Pancreatic ductal adenocarcinoma (PDAC) correlates with high mortality and is about to become one of the major reasons for cancer-related mortality in the next decades. One reason for that high mortality is the limited availability of effective chemotherapy as well as the intrinsic or acquired resistance against it. Here, we report the impact of nab-paclitaxel on the cellular metabolome of PDAC cell lines. After establishment of nab-paclitaxel resistant cell lines, comparison of parental and resistant PDAC cell lines by metabolomics and biochemical assessments revealed altered metabolism, enhanced viability and reduced apoptosis. The results unveiled that acute nab-paclitaxel treatment affected primary metabolism to a minor extent. However, acquisition of resistance led to altered metabolites in both cell lines tested. Specifically, aspartic acid and carbamoyl-aspartic acid were differentially abundant, which might indicate an increased de novo pyrimidine synthesis. This pathway has already shown a similar behavior in other cancerous entities and thus might serve in the future as vulnerable target fighting resistance acquisition occurring in common malignancies. Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive and fast growing type of cancer , which iThere are different therapies for PDAC treatment available, but effects are limited . Three oUnlike inhibition of DNA synthesis and DNA damage induction by 5-FU  and gemcWe investigated metabolic alterations in PDAC cell lines upon exposure to the chemotherapeutic compound nab-paclitaxel. Moreover, we analyzed the metabolic adaptations of nab-paclitaxel-resistant PDAC cell lines, as shown for gemcitabine resistance in previous publications ,38. Our 2 in a humidified atmosphere. Characteristics of parental cell lines are summarized in Lagies et al. [MiaPaCa-2 (CRL-1420™) and Panc-1 (CRL-1469™) were purchased from the American Type Culture Collection . Resistant cell lines were established as described below. All cell lines were cultured in DMEM high-glucose medium  supplemented with 10% fetal bovine serum (Gibco). Culture medium for Panc-1/NPR cells was supplemented with 10 nM nab-paclitaxel  and culture medium for MiaPaCa-2/NPR cells was supplemented with 5 nM nab-paclitaxel. All cell lines were grown at 37 °C and 5% COs et al. .Three thousand cells/well were seeded in a total volume of 100 µL culture medium in 96-well plates and incubated o/n at the respective culture conditions. The next day, cells were treated as indicated and incubated for 72 h before addition of 10 µL resazurin  for 4 h. Fluorescence was read at 544 nm excitation and 590 nm emission. Cell viability was calculated as relative viability with reference to untreated control (100%). The IC50 values were calculated using GraphPad Prism software V5 .2 tissue culture flasks for further culturing, split when approximately 75% confluent and directly seeded in medium supplemented with chemotherapy. All resistant cell lines were cultured in culture medium with chemotherapy for at least 100 days before IC50 determination.Chemotherapy resistant cell lines were established from MiaPaCa-2 and Panc-1 cells. 100,000 cells were seeded in 1 mL in 12-well plates and incubated o/n before treatment. Cells were shock-treated with high-dose chemotherapy (100 × IC50-concentration) for 24 h. The next day, cells were treated with 0.5 × IC50 chemotherapy concentrations. Culture medium and treatment were replaced twice a week and gradually increased when cells were growing well. The cells were transferred into 75-cm5 cells were seeded in 6-well plates in 2 mL culture medium o/n and treated with chemotherapy for 72 h. Cells were harvested, washed with 1 × PBS and incubated with 50 µL annexin-V Ax647  ) on ice for 15 min. 450 µL of 20 µg/mL propidium iodide  was added to each sample. The samples were acquired using a BD LSR Fortessa  and analyzed using the FlowJo™V10 software.For the cell death assay, 2.5 × 106 cells were seeded in 10-cm culture dishes in 8 mL culture medium and treated with chemotherapy o/n for 72 h before metabolomics analysis. Metabolic profiling was conducted as reported previously [2O 9:1 (v/v)) containing internal standards; then, cells were scraped off and snap-frozen in liquid nitrogen. After mechanical lysis, metabolite pellets were derivatized by silylation and methoxyamination. Samples were analyzed in randomized order by GC-EI-MS (Agilent 7890A/5975C). Conserved mass spectral features [For the untargeted GC/MS-based metabolic profiling, 1 × 10eviously . In shorfeatures  were matfeatures . For prit-test. Data are displayed as mean ± standard error of the mean. A p-value < 0.05 was considered as statistically significant. Significances were shown with symbols . No repeated measurements from the same sample were performed with the exception of QC samples in GC/MS analyses.For other than the untargeted metabolomics profiling data, statistics were performed using the GraphPad prism software V5. Normally distributed data were compared using an unpaired Results of GC/MS analyses are provided in Supplementary Data files SD1 and SD2. To investigate the metabolic effects of chemotherapy treatment in pancreatic cancer cells lines, the IC50 concentrations of nab-paclitaxel were determined in the PDAC cell lines MiaPaCa-2 and Panc-1 (4.1 pM and 7.3 pM). The cells were treated with increasing concentrations of chemotherapy  and cell viability was measured 72 h after treatment. The viability of both cell lines significantly decreased in a dose-dependent manner compared to the control treatment A. The coFollowing, chemotherapy treated cells were subjected to untargeted GC/MS-based metabolic profiling. Applying two-dimensional principal component analysis (PCA), revealed global changes between the cell lines B. DespitNab-paclitaxel treatment did only have slight effects on cellular metabolism. Intracellular levels of polyol-pathway intermediates revealed its inactivation upon nab-paclitaxel application. Additionally, an increase of taurine upon chemotherapy application confirms this hypothesis . Within Application of the anti-metabolite gemcitabine to MiaPaCa2 and Panc-1 cells resulted in substantial metabolic changes, as shown by starker differentiation in the PCA  and diffThe nab-paclitaxel-resistant cell lines MiaPaCa-2/NPR and Panc-1/NPR were selected for at least 100 days, as described in the methods section. The resistant cells and their corresponding parental cell lines were treated with increasing doses of chemotherapy for 72 h before viability analysis. In all cell lines, the viability decreased in a dose-dependent manner, whereas the viability of the resistant cell lines was always higher than the viability of the parental cell line A,B. The + and Annexin V+/PI+ cells were summarized as dead cells and are given in percent of all analyzed cells after removal of doublets. Ten nanomolar (10 nM) nab-paclitaxel was used as the viability assay measures the redox state and disturbances in this may be insufficient to induce apoptosis. In MiaPaCa-2 cells treated with nab-paclitaxel, 90% of all cells underwent apoptosis, with only 20% dead cells in the MiaPaCa-2/NPR cell line . Annexin Vell line E. Treatmell line F. Taken The cell lines MiaPaCa-2/NPR and Panc-1/NPR, as well as MiaPaCa-2 and Panc-1, were treated with control or nab-paclitaxel for 72 h. Afterwards, primary metabolites were assessed as mentioned previously. PCA revealed again that cell line differences were more prominent than the impact of nab-paclitaxel . The heaElevation of lactate as well as the decrease of citrate and malate in the MiaPaCa-2/NPR cell line compared to its parental cell line suggests a switch to aerobic glycolysis. Putrescine and spermidine levels showed that Panc-1 cells had an activated polyamine pathway, however, the levels are even higher in the nab-paclitaxel resistant cell line Panc-1/NPR . IncreasMost importantly, the acquisition of resistance against nab-paclitaxel had a higher impact on the metabolome than acute nab-paclitaxel treatment did. Despite the cell line-specific alteration to the prolonged application during the generation of resistant cell lines, three metabolites were likewise regulated in resistant cells, regardless of the acute treatment. While aspartic acid was reduced in the resistant cell lines, carbamoyl-aspartate was increased . The oppIn summary, there were only small metabolic differences between Panc-1/NPR treated and untreated cells, as well as between MiaPaCa-2/NPR treated and untreated cells supporting that acute nab-paclitaxel does not have a high impact on cellular metabolism. In contrast, resistance against nab-paclitaxel was accompanied by changes in some metabolic pathways. These could be cell line-specific as well as cell line independent.Metabolic alterations in cancer cells are either necessary to cover the energetic demand of the cells or they contribute to the aggressiveness of cancer cells ,56. GlucIn general, nab-paclitaxel led to only a few metabolic alterations. This could be explained by the mode of action. Nab-paclitaxel arrests cancer cells in mitosis, whereas other chemotherapeutic agents such as 5-FU and gemcitabine interfere directly with DNA synthesis by acting as an anti-metabolite ,59. HoweAnother aspect that has to be mentioned is the possible existence of sub-populations in cancer cell lines, which could have been selected during the prolonged chemotherapy application . In summary, we investigated the metabolic fingerprint of different pancreatic cancer cell lines and showed how metabolism changed after acquisition of chemotherapy resistance. We are the first group evaluating the metabolic adaptions and changes in Panc-1 and MiaPaCa-2 cells being resistant to nab-Paclitaxel using untargeted GC/MS-Analysis. Since it is not clear whether the metabolic alterations are causative, supportive or an epiphenomenon, the putative vulnerabilities of the affected pathways have to be functionally validated in further studies. Such studies might include the establishment of nab-paclitaxel resistant pancreatic cancer cell lines with and without simultaneous inhibition of aspartate transcarbamoylase. Such inhibition can be achieved by the anti-metabolite N-phosphonacetyl-L-aspartic acid . Time-reThese are important questions to address in further validation studies. However, our results might already aid in better understanding the mechanisms of chemotherapy resistance in pancreatic cancer cells.


Rationale: Tumors have significant abnormalities in various biological properties. In renal cell carcinoma (RCC), metabolic abnormalities are characteristic biological dysfunction that cannot be ignored. Despite this, many aspects of this dysfunction have not been fully explained. The purpose of this study was to reveal a new mechanism of metabolic and energy-related biological abnormalities in RCC.Methods: Molecular screening and bioinformatics analysis were performed in RCC based on data from The Cancer Genome Atlas (TCGA) database. Regulated pathways were investigated by qRT-PCR, immunoblot analysis and immunohistochemistry. A series of functional analyses was performed in cell lines and xenograft models.Results: By screening the biological abnormality core dataset-mitochondria-related dataset and the metabolic abnormality core dataset-energy metabolism-related dataset in public RCC databases, PCK2 was found to be differentially expressed in RCC compared with normal tissue. Further analysis by the TCGA database showed that PCK2 was significantly downregulated in RCC and predicted a poor prognosis. Through additional studies, it was found that a low expression of PCK2 in RCC was caused by methylation of its promoter region. Restoration of PCK2 expression in RCC cells repressed tumor progression and increased their sensitivity to sunitinib. Finally, mechanistic investigations indicated that PCK2 mediated the above processes by promoting endoplasmic reticulum stress.Conclusions: Collectively, our results identify a specific mechanism by which PCK2 suppresses the progression of renal cell carcinoma (RCC) and increases sensitivity to sunitinib by promoting endoplasmic reticulum stress. This finding provides a new biomarker for RCC as well as novel targets and strategies for the treatment of RCC. Cancer is a disease that is always accompanied by a variety of characteristic biological dysfunctions PCK2 (phosphoenolpyruvate carboxykinase 2) is a mitochondrial isoform of the key gluconeogenesis enzyme phosphoenolpyruvate carboxykinase (PEPCK) The endoplasmic reticulum (ER) is widely involved in a variety of biological functions, including maintaining calcium homeostasis, mediating protein synthesis and transport, and regulating lipid and glucose metabolism. It constitutes a huge network connected to a variety of organelles in terms of function and physical structure With the development of RCC treatments, anti-angiogenesis therapy has become the preferred drug treatment for RCC and of these treatments, sunitinib is the most commonly used clinical first-line drug In this study, PCK2 was found to be a novel biomarker that can predict the prognosis of RCC. It was also found that low expression of PCK2 in RCC was caused by methylation of its promoter region. At the same time, PCK2 was shown to inhibit RCC progression and alleviate sunitinib resistance. Mechanistic studies showed that this process is mediated by PCK2-activated ER stress. These findings provide a new biomarker for RCC as well as novel targets and strategies for the treatment of RCC.The normal cell line HK2 and the human kidney cancer cell lines 786-0, A498, ACHN, CAKI, and OSRC were obtained from The American Type Culture Collection . Sunitinib-resistant kidney cancer cell lines were provided by the Department of Urology, Wuhan Union Hospital. Cells were cultured in an incubator at 37 ºC and 5% CO2 in DMEM high-glucose medium supplemented with 10% FBS and 1% penicillin-streptomycin.The expression lentivirus for PCK2 and the corresponding control vector were purchased from Genechem, China. 5-AZA was obtained from MCE. ER-Tracker Red was obtained from Beyotime Biotechnology, China. Tauroursodeoxycholic Acid (TUDCA) was purchased from Selleckchem, S3654. The CRISPR/dCas9-mediated editing system for PCK2 specific demethylation was purchased from Genechem.Thirty-two pairs of human RCC tissues were obtained from the Department of Urology, Union Hospital, Tongji Medical College  from 2017-2018. Adjacent normal tissues, which were located at least 5 cm away from the tumor site, were obtained and used as normal control tissues corresponding to the RCC tissues. And liquid nitrogen was used to store the tissues at -80 °C for RNA and protein extraction. All patients whose tissues were used were fully informed as to the content of the experiments. The Institutional Review Board of Huazhong University of Science and Technology approved the implementation of this study.For immunohistochemical staining, tissue samples embedded in formalin-fixed paraffin were first made into 4-μm sections. Subsequently, paraffin was removed using EDTA, rehydrated, and sections were incubated at 120 °C for 5 minutes for antigen retrieval. After completion, incubate for 15 minutes at room temperature with 3% H2O2. Sections were then blocked with fetal bovine serum. Next, the tissue sections were incubated overnight at 4 °C in primary antibody solution after which they were incubated with an appropriate HRP-conjugated secondary antibody for 1 h at room temperature. Finally, the immune complexes were visualized by DAB and sections were counterstained in hematoxylin.To visualize the endoplasmic reticulum, ER-Tracker Red was used according to the manufacturer's instructions. Staining was captured using confocal microscopy, after which the acquired images were processed and quantified using ImageJ software.According to the manufacturer's instructions, TRizol reagent  was used to extract total RNA of tissues and cells. The purity and concentration of the RNA solution was measured using a NanoDrop 2000 spectrophotometer . In all, 1 μg of RNA was needed for reverse transcription. qPCR analysis was performed with a SYBR Green master mix  in StepOnePlus™ Real-Time PCR System . GAPDH was used to normalize the samples. The gene primers used are listed in table Genomic DNA from RCC tissues was prepared following the manufacturer's instructions. The following MSP primers were designed according to the genomic sequences flanking the transcription start sites: PCK2-M-sense, 5'- GTTTGGAGTTTCGGGGTCGA -3'; PCK2-M-antisense, 5'- CGAAACGAACCACAACAAAAAC -3'; PCK2-U-sense, 5'- GGAGTTTGGAGTTTTGGGGTTG -3' and PCK2-U-antisense, 5'- AACAAAACAAACCACAACAAAAACT -3'. The primer sequences were oligosynthesized  for MSP to detect bisulfate-induced changes affecting unmethylated (U) and methylated (M) alleles.Genomic DNA from RCC tissues and cell lines were prepared following the manufacturer's instructions. The following BSP primers were designed according to genomic sequences flanking the transcription start sites: PCK2-BSP-F, 5'-TAGTATTTTGGGAGGTTAAGGTAGGC -3'; PCK2-BSP-R, 5'-CTCCTCCTAATCAAAACTCCTTTCTAC -3'. Subsequently, carry out Bisulfite treatment of DNA, PCR amplification, Electrophoresis, Purify DNA from agarose gel, Transformation experiment using DH5α, PCR amplification identification, and DNA sequencing. Specific technical support is provided by Servicebio, China.For the Western blot analysis, protein extraction was performed using RIPA protein lysis buffer  containing a protease inhibitor cocktail and PMSF. The extracted protein was subjected to SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was then blocked with 5% skim milk for 1 hour at room temperature. Subsequently, the primary antibody was used for incubation overnight. Prior to detection, membranes were washed with PBST and incubated with secondary antibodies in blocking buffer for 2 hours. The antibodies used for western blots were as follows: PCK2 , IREa , P-IREa , PERK , P-PERK , ATF6 , and GAPDH .3 cells were first seeded in a 96-well plate, and then a CCK8 assay  was used to test the cell proliferation capacity. Cell viability was measured at 0, 24, 48, 72, and 96 hours.According to the manufacturer's instructions, 2 × 10In order to perform colony formation assays, 1,000 cells were first seeded into a 6-well plate, and cell colony formation was evaluated after 14 days of culture. The cells were then fixed in methanol during the assay, and then viable colonies (> 50 cells/colonies) were observed by staining with 0.05% crystal violet.When the cells reached 90% confluence in a 6-well plate in serum-free medium, a 10-µl pipette tip was used to generate a wound and was then washed with PBS to remove cell debris. Images were obtained at 0 and 24 hours after the injury. The images were processed by Photoshop 7.0 software.A Transwell assay was used to assess the ability of cells to migrate and invade. The cells to be tested were first cultured in serum-free medium for 24 hours, after which they were seeded in a Transwell chamber. An additional 1: 8 ratio of Matrigel was added to evaluate the invasive capacity of the cells. After 24 hours, the cells were fixed in methanol and stained with 0.05% crystal violet. The resulting images were captured by a microscope, which randomly selected multiple fields for cell counting.6 cells were injected subcutaneously into the axilla of these mice to construct a xenograft tumor model. A metastasis model was constructed by injecting tumor cells into the tail vein to assess their metastatic ability. Tumor size was measured every 4 days. Immunohistochemical staining was performed according to a standard procedure as previously described. All animal experiments were performed in accordance with animal protocols approved by the Institutional Animal Use and Care Committee of Tongji Medical College, Huazhong University of Science and Technology.The 6-week-old male nude mice used in this study were purchased from Vital River Laboratory Animal Technology Co. Ltd. In all, 2 × 10https://www.oncomine.org) and the European Bioinformatics Institute (EMBL-EBI) (https://www.ebi.ac.uk). (Gene set (1) is derived from the mitochondrion (goterm) project in the "Gene array analysis of clear cell renal cell carcinoma tissue versus matched normal kidney tissue" data subset of the EMBL-EBI database; Gene set (2) is derived from the mitochondrion (goterm) project in the “Transcription profiling of clear cell renal carcinomas and normal kidney cortical tissues” data subset of the EMBL-EBI database; Gene set (3) is derived from the mitochondrion Go Cellular Component (GO) project in the “differentially expressed genes in renal cell carcinoma in Lenburg Renal” data subset of the Ocomine database; Gene set (4) is derived from the mitochondrion Go Cellular Component (GO) project in the “differentially expressed genes in renal cell carcinoma in Beroukhim Renal” data subset of the Ocomine database; Gene set (5) is derived from the Glucose metabolism (pathwayname) project in the “Gene array analysis of clear cell renal cell carcinoma tissue versus matched normal kidney tissue” data subset of the EMBL-EBI database; Gene set (6) is derived from the Glucose metabolism (pathwayname) project in the “Transcription profiling of clear cell renal carcinomas and normal kidney cortical tissues” data subset of the EMBL-EBI database). A clinical prognosis analysis of related molecules derived from data in the TCGA database . CpG island predictive analysis was performed by http://www.urogene.org/. Gene function enrichment analysis was performed by gene set enrichment analysis (GSEA). STRING (https://string-db.org/) was used to create the interaction network of genes.Data for genetic screening were obtained from the Oncomine database  and SPSS Statistics 22.0 . All Figure ). To further assess the expression of these two genes in RCC, we analyzed data from the TCGA-KIRC database which contains 533 ccRCC cases and 72 paired normal cases. As shown in Figure , these two genes were both significantly downregulated in RCC and PCK2 has a relatively more pronounced low expression trend. Further bioinformatics analysis showed that PCK2 expression had a significant predictive value for the survival time of RCC patients, as lower PCK2 expression predicted a shorter overall survival time. However, no significant difference was observed in the survival times between those with high expression of SLC25A10 and those with low expression of SLC25A10 . Subsequently, a ROC curve was used to assess the ability of these two genes to identify clinical RCC patients. The results showed that both genes could identify clinical RCC cases, and PCK2 has a higher AUC (area under the curve), which indicated that PCK2 had a relatively better diagnostic value for RCC . Based on the above results, PCK2 was selected as a target molecule for further study. To further validate the value of PCK2 in RCC, we performed a more in-depth bioinformatics analysis. The low expression of PCK2 in RCC was confirmed again in four independent data sets from the Oncomine database . The detailed analysis of the TCGA database also found that PCK2 not only predicted the survival time of RCC patients overall, but that it could also predict the survival time of patients in different clinical subgroups . In addition, univariate and multivariate Cox regression analyses based on the TCGA database also confirmed that PCK2 is an independent prognostic marker for RCC (Table 1).Tumors are accompanied by dysfunctions in a variety of biological processes. Previous studies have confirmed that mitochondria are the central organelles that mediate the functions of cell biology, and the disorders of mitochondrial function lead to abnormalities in various cellular biological functions Figure  the protein and mRNA levels of PCK2 were significantly lower in RCC tissues compared with normal tissues. Similar results were obtained in cell lines, in which the protein and mRNA levels of PCK2 were also greatly downregulated in the RCC cell lines compared with the control cell line .To verify the results of the bioinformatics analysis, we examined the expression of PCK2 at both the mRNA and protein levels in RCC tissues and cell lines. As shown in Figure ). In order to clarify the changes of PCK2 methylation level in RCC, we performed Bisulfite Sequencing PCR (BSP) on 5 pairs of renal cell carcinoma tissues and adjacent tissues. After quantitatively detecting the methylation level, it was found that the methylation level in RCC of the PCK2 promoter region was significantly higher than its corresponding para-cancerous tissues . Similarly, MSP (Methylation-Specific Polymerase Chain Reaction) experiments also confirmed that PCK2 was highly methylated in RCC .Based on the above results, we found a significant epigenetic difference in PCK2 between RCC and normal tissues, and this difference is responsible for the significant downregulation of PCK2 in RCC. Epigenetics, which has become an important field of biomedical research, can reveal new underlying mechanisms of biological processes ). Subsequently, the demethylating drug 5-AZA was used to treat RCC cell lines to demonstrate the effect of methylation on the expression of PCK2. The results showed that 5-AZA could significantly increase the expression of PCK2 in RCC cell lines at both mRNA and protein levels . In order to illustrate the above results more specifically, a CRISPR/dCas9-mediated editing system for PCK2 specific demethylation was constructed . Similar results could be found, specific demethylation also significantly increased the expression levels of PCK2 in both mRNA and protein levels . Based on the above results, conclusion could be drawn that the epigenetic changes in PCK2 in RCC, by which PCK2 was significantly downregulated were due to DNA methylation.In order to further explore the effect of methylation in the PCK2 promoter region on its expression in RCC, experiments on cell lines were performed. Firstly, Bisulfite Sequencing PCR (BSP) was also carried out to detect the methylation level of PCK2 in 5 different kinds of RCC cell lines. The quantitative results showed that their methylation percentages were all over 90%, which meant that they were all in a highly methylated state . As shown in Figure , CCK8 proliferation curves were used to assess the proliferative capacity of the cells, and the results clearly showed that PCK2 overexpression significantly suppressed the proliferation of RCC cell lines. In addition, the migration and invasion abilities of tumors are also important characteristics to measure the degree of tumor malignancy and affect tumor progression. To measure the abilities of tumor migration and invasion, Transwell assays were carried out. Similarly, the results showed that PCK2 overexpression could significantly inhibit the migration and invasion of RCC cell lines in both CAKI and ACHN . To more thoroughly verify the above experimental results, we subsequently performed a colony formation assay and wound healing assay. As expected, similar results were shown in Figure  in which PCK2 overexpression significantly inhibited the colony formation ability and reduced the migration capacity of RCC cells.Based on the finding that PCK2 is greatly downregulated in RCC, we speculate that PCK2 is likely to have a certain degree of influence on the occurrence and progression of RCC. To confirm the above hypothesis, we successfully constructed CAKI and ACHN cell lines with PCK2 stably overexpressed by lentivirus . CCK8 assay showed that PCK2 expression increased by specific demethylation could still significantly inhibit RCC cell proliferation . Similarly, the ability of migration and invasion could be also inhibited by the specific demethylation of PCK2 . Taken together, the above experimental results allow us to conclude that PCK2 acts as a tumor suppressor to repress RCC progression.Subsequently, in order to further verify whether the changes in PCK2 expression caused by methylation have the same biological effects, CRISPR/dCas9-mediated editing system specifically demethylated RCC cell lines and sunitinib resistance RCC cell lines were constructed . Subsequently, a correlation analysis based on the TCGA database also showed that PCK2 was highly negatively correlated with multiple angiogenic factors and angiopoietins . These results imply that PCK2 is highly likely to be associated with RCC anti-angiogenic therapy. To test the above hypothesis, we first assessed the expression of PCK2 in ACHN and 786-0 sunitinib-resistant cell lines. As shown in Figure , the expression of PCK2 was significantly decreased in sunitinib-resistant RCC cell lines in both protein and mRNA levels. Then, we performed a dosing experiment on common RCC cell lines and found that PCK2 expression gradually increased as the sunitinib concentration increased as a short-term effect of sunitinib . Based on the response of PCK2 expression to sunitinib and its expression differences in drug-resistant RCC cell lines, we speculated that PCK2 likely exerted a synergistic effect with sunitinib in RCC. To directly show the effect of PCK2 on the drug sensitivity of RCC cell lines, we performed CCK8 assay to construct drug sensitivity curves for RCC cell lines against sunitinib. First, we constructed drug sensitivity curves of ACHN and 786-0 drug-resistant cell lines to verify the efficiency of drug resistance. The results showed that the IC50 of sunitinib in drug-resistant cells was greatly higher than control cells and exceeded 2-fold, which meant that the construction of drug-resistant cell lines was successful . Subsequently, we examined the effects of PCK2 on sunitinib-resistant RCC cell lines and normal RCC cell lines on the sensitivity of sunitinib, respectively. As shown in Figure , the drug sensitivity curve obtained in the CCK8 assay revealed a faster rate of decline in the PCK2 overexpression group, regardless if it is in resistant RCC cells or normal RCC cells.The above experimental results confirmed that PCK2 can largely inhibit the progression of RCC, and these characteristics are mainly focused on the inhibition of the basic characteristics of tumors. In the actual process of tumor progression, clinical treatment is an important factor that cannot be ignored, among which drug therapy is especially important. For RCC, angiogenesis has become an important process affects the progression of RCC due to the extensive loss of VHL, which leads to the activation of its downstream angiogenic target genes Figure ). From these data, we can conclude that PCK2 exerts synergistic toxicity with sunitinib in RCC and can increase the sensitivity of RCC cells to sunitinib.In order to verify whether the specific demethylation of PCK2 had the same effect, drug sensitivity experiments were carried out in specific demethylated RCC cell lines and sunitinib-resistant cell lines. The results showed that increasing the expression of PCK2 through specific demethylation could also increase the sensitivity of cells to sunitinib . It is well known that the ordering of the three basic basal metabolisms maintains the normal physiological activities of cells. Once this balance is destroyed, homeostasis in the intracellular environment will become disordered Figure ). Similarly, data analysis based on TCGA also indicated that PCK2 was positively correlated with unfolded protein response related proteins . Subsequently, we examined the expression of endoplasmic reticulum stress sensors in RCC cell lines, and the results showed that these sensors of endoplasmic reticulum stress were greatly increased in RCC cell lines with stably overexpressed PCK2 . Similarly, as shown in Figure -G, endoplasmic reticulum stress sensors were also greatly increased in the sunitinib-resistant RCC cell lines with stably overexpressed PCK2. Furthermore, ER Tracker imaging was conducted to visualize structural changes in the endoplasmic reticulum, and the results showed that ER tracker staining was significantly enhanced in RCC cell lines with stably overexpressed PCK2 .Based on the finding that PCK2 has a significant impact on biological functions of RCC, its specific mechanism and biological effects are worthy of further discussion and research. The GSEA gene enrichment analysis, which was based on the TCGA database, showed that PCK2 is highly correlated with the three basal metabolisms in RCC, including glucose metabolism, lipid metabolism, amino acid metabolism and glutathione metabolism . The above results indicate that PCK2 can promote endoplasmic reticulum stress in RCC.To confirm whether PCK2 specific demethylation had the same biological effect, we tested the level of endoplasmic reticulum stress sensors in RCC cell lines with CRISPR/dCas9-mediated specific demethylation. The results showed that the endoplasmic reticulum stress sensors were also significantly improved in these cell lines, which meant that the endoplasmic reticulum stress was also activated . As shown in Figure  inhibition of endoplasmic reticulum stress could reverse the decline in cell proliferative capacity caused by overexpression of PCK2 in RCC. As for the migration and invasion ability of tumor cells, similar findings could be seen in Figure , in which the inhibition of endoplasmic reticulum stress significantly attenuated the weakened migration and invasion capacities of tumor cells induced by PCK2.To further illustrate the role of endoplasmic reticulum stress in the PCK2-mediated biological functions in RCC, functional rescue experiments were conducted. We constructed cell lines in which endoplasmic reticulum stress was suppressed by using the endoplasmic reticulum stress-specific inhibitor TUDCA in cell lines with PCK2 overexpression and in the corresponding control cell lines . In summary, conclusion could be drawn that endoplasmic reticulum stress played an important role in the inhibition of tumor progression caused by PCK2.In order to further verify whether PCK2 specific demethylated cell lines had the same biological effects, functional rescue experiments were carried out in these cell lines. The results showed that inhibition of endoplasmic reticulum stress could also alleviate the inhibition of RCC proliferation, migration and invasion caused by PCK2 specific demethylation  and western blot to assess the level of endoplasmic reticulum stress in vivo and to evaluate the malignancy of the subcutaneous tumors formed. The results showed that the levels of endoplasmic reticulum stress markers were greatly increased in the PCK2 overexpression group, while the marker of tumor malignancy, KI67, was decreased in the PCK2 overexpression group . Subsequently, a tail vein metastasis model in nude mice was used to assess tumor metastatic capacity in vivo. As shown in Figure , a similar conclusion could be drawn that overexpression of PCK2 reduced the ability of tumors to metastasize in vivo. To clarify the impact of PCK2 specific demethylation in vivo, models of subcutaneous xenografts and tail vein metastases were also constructed by cell line with PCK2 specific demethylation. The results showed that PCK2 specific demethylation could also significantly inhibit the progression of RCC in vivo .Based on the results of the above cell experiments, we further evaluated the role of PCK2 in RCC Figure ).Based on the above findings, we proposed a model in which methylation of the PCK2 promoter region in RCC inhibits the expression of PCK2, which reduced endoplasmic reticulum stress resulting in the tumor progression. Restoration of PCK2 expression could activate endoplasmic reticulum stress in RCC, thereby inhibiting tumor progression and increasing tumor sensitivity to sunitinib  is associated with significant biological function abnormalities. Although research on RCC is gradually deepening, many enigmas and doubts about the biological function abnormalities in RCC still exist. By screening the public database, we found a protein named PCK2 that is differentially expressed in RCC and highly related to the biological function of RCC. Through in-depth research of PCK2, we found that PCK2 could inhibit RCC progression and increase the sensitivity of RCC to the targeted drug sunitinib. These findings can provide new targets and novel drug combinations for RCC treatment.The endoplasmic reticulum is an important organelle which functions primarily in protein metabolism, while maintaining a comprehensive balance of glucose, lipids and intracellular ions in the cell The process of genetic information from DNA to protein is called gene expression. Due to differences in tissues, cell functions and environmental stimuli, differences in gene expression will occur under the same large genetic background, which is called the regulation of gene expression. Generally, the regulation of gene expression includes four aspects, namely, pre-transcriptional modification, transcriptional modification, post-transcriptional modification and modification of translation level. Normally, for eukaryotes, pre-translational modification has become the main way of regulating gene expression due to the particularity of their DNA structure Methylation has a certain foundation in the research of RCC. Previous studies have reported that methylation of some genes can affect the occurrence and development of RCC. The promoter of OVOLl is methylated in 40% of RCC, which can increase the expression of c-Myc, activate a variety of oncogenes, and promote the occurrence of RCC Sunitinib is a first-line targeted drug for RCC treatment, and its use has greatly improved the prognosis of patients with RCC In addition, our study also found significant epigenetic differences in PCK2 in RCC which are manifested as significantly low expression due to high methylation of its promoter region. Combined with the finding that PCK2 inhibits the progression of RCC and increases the sensitivity to sunitinib, we can use specific drugs to reduce the methylation level of the PCK2 promoter region, thereby restoring epigenetically silenced PCK2 expression to inhibit RCC progression. This also provides a potential new direction for RCC drug development.Here, we report a marker gene that is greatly differentially expressed between RCC and normal tissues and that functionally inhibits the progression of RCC and increases the sensitivity of RCC to sunitinib by activating endoplasmic reticulum stress. It is highly likely that this gene may serve as a new target and that novel combined treatment options for RCC treatment will be developed.Supplementary figures and tables.Click here for additional data file.


Human breast cancer is commonly treated with radiotherapy; however, tumor response depends on both innate radiosensitivity and on tumor repopulation by cells that develop radioresistance. Comparative canine and human studies investigating the mechanisms of radioresistance may lead to novel cancer treatments that benefit both species. In this study, we developed a canine mammary cancer (REM-134) radioresistant (RR) cell line and investigated the cellular mechanisms related to the development of acquired radioresistance. We performed a comparative analysis of this resistant model with our previously developed human breast cancer radioresistant cell lines , characterizing inherent differences through genetic, molecular, and cell biology approaches. RR cells demonstrated enhanced invasion/migration capabilities, with phenotypic evidence suggestive of epithelial-to-mesenchymal transition. Similarities were identified between the REM-134 RR, MCF-7 RR, and ZR-751 RR cell lines in relation to the pattern of expression of both epithelial and mesenchymal genes, in addition to WNT, PI3K, and MAPK pathway activation. Following the development of radioresistance, transcriptomic data indicated that parental MCF-7 and ZR-751 cell lines changed from a luminal A classification to basal/HER2-overexpressing (MCF-7 RR) and normal-like/HER2-overexpressing (ZR-751 RR). These radioresistant subtypes were similar to the REM-134 and REM-134 RR cell lines, which were classified as HER2-overexpressing. To our knowledge, our study is the first to generate a canine mammary cancer RR cell line model and provide a comparative genetic and phenotypic analysis of the mechanisms of acquired radioresistance between canine and human cancer cell lines. We demonstrate that the cellular processes that occur with the development of acquired radioresistance are similar between the human and canine cell lines; our results therefore suggest that the canine model is appropriate to study both human and canine radioresistant mammary cancers, and that treatment strategies used in human medicine may also be applicable to veterinary patients.Research using  Naturally-occurring mammary tumors are the most frequently diagnosed cancer in bitches. These neoplasms represent ~50% of all canine tumors , of whicHBC is routinely managed with surgery followed by combinations of adjuvant chemotherapy, radiotherapy (RT), endocrine therapy or targeted therapy. RT is a commonly used breast cancer treatment; estimates indicate that curative or palliative RT can benefit ~83% of breast cancer patients . The use+, basal and “claudin-low”  and hormone-independent ER−/PR−/HER2− (MDA-MB-231) cell lines that represented different molecular HBC subtypes. In this new study we now go on to develop a novel CMT radioresistant cell line (REM-134), investigate the mechanisms of CMT radioresistance and perform a comparative analysis with our HBC RR cell line models. Although multiple CMT cell lines have previously been generated  cell lines . These ienerated –38, the enerated , a commo−1 penicillin and 50 mg ml−1 streptomycin. The CMT cell line REM-134 was cultured in DMEM (high glucose) with the same additions. All cells were incubated at 37°C in a humidified atmosphere with 5% CO2. The HBC cell lines were obtained from the American Type Culture Collection ; the REM-134 cell line was a kind gift from Professor R.W. Else . All cell lines were authenticated at Health England  by short tandem repeat (STR) profiling. All experiments were performed using cell lines maintained at low passage numbers with new cells acquired from frozen stocks after 10 passages.Unless otherwise stated, reagents used for cell culture were acquired from Gibco Thermo Fisher Scientific . The HBC cell lines MCF-7, ZR-751 and MDA-MB-231 were cultured using Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum, 50 U mlA Faxitron cabinet X-ray system 43855D  was used to irradiate cells. Radioresistant cells were established from parental cell lines through exposure to weekly doses of radiation. Beginning with an initial dose of 2 Gy, cells were irradiated weekly with incremental doses of 0.5 Gy for 12-weeks. After this development period cells were maintained with additional doses of 5 Gy given every week. Cells were routinely passaged 24 h after each radiation dose.2O and dried. Fifty microliter SRB dye [0.4% SRB dissolved in 1% glacial acetic acid ] was added to the wells and the cells were incubated for 30 min. The cells were then washed 4 times in 1% glacial acetic acid and incubated for 60 min after the addition of 150 μl 10 mM Tris-NaOH buffer (pH 10.5). A Biohit BP800 spectrophotometer  and Wallac 1,420 Manager program  were used to analyze optical density at 540 nm.Cells were seeded into 96 well plates (500 cells/well). After 24 h of incubation, cells were irradiated and then fixed up to 120 h post-treatment by adding 50 μl 25% trichloracetic acid  at 4°C for 1 h. Wells were washed in dHCells were irradiated with varying doses 24 h after seeding into 75 mm plates . 1,9-dimethyl-methylene blue zinc chloride double salt  was used to fix and stain the cells between 10 and 14 days after seeding, when colonies consisting of at least 50 cells/colony had formed in the untreated control group. The plating efficiencies and survival fractions for treatment and control colony formation (CF) groups were calculated .Cells were seeded into 6 well plates at densities that led to 100% confluence after 24 h. Scratch assays were carried out as previously described  followinA single cell suspension of ~15,000,000 cells was placed into a spinner flask  containing 100 ml of standard DMEM. The flask was placed onto a magnetic stirrer platform  with MTS forming over a period of 7 days under routine incubation conditions. Hypoxyprobe-1  was used to detect hypoxic areas within MTS. MTS were incubated with 100 μM hypoxyprobe-1 for 1 h before fixation.MTS were transferred into the wells of a 24-well plate with 500 μl of collagen mix , fetal calf serum, 10x DMEM , cell matrix type 1-A  and ice cold 0.1% filtered acetic acid at concentrations of 10, 10, 10, 25, and 45% respectively). The plates were incubated at 37°C for 1 h to allow collagen polymerization, after which 500 μl of routine DMEM was added. Phase-contrast images were taken at 24 h time-points up to 96 h . Invasion was measured at each time point with a FIJI macro developed by Matthew Pearson , with invasion expressed as a % of the initial MTS area.Whole cell lysates were prepared as described previously . Equal pMTS were fixed in 4% formaldehyde  for 24 h. After fixation, the MTS were placed in 2% agarose and processed using the Thermo Scientific Excelsior AS Tissue Processor  and embedded in paraffin. Blocks containing MTS were cut with a Leica RM2235 rotary microtome ; 4 μm sections were placed on SuperFrost Plus glass slides  and dried for 4 h at 53°C.2O2 solution  was performed for 10 min to block endogenous peroxidase activity. Total Protein Block  was used for 10 min to block non-specific antibody staining with the MTS subsequently incubated with primary antibodies for 1 h  using the Spliced Transcripts Alignment to a Reference (STAR) algorithm . After 24 h of incubation, pellets containing up to 10,000,000 cells were collected by trypsinisation, snap-frozen on dry ice and stored at −70°C for RNA extraction. The RNeasy Mini Kit using QIAshredder technology  was used to extract RNA from the cells. Total RNA was purified from animal cells using spin technology, as per the manufacturer's protocol. The NanoDropTM Spectrophotometer ND1000 (Thermo Fischer Scientific) was used to quantify the RNA and assess for contaminants. RNA quality was evaluated by producing an RNA integrity number (RIN) for each sample ; each of the samples had RIN values above 9.7 . Lexogenlgorithm .genefu R package  Parental cell line intrinsic radiosensitivity and confirmation of acquired radioresistance in the developed cell lines were investigated through CF and SRB assays. Using the survival fraction of cells that were given a 2 Gy dose of radiation , a range of intrinsic radiosensitivities was found to be present in the parental cell lines, with the REM-134 cell line showing significantly greater radioresistance compared with the human cell lines. Within the parental human cell lines SF2 was not associated with molecular subtype. The ability of RR cells to form colonies was significantly higher than that of parental cells when subjected to a single radiation dose of up to 2 Gy, confirming the acquisition of radioresistance . SignifiUsing an unsupervised analysis, a large number of genes were found to be inherently differentially expressed  between the REM-134 and REM-134 RR cell lines . Higher + HBC and REM-134 cell lines. Cluster 2 expression appears to be specific to the REM-134 cell line; the expression of these genes does not appear to change in the MDA-MB-231 cells, suggesting that a different mechanism may underlie the development of radioresistance in this triple-negative model  exhibited an epithelial-like morphology, comprising of tightly packed cells that form cobblestone-like monolayers, typical of luminal subtypes. However, their RR derivatives showed mesenchymal-type characteristics, with cells gaining a spindle-shaped morphology that contacted neighboring cells through focal points rather than the entire cellular circumference. The parental human ER− (MDA-MB-231) and the canine (REM-134) cell lines exhibited a mesenchymal-like phenotype, typical of basal and HER2-overexpressing subtypes; morphological changes in their RR derivatives were not as obvious as those observed in the RR cell lines derived from ER+ cells  in ER+ HBC RR and REM-134 RR cell lines compared to their parental lines.These observed changes in cell morphology provided evidence that the cells were undergoing EMT. We therefore assessed the protein expression levels of EMT markers through IHC. MTS formed from MCF-7 RR and ZR-751 RR cells demonstrated higher expression levels of N-cadherin, vimentin, and SNAIL, in addition to a partial downregulation of E-cadherin. The MDA-MB-231 cell line exhibited high levels of vimentin and SNAIL along with low E-cadherin and N-cadherin expression; no differences between the parental and RR MDA-MB-231 cell lines were identified . AlthougFollowing the identification of cellular changes suggestive of EMT, we investigated the invasive and migratory characteristics of the cell lines. 2D migration assay results demonstrated that the RR cell lines all had significantly increased migratory ability in comparison to their parental cells. Similarly, using 3D invasion assays, the MCF-7 RR, ZR-751 RR, and REM-134 RR cells had increased invasive potential compared to their parental cells .+ HCB RR cell lines and both the REM-134 parental and RR lines, potentially suggesting that different WNT signaling pathways in these models may be responsible for the downstream WNT target activation. When the REM-134 and REM-134 RR cell lines were analyzed separately for key differentially expressed  members of the WNT pathway represented in the dataset, clear differences were observed, with the REM-134 RR cell line showing overall higher expression of frizzled family members 1/3/6 and lower expression of frizzled members 2/4, in addition to around 4-fold higher expression of WNT5a , a difference was seen in the MCF-7 and ZR-751 cell lines. Both MCF-7 and ZR-751 cell lines were classified as ER+/PR+/HER2−; however, their RR derivatives lost ER and PR expression, becoming ER−/PR−/HER2−  were characterized by high expression of all of these genes, while in comparison their RR derivatives, together with the REM-134 and REM-134 RR cell lines, were found overall to have lower expression levels of these genes   . Our pro8–10 Gy) ; this in8–10 Gy) . These din vivo tumor microenvironment, including low oxygen levels, creating necrotic, peri-necrotic, and hypoxic regions. Proliferative gradients found within cancers are also present in MTS  and death , 71. A sUsing an initial unsupervised analysis, downregulation of genes involved in HIPPO signaling was identified in the REM-134 RR cell line. This pathway regulates cellular proliferation and apoptosis and has multiple family members that act as tumor suppressors . Activat+ cell lines, with the former exhibiting a typical mesenchymal phenotype. Although no significant differences were seen in their RR derivatives, their morphology was similar to the MCF-7 RR and ZR-751 RR cell lines. IHC and gene expression analysis were used to investigate these results further. Firstly, using IHC to investigate established EMT breast cancer biomarkers  or subcutaneous implantation of paired sensitive and RR REM-134 cells could be used for a multitude of pre-clinical research opportunities. Studies evaluating the effectiveness of radiosensitizing agents, the development of metastatic models or the detection of serum-based biomarkers of radiation response are all achievable with the generation of RR cell lines such as ours.The development of novel RR CMT cell lines opens up the possibility of future + cell lines and the canine parental and RR cell lines. These results suggest that the mechanisms involved in the acquisition of radioresistance may be similar in the 2 species. As we continue to appreciate the significant similarities between human and canine mammary tumors, comparative studies will become more important for the investigation of carcinogenesis in both species. We believe that comparative studies of resistant disease will be fundamental for future research, leading to the development of novel treatment strategies that are equally applicable to both human and veterinary patients.This study is the first to report the development and characterization of a novel canine mammary cancer RR cell line which was used as a comparative model for HBC. The generation of new radioresistant models is important, as these will aid the understanding of the molecular mechanisms that drive the development of radioresistance. Similarities in terms of EMT, WNT signaling, estrogen regulation, HER signaling, and subtype classification were identified between the RR cell lines derived from the human ERhttps://www.ncbi.nlm.nih.gov/geo/, GSE149988.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: DA secured funding for this research. MG conceptualized, performed, analyzed, interpreted the laboratory work, wrote the majority of the manuscript, and composed the figures, with significant contributions from AT and JM. AT performed all bioinformatic analysis. CM-P uploaded data to GEO. Critical revisions were made by MG, AT, JM, CM-P, CK, LP, and DA. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The efficiency of anti-cancer drugs is commonly determined by endpoint assays after extended incubation times, often after days. Here we demonstrate that curcumin encapsulated in crosslinked cyclodextrin nanoparticles (CD-NP) acts extremely rapidly on cell metabolism resulting in an immediate and complete inhibition of cell growth and in efficient cancer-cell killing only few hours after incubation. This early onset of anti-cancer action was discovered by live-cell high-throughput fluorescence microscopy using an environmental stage. To date, only very few examples of covalently crosslinked nanoscale CD-based (CD-NP) drug carriers exist. Crosslinking cyclodextrins enables the adsorption of unusually high payloads of hydrophobic curcumin (762 µg CC/mg CD-NP) reflecting a molar ratio of 2.3:1 curcumin to cyclodextrin. We have investigated the effect of CD-NP encapsulated curcumin (CD-CC-NP) in comparison to free, DMSO-derived curcumin nanoparticles (CC-NP) on 4 different cell lines. Very short incubations times as low as 1 h were applied and cell responses after medium change were subsequently followed over two days. We show that cell proliferation is inhibited nearly immediately in all cell lines and that a cell- and concentration dependent cancer-cell killing occurs. Anti-cancer effects were similar with free and encapsulated curcumin, however, encapsulation in CD-NP drastically extends the long-term photostability and anti-cancer activity of curcumin. Curcumin-sensitivity is highest in HeLa cells reaching up to 90% cell death under these conditions. Sensitivity decreased from HeLa to T24 to MDA MB-231 cells. Strikingly, the immortalized non-cancerous cell line MCF-10A was robust against curcumin concentrations that were highly toxic to the other cell lines. Our results underline the potential of curcumin as gentle and yet effective natural anti-cancer agent when delivered solvent-free in stabilizing and biocompatible drug carriers such as CD-NP that enable efficient cellular delivery. Many potent anti-cancer drugs are too hydrophobic for direct pharmaceutical use . As a reCDs are cyclic oligosaccharides composed of 6, 7 or 8 D-glucopyranose subunits which constitute hydrophobic cavities with diameters of about 5, 7 and 9 Å. They are able to readily absorb numerous APIs, most of them being poorly soluble in aqueous media and/or are highly toxic . Thus, cIn contrast, direct covalent crosslinking of the CD building blocks can substantially improve the carrier stability and avoid the exposure to potentially toxic metal ions. Moreover, such CD nanoparticles could potentially achieve higher loading capacities (within the network constituting the NP) and allow dual delivery of components within a single host. Reports on crosslinked CD nanoparticles are scarce. Suitable crosslinking agents such as hexamethylene diisocyanate, diphenyl- or dimethyl-carbonates, pyromellitic dianhydride or carbonyldiimidazole were reported to generate crosslinked CD bulk materials, which were fabricated into particles usually larger than 400 nm in size by crushing the dried polymer materials ,24. The Our group has recently reported on a new synthesis of small β-cyclodextrin nanoparticles sized about 200 nm by crosslinking with the rigid spacer tetrafluoroterephthalonitrile (TFTN) . This TFCurcumin (CC), a naturally occurring polyphenol in turmeric roots is acknowledged in numerous reports for showing a number of health benefits, ranging from being an anti-oxidant, anti-inflammatory, inhibitor of amyloid fibrillation, potent anti-carcinogenic and even anti-metastatic agent. The multi-faceted health benefits of curcumin were recently showcased in a special issue on curcumin including several reviews concerning its role in cancer applications ,34,35,36Here, we demonstrate that small crosslinked cyclodextrin nanoparticles (CD-NP) can function as a promising carrier for curcumin to address all these critical aspects. We highlight the immediate and dramatic impact of the NP-packaged curcumin on cell proliferation and demonstrate its efficient anti-cancer activity when delivered protected, solvent-free and highly concentrated in these CD-NP. We obtained similar anti-cancer effects also with free, DMSO-derived curcumin nanoparticles CC-NP, however, we show that the inherent limited stability of free curcumin is dramatically enhanced upon adsorption and encapsulation in our cyclodextrin nanoparticles. The discovery of the fast onset of cytotoxic action of curcumin was facilitated by applying label-free, live-cell imaging using high-throughput fluorescence microscopy equipped with an environmental stage. The cell response following curcumin exposure was tracked over extended time using high-content screening. Short primary incubation times of 1 to 3 h were chosen to approach realistic delivery conditions and were evaluated on four different cell lines. We show that the cancer-cell lines HeLa, MDA MB-231 and T24 are strongly affected by curcumin while the non-cancerous cell line MCF-10A is very resistant, surviving even high curcumin concentrations.2CO3 and CTAB and PEG2000 in DMSO [The nanoparticle synthesis was performed by covalently coupling β-cyclodextrin units via the rigid spacer TFTN in order to create particles with a high absorption capacity that conceptually could exceed the inherent cavity space of the individual CD units. This linker is further equipped with nitrile groups that are ready for additional functionalization via click reactions. We used an optimized synthesis route as reported before by our group by combining β-CD, TFTN, K in DMSO  and incr2/g , T24 (urinary bladder carcinoma cells) and MDA MB-231 cells (a triple negative breast cancer cell line). For comparison we included also the non-tumorigenic breast epithelial cell line MCF-10A in our study. In general, to 100 µL medium containing the respective cells seeded in 96-well plates, we added 0.6, 1.2, 1.8, 2.4 and 3 µg curcumin as CC-NP or adsorbed in CD-CC-NP. The originally offered curcumin concentrations are displayed in all graphs, however, since typically only 90% of the initially offered curcumin was absorbed in the CD-NP host we adjusted the listed IC50 values for these differences in the text. Cells were incubated with curcumin for 1 to 3 h (in the following termed “incubation time”), followed by a medium exchange, medium without curcumin. The cell status was further microscopically monitored for up to 3 days; this time span is referred to as “post-incubation time”.In the following we report on the influence of curcumin on the cell behavior of a number of different cancer cell lines as studied by high throughput microscopy using an environmental stage, maintained at 37 °C under exposure of 5% COWe will describe the processes occurring in HeLa cells in greater detail, but observations made with this cell line were also made with the other cell lines. Due to the convenient auto-fluorescence of curcumin, its uptake into cells is easily followed in the microscope. This process happens nearly instantaneously in all cell lines, taking less than 1–2 min, in fact is even too fast to be captured in a movie in our setup. The green fluorescence is visible throughout the cells indicating that CC is immediately dissolved in the cell membranes and organelles, regardless if it was delivered as pure CC-NP or adsorbed in CD-CC-NP. The highest curcumin intensities were observed in areas of the endoplasmic reticulum and the nuclear envelope see .The fast uptake of curcumin in the cells prompted us to study the release of curcumin from the CD-NP in different media. CD-CC-NP with a mole ratio of CD:CC 1:1 were stirred in water, DPBS and in the DMEM cell medium either with or without 10% FBS. When supernatants of these solutions were analyzed with UV-Vis or fluorescence spectroscopy, we could detect only minor amounts of curcumin in water, DPBS or in cell medium without FBS, even when samples had been stirred in these solutions for extended times. In contrast, when FBS was present in DMEM, substantial amounts, up to 36% of the total curcumin content in the CD-CC-NP was released after 15-min stirring see . The relIn our systematic cell studies, HeLa cells were incubated with curcumin absorbed in cyclodextrin nanoparticles (CD-CC-NP) with a molar ratio CD:CC 1:1. Concentrations between 2 to 10 µg of these nanoparticles were added to 100 µL well volume, resulting in wells containing 0.6 to 3 µg CC. After an incubation time of 1 h we performed a medium change and continued to monitor the cells over the next 20 h post-incubation time, successively taking images of identical well areas. Three to six of these images from 3 different wells were visually inspected and evaluated by counting the total number of cells as well as by assessing the number of living and dead cells after each time step. When comparing the images of cells directly after medium change (post-incubation time 0) and after a 5-h post-incubation time in 50 between 73 to 43 µM curcumin (3.0 to 1.8 µg CC/100 µL) is established within 0 to 3 h post-incubation or of 29 µM (1.2 CC/100 µL) after 5-10 h, notably after a very short incubation time of only 1 h. Similar, slightly less effective kinetic response curves were obtained when these HeLa cells were exposed to identical curcumin concentrations in form of DMSO-derived pure CC-NP . Large vacuoles appeared, predominantly around the nucleus and the site of the endoplasmic reticulum, an area where curcumin had accumulated mostly before. In The induction of autophagy can be activated by the unfolded protein response (UPR) associated with the endoplasmic reticulum (ER) stress . ER streSince we have shown above that the photostability of curcumin is markedly extended when it is encapsulated in the CD-NP carrier, we have also compared the toxicity of 5 day-aged CC-NP to 5 day-aged CD-CC-NP samples see . We obseTraditionally, cell viability endpoint tests are performed using dye indicators based on tetrazolium salts (MTT assay). We have tested both MTT and CCK-8 indicators, the latter is based on a tetrazolium-analog forming water-soluble formazan upon reduction, which resulted in more consistent results in this study. Here, we applied the CCK-8 assay as endpoint assay in all our cell experiments. Representative results for HeLa cells are displayed in The viability assays establish the non-toxicity of the CD-NP carrier system over the whole concentration range, confirming our findings from image analysis. However, we do find that even CCK-8 assays generally display an apparently higher cell viability than the results from image analysis after treatment with CD-CC-NP or CC-NP, as documented in Similar to the studies discussed above with HeLa cells, we also performed experiments with other cancer cell lines. Bladder cancer is commonly treated with cis-platin but treatment is often impaired by developing tumor resistance. Only scarce information exists about the activity of curcumin with respect to T24 bladder cancer cells, but a diminished cell proliferation and apoptosis was reported, caused presumably by suppression of the matrix metalloproteinase MMP pathway . A combiOne reason for this extended survival of T24 cells might be found in the successful exocytosis of curcumin-affected cell organelles. We observed a time-dependent accumulation of auto-fluorescing particles in the cell medium when T24 cells were treated with the high concentration of 3 µg CC in CD-CC-NP see . VacuoliThe MDA MB-231 cell line is an aggressive triple negative breast cancer line prone to develop chemo-resistance against powerful therapeutics such as doxorubicin or paclitaxel. Curcumin was shown to reverse this resistance when delivered in combination with doxorubicin to MDA MB-231 cells and to increase the sensitivity of breast cancer stem cells to paclitaxel, cis-platin and mitomycin C ,83. We e50 values of 20 µM curcumin were reported for this cell line when curcumin was micellized in amphiphilic block-co-polymers with high loading capacities [However, a retardation of cell growth was observed even here when treated with the highest curcumin concentration encapsulated in CD-CC-NP see . It requpacities  or applipacities , and it pacities . HoweverCommon anti-cancer treatments are usually non-discriminately aggressive against all cell lines and often induce severe side-effects. We have included a non-cancerous cell line in our studies to evaluate the cytotoxic effect of curcumin towards healthy cells. Exposure conditions to CD-CC-NP and CC-NP were set to 3 h incubation and 48 h post-incubation, the maximum values used for the cancer cell lines discussed above. In none of the four independent experiments performed did we encounter comparable mortality rates as observed for any of the other cell lines described above. In fact, even with the highest concentration of curcumin of 3 µg/well, we encountered at most a 30–40% mortality. In The notable resistance of non-cancerous cells towards curcumin in contrast to its remarkable killing efficiency with respect to cancer cell lines has now been documented in several studies for a number of different cell lines. For instance, human mesenchymal stem cells (MSC) showed higher resistance to curcumin delivered in a cyclodextrin/liposomal formulation than KHOS cells  . SimilarNumerous articles have documented the health benefits and anticancer activities of curcumin, and excellent review articles summarize the collected knowledge about its mechanistic action, such as the binding to cancer associated targets  and CX-2, both connected to tumor progression), ,86 the iHere, we complement these findings by performing cytotoxicity assessments through microscopy observations of transient cell responses towards curcumin exposure directly from the start. In vitro cell experiments represent the most basic set-up for determining the potential of a successful anti-cancer agent. Our work was thus designed to explore the minimum requirements for efficient curcumin toxicity towards cancer cell lines. Incubation times were accordingly chosen as short as possible to approach realistic in vivo conditions. By following the response of cancer cell lines directly without employing disturbing labelling procedures and by evaluating cell images by visual inspection in time, we were able to determine the cell-specific conditions for achieving cancer cell growth inhibition and the induction of apoptosis.We present a novel carrier system based on covalently cross-linked cyclodextrin units that is able to absorb very high payloads of small hydrophobic anti-cancer agents as demonstrated here using polyphenolic curcumin. The encapsulation of curcumin in CD-NP is advantageous for a number of reasons even though comparable results were obtained with free (fresh) curcumin nanoparticles in this study: CD-nanoparticles are stable against centrifugation and redispersion, ease handling and storage requirements and simultaneously offer a pronounced solubility enhancement of the curcumin guest molecules. The nanoparticles stabilize curcumin against photodegradation and more importantly, prolong the cytotoxic potential of curcumin as shown in cell experiments using aged samples. Furthermore, by absorbing curcumin into these carriers, cell exposure to toxic  solvents such as DMSO is eliminated.50 value which decreases upon prolonged cell exposure. Cell responses to curcumin were directly imaged in time over periods of 24 to 48 h, enabled by a high-throughput environmental stage attached to the fluorescence microscope. The resulting cell growth and cell death histograms revealed the prompt and pronounced cytotoxic effect of curcumin, which would escape detection by the sole application of endpoint assays. We document a nearly complete killing of HeLa cells already after 5 h post-incubation, even when cells were exposed for only 1 h to curcumin before.Our study highlights the real-time label-free microscopic cell observation as a valuable tool to assess the optimal conditions for curcumin anti-cancer activity. Cell incubation was minimized to only 1–3 h in order to approach realistic delivery conditions. Notably, this incubation time influences the value of the apparent ICBladder cancer cells T24 and breast cancer cells MDA MB-231 require longer exposure times to curcumin, but a 3 h incubation period showed successful cell killing also with these cell lines. In contrast, non-cancerous MC-10A cells display a profound resistance against curcumin, supporting the promising biocompatibility of curcumin. Importantly, cell proliferation was completely stopped in all cell lines at low concentrations and at early stages of post-incubation, an effect that has been related to the induction of a G2/M cell cycle arrest . This ph


It has been implicated in the pathogenesis of a number of cancer types, including oesophageal and ovarian cancer, while its closely associated family member, iRhom1, is implicated in head and neck cancer. However, a role for iRhom2 in head and neck cancer has not been investigated. Immunoblotting for iRhom2 in 54 oral squamous cell carcinoma (OSCC) and 24 paired normal tissues demonstrated higher levels of iRhom2 protein in tumour compared with normal samples (P < 0.05). iRhom2 over-expression correlated with poor patient survival (P < 0.0005) but with no other clinicopathological variable. Increased cell migration was observed in stably over-expressing iRhom2 clones of OSCC cell lines in the absence of increased cell proliferation, but not in the normal oral keratinocyte cell line, NOK-hTERT, and this was abrogated by knock-down of iRhom2. iRhom2 protein expression is increased in a proportion of OSCC and this up-regulation is associated with faster cell migration and decreased patient survival. These data implicate iRhom2-controlled signalling events in the pathogenesis of this cancer.The online version of this article (10.1007/s11033-020-05381-y) contains supplementary material, which is available to authorized users. Rhomboids are intramembrane serine proteases, with their active and catalytic residues located within the cell membrane lipid bilayer, enabling them to easily cleave and activate / inactivate membrane proteins within their transmembrane helices . AlthougHNSCC was reported to be the eighth most common cancer type in the UK in 2014. Worldwide, it is the sixth leading cancer type by incidence, with more than 550,000 cases and about 300,000 deaths in 2014 . The 5 yThis investigation focuses on the possible oncogenic role of iRhom2 in oral squamous cell carcinomas, the most common form of HNSCC, and its possible utility as a biomarker for survival.Fresh, frozen tissues were obtained from 54 OSCC tumours and 24 paired normal adjacent tissues. Written consent was obtained from all patients (REC numbers: EC.47.01 & 10/H1002/53). The tissues were snap frozen at point of surgery and stored at -80°C prior to use. Demographic and clinicopathological data was collected.Frozen tissues were embedded in OCT and a 5 μm section assessed for tumour presence. Tumour samples with less than 60% tumour content were excluded. Normal samples with more than 5% tumour content were also excluded.The oral cancer cell lines, PE/CA-PJ15   and were mycoplasma negative by E-Myco PCR detection kit (CHEMBIO Ltd).3 pieces of tissue were excised from areas with an increased number of tumour cells or lack of tumour as appropriate (determined from H&E sections) and re-suspended in protein extraction buffer , and sonicated for 30 s to extract total protein which was stored at − 80 °C until use. Recovered cell pellets were re-suspended in protein extraction buffer and similarly processed.5 mmProtein samples were loaded unto 10% SDS/PAGE gels. TE3-RHBDF2, an oesophageal cancer cell line constitutively expressing iRhom2, was used as a positive control. Following electrophoresis, gels were transferred to 0.45 μm PVDF membranes  and western blotting carried out. Membranes were blocked for one hour in PBS/Licor Blocking Buffer, ratio 1:1 then probed, using rabbit polyclonal anti-RHBDF2 (1:500: Sigma Aldrich: HPA018080) or mouse monoclonal anti-β-actin (1:1000: SantaCruz: sc-4778) at room temperature for 2 h. Fluorescently labelled secondary antibodies; IRDye@800 goat anti-rabbit (Licor) and Alexa Flour@680 rabbit anti-mouse (Life Technologies), were used at a concentration of 1: 10,000. Visualisation of fluorescent secondary antibody localisation was undertaken on a Licor Odyssey 3.0 imager (Licor Biosciences UK Ltd).th percentile from the cumulative frequency curve of the expressing samples. Samples demonstrating no measurable expression were classified as no expression.Densitometry was used to normalise and semi-quantitate the levels of protein expression in comparison with the actin control and the positive control sample run on each gel. Samples were ascribed a status of high (or overexpression) or moderate  based on a cut-off of 19.9 relative absorbance units, which was determined to be the boundary of the 75RHBDF2 were created by transfection with a 5.3 kb pIRESneo vector (Clontech) containing isoform 2 of RHBDF2 (Epoch Life Science Inc). shRNA knock-down of RHBDF2 gene was achieved using Mission®shRNA (SIGMA—09051604MN) with Mission®pLKO.1-puro B2M shRNA control (SIGMA-SHC008-04021321MN)( Sigma-Aldrich/Merck).Clones of PE/CA-PJ15, Liv37K and NOK-hTERT that stably over-expressed A crystal violet assay was used to assess proliferation. Briefly, cells were seeded into 6 well plates and harvested daily for 5 days, then stained with 0.05% crystal violet solubilised in 10% acetic acid and absorbance read at 570 nm using a Spectramax-Plus284 absorbance microplate reader (Molecular Devices) against a 10% acetic acid blank.https://imagej.nih.gov/ij/) and T-Scratch [Cells were seeded in triplicate into removable chambers  placed in 6 well plates, then cultured until they reached approximately 85% confluency. The removable inserts were withdrawn and the plates transferred to culture chamber attached to a Nikon Eclipse TE300 microscope for image capture at ten minutes intervals for 30 h. Gap closure images were analysed on Image J . Chi-squared and the Kaplan–Meier survival tests were used to analyse associations between tissue expression and clinicopathological features.iRhom2 protein was highly expressed in 19/54 (35%) tumour samples, moderately expressed in 17/54 (31%) tumour samples, and undetected in the remaining 18/54 (33%)  , but was reduced in Liv37K and, to a lesser degree, in NOK-hTERT, while having no apparent effect on PE/CA-PJ15.Despite the lack of increase in cell proliferation, PE/CA-PJ15 and Liv37K clones with up-regulated iRhom2 migrated approximately twice as fast as their respective wild-type variants  . ADAM17,To augment our observational study on clinical samples, we both over-expressed and knocked down iRhom2 expression in two oral cancer cell lines and one immortalised normal oral keratinocyte cell line. Interestingly, cell proliferation was not shown to be augmented by increased expression of iRhom2 and these data are in contrast to the literature that suggests a proliferative role for iRhom2 , 17. UprThese data implicate activation of the iRhom2-ADAM17 pathway in the pathogenesis of OSCC, and suggest that this system works by influencing or increasing rate of cell migration. Deregulation of ADAM17 has previously been implicated in head and neck squamous cell carcinoma (HNSCC) , with upSupplementary file1 (GIF 80 kb)Supplementary file2 (GIF 13 kb)Supplementary file3 (DOCX 12 kb)Below is the link to the electronic supplementary material.


Plasmablastic lymphoma (PBL) is an aggressive high-grade B cell lymphoma, considered a variant of diffuse large B cell lymphoma with approximately 75% mortality within 6-7 months. We describe an unusual case of PBL arising as a maxillary mass in an HIV-negative, nontransplanted 78-year-old female. Histologic examination revealed a diffuse infiltrate of anaplastic appearing cells exhibiting plasmablastic morphology with an adjacent contiguous infiltrate of mature appearing plasma cells. The PBL and mature plasma cell components both demonstrated an immunophenotype of CD20(-), CD38(+), and CD138(+). The two populations differed by the PBL featuring a high proliferation rate by Ki-67 (~95%) with coexpression of both c-MYC and EBV, while the mature plasma cell component featured a low proliferation rate by Ki-67 (~5%) without coexpression of c-MYC or EBV. Kappa/lambda staining demonstrated lambda light chain restriction involving the PBL, while the mature plasma cell infiltrate revealed kappa light chain restriction. Our findings describe the rare association of PBL with a synchronous distinct population of mature plasma cells exhibiting opposite light chain restriction. Plasmablastic lymphoma (PBL) was first described in 1997 as an aggressive high-grade B cell lymphoma with predilection of involving the oral cavity and strongly associated with HIV and EBV infection , 3. EBV Here, we describe an HIV-negative, nontransplanted 78-year-old female diagnosed with a lambda light chain restricted PBL involving the maxillary sinus with a concurrent mature plasma cell population distributed predominately in a contiguous circumferential pattern around the PBL exhibiting kappa monoclonal light chain restriction. We investigated the significance of these two populations by morphologic, immunophenotypic, and molecular methods including next-generation sequencing (NGS) targeted gene panel (596 gene panel) and immunoglobulin heavy (IGH) chain clonality testing by NGS methods.A 78-year-old female with no significant past medical history presented with chronic right-sided nasal obstruction with new-onset facial swelling. Computed tomography (CT) revealed a 3.3 cm mass centered in the right maxillary sinus extending into the right nasal cavity, right retro maxillary space, and along the inferior extraconal compartment of the right orbit .The patient underwent nasal endoscopy with bioptic material revealing effacement of architecture by a diffuse infiltrate composed primarily of large plasmablastic appearing cells with amphophilic cytoplasm, irregular nuclei, fine reticular chromatin, and occasional single, central nucleolus Figures . Brisk mA second distinct population of mature-appearing plasma cells without frank anaplastic/plasmablastic features was surrounding and contiguous to the PBL Figures . A carefWe subjected the two populations to further testing by mutation screening using a 596 gene panel and immunoglobulin heavy (IGH) chain clonality testing by next-generation sequencing (NGS) methods. The NGS targeted panel testing was performed after careful macrodissection, with DNA and RNA extracted from unstained formalin-fixed, paraffin-embedded sections. 100 nanograms (ng) of DNA for each tumor was mechanically sheared to an average size of 200 base pairs (bp) using a Covaris ultrasonicator. DNA libraries were prepared using the KAPA Hyper Prep Kit (Roche), hybridized to the xT probe set, and amplified with the KAPA HiFi HotStart ReadyMix (Roche). The amplified target-captured DNA tumor libraries were sequenced to an average unique on target depth of 500x on an Illumina HiSeq 4000. Samples were further assessed for uniformity with each sample required to have 95% of all targeted bp sequenced to a minimum depth of 300x. The NGS data for both the PBL and the light chain restricted mature appearing plasma cell population revealed no definitive pathologic variants, but multiple variants of unknown significance were noted Tables .Immunoglobulin heavy chain clonality testing by next-generation sequencing was performed in triplicate in both PBL and mature plasma cell populations after careful macrodissection. Clonality analysis was performed using the Lymphotrack IgH assay . DNA was extracted from three separate regions of PBL and two distinct regions containing the mature plasma cell component from a single FFPE block. After PCR with FR1/2/3 and J primers, libraries were pooled and sequenced on a Miseq instrument according to the manufacturer's protocol. Fastq files were analyzed by the Lymphotrack software. A clonal read was defined if it was >2.5% of the total reads and was >10X the polyclonal background. Further characterization of the rearrangements was performed using the web-based IMGT/V-Quest and IgBLAST programs. Results of the PBL contained two clonal rearrangements with similar clonal frequencies. The FR1 primer set amplified both rearrangements much better than FR2 or FR3, but all three demonstrated similar findings. One rearrangement was productive (V3-20∗03/D3-22∗01/J3∗02), whereas the other was nonproductive  suggesting a malignant clone with biallelic rearrangements. The V regions of both rearrangements contained extensive somatic hypermutation, indicating a postgerminal center cell origin of the tumor. The three separate regions of PBL that were analyzed contained the same clonal rearrangements. The mature plasma cell component showed an oligoclonal pattern of rearrangements suggesting either paucity of plasma cells with reduced diversity in the regions analyzed or inability of FR1/FR2/FR3 primers to amplify rearrangements due to somatic hypermutation. In contrast to the adequate number of reads obtained with the FR1 primer set in the PBL, the mature plasma cell component did not amplify well with the FR1 primer set and generated a low number of reads. Clonal rearrangements were not identified in both mature plasma cell regions when the criteria of >2.5% read frequency and >10X the background were applied . NonetheSubsequent bone marrow staging revealed trilineage hematopoiesis and was negative for involvement by plasmablastic lymphoma, but positive for a small plasma cell population (approximately 5-9% involvement) exhibiting kappa light chain restriction by both flow cytometric and in situ hybridization studies. No additional aberrant antigen expression by flow cytometry was detected. Morphology revealed mature plasma cells without frank anaplastic/plasmablastic features Figures . EBER-ISLaboratory studies at the time of diagnosis revealed a serum protein electrophoresis (SPEP) with an elevated kappa/lambda ratio  without definitive paraprotein (confirmed by immunofixation), with similar findings demonstrated in the urine. Complete blood count, calcium, liver function, and lactate dehydrogenase (LDH) studies were within the normal range. CT imaging demonstrated no masses or enlarged abdominal, pelvic, or thoracic lymph nodes. The patient ultimately opted to be treated by her local outside oncologist who reported that the patient was treated with 6 cycles of R-CHOP and was asymptomatic at a six-month follow-up. No further follow-up information is available in the electronic medical record.PBL is a rare and aggressive variant of diffuse large B cell lymphoma with the vast majority of cases associated with various states of immunodeficiency. A recent study by the Lysa group demonstrated in their cohort of 135 PBL cases that HIV-positive cases represented 42% of the cases, posttransplant 12%, and HIV-negative/nontransplanted 46% of the cases , 11.The current case was associated with a unique finding of a concurrent distinct population of opposite light chain restricted mature plasma cells. Therefore, we sought to further understand the biology of these two populations by both a next-generation sequencing (NGS) targeted panel (596 gene panel) and immunoglobulin heavy (IGH) chain clonality testing by NGS methods. In the NGS targeted panel, both the PBL and the mature plasma cell population demonstrated among others a STAT3 missense mutation  at 38.9% and 14.6% variant allele frequencies, respectively. STAT3—typically implicated in T cell malignancy—is only occasionally reported in B cell-derived tumors. It is known to upregulate MYC protein expression and has also been implicated in the pathogenesis of ALK-positive large B cell lymphoma, another postgerminal center B cell malignancy that exhibits “plasmablastic” morphology [PBL often features plasmacytic differentiation, which is enriched in cells with rounder nuclei, coarse chromatin, eccentric nuclei, and abundant basophilic cytoplasm with a paranuclear hof. These cases are found typically in the setting of nodal or nonoral involvement and are more likely in HIV-negative patients , 6, 15. We hypothesized that our case might represent a transformation of an underlying plasmacytoma into a PBL, possibly with the addition of MYC and EBV coinfection. As such, the mature plasma cell component possibly underwent a second round of extramedullary VJ recombination to express the lambda light chain at the same time that it gained copies of the MYC locus, and then subsequently underwent a germinal center type reaction to attain evident somatic hypermutation. An alternative hypothesis that is partially supported by the IgH heavy chain analysis by NGS suggests the mature plasma cells were potentially recruited in a reactive phenomenon that exhibits monotypic light chain restriction, yet are oligoclonal. Of note, the bone marrow findings revealed kappa monotypic plasma cells of the same light chain restriction noted in the mature plasma cell component of the maxillary mass. SPEP and urine protein electrophoresis both reported an elevated kappa/lambda ratio , but without paraprotein.Transformation to PBL has only been rarely described and typically attributed to a preexisting mature B cell neoplasm most often implicating chronic lymphocytic leukemia and follicular lymphoma . Ambrosi


Through a salt metathesis reaction, the chloride ligand in 1 was replaced by potassium benzothiazole-2-thiolate (Kbt) and potassium benzoimidazole-2-thiolate (Kbi) to afford complexes (PhCH2NC)Au(κ1-S-bt)], 2a and (PhCH2NC)Au(κ1-S-bi)], 2b, respectively, which were characterized by NMR spectroscopy. The cytotoxic activities of 2a and 2b were evaluated against three human cancer cell lines, including A549 (lung), SKOV3 (ovary), and MCF-7 (breast). Our results indicated that 2a exhibited comparable cytotoxicity on investigated cell lines with cisplatin. It showed a good anti-proliferative activity with IC50 of 19.46, 11.76 and 13.27 μM against A549, SKOV3 and MCF-7 cell lines, respectively. The effects of these complexes on the proliferation of the non-tumorigenic epithelial breast cell line (MCF-10A) showed their good selectivity between the tumorigenic and non-tumorigenic cell lines. Molecular docking simulation studies were also conducted to determine the specific binding site and binding mode of the synthesized gold complexes to DNA and thioredoxinreductase (TrxR) as their proposed targets.The complex [(PhCH The commercially available platinum-based drugs such as cisplatin, carboplatin, and oxaliplatin suffer from severe side effects and resistance in a wide range of cancers , 2. ReceTwo significant oxidation states of gold is +1 and +3 . Au(I) aI-SR have been synthesized, so that ligand(L) could be different neutral ligands such as isocyanides (Thiol group (RSH) and their corresponding anion forms, called thiolate (RS-), as a chemically unique ligands, can be used as a source of sulfur donor ligands in a variety of organometallic complexes -27. To dcyanides -31, phoscyanides  and 33, cyanides -36. Genecyanides  and 36.Recent studies demonstrated that the thioredoxin reductase (TrxR) which catalyzed the NADPH-dependent reduction of oxidized thioredoxins, is an effective target for the development of novel antitumor agents , 38. SevHere, in this study, the synthesis, structural characterization, and cytotoxic activity of two new Au(I) complexes with benzyl isocyanide and thiolate ligands (benzothiazole2-thiolate (bt) and benzoimidazole-2-thiolate (bi)) were demonstrated. The cytotoxic activity was evaluated on three different cancer cell lines including human lung (A549), ovarian (SKOV3), and breast (MCF-7) cancer cell lines. Molecular docking simulation studies were also conducted to determine the specific binding site and binding mode of the synthesized gold complexes to DNA and thioredoxinreductase (TrxR) as their proposed targets.Materials and methodsGeneral procedures and materials1H and 13C{1H}) were recorded on a Bruker Avance DPX 400 MHz instrument and referenced to the residual peak of the solvent, i.e. CDCl3 (1H and 13C). The chemical shifts (δ) were reported as ppm and coupling constants (J) were expressed in Hz. The melting point values were measured by a Buchi 510. The microanalyses were performed using a vario EL CHNS elemental analyzer. Benzyl isocyanide (PhCH2NC), benzothiazole-2-thiol (Hbt), and benzoimidazole-2-thiol (Hbi) as well as all the solvents were purchased from Aldrich and used without further purification. Complex [(Me2S)AuCl], A, was prepared according to literature method (40).All reactions were carried out under a nitrogen atmosphere using standard Schlenk techniques. NMR spectra ( Synthesis of potassium benzothiazole-2-thiolate (Kbt)iPrOH (2 mL) and the resulting yellow solid was filtered and dried. This procedure was also used for preparation of potassium benzoimidazole-2-thiolate (Kbi).A solution of benzothiazole-2-thiol  in MeOH (10 mL) was added to a solution of KOH  in MeOH (5 mL). The resulting yellow solution was stirred at room temperature for 1 h, and then the solvent was completely evaporated. The residue was treated with Synthesis of [(PhCH2NC)AuCl], 1 2S)AuCl], A  in CH2Cl2 (20 mL), 1 equivalent of PhCH2NC  was added. The mixture was stirred at room temperature for 1 h and then concentrated (~1 mL) under vacuum, and n-pentane (5 mL) was added to give 1 as a white solid, which was filtered and washed with n-pentane (2 × 3 mL) and dried. Yield: 197 mg, 83%; m.p. = 138 °C. Elem. Anal. Calcd. for C8H7AuClN (349.57): C, 27.49; H, 2.02; N, 4.01. Found: C, 27.61; H, 2.05; N, 4.06. IR : 2260 . NMR data in CDCl3: δ (1H) 4.87 , 7.35 , 7.46-7.43 ; δ (13C{H}) 48.2 , 127.5 , 129.4 , 129.6 , 129.7 , 135.6 .To a solution of [(MeSynthesis of(PhCH2NC)Au(κ1-S-bt)], 2a 1  in CH2Cl2 (15 mL). The reaction mixture was stirred at room temperature for 15 h. Then, the solvent was removed under reduced pressure and the residue was extracted with CH2Cl2 (10 mL). The obtained colorless solution was filtered through celite and the filtrate was concentrated (~1 mL) under vacuum, and n-pentane (5 mL) was added to give 2 as a white solid, which was filtered and washed with n-pentane (3 × 3 mL) and dried. Yield: 87 mg, 74%; m.p. = 172 °C. Elem. Anal. Calcd. for C15H11AuN2S2 (480.35): C, 37.51; H, 2.31; N, 5.83. Found: C, 37.46; H, 2.34; N, 5.85. NMR data in CDCl3: δ (1H) δ 8.04 – 7.93 , 7.39 , 7.27 – 7.16 , 7.08 – 6.94 , 4.15 . δ (13C{H}) 168.9, 152.0, 143.0, 139.0, 134.6, 128.5, 128.2, 126.2, 124.5, 121.5, 121.1, 41.3.An equimolar amount of Kbt  was dissolved in mixture of MeOH/acetone (2/8 mL) and added to a solution of Synthesis of (PhCH2NC)Au(κ1-S-bi)], 2b 2a. Yield: 91 mg, 75%; m.p. = 186 °C. Elem. Anal. Calcd. for C15H12AuN3S (463.04): C, 38.89; H, 2.61; N, 9.07. Found: C, 38.82; H, 2.67; N, 9.11. NMR data in CDCl3: δ (1H) δ 7.98 – 7.87 , 7.47 – 7.40 , 7.25 – 7.12 , 7.08 – 6.94 , 4.15 . NMR data in CDCl3: δ (1H) 153.4, 143.0, 140.4, 139.0, 137.5, 128.5, 128.2, 126.1, 124.6, 122.2, 117.4, 111.0, 41.3.The synthesis procedure was as the same as Biological AssayCell Lines and Cell Culture2 incubator. MCF10A cells  were cultured in DMEM/Ham’s F-12  supplemented with 100 ng/mL cholera toxin, 20 ng/mL epidermal growth factor (EGF), 0.01 mg/mL insulin, 500 ng/mL hydrocortisone, and 5% chelex-treated horse serum.Human cancer cell lines, MCF-7 (breast cancer), SKOV3 (ovarian cancer), and A549  were purchased from National Cell Bank of Iran . All the cells were cultured in RPMI 1640 medium (Biosera), supplemented with 10% fetal bovine serum  and 1% pencilin−streptomycin and were incubated at 37 °C in humidified COMTT Assay2a and 2b were evaluated using standard 3--2,5-diphenyl-tetrazolium bromide (MTT) assay according to a known protocol  using HyperChem 8, followed by energy minimization calculations at Hartree-Fock (HF) level, using Gaussian 09. The output structures were then converted to PDBQT using MGLtools 1.5.6. The ligands, thereafter, were docked in the active site of DNA and TrxR using an in-house batch script (DOCKFACE) of AutoDock 4.2, based on Lamarckian genetic algorithm  and TrxR (PDB ID: 4CBQ) were retrieved from protein data bank (www.rcsb.org/pdb). Co-crystal ligands were excluded from the structures and the PDBs were checked in terms of missing atom types. Subsequently, MGLtools 1.5.6 was applied to convert these corrected PDB files to PDBQT. The structure of each gold(I) complexes was created by HyperChem Professional . Each complex was optimized by molecular mechanic methods AuCl], A, was prepared  and afforded the corresponding complex [(PhCH2NC)AuCl], 1. Complex 1 was also treated with potassium benzothiazole-2-thiolate (Kbt) and potassium benzoimidazole-2-thiolate (Kbi) in a 1:1 molar ratio and yielded complex (PhCH2NC)Au(κ1-S-bt)], 2a, and (PhCH2NC)Au(κ1-S-bi)], 2b, respectively, through a salt metathesis reaction  displayed signal resonances due to the PhCH2NC ligand in expected regions  and a simple pattern for the thiolate ligands (e and Cf of PhCH2NC ligand indicated a resolving coupling with nitrogen nucleus (14N) in the 13C{1H} NMR spectra of synthesized complexes which is characteristic feature for numerous isocyanide ligand and their complexes , lung (A549), and breast (MCF-7) carcinoma. As shown in 2a, the exhibited comparable cytotoxicity on the investigated cell lines with cisplatin. It showed a good anti-proliferative activity with IC50 of 19.46, 11.76, and 13.27 μM compared with those measured for cisplatin . 2b also showed generally a moderate antitumor activity especially against MCF-7 cell line with IC50 of 19.14 μM. Interestingly, 1 showed better antitumor activity against all the studied cancer cell lines compared to 2b.The To investigate the selectivity between cancer and the normal cell line, the effects of the synthesized gold(I) complexes on the proliferation of the nontumoral cell line  was also acquired. The results showed good selectivity between the tumorigenic and non-tumorigenic cell lines.Molecular docking analysisIt was observed that gold acts as an anticancer agent through different mechanisms such as inhibition of the thioredoxinreductase (TrxR), gluthationereductase (GR) enzymes, and intercalation with the DNA . Hence, 2a, the best compound in the cytotoxic activity, showed also better energy regarding binding to TrxR active site compared to 2a. These results suggest TrxR as the main target of these gold(I) anticancer agents. The ΔGbind values of the best docked poses of these compounds are within the range of -7.71 to -13.86 kcal.mol-1 for DNA and -7.80 to -12.26 kcal.mol-1 for TrxR. The validity of the docking procedure was maintained by re-docking of Auronofin, the co-crystal ligand of TrxR, into 3D structure of TrxR. All the docking protocols were done on validated structures with RMSD values below 2 Å. The docking binding energies of the synthesized Au(I) complexes with DNA and TrxR targets are shown in 1 interacted with the minor groove of DNA with -7.71 kcal/mol binding energy through its chloro group with A6, and the benzyl CH2 group with T7 and T8 base pairs in the minor groove of DNA (PDB ID: 1BNA) (2a was through hydrogen bonding of benzothiazole sulfur group with T7 in the minor groove of DNA (2b interacted with the minor groove of DNA (PDB ID: 3CO3) with -8.55 Kcal/mol binding energy through its sulfur group attached to the gold atom with G9, benzothiazole nitrogen group with G9, gold atom with G7 and the benzyl CH2 group with T8 base pairs (The docked model suggested that the compound D: 1BNA) . The maie of DNA . 2b intese pairs .2a in binding to TrxR were the interaction of gold atom with Asp287, isocyande nitrogen group with Asp284, sulfur group attached to the gold atom with Ser299 and benzothiazole nitrogen group with Cys286  or potassium benzoimidazole-2-thiolate (Kbi), led to formation 2a or 2b, respectively. These complexes are fully characterized by NMR and elemental analysis. The cytotoxic activities of 2a and 2b against various cancer cell lines revealed that 2a has reasonable IC50 with higher potency than 2b. The evaluation of their cytotoxicity against the non-tumorigenic epithelial breast cell line (MCF-10A), showed good selectivity between the tumorigenic and non-tumorigenic cell lines. Complex 2a showed a good anti-proliferative effect which is even higher than cisplatin against MCF-7 cell line.In this study, two novel sulfur-based gold complexes are reported. Complex