligands and formula [PdCl The aim of the study was to check the potential anticarcinogenic ability of these complexes in Hela, HL-60 and U937 cell lines via MTS  method and verify if the presence of bulky ligands influences the biological activity. Moreover, for the complex with better results, the accumulation of Pd(II) ion via ICP-MS and the induction of apoptosis by flow cytometry were checked.Considering the relevance of the ligand structure in the complexes, our group recently published an article with some Pt(II) complexes where the presence of aromatic groups in the ligand augmented the accumulation and cellular uptake of the metallodrugs, therefore increasing the complexes’ cytotoxicity against HeLa , HL-60 and U-937 cell lines . Thus, wThe human histiocytic lymphoma (U-937), human promyelocytic leukemia (HL- 60), and human epithelial cervix adenocarcinoma (HeLa) cell lines were purchased from the European Collection of Authenticated Cell Cultures . All chemical reagents were acquired from commercial sources. The synthesis of the ligands 2-(1-pyrazolyl)-2-thiazoline (PzTn), 2--2-thiazoline (DMPzTn), 2--2-thiazoline (DPhPzTn), 2-(1-pyrazolyl)-1,3-thiazine (PzTz), 2--1,3-thiazine (DMPzTz) and 2--1,3-thiazine (DPhPzTz) was carried out as reported elsewhere ,17,18.−1 range. To obtain 1H NMR spectra, it was used a Bruker 48 instrument at 300 MHz for PdPzTn and PdPzTz, an AV300 instrument at 300 MHz for PdDMPzTn, and a Bruker Avance III 500 instrument at 500 MHz for PdDPhPzTn and PdDPhPzTz, in DMF-d7 or DMSO-d6 according to the complex solubility. 1H NMR data could not be obtained for PdDMPzTz due to its low solubility. 1H NMR signals were referenced to residual proton resonances in deuterated solvents.The elemental analysis was performed with a microanalyzer (Leco CHNS-932). IR spectra were recorded on a Perkin-Elmer 100 FTIR spectrophotometer from KBr pellets in the 4000–400 cm2[PdCl4]·H2O, both previously solved in ethanol , was stirred at room temperature for 24 h and then filtered to provide a powder. The crude product was recrystallized with DMF for PdPzTn and PdPzTz, with DMSO for PdDMPzTn and PdDMPzTz and with acetonitrile for PdDPhPzTn and PdDPhPzTz. Crystals suitable for X-ray diffraction were gather up by filtration and washed with distilled water and cold diethyl ether.A mixture of the corresponding ligand and Na2[PdCl4]·H2O  were used to synthesize PdPzTn. Yield 189.8 mg (88.0%). Elemental analysis (%): cal. for C6H7Cl2PdN3S: C, 21.80; H, 2.14; N, 12.72; S, 9.70%. Found: C, 21.73; H, 2.08; N, 12.53; S, 9.39%. Selected IR data : thiazoline ring vibration 1608 [υ(C=N)]; pyrazole ring vibrations 1532, 1413, 1371, 1001. 1H NMR  δ 8.92 , 8.16 , 6.99 , 4.30 , 4.13 , ppm.PzTn  and Na2[PdCl4]·H2O  were used to synthesize PdPzTz. Yield 175.5 mg (85.2%). Elemental analysis (%): cal. for C7H9Cl2PdN3S: C, 24.39; H, 2.64; N, 12.20; S, 9.30%. Found: C, 24.52; H, 2.65; N, 12.10; S, 9.28%. Selected IR data : thiazine ring vibration 1596 [υ(C=N)]; pyrazole ring vibrations 1524, 1413, 1358, 1003. 1H NMR  δ 8.81 , 8.14 , 6.92 , 3.98 , 3.58 , 2.22  ppm.PzTz  and Na2[PdCl4]·H2O  were used to synthesize PdDMPzTn. Yield 199.4 mg (95.3%). Elemental analysis (%): cal. for C8H11Cl2PdN3S: C, 26.79; H, 3.10; N, 11.73; S, 8.94%. Found: C, 26.83; H, 3.08; N, 11.34; S, 9.24%. Selected IR data : thiazoline ring vibration 1601 [υ(C=N)]; pyrazole ring vibrations 1571, 1413, 1397, 1378, 973. 1H NMR  δ 6.60 , 4.22 , 3.98 , 2.65 , 2.63 , ppm.DMPzTn  and Na2[PdCl4]·H2O  were utilized to synthesize PdDMPzTz. Yield 158.2 mg (77.9%). Elemental analysis. (%): cal. for C9H13Cl2PdN3S: C, 29.03; H, 3.52; N, 11.29; S, 8.60%. Found: C, 28.85; H, 3.57; N, 11.17; S, 8.71%. Selected IR data : thiazine ring vibration 1592 [υ(C=N)]; pyrazole ring vibrations 1564, 1404, 1382, 1341, 991.DMPzTz  and Na2[PdCl4]·H2O  were used to synthesize PdDPhPzTn. Yield: 76.2 mg (48.2%). Elemental analysis (%): cal. for C18H15Cl2PdN3S: C, 44.78; H, 3.14; N, 8.71; S, 6.64%. Found: C, 44.59; H, 3.02; N, 8.76; S, 6.67%. Selected IR data : thiazoline ring vibration 1587 [υ(C=N)]; pyrazole ring vibrations 1556, 1411, 1314, 996. 1H NMR  δ 7.84 , 7.66 , 7.48 , 7.12 , 4.13 , 3.78  ppm.DPhPzTn  and Na2[PdCl4]·H2O  were used to synthesize PdDPhPzTz. Yield: 123.9 mg (79.7%). Elemental analysis (%): cal. for C19H17Cl2PdN3S: C, 45.93; H, 3.46; N, 8.47; S, 6.45%. Found: C, 45.90; H, 3.41; N, 8.92; S, 6.24%. Selected IR data : thiazine ring vibration 1606 [υ(C=N)]; pyrazole ring vibrations 1556, 1404, 1313, 1000. 1H NMR  δ 7.86 , 7.63 , 7.47 , 7.07 , 4.02 , 3.28 , 2.09  ppm.DPhPzTz  and NaCrystallographic data and experimental information of the six complexes are summarized in www.ccdc.cam.ac.uk/data_request/cif (accessed on 3 January 2023).X-ray data were collected in a Bruker D8 VENTURE PHOTON III-14 diffractometer using graphite monochromated Mo Kα radiation (λ = 0.71073 Å) and the ω scan technique at 100 K. The collected frames were processed with the SAINT  and corrv/v).Human cancer cell lines  were challenged with the free ligands DMPzTn and DMPzTz  and the3, 1.5 × 104, 2.5 × 104 cells/well, respectively), and challenged with different concentrations of the compounds for 24, 48, or 72 h. After that time, 10 µL of the CellTiter 96 AQueous One Solution Reagent were added directly to culture wells and then incubated at 37 °C at different times according to the cell type . In the end, a microplate reader  was used to record the absorbance at CellTiter 96 AQueous One Solution Cell Proliferation Assay  was used to estimate the cytotoxicity of the complexes synthesized on the tumor cell lines selected. This assay is based on the reduction of an MTS tetrazolium compound. To perform the experiment, HeLa, U-937, and HL-60 cells were seeded in 96-well plates , was employed to measure the induction of apoptosis, as reported elsewhere .6 cells were seeded in 100 mm Petri dishes and treated with 67.55 µM of PdPzTn, 77.75 µM of PdPzTz, 62.74 µM of PdDPhPzTn or 57.83 µM of PdDPhPzTz for 4 h. After trypsinization and washing with PBS, cells were centrifuged at 500× g for 5 min and then lysed with RIPA buffer ·H2O in an ethanol solution under agitation for 24 h. Complexes’ characterization was made using single-crystal X-ray diffraction analysis, elemental analysis, IR spectroscopy, and 1H NMR spectroscopy.The Pd(II) complexes synthesis was carried out as described in 2L]  or [PdCl2L]·C2H3N . Crystal structures of all complexes can be observed in Crystal structures of Pd(II) complexes consist of monomeric units , like what happens for Pd-Npyrazole bond distances with respect to the mean value calculated [2.031(19) Å for 109 Pd(II) complexes] in CSD  vibration in thiazoline and Ψ1[υ(C=N)] vibration in thiazine suffers a shift to lower wavenumber in the complexes with respect to their respective ligands. It has been described that the reason for this displacement is related to the presence of the C-N bond as part of a chelate ring where the back-coordination from the metal to the ligand compensates for the loss of electron density on the nitrogen atom. In addition, the bands assigned to pyrazole ring vibrations are shifted to a higher wavenumber for PdPzTn and PdPzTz, not showing so well define patron for the other four complexes, which can be explained by the presence of the substituents. So, the palladium coordination to both heterocycles through the nitrogen atoms was confirmed for these frequency shifts in the infrared spectra [On the other hand, the IR spectra of complexes PdPzTn, PdPzTz, PdDMPzTn, PdDMPzTz, PdDPhPzTn, and PdDPhPzTz  showed t spectra ,18.50 values > 100 µM)  complexes were tested at concentrations from 1 to 100 µM on the three tumor cell lines selected during 24 h . Data ob 100 µM) . Moreovell lines . As repoong time . The dec50 values achieved with other palladium(II) compounds, thereby decreasing the concentration of some of them at the same incubation times, and even having lower IC50 at shorter incubation times (24 h) [50 values in all three cell lines used in the present work [50 in the HL-60 cell line at 50 µM.With respect to other palladium(II) complexes reported, some of them have better biological results than ours at the same incubation times ,30,32. Is (24 h) ,31,32. Ms (24 h) . In content work . ParticuOn the other hand, it was checked that none of the free ligands generated relevant cytotoxicity on any of the tumor cell lines studied, except for DPhPzTn and DPhPzTz in HeLa cells at high doses  complexes with the same ligands in previous studies [ICP-MS was used to verify Pd(II) accumulation into HeLa cells. To do that, the tumor cells were incubated the ICactivity . As it c studies .50 of the complexes with phenyl rings during 24 h produced significant changes in the percentage of cell populations, particularly in the case of live (decrease) and late apoptotic (increase) cells for both complexes, in comparison to the vehicle-treated  were observed for PdDPhPzTz and PdDPhPzTn. Thus, the presence of aromatic rings in the ligand may influence the accumulation and biological activity of these complexes, improving their effectivity in solid and non-solid tumors.In this study, we have synthesized and characterized six new Pd(II) complexes with ligands containing pyrazole and thiazine/thiazoline heterocycles. The cytotoxic effect produced by these complexes was checked in three different tumor cell lines . Our results showed no modification of cell viability for complexes with methyl substituents on any of the three lines and not even at longer incubation times. On the other hand, moderate cytotoxicity was found for complexes with no extra substituents or with phenyl rings, in both cases improving the biological effects of their respective ligands. Interestingly, longer incubation times strongly improved the cytotoxic effects of these complexes, thereby reducing their IC