Fucus vesiculosus, F. serratus, F. distichus subsp. evanescens, Dictyosiphon foeniculaceus, Laminaria digitata, Saccharina latissima) as well as three reference compounds  on tumor  and non-tumor  cell lines. All fucoidans were extracted according to a standardized procedure and tested in a commercially available MTS assay. Cell viability was measured after 24 h incubation with test compounds (1–100 µg/mL). Apart from few exceptions, fucoidans and heparins did not impair cell viability. In contrast, fucoidans significantly increased cell viability of suspension cell lines, but not of adherent cells. Fucoidans slightly increased viability of tumor cells and had no impact on the viability of non-tumor cells. The cell viability of HeLa and ARPE-19 cells negatively correlated with protein content and total phenolic content (TPC) of fucoidans, respectively. In summary, none of the tested fucoidans turned out to be anti-proliferative, rendering them interesting for future studies and applications. Fucoidans extracted from brown algae exert manifold biological activities paving the way for the development of numerous applications including treatments outside tumor therapy such as age-related macular degeneration or tissue engineering. In this study, we investigated the antiproliferative effects of fucoidans extracted from six different algae ( Cancer is one of the leading causes of death in industrial nations and a wide variety of types of cancer exists. Despite enhanced research interest and already existing medical treatments, the mortality rate is still very high, depending on the cancer type. A common complication in cancer patients and one of the leading causes of death is venous thromboembolism (VTE) . The firA potential alternative may be fucose-containing sulfated polysaccharides, the so-called fucoidans, extracted from brown algae. Fucoidans display an attractive array of bioactivities and potential applications including not only cancer inhibition, but also immune modulation, pathogen defense and numerous others  ,10. ManyIn general, one of the pivotal initial in vitro tests is the examination of the influence of potentially active agents such as fucoidans on cell viability. Whereas antiproliferative effects on tumor cell lines are favorable for antitumor therapies, cytotoxicity on non-cancerous cells is a big limitation for any lead compound, including fucoidans. Such toxicity may hamper other potential applications of fucoidans, for example their use in tissue engineering for improvement of bone vascularization during bone repair ,17. AnotSaccharina japonica inhibited the proliferation of human myeloma and T-cell lymphoma cells as well as having cell growth inhibitory effects on SH-SY-5Y (neuroblastoma cells) and MKN-28 (adenocarcinoma cells) , thus including a total of seven tumor cell lines  and two non-cancerous cell lines (ARPE-19 and HaCaT). After incubation for 24 h with the different fucoidans, the cell viability was measured using a commercial MTS assay according to a standardized protocol. As reference compounds three different commercially available substances, namely reference heparin as well as reference enoxaparin (a LMWH) and the commercial Fucus vesiculosus fucoidan, were additionally tested. The tests were performed to obtain comparative information on the suitability of the different fucoidans for further investigations. In the present study, we tested the cell viability effects of six fucoidans on nine different cell lines. The fucoidans were extracted from six different brown algal species by using the same extraction method. Cell viability effects were assessed using one standardized assay enabling direct comparison of the results. The extracted algal species were The details of the extraction procedure of the six fucoidans as well as their basic characteristics namely degree of sulfation (DS), molecular mass, monosaccharide composition, their contents of polyphenols, laminarin and protein as well as their radical-scavenging capacity, three exemplary bioactivities and their fluorescence intensity (FI) increasing effect on the sulfated glycan sensor Polymer-H are described elsewhere (unpublished data until accepted). To compare the effects of the different fucoidans and reference substances on cell viability, we determined their effects at concentrations up to 100 µg/mL  on nine different cell lines in relation to the untreated control (100%) . p < 0.05), heparin only at 50 µg/mL (p < 0.05) and the LMWH enoxaparin only at 100 µg/mL (p < 0.001). A questionable exception was the significant lower cell viability  after treatment with 10 µg/mL enoxaparin, whereas 100 µg/mL increased the cell viability significantly (p < 0.001). The commercial fucoidan had a stimulating effect at 50 and 100 µg/mL (p < 0.05). In the leukemic cell line HL-60, all test compounds apart from the DF fucoidan increased the cell viability a. The efp < 0.01). The other fucoidans and the reference substances showed no significant effect.In the Burkitt lymphoma cell line Raji, no inhibitory but rather a stimulatory effect was observed b. FV andp < 0.01; 10 µg/mL 65.7 ± 17.8%, p < 0.01; 50 µg/mL 68.7 ± 19.2%, p < 0.01; 100 µg/mL 63.6 ± 24.3%, p < 0.05). FE fucoidan decreased the cell viability at 1 µg/mL (p < 0.01). The reference substance heparin showed significant reduction in cell viability at concentrations of 50 and 100 µg/mL .The cervical adenocarcinoma cell line HeLa was one of the four cell lines, in which the compounds partly reduced the cell viability c. This wp < 0.001; 90.9 ± 2.6%, p < 0.001; 87.4 ± 8.7%, p < 0.01; 79.3 ± 14.7%, p < 0.01) . The cell viability of the ARPE-19 cells incubated with the other compounds was comparable to the untreated control, although some small changes turned out to be significant  d. In connificant d.p < 0.001, 87.1 ± 11.9%, p < 0.01) and 100 µg/mL  as well as by FE and SL at 100 µg/mL . Significant increase in the cell viability was observed for FV, LD and SL fucoidans at 10 µg/mL. A trend of increase was further seen with heparin at concentrations of 1–50 µg/mL. Similar to ARPE-19 cells, the uveal melanoma cell line OMM-1 responded differently to the treatment with the compounds, whereby FE and DF led to concentration-dependent effects e. The cep < 0.001, and 100 µg/mL, 105.2 ± 0.9%, p < 0.001) as well as DF at the highest concentration  and LD at the lowest concentration . Of note, slightly significant reduction of cell viability for A-375 was measured only for SL . In the skin melanoma cell line A-375 f a signip < 0.01; 50 µg/mL 108.7 ± 5.4%, p < 0.05; 100 µg/mL, 111.9 ± 3.8%, p < 0.001) and LD , as well as heparin at 100 µg/mL (p < 0.001) showed significantly increased cell viability. Lower but still slightly significant increasing effects on cell viability were observed for fucoidans from FV , FE , SL at the lowest three tested concentrations . A slight, but significant increase in cell viability was also measured for the Sigma fucoidan at 10 µg/mL and 100 µg/mL, p < 0.01; heparin 100 µg/mL, p < 0.05). The colon carcinoma cell line HCT-116 treated with DF . A slightly significant increase in cell viability was however measured for fucoidans from all Fucus species, namely FV with increasing concentration , FS at the two highest test concentrations , FE  as well as LD . The hepatocellular carcinoma cell line Hep G2 showed no significant change of viability after application of almost all reference compounds at all concentrations with one exception: enoxaparin at 1 µg/mL, p < 0.05; 10 µg/mL 91.8 ± 3.0%, p < 0.001; 50 µg/mL 87.2 ± 2.6%, p < 0.001; 100 µg/mL 87.9 ± 3.9%, p < 0.001). The heparins had differential effects: whereas no significant change in HaCaT viability was detected for LMWH, heparin showed a significant decrease at the three highest test concentrations . In the non-cancerous keratinocyte cell line HaCaT, commercial fucoidan lowered the cell viability significantly at all used concentrations  and SL . Notably, DF fucoidan significantly reduced the proliferation at 50 µg/mL .Slight, but significant reduction of cell viability was further found for fucoidan extracts from LD . In the HeLa cell line, the cell viability was significantly decreased with 10 µg/mL DF fucoidan . UFH decreased viability compared to control at 50 and 100 µg/mL . SL fucoidan significantly increased viability  as well as 1 µg/mL Sigma fucoidan . The differences of Raji and HeLa cell viability between 24 h and 72 h incubation time compared to control are minor.In addition to the incubation for 24 h, cell viability was estimated after 72 h for selected cell lines . In geneBesides the comparison of individual effects of fucoidans on the nine cell lines, we aimed to evaluate whether the differently sourced fucoidans show different effects on cell viability as well as whether the cell lines differ in their response to all fucoidans and heparins . Regarding the medians (ranging from 99% to 107%), neither the fucoidans nor the heparins modified the cell viability a. The laThe effect of the fucoidans and heparins on cell viability differed stronger depending on the cell line than from each used fucoidan or heparin b. Concerp < 0.05), which further increased to 11 and 10% at 10 and 50 µg/mL (p < 0.001). Interestingly, the increasing effect of 6% at the highest concentration (100 µg/mL) was not significant , to 123 ± 8% at 50 µg/mL (p < 0.001) and to 120 ± 5% at 100 µg/mL (p < 0.001). The comparison of the suspension cell lines (HL-60 and Raji) with the adherent cell lines  revealed a significant difference as well b. In conBoth comparisons a,b did np < 0.05)  d. ortho-aminomethyl-phenylboronate units, ethylammonium units and dansyl units) is specifically increased in the presence of sulfated glycans. The so-called Polymer-H assay was shown to be an useful parameter for an initial quality screening of fucoidans [In order to get indications of potential causes for the divergent reactions of the different cell lines in response to application of the different fucoidans, we performed correlation analyses between the cell viability at the highest fucoidan concentration (100 µg/mL) and the fucoidan characteristics protein content, total phenolic content (TPC) and fluorescence intensity (FI) increase of the glycan sulfate sensor Polymer H (unpublished data) for each cell line . The fluucoidans ,32  (2 = 0.6278) (2 = 0.7644) by the fucoidans. Additionally, HL-60 viability correlated with fucose content (R2 = 0.8994) and their DS (R2 = 0.9358)  a as well 0.6278) b. In con 0.9358) .Fucus vesiculosus fucoidan from Sigma Aldrich [Fucoidans extracted from brown algae have been described to exhibit antitumor effects ,23,35, b Aldrich ,38,39,40 Aldrich . Consequ Aldrich ,37, whicMoreover, for the cell viability analyses reported in literature, many different assays were used such as WST-1, MTT, Alamar Blue, Annexin V/PI staining, cell cycle analysis, neutral red release assay, trypan blue exclusion assay, which not always deliver similar results ,36,37,41To come to a general statement about the influence of all tested fucoidans and the reference heparins on cell viability, we calculated the overall mean of all cell viability values after incubation with fucoidans (n = 7 × 9 × 4) and heparins (n = 2 × 9 × 4), respectively. The resulting means of 104.1 ± 13.1% and 100.5 ± 10.1% did not significantly differ from the control (data not shown). Additionally, in previous studies we did not find any cytotoxic effect of fucoidans measured by the release of lactate dehydrogenase (LDH) (data not shown). This leads to the conclusion that fucoidans just as heparins are non-toxic. To obtain an indication about longer incubation times, selected cell lines were treated with fucoidans and heparins for 72 h. Here, the effects were very comparable to those after 24 h of incubation, i.e., similar minor stimulating or decreasing effects on cell viability, respectively. Hence, our data show no indication of long term toxicity. However, as demonstrated by the results of this study, certain fucoidans and certain cell lines, respectively, may deviate from this general conclusion.The results of the present study are compared with published studies investigating the effects of fucoidans on the same cell lines. As cancer cell lines are genetically unstable and often differ between laboratories, results obtained with these cell lines can be heterogeneous . TherefoF.v.) for 48 h instead of 24 h, whereby they used neutral red release assay, MTT and WST-8 proliferation assay [In the leukemia cell line HL-60 treated with fucoidans, no reduction but rather an increase of cell viability was detected a. In conon assay ,39,43. Won assay , or crudon assay . After 22O2 did not [Fucus sp. and did not influence the proliferation [The influence of fucoidan purity on cell viability has previously been demonstrated by experiments with the Burkitt lymphoma cell line Raji: Crude Sigma fucoidan exerted an antiproliferative effect on Raji cells, whereas the fraction purified by ion exchange chromatography did not show such an effect . Similar did not . Moreove did not . Like thferation . Saccharina japonica and Ascophyllum nodosum on the HeLa cell line which showed no significant effect on cell viability [Cladosiphon novae-caledoniae inhibited the cell proliferation [Saravana et al. and Jiang et al. tested fucoidan from iability ,21, wherferation ,35. The feration . Ascophyllum nodosum [In line with our data on ARPE-19 cells, Dithmer et al. and Li et al. observed no toxic effect of 100 µg/mL Sigma fucoidan on ARPE-19 cells ,44. Prol nodosum . In EPC  nodosum  which fiIn the OMM-1 cell line, neither pure Sigma fucoidan  nor crudS. cichorioides, F. evanescens and U. pinnatifida on the skin melanoma cell line RPMI-7951 and showed a slight reduction of cell viability after 24 h using MTS assay, which could be due to the higher concentration [To our knowledge, the present study is the first one testing fucoidans on cell viability of A-375 skin melanoma cells, but there are some studies using B16 and RPMI-7951 skin melanoma cells. Similar to our results, Wang et al. found no effect on the cell viability of B16 cells treated with 100 µg/mL Sigma fucoidan (of undefined purity) for 48 h using the cell counting kit-8 (CCK8). This was confirmed by Ale et al. 2011, who tested crude Sigma fucoidan using MTT assay. However, 6–10 times higher concentrations reduced the B16 viability in both studies ,47. Vishntration . Furtherntration .F.v.) on HCT-116 cells [A recent study of Park et al. reported the time- and concentration-dependent effects of crude Sigma fucoidan  did not affect the cell viability up to 50 µg/mL, whereas 100 µg/mL fucoidan reduced it by about 10%, which corresponds to our findings on Sigma fucoidan [00 µg/mL .Overall, the literature confirms that different cell lines react differently to fucoidans regarding cell viability b and theL. japonica, L. cichorioides and F. evanescens, respectively, were required to induce apoptosis on human peripheral blood lymphocytes (PBL) [The viability of the two non-tumor cell lines ARPE-19 and HaCaT turned out to be not modified by the tested fucoidans a. This ies (PBL) . 0 or SubG1 or G1 phase arrest [In contrast to the non-tumor cell lines, the fucoidans slightly increased the viability of the seven tumor cell lines a. Howevee arrest ,28,51. Re arrest . The dise arrest , concente arrest ,41,51,55e arrest ,55. The S. latissima and purified Sigma fucoidan stimulated the cell proliferation measured using a BrdU-based proliferation ELISA, whereas the Sigma fucoidan (other batch than used in the present study) reduced the proliferation [In total, we detected a stimulating effect of the fucoidans on the viability of the two suspension cell lines HL-60 and Raji, whereas the mean viability of the seven adherent cell lines was not modified b. There feration . The stiferation ,56.Regarding the adherent cells, the detection of any proliferation stimulating effect is impeded by the fact that further proliferation of confluent cells is restricted because of the limited growth area. According to the overall mean of the cell viability, the heparins  showed no significant effects (100.5 ± 10.1%). Similar to the fucoidans, there were certain differences depending on the cell line, but even significant effects amounted to less than 22%. Heparin and enoxaparin, respectively, showed no effect on the proliferation on endothelial cells , human pAnalysis of the correlation between the cell viability and various characteristics of the six different fucoidans suggest that highly pure fucoidans do not impair the cell viability. The viability of HeLa cells negatively correlated with the protein content of the fucoidans a and thaDepending on the algae species the purity of the extracted crude fucoidans was shown to differ (manuscript submitted) and this characteristic turned out as the most important one for changes of the viability of the used cell lines. Due to the overall only small effects of the fucoidans on the cell viability, its impact was only observed in those three cell lines with the largest fucoidan-dependent variability. This may be also the reason for missing correlations between the cell viability and other parameters such as the molecular weight (data not shown), although that is generally considered an important parameter for the biological activities of fucoidans ,70,71. v/v) ethanol was applied for defatting of the pulverized algal material. Then, the algal material was extracted using aqueous 2% calcium chloride for 2 h at 85 °C under reflux conditions. The supernatants of the raw extracts were concentrated by evaporation and precipitated with ice-cold ethanol in a final concentration of 60% at 4 °C. The precipitate was centrifuged, dissolved in demineralized water, dialyzed and lyophilized.The algal material was kindly provided by the company Coastal Research & Management GmbH . The used algae species for extraction, their harvest date and place are shown in Fucus vesiculosus) from Sigma Aldrich , heparin (Cat. No. Y0001282) and enoxaparin (Cat. No. E0180000) from EDQM were purchased and solved in Ampuwa water .As reference compounds fucoidan , HL-60 , HCT-116  and Hep G2  were purchased from the German Collection of Microorganisms and Cell Cultures . The HeLa cell line  was a kind gift of Prof. Dr. Clement . ARPE-19 cells  were bought from ATCC . The OMM-1 cell line  was a kind gift of Dr. Sarah Coupland . The human malignant melanoma cell line A375 (skin melanoma) and the immortalized HaCaT cell line (keratinocytes) were purchased from Cell Line Service .The characteristics of the used cell lines are shown in 2.Raji, HL-60 and HeLa cell lines were cultivated in RPMI-1640  with 10% heat inactivated fetal bovine serum (FBS). The ARPE-19 cell line was cultivated in DMEM  with 10% FBS and 1% penicillin/streptomycin and 1% non-essential amino acids. OMM-1 cells were cultivated in RPMI-1640  with 10% FBS and 1% penicillin/streptomycin. HaCaT and Hep G2 cell lines were cultivated in Gibco RPMI-1640  medium supplemented with 10% FBS and 1% penicillin/streptomycin. The A-375 and HCT-116 cell line were cultivated in DMEM  after adding 10% FBS and 1% penicillin/streptomycin. All cell lines were incubated in a humidified incubator at 37 °C and 5% CO® AQueous One Solution Cell Proliferation Assay  was used to determine cell viability. The assay was performed in 96-well microplates in phenol red-free medium ). For HL-60, HeLa and Raji cells 1X Gibco GlutaMAX™-I  was added. The medium for ARPE-19 and OMM-1 was supplemented as described above in cell culture. For OMM-1 cells 2 mM L-glutamine  was added. For Hep G2, HaCaT, A-375 and HCT-116 cell lines the cell viability assay was also performed using Gibco RPMI 1640 medium as described above for cell culture. Extracts in medium were filtered through a 0.2 µm filter before treatment . The Raji (10.000 cells/100 µL) and HL-60 (15.000 cells/100 µL) cells were seeded and treated with fucoidan extracts on the same day. ARPE-19 cells (10.000 cells/100 µL) were seeded and treated at 100% confluence. OMM-1 cells were treated at 80% confluence. HeLa (3000 cells/100 µL), Hep G2, HaCaT, A-375 and HCT-116 cells (each 10.000 cells/100µL) were seeded the day before treatment with fucoidan.The CellTiter 962. The absorbance was measured at 490 nm. Cells were treated with the six different fucoidans as well as with the reference compounds  in four different concentrations  for 24 h. As control, cells treated with 100 µL medium were used. After the incubation time 20 µL of the MTS solution were added to each well and incubated for 1 h at 37 °C, 5% COt-test. A Student’s t-test with p < 0.05 was considered as significant. All bars represent mean and standard deviation.Each experiment was performed in triplicates at least four times. Data of the experiments were analyzed statistically in Microsoft Excel using the mean, standard deviation and Student’s Fucus vesiculosus, F. serratus, F. distichus subsp. evanescens, Dictyosiphon foeniculaceus, Laminaria digitata, and Saccharina latissima) as well as three reference compounds  were tested on nine different human cell lines . The cell viability was determined by the commercially available MTS assay according to a standardized test and analysis protocol for comparability of the results. After 24 h incubation of the cells with the fucoidans (1–100 µg/mL), the overall mean of the cell viability amounted to 104.1 ± 13.1% and was quite similar to that of the heparins (100.5 ± 10.1%), which are known to exhibit no antiproliferative effects. Although some individual cell viability values were significantly reduced compared to the control , no consistent antiproliferative effect was found for any fucoidan. The cell viability medians of the different fucoidans ranged from 99% to 107%, whereby the largest cell line- and concentration-dependent variability was observed for SL (86–158%) and DF (61%–129%). As indicated by the medians of the different cell lines ranging from 94% (HeLa and HaCaT) to 115% (HL-60), the cell lines differently reacted to fucoidans. Whereas cell viability of the adherent cell lines was not influenced by the fucoidans, that of the suspension cell lines  was significantly increased. In contrast to the non-tumor cell lines , the viability of the tumor cell lines was also slightly increased, but this was mainly due to the increased viability of HL-60 and Raji cells. Analyses on the role of the various analytical fucoidan characteristics for their marginally different cellular effects revealed that the effect on the cell viability is primarily dependent on the purity of the fucoidans including co-extracted compounds. The cell viability of HeLa and ARPE-19 cells negatively correlated with protein content and TPC, respectively, whereas that of HL-60 cells positively correlated with FI increase of Polymer H, fucose content and DS.Due to the heterogeneous and partly conflicting data in the literature, the aim of the present study was to investigate the effects of different fucoidans on cell viability and to compare them depending on cell line and type . For this, six fucoidans extracted from different brown algal species .


PIK3A, PTEN, and KRAS. Different gene mutations in endometrial cancer cells have varied responses to anticancer drugs and cancer therapies. The most frequently altered gene in endometrioid endometrial carcinoma tumors is PTEN. PTEN protein has lipid phosphatase and protein phosphatase activity, as well as other functions in the nucleus. Although the tumor-suppressive function of PTEN has mainly been attributed to its lipid phosphatase activity, a role for PTEN protein phosphatase activity in cell cycle regulation has also been suggested. Various tumor type-specific PTEN mutations are well documented. Here, nine endometrioid endometrial cancer cell lines with PIK3A, PTEN, and KRAS gene mutations were treated with docetaxel and radiation. One mutation with a docetaxel drug-resistant effect was a truncated form of PTEN. Among PTEN mutations in endometrial cancer cells, the Y68 frame shift mutation of PTEN constitutes a major mechanism of resistance to docetaxel treatment. The molecular mechanism involves truncation of the 403 amino acid PTEN protein at amino acid 68 by the Y68 frame shift, leading to the loss of PTEN protein phosphatase and lipid phosphatase activities.In this study, we aimed to identify mutations of key genes associated with docetaxel resistance in nine endometrial cancer cell lines. Endometrial cancers are associated with several critical gene mutations, including  Endometrial cancers are divided two types, estrogen dependent (type I) and estrogen independent (type II). Type I is the most common type of endometrial cancer. Type II cancers include clear cell carcinoma, mucinous adenocarcinoma, and papillary serous adenocarcinoma, which are less common types of endometrial adenocarcinomas.Endometrial cancer is one of the most common gynecological malignancies in women worldwide6. However, no standard management modality is available. Adjuvant chemotherapy and radiotherapy in the “sandwich” sequence were adopted to help identify the most effective adjuvant method for patients with advanced disease11.Early stage diseases can have good outcomes through surgery, chemotherapy, radiotherapy or hormonal therapy, while advanced diseases are more likely to recur and require adjuvant chemotherapy and radiotherapy. The combination of chemotherapy and postoperative radiotherapy has been used in the treatment of advanced endometrial cancer14. Currently, chemotherapeutics remains the primary treatment for endometrial cancer. However, a major problem with chemotherapeutics is drug resistance. Therefore, the identification of genetic mechanisms involved in the chemotherapeutic response is critical for predicting the drug response of tumors with gene mutations. We propose that critical mutations of the tumor suppressor gene PTEN may be the major chemotherapeutic resistant factor in the treatment of patients with docetaxel-resistant endometrial cancer.Type I and type II endometrial cancers contain more than 20 gene mutations. Thus, improving our understanding of the disease at the molecular level and finding more effective strategies are importantPIK3A, PTEN, KRAS and FGFR2 might affect adjuvant treatment of endometrial tumors18. Radiation therapy is a key therapeutic strategy for endometrial carcinomas. However, how different gene mutations affect radiation sensitivity and drug responses remains unknown. Currently, treatment for metastatic or recurrent disease is based on the conventional chemotherapy method. Despite the different gene mutations in endometrial cancers, most clinical treatments have not taken this diversity into account20.Frequent mutations in PTEN lead to deregulation of the cell cycle21. PTEN suppresses the progression of the cell cycle through reduced cyclin D1 and increased p27.Gene mutations in PIK3A, PTEN, KRAS and FGFR2 gene mutations and five different mutations of PTEN in endometrioid endometrial carcinoma (EEC) cells to identify the mechanisms of docetaxel chemotherapy and radiation therapy resistance for different mutations in endometrial carcinomas. Cells were exposed to a chemotherapy drug (docetaxel), ionizing radiation (2 Gy) or a combination of both (sandwich method). Drug responses and radiosensitizing effects were evaluated using MTT assays and xCELLigence Real-Time Cell Analysis (RTCA). The effects of treatment with different doses of the chemotherapy drug (docetaxel) were evaluated following exposure to ionizing radiation (2 Gy).Here, we aimed to investigate the roles of We present multiple analyses of MTT assays and xCELLigence RTCA of 9 EEC cell lines treated with docetaxel chemotherapy and radiation. This integrated analysis provides the molecular parameters of different responses of endometrial carcinoma cells with various gene alterations, which may have a direct effect on treatment recommendations for patients. Our analysis also provides references for gene mutation-based clinical practice and novel treatments involving docetaxel chemotherapy and radiation.21.The effects of docetaxel on malignant cell growth were studied in a panel of 9 established human endometrial cancer cell lines. The individuality of each cell line was confirmed by mitochondrial DNA sequencing immediately after receipt from the collaborating research laboratory. Cell lines were passaged for less than 6 months after authentication and SPAC-1-L cell line was confirmed by PCR and sequencing experiments. Ishikawa cells were obtained from the European Collection of Animal Cell Cultures. The established human endometrial carcinoma cell line HEC155 was obtained from the Japanese Health Science Research Resources Bank. The cell line SPAC-1-L was provided by the laboratory of Dr. Y. Hirai from the Department of Gynecology, Cancer Institute Hospital . Dr. A. Santin provided ARK1 (USPC1) and ARK2 (USPC2) cells from the Division of Gynecologic Oncology at the University of Arkansas . The cell lines were cultured in Modified Eagle’s Medium (MEM) supplemented with 10% heat-inactivated FBS, 2 mmol/L glutamine and Antibiotic-Antimycotic Solution 50 for a given drug were nominally assigned the highest concentration screened . Experiments were performed in triplicate for each cell line. The association between a cell line and the drug response was determined using a two-tailed P value less than 0.05, which was considered statistically significant.Cells were plated in 96-well plates at a density of 2,000 cells per well, and untreated control cells were optimized to 85 to 95% confluence at the endpoint of the experiment. After 24 hours, cells were treated with radiation doses (2 Gy) and different concentrations  was used according to the manufacturer’s instructions. Briefly, the working mechanism of the system can be summarized as follows. The xCELLigence System allows continuous measurement and quantification of cell adhesion and proliferation. This system uses 96-well E-plates, and these disposable plates are suitable for single use. On the bottom of the wells, the plates have a base containing gold microelectrodes. There is 70% electrode coverage on the bottom of the wells. The electrical impedance of these sensor electrodes is recorded to monitor the changes in the cells. Changes in electrical impedance are presented with a parameter called the “cell index (CI)”. When there are no cells in the wells, electrode impedance and the CI are zero. After seeding cells, the CI will increase. The increase in the CI correlates with the increase in attached cell numbers. When more cells are attached to the surface of the E-Plate, the CI increases. Additionally, instead of cell numbers, cell viability and the strength of cell adhesion can cause changes in the CIA cell proliferation experiment was performed using an xCELLigence System. When EEC cells reached 80% confluence in a 6-well plate, the cells were washed with PBS. Then, the cells were treated with 0.05% trypsin/EDTA. After 2 min, 5 mL complete medium was added to the wells. The cell resuspension was centrifuged at 400 × g for 5 min. The pellet was resuspended in 5 mL media, and the cells were counted by using a hemocytometer. A total of 5,000 cells per well were seeded in an E-Plate, and the total volume of the wells was adjusted to 100 µL with media. The E-Plate was incubated overnight in a cell culture incubator. Before measuring the cells in the E-Plate, a standard background was measured by adding 50 μL complete medium at 37 °C to the wells. EEC cells were monitored every 15 min for 144 hours. Data are presented as a normalized CI.PIK3A, PTEN and KRAS mutations. Four cell lines  were obtained from patients with type II uterine serous papillary endometrial carcinoma. The MFE319 cell line originates from an endometrial adenosquamous carcinoma. The remaining cell lines are derived from type I EECs24. Among these cell lines, five out of nine (56%) EEC cell lines harbor PTEN gene mutations in different positions . We irradiated each of the 9 EEC cell lines with 0, 1, 2, 4, 6, 8, 11, or 15 Gy of X-rays and measured by MTT. The results showed the irradiation dose of 2 Gy not only partially reduced survived but also reserved a fraction room for docetaxel chemotherapeutics. Therefore the irradiation 2 Gy is an appropriate dose for the studying combination of irradiation and chemotherapeutics of docetaxel to EEC cells. We exposed nine EEC cell lines to different concentrations of the chemotherapy drug docetaxel, radiation (2 Gy) and both treatments for two or five days (mean of at least 3 independent experiments performed in triplicate), followed by MTT assays.PTEN gene mutations are common in EEC cell lines. We investigated the responses of nine EEC cell line panels to docetaxel chemotherapy and radiation to determine docetaxel drug resistance in association with 10-IC90) was identified using a wide range of drug concentrations (0.5–100 ng/ml). Docetaxel inhibited the proliferation of all endometrial cancer cell lines investigated in a concentration-dependent manner. However, the IC50 values significantly varied between individual cell lines with up to a 50-fold difference in the IC50 values and ranged between 1.95 in ARK1 endometrial cells with a PI3K gene mutation and 105.82 in SAPC-1-L cells that harbor a PTEN mutation (E288 frame shift /Y68 frame shift)  were less sensitive to docetaxel with a mean IC50 of 18.2 ng/ml, while ECC1 cells were sensitive to 2 Gy radiation with a relative viability ratio of 0.29. HEC155 cells were less sensitive to docetaxel with an IC50 value of 19.7 ng/ml, but these cells were sensitive to radiation with a relative viability ratio of 0.48 with a 2 Gy irradiation dose.The effective dose range for drug docetaxel  responded to docetaxel with low IC50 values of 5.28 and 7.36 to higher values of 18.21 ng/ml. ISHIKAWA and MEF319 cells were sensitive to docetaxel in a dose-dependent fashion from a low drug concentration of 1 ng/ml  were sensitive to docetaxel with a dose-dependent reduction from a low drug concentration of 1 ng/ml, with a drug IC50 value of 2.63 ng/ml. Additionally, SAPC-1-L cells that harbor a PTEN mutation (E288 frame shift/Y68 frame shift) were resistant to docetaxel, exhibiting a dose-dependent reduction with a high drug IC50 value of 105.8 ng/ml, which suggests that the Y68 frame shift mutation of PTEN may be associated with docetaxel resistance in endometrial cancer cells.Exposure of ECC-1, ISHIKAWA, MFE319, SNG-II and SPAC-1-L human endometrial cancer cells with Because SPAC-1-L cells were highly resistant to docetaxel, we sought other methods (radiation) to more effectively kill these cells. The survival rates of ARK1, ARK2, ECC-1, HEC155, HEC-1-A, ISHIKAWA, SNG-II and SPAC-1-L cells ranged from 0.65 to 0.96 with a 2 Gy dose for two days and from 0.28 to 0.57 with a 2 Gy dose for five days. MFE319 cells were resistant to radiation (2 Gy), with survival rates of 0.98 for two days and 0.75 for five days  EC cell lines harbored PTEN gene mutations in different amino acid change positions (ECC-1/V317 frame shift). The docetaxel IC50 values of the ECC-1, ISHIKAWA, MFE319, and SNG-II cell lines with PTEN mutations were 18.21 ng/ml, 7.36 ng/ml, 5.28 ng/ml and 2.63 ng/ml, respectively. These IC50 values were similar to those of cell lines with other gene mutations . The docetaxel IC50 value of SPAC-1-L with the Y68 frame shift mutation of PTEN was 105.8 ng/ml, which was five times greater than the IC50 values of cell lines with other PTEN mutations and other gene mutations.Due to the loss of the core motif  and C2 tensin-type domain (residues 186–351), the Y68 frame shift leads to the loss of the protein phosphatase and lipid phosphatase activities of in vitro findings demonstrate that loss of PTEN expression with the Y68 frame shift mutation predicts a lack of benefit from docetaxel in patients during endometrial cancer chemotherapy. Therefore, other chemotherapy drugs should be considered instead of docetaxel. This novel potent biomarker might lead to further refinement of therapy for patients with endometrial cancer with a Y68 frame shift mutation of PTEN, facilitating better outcomes, a better toxicity profile, and more effective health care delivery. Due to the relevance of the Y68 frame shift mutation and docetaxel resistance in endometrial cancer, this mutation is a predictive marker worth further preclinical and clinical evaluation.In this study, we found an association between the Y68 frame shift and increased resistance to docetaxel. Our preclinical Since cancer is one of the leading causes of death worldwide, finding better treatments is an urgent need. Although remarkable progress has been made towards understanding the molecular and cellular mechanisms of cancer development and treatment in recent years, the clinical management of cancer remains a challenge. Currently, chemotherapeutics and radiation therapy remain the predominant options for cancer therapy. However, resistance to chemotherapy in cancer is common. Therefore, the identification of gene mutations involved in the chemotherapeutic response is critical for predicting tumor responses and treating patients with drug-resistant cancer with different gene mutations. In this study, we sought to identify genetic mutations associated with resistance to facilitate effective combination therapy.28. Mutations of the RAS gene are also very common, accounting for more than 20% of mutations in human tumors29. Moreover, the fibroblast growth factor receptor (FGFR) pathway is involved in the pathogenesis and progression of endometrial cancer32. These gene mutations hold great promise for more effective treatment strategies for this disease.Therapeutic strategies could be improved according to appropriate biomarkers and patient-specific mutation profiles to achieve more benefits from combination therapies. The PI3K pathway is frequently activated in most EECsMost endometrial carcinomas are diagnosed at an early stage. Nevertheless, 15–20% of these carcinomas recur with limited effects of systemic therapies in metastatic disease. Recent progress in the identification of genetic abnormalities in endometrial cancer has spurred the development of novel strategies for the treatment of patients with endometrial cancer. Novel strategies involving therapies with greater efficacy and reduced toxicity are needed. Currently, mutation-based treatments are not available in the clinic; thus, more systematic approaches to the integration of biomarkers in clinical trials of therapeutic agents are needed for clinical implementation.50 values. Surprisingly, significantly higher IC50 values were associated with a Y68 frame shift mutation of PTEN in SPAC-1-L cells. The role of the Y68 frame shift mutation of PTEN in endometrial cancer has not yet been studied. However, our preclinical findings suggest that the PTEN Y68 frame shift mutation is associated with resistance to docetaxel in endometrial cancer.To date, no studies have investigated the responses of endometrial cancer cells with different gene mutations to docetaxel chemotherapy. Here, we show a concentration-dependent reduction in the viability of nine endometrial cancer cell lines in response to docetaxel. However, the response significantly varied among individual cell lines, with differences of up to 40 times in IC33.PTEN is an essential tumor suppressor gene that antagonizes the PI3K/Akt/mTOR antiapoptotic pathway and is often abnormal in endometrial cancers. Impairment of this tumor suppressor pathway potentially becomes a causal factor for the development of malignancies. Activation of the PI3K/Akt/mTOR pathway is implicated in the pathogenesis of malignancies and the development of resistance to anticancer therapiesPI3K gene mutation are sensitive to docetaxel with an IC50 of 1.95 ng/ml, while ECC-1 cells with a PTEN mutation are nine times more resistant to docetaxel with an IC50 of 18.21 ng/ml  based on the real-time response curves of radiation. When docetaxel concentrations were higher than 1 ng/ml, the drug achieved a greater therapeutic effect than did the combination of drug and radiation , SNG-II (E288 frame shift) and SPAC-1-L (E288 frame shift/Y68 frame shift) cells with a ion Fig. . CompareThe main goal of radiation therapy is to kill cancer cells by destroying their multiplication (cell division) potential. Resistance to chemotherapy and radiation therapy is a major problem facing current cancer research. In the current study, we evaluated the therapeutic effect of docetaxel with or without the addition of radiation  in nine endometrial cancer cell lines.37. Given the known molecular mutations, we hypothesized that EEC cell lines with activating the Y68 frame shift mutation of PTEN are resistant to irradiation therapy compared to EEC cell lines with wild-type PTEN.An antagonistic interaction was observed for docetaxel plus radiation 2 Gy) in ECC-1, HEC155, HEC-1-A, ISHIKAWA, MFE319, SNG-II and SPAC-1-L cells. In Fig.  Gy in EC50 value of 17.3 ng/ml. Since HEC-1-A cells were with PI3K and KRAS mutations and wild type PTEN, they were more sensitive to radiation with a relative viability ratio of 0.28 with a 2 Gy irradiation dose. SPAC-1-L is mutated with the Y68 frame shift mutation of PTEN, and then it is more resistant to radiation. This result supports hypothesis that SPAC-1-L is mutated with the Y68 frame shift mutation of PTEN induces docetaxel resistance in endometrial carcinoma cells.HEC-1-A cells were less sensitive to docetaxel with an ICBased on data from the xCELLigence Real-Time Cell Analyzer System, an antagonistic interaction was observed for docetaxel plus irradiation (2 Gy) in a dose-dependent manner with drug concentrations of 0, 1, 5, and 10 ng/ml in the HEC155 cell line Fig.  for fivePTEN gene led to docetaxel drug resistance in chemotherapy of endometrial cancer cells by destroying the protein phosphatase and lipid phosphatase activities of PTEN. Compared with treatment with docetaxel alone, the combination of docetaxel and X-ray (2 Gy) radiation resulted in a significant decrease in overall cytotoxicity for most endometrial cancer cells. The radiation sensitizing effects of docetaxel on endometrial cancer cells were observed at very low drug levels, far below the levels required for cytotoxic effects. Our results demonstrated that the addition of X-ray radiation interfered with the cytotoxic effects of docetaxel and might provide further proof for the cell cycle-dependent interaction.Our results showed that the Y68 frame shift mutation of the


Intervertebral disc degeneration (IDD) is characterized by excessive apoptosis of nucleus pulposus (NP) cells and hyperactive extracellular matrix (ECM) catabolism. Our previous studies revealed the relationship between human islet amyloid polypeptide (hIAPP) and NP cell apoptosis. However, the role of hIAPP aggregates in IDD has not yet been investigated. This study aimed to determine whether the accumulation of hIAPP aggregates promotes IDD progression. The aggregation of hIAPP increased in human NP tissues during IDD. The deposition of hIAPP aggravated the compression-induced IDD that promoted NP cell apoptosis and ECM degradation via IL-1β/IL-1Ra signaling in an ex vivo rat disc model. Moreover, neutralizing IL-1β augmented the protective effects of hIAPP overexpression by decreasing hIAPP aggregation in human NP cells. These results suggest that the aggregation of hIAPP promotes NP cell apoptosis and ECM degradation ex vivo and in vitro by disrupting the balance of IL-1β/IL-1Ra signaling. Intervertebral disc degeneration (IDD), is caused in part by a protein, hIAPP, that is secreted by the cells that make insulin, a blood sugar regulator. In IDD, the nucleus pulposus in disc degenerate, causing low back pain. Recent research linked formation of hIAPP aggregates with death of intervertebral disc cells. Given inflammation’s known role in IDD, Cao Yang at the Huazhong University of Science and Technology, Wuhan, China, and colleagues investigated how hIAPP and inflammation interact in IDD. Using a method they developed to model IDD by applying compression to cultured rat intervertebral disc cells, they showed that accumulation of hIAPP aggregates triggered inflammation and accelerated cell death. Blocking the inflammation decreased hIAPP aggregation and slowed IDD progression. Methods to disrupt hIAPP aggregation hold promise for treatment of IDD. Many factors, such as obesity, smoking and diabetes mellitus (DM), are thought to be involved in IDD progression3. However, the etiology and pathogenesis of IDD remain unknown. The intervertebral disc (IVD) functions as a buffering and conjunction organ for bearing mechanical loads and maintaining spinal stability; it is composed of the inner nucleus pulposus (NP) and the outer annulus fibrous (AF)4. IDD is characterized by the depletion of resident cells and the degradation of the extracellular matrix (ECM)5. An increase in the apoptosis level of NP cells contributes to excessive cell death, which accelerates the progression of IDD. Recent studies have reported that the amelioration of NP cell apoptosis exerts a therapeutic effect on the progression of IDD8. However, further research on the prevention and reversal of IDD progression is necessary.Low back pain (LBP) associated with intervertebral disc degeneration (IDD) is a common cause of disability worldwide; ~80% of people will suffer from LBP at some point in their lives10. Recent studies have revealed that the aggregation of hIAPP contributes to cell death by inhibiting autophagy, inducing oxidative stress, and promoting inflammatory cytokine release and cell membrane disruption15. IAPP is a normally soluble protein that is released by cells and can form insoluble toxic aggregates11. Previously, we showed that hIAPP expression is related to the development of IDD and that IAPP may regulate ECM metabolism and control the crosstalk between apoptosis and autophagy in NP cells17. Many studies have reported that hIAPP aggregates are toxic and result in islet cell apoptosis in vitro and in vivo. The aggregation of hIAPP has been identified in the pancreas and other extra-pancreatic tissues18. Previous studies have revealed that extracellular components, such as glycosaminoglycan, can accelerate the deposition of hIAPP, especially in an acidic environment20. NP tissues are abundant in the ECM, and a relatively low pH has been confirmed in NP tissues during IDD21. This suggests that hIAPP aggregates may play an important role in IDD progression. However, the aggregation and deposition of hIAPP in NP tissues during IDD has not yet been investigated.Human islet amyloid polypeptide (hIAPP), a 37–amino acid polypeptide, has been confirmed to damage cells through the formation of aggregates24. The proinflammatory cytokine interleukin-1β (IL-1β) is highly expressed in degenerative IVD tissues and has been proposed to be a major mediator of inflammation in the progression of IDD25. The stimulation of IL-1β leads to a significant increase in the apoptotic rate in NP cells and AF cells26. Moreover, as a proinflammatory mediator, IL-1β regulates the production of various cytokines, chemokines, and matrix metalloproteinases (MMPs), as well as a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs), resulting in inflammation and ECM degradation during IDD26. In degenerative disc tissues, IL-1β increases rapidly, while IL-1 receptor antagonist (IL-1Ra) decreases consistently28. An imbalance between IL-1β and IL-1Ra accelerates the progression of IDD to some degree. A recent study showed that hIAPP aggregates induce FAS upregulation, caspase activation, and cell apoptosis by stimulating the expression of IL-1β and reducing the release of IL-1Ra29. Moreover, IL-1β impaired IAPP processing, thereby potentiating the formation of aggregates and facilitating the activation of cell death signaling29. We surmise that IL-1β/IL-1Ra signaling may be involved in IDD by interacting with the deposition and aggregation of hIAPP.Accumulating evidence suggests that inflammation plays a critical role in the pathogenesis of IDDAs of now, the role of the deleterious hIAPP aggregates in IDD progression has not yet been investigated. We hypothesized that hIAPP aggregates may accelerate NP cell apoptosis and ECM degradation and may also be related to IL-1β/IL-1Ra signaling. To test this hypothesis, we conducted our experiments using tissue samples and an ex vivo rat disc model Fig. . The depThe experimental protocols were approved by the Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology. Written informed consent was obtained from all participants involved in our study. Degenerative human NP tissues were collected from patients who underwent disc excision and spinal fusion surgery for IDD, while normal human NP tissues were obtained from patients who underwent deformity correction surgery for scoliosis. Detailed information for each patient is listed in Supplemental Table 2 to simulate the physiologically hypoxic disc environment. The culture medium was replaced twice each week, and the cells from the second or third passage were prepared for the following experiments.Isolated NP tissues were cut into pieces and enzymatically digested in 0.2% type II collagenase and 0.25% trypsin  for 3 h. After being filtered and washed in PBS, the suspension was centrifuged, and the isolated cells were cultured in Dulbecco’s modified Eagle medium (DMEM) containing 15% fetal bovine serum  and 1% penicillin/streptomycin . The cells were incubated at 37 °C under 5% O2) with saturated humidity. The culture medium was replaced twice each week.IVDs were collected from Sprague-Dawley rats  with the ethical approval of the Animal Experimentation Committee of Huazhong University of Science and Technology. Caudal discs with complete endplates were isolated and cultured in DMEM containing 15% fetal bovine serum and 1% penicillin/streptomycin . The osmolarity of the culture medium was adjusted to 400 mOsm, and physiological conditions were approximated by the addition of 1.5% of a 5 M NaCl and 0.4 M KCl solution. Samples were incubated under a hypoxic atmosphere  of double-distilled water to maintain a humidified atmosphere. After mixed compressed air was pumped into the compression apparatus, it was applied to deliver 1.0 MPa static compression for 2 weeks.IVD organs or NP cells were cultured in a compression apparatus with a pressure of 1.0 MPa, as described previously32.The NP tissues or rat discs were washed in PBS, fixed with buffered formaldehyde  for 12 h, decalcified in a 10% formic acid solution, dehydrated by ethanol, and embedded in paraffin. The specimens were sectioned at 4 µm, and the sections were deparaffinized, rehydrated, and stained with hematoxylin and eosin (HE), safranin-O/fast green, Masson’s trichrome or alcian blue. The histological grades of the specimens were assessed according to standards established previously17. Briefly, NP cells or tissues attached to slides were fixed with 4% paraformaldehyde and then permeabilized with 0.2% Triton X-100  in PBS. The slides were washed in PBS, blocked with 2% bovine serum albumin in PBS for 2 h at 37 °C and then incubated with primary antibodies against caspase-3 , caspase-9 , IL-1β , IL-1Ra , COL2A1 , and FAS . After being washed twice, the slides were subsequently treated with a secondary goat anti-rabbit antibody  at 37 °C for 2 h. Nuclei were costained for 5 min with 0.1 g/mL 4-6-diamidino-2-phenylindole . Images were captured under a microscope .Immunofluorescence analysis was performed, as previously describedTerminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was used to assess cell apoptosis. Cells were fixed in 4% paraformaldehyde for 1 h and then treated with 0.5% Triton X-100 for 10 min. After being washed with PBS, the cells were incubated with reagents from a TUNEL reaction kit  according to the manufacturer’s instructions. Thioflavin S (Th-S) staining was used to evaluate the deposition of hIAPP aggregates. After being washed with PBS, the fixed cells were incubated with Th-S solution . All of the images were captured under a fluorescence microscope .Total RNA was extracted from NP cells with Trizol reagent  and then reverse-transcribed and amplified by RT-qPCR according to standard protocols. The sequences of the primers used for RT-qPCR are listed in Supplemental Table 16. The following antibodies were used: hIAPP , FAS , FASL , Bax , Bcl-2 , VDAC1 , cyto-c , cleaved caspase-3 , cleaved caspase-9 , MMP3 , MMP9 , MMP13 , ADAMTS5 , SOX9 , aggrecan , COL2A1  and GAPDH . Horseradish peroxidase-conjugated secondary antibodies  were used, and the protein bands were visualized and detected using an enhanced chemiluminescence system. GAPDH was used as a loading control. The experiment was performed in triplicate.Proteins were isolated from cells or tissues using a Western cell lysis kit (Beyotime) according to the manufacturer’s instructions. The procedures and analysis were performed using standard protocolsg for 20 min to remove dead cells and debris. The IL-1β or IL-1Ra content in the supernatant was analyzed using an ELISA kit for IL-1β or IL-1Ra  according to the standard protocol. The experiment was performed in triplicate.The cell culture supernatant was collected and centrifuged at 1000 × 33. The shRNA sequences are listed in Supplemental Table NP cells were seeded and cultured in six-well plates and reached 70–80% confluence before transfection. For hIAPP overexpression, NP cells were infected with pLVX-mCMV-ZsGreen-PGK-Puro or pLVX-mCMV-ZsGreen-PGK-Puro-hIAPP according to the manufacturer’s instructions. For shRNA knockdown, three shRNA plasmids were designed, and their efficiencies were tested Fig. . To exclt-test and one-way or two-way analysis of variance (ANOVA) with Tukey’s post hoc test were used to assess the changes in the effects for each experimental group. Statistical significance was set to P < 0.05 and determined using GraphPad Prism 7 software .All experiments were performed independently at least in triplicate, and the data are presented as the mean ± standard deviation (SD). Student’s Histologic staining was used to evaluate the specimens from patients with scoliosis (IS) or IDD diseases. The results indicated that, compared to IS tissues, IDD tissues showed degenerative changes by HE, Masson’s, and alcian blue staining Fig. . To deteTo evaluate the effects of hIAPP aggregates on IDD, we used an ex vivo model of IVD organ culture. rat discs were treated with exogenous hIAPP  and cultured in a compression apparatus to induce IDD progression Fig. . Th-S stTo further investigate the effects of hIAPP aggregates on IDD, the levels of proteins involved in ECM metabolism and cell apoptosis were measured by Western blot analysis Fig. . hIAPP d17. In addition, the effect of neutralizing IL-1β on ECM remodeling and cell apoptosis was evaluated by Western blot analysis  and the downregulation of the expression of anabolism genes  were observed in IDD in our compression-induced degeneration model. Meanwhile, pro-apoptotic proteins  were increased and the anti-apoptotic protein Bcl-2 decreased in compression-induced degeneration. These results indicated that mechanical loading effectively induced IDD in this ex vivo model. Studies have shown that the structure of mouse IAPP is different from that of human IAPP and that mouse IAPP does not form toxic aggregates44. In an ex vivo rat model, exogenous hIAPP was applied to rat discs during compression-induced IDD. The disc samples from the exogenous hIAPP group showed a higher histological grade and obvious characteristics of degeneration.The ex vivo culture system in our study simulated the in vivo IVD environment. The IVD organ culture, including the intervertebral disc and adjacent endplates, allowed nutrients and outer substances to penetrate into the inner disc while leaving the IVD structure and native ECM intact45. Accumulating evidence has suggested that the oligomers rather than the monomers of hIAPP are responsible for cell apoptosis11. The mature monomer form of hIAPP has been found to be nontoxic to cells while prone to aggregation in T2D individuals; however, the underlying mechanism is currently unknown48. Our previous studies showed that the overexpression of hIAPP in human AF or NP cells inhibits apoptosis and that the downregulation of hIAPP expression induces the death of disc cells17. In the present study, the analysis of IVD tissue samples suggested that hIAPP deposited and aggregated in degenerative IVD tissues. In an ex vivo model, the catabolism- and apoptosis-related genes were upregulated, while ECM degradation and excessive NP cell apoptosis were observed to increase along with the number of hIAPP aggregates. These results demonstrated that the formation of toxic hIAPP aggregates might occur as a consequence or as a cause of IDD. Several studies have reported that hIAPP aggregates faster in acidic environments and maintains a soluble nontoxic form at a higher pH44. IDD has been confirmed to be related to an increase in lactic acid levels and the intradiscal pH49. This partly explains the significant deposition of hIAPP aggregates in IDD tissues. However, the underlying mechanisms of hIAPP aggregation in IVD tissues remain unclear and require further study.Human IAPP is always deposited in matrix-abundant tissues and has been confirmed as the primary culprit in pancreatic cell loss12. A recent study showed that IL-1β plays a dual role in hIAPP-induced cell damage29. IL-1β impairs the processing of hIAPP, resulting in the aggregation of its immature form, which is easily deposited in human organs or tissues. Moreover, hIAPP aggregates also stimulate the expression of IL-1β, causing a vicious cycle. During the progression of IDD, the increased level of IL-1β expression has also been detected, and IL-1β has been confirmed as a critical mediator of IDD51. It has been suggested that excessive IL-1β promotes the aggregation of hIAPP during IDD progression and impairs the formation of nontoxic mature hIAPP monomers. Inflammation and ECM degradation play critical roles in the IDD process. Hyperactive ECM catabolism and inflammation have been characterized as the major phenotypes in degenerative IVD tissues24. Accumulating evidence has shown that mechanical compression affects IVD biology and that static compression promotes the progression of IDD52. Moreover, an overload of mechanical compression has been confirmed to induce the expression of proinflammatory cytokines and promote ECM catabolism in NP cells53. During the process of disc degeneration, IL-1β is upregulated, while IL-1Ra levels decrease, leading to hyperactive IL-1β signaling and accelerating the progression of IDD27. Park et al. reported that increased IL-1β levels impair the processing of pro-hIAPP and promote the deposition of immature hIAPP aggregates, which ultimately damages islet cell function29. In our ex vivo disc model, cotreatment with neutralizing IL-1β antibodies (nIL-1β) and hIAPP ameliorated IDD progression under static compression, while cotreatment with IL-1Ra (nIL-1Ra) antibodies and hIAPP accelerated the progression of IDD. Our results suggested that mechanical loading may promote the aggregation of hIAPP in NP tissues by regulating IL-1β/IL-1Ra signaling.The pathogenic mechanism of hIAPP aggregates has been confirmed to be closely related to the production of IL-1β16. This finding may be explained by the observation that hIAPP overexpression increases the formation of nontoxic hIAPP monomers and decreases the ratio of monomeric hIAPP aggregates. The number of hIAPP aggregates and the ratio of IL-1β/IL-1Ra both increased during compression-induced NP cell degeneration. However, the levels of IL-1β and IL-1Ra both decreased when NP cells directly overexpressed with hIAPP, indicating a balance in IL-1β signaling. Moreover, the blockade of IL-1β signaling with nIL-1β reduced the deposition and aggregation of hIAPP, which ameliorated the toxicity of hIAPP aggregates in the nIL-1β group compared to the control group. In contrast, the toxicity of hIAPP aggregates in NP cells was enhanced upon treatment with nIL-1Ra. These results suggested that hIAPP aggregates exacerbated the IDD process by accelerating the imbalance in IL-1β/IL-1Ra signaling.Interestingly, we observed that the overexpression of hIAPP decreased the level of IL-1β, reduced ECM degradation and attenuated apoptosis in NP cells. Consistent with our previous study, the overexpression of hIAPP exerted a mostly protective effect in NP cells in vitro11. A previous study showed that treatment with hIAPP induces FAS expression as well as the transcription of downstream genes54. The classical pathway is mediated by FAS receptor binding, resulting in the activation of the FAS-associated death receptor pathway. Caspase-8 is then activated and released into the cytoplasm, leading to the activation of caspase-9 and caspase-3 or the release of other pro-apoptotic factors, such as Bax, VDAC1, and cyto-C55. Moreover, the intrinsic apoptotic pathway can be elicited by various kinds of cellular stresses. The abnormal aggregation of hIAPP has been demonstrated to induce severe endoplasmic reticulum (ER) stress and oxidative stress as well as the unfolded protein response (UPR)48. Prolonged or severe UPR promotes the degradation of abnormal and normal cellular proteins and the expression of pro-apoptotic genes, ultimately causing cell apoptosis57. Inhibiting ER stress reduces the formation of hIAPP aggregates and ameliorates cellular dysfunction9. Therefore, severe cellular stresses, such as the mechanical loading-induced cellular stress used in our study, are potential mediators of hIAPP-induced NP cell apoptosis. In addition, several studies have reported that hIAPP aggregates enhance cell death by impairing autophagic flux; the activation of autophagy can attenuate the detrimental effects of hIAPP aggregates59. Autophagy is considered to be a cell self-protective mechanism, and impaired autophagic flux has been confirmed to promote the deposition of hIAPP aggregates13. Consequently, we hypothesize that hIAPP damages cells by impairing the balance of cellular protein processing, such as through autophagic degradation and ER stress, which accelerates the formation of hIAPP aggregates. In fact, the pathogenic mechanisms of hIAPP may be involved in the activation of the apoptotic signaling pathway, excessive cellular stresses, autophagic dysfunction, and more. A great deal of research is necessary to explain the underlying mechanisms.Our results showed that hIAPP aggregates accelerated IDD progression and caused excessive cell apoptosis. During the IDD process, hIAPP aggregates may induce NP cell death by activating apoptotic signaling. The immature form of hIAPP, which presents as a toxic molecule that induces death signaling, including the FAS and caspase-3-dependent signaling pathways, is more likely to aggregate and be depositedThe present study has certain limitations. First, the ex vivo model used in our experiments to mimic the IDD process has more advantages than monolayer cell culture, but it still does not fully represent in vivo conditions, especially during long-term IDD progression. Second, exogenous hIAPP treatment was used in our ex vivo experiments because mouse hIAPP cannot form aggregates spontaneously. However, the results would be more convincing if hIAPP transgenic mice has been used in this study. Third, the pathogenic mechanisms of hIAPP aggregates discussed in our study may represent only a small fraction of the mechanisms at play. More specific mechanisms of hIAPP aggregates in the progression of IDD still require further investigation.In summary, we demonstrated that hIAPP aggregation in NP cells is associated with IDD progression. The deposition of the toxic hIAPP aggregates promoted NP cell apoptosis and ECM degradation by disturbing the balance of IL-1β/IL-1Ra signaling in ex vivo and in vitro systems. Therefore, methods aimed at inhibiting the formation of hIAPP aggregates in IVD tissues may prove beneficial for the treatment of IDD.Supplementary Information


This study investigated the impact of immune-related pathway inhibition, among them indolamine 2,3-dioxygenase (IDO), alone and together with immune cells on growth and viability of colorectal cancer (CRC) cells. A panel of patient-derived CRC cell lines with different molecular characteristics  was included. Initial phenotyping of CRC cell lines (n=17) revealed high abundance of immunosuppressive checkpoint-molecules in general, but an individual profile for IDO. Presence of immune-related molecules was independent of the molecular subtype. Selective treatment of CRC cell lines showing high or low IDO expression (n=2 cell lines each) was performed with single agents and combinations of Indoximod, Curcumin, and Gemcitabine with and without the addition of peripheral blood lymphocytes (PBL) in an allogeneic setting. All substances affected CRC cell growth in a cell line specific manner. The combination of Curcumin and Gemcitabine proved to be most effective in tumor cell elimination. Functional read-out analyses identified cellular senescence, after both single and combined treatment. Curcumin alone exerted strong cytotoxic effects by inducing early and late apoptosis. Necrosis was not detectable at all. Addition of lymphocytes generally boosted antitumoral effects of all IDO-inhibitors, with up to 80 % cytotoxicity for the Curcumin treatment. Here, no obvious differences became apparent between individual cell lines. Combined application of Curcumin and low-dose chemotherapy is a promising strategy to kill tumor target cells and to stimulate antitumoral immune responses. Vice versa; their high immunogenicity forces MSI+ tumor cells to escape the otherwise effective immune attack by creating an immunosuppressive microenvironment. Additionally to downregulate major histocompatibility complex (MHC) class I on the tumor cells' surface and inactivation of the antigen-processing machinery, upregulation of immune-checkpoint-molecules (such as programmed cell death 1 (PD-1)/programmed cell death 1 ligand 1(PD-L1)) represents another escape mechanism . Authenticity for these lines was verified by means of short tandem repeat fingerprinting  from genomic DNA according to  and cho+ cell lines. CIMP-associated cell lines' response was weaker.Substance combinations enhanced antitumoral effects in almost all cases . Best kiγ pretreatment, described to induce IDO expression and rendering cells more vulnerable to cytolysis [data not shown).Sequential treatment (preincubation with Indoximod for 72 h) did not boost effects of the monotherapies. Thus, the direct cytotoxic potential of Indoximod on CRC cells was rather low. Even interferon-ytolysis , did notμM led to a heterogeneous expression profile, with an increased expression of DNA-damage response gene ATM in MSI+ cell lines HROC257 T0 M1 and HROC50 T1 M5. Expression of CDKN2A and CCNE1, both being involved in cell cycle regulation, was largely unchanged ( ATM and CDKN2A (p < 0.05 versus control). CCNE1 and MDM2 were also upregulated in this combination  and HROC257 T0 M1 (IDOhigh) cells.We next aimed to unravel the underlying mechanisms of biomass reduction under treatment. Autophagy induction was studied by immunofluorescence based on staining of acidic vesicles with acridine orange. Frequency of autophagy was quite low . By contreatment . Most prhigh) cells; sub-G1 defines dead cells). The best antiproliferative effect was inducible in HROC183 T0 M2 (IDOlow) cells after Indoximod/Curcumin treatment  cells, apoptosis was two to three times higher than in controls (early apoptosis: 36 ± 20 % versus 11 % ± 2 % control and late apoptosis: 26 % ± 10 % versus 15 % ± 8 % control). Adding Gemcitabine to Curcumin further enhanced cytotoxic effects  cell line.All substances reduced tumor cell numbers in this test system. The best cytotoxic effect could be induced by Curcumin, resulting in a massive tumor cell reduction in all four cell lines (> 80 % versus control). Of note, combining Curcumin either with Indoximod or Gemcitabine even enhanced this toxic effect with nearly complete elimination of tumor cells . Best tuHowever, even with 72-hour incubation time, a specific antigenic activation is unlikely to occur and thus the observed effects are most likely due to a more unspecific stimulation of lymphocytes by the tested drugs.In this study, we describe (I) the expression profile of immune-modulating molecules on a panel of molecularly well-characterized patient-derived CRC cell lines and (II) the effects of IDO-inhibition directly on CRC cells as well as in coculture experiments with immune cells. in vivo [+ lymphocytes and clinical patients' outcome [ In vitro, IDO decreases tumor cell proliferation and mediates mediating drug resistance [data not shown), we indeed found some overlap between IDO level and sensitivity towards clinically used drugs, such as 5-fluorouracil .Specific IDO blockade with Indoximod identified an inverse correlation between biomass reduction and IDO1 abundance in our patient-derived cell lines. Of note, IDOmembrane . Additiomembrane . However in vitro encouraged us to use combinations with other IDO-blocking substances. Curcumin and Gemcitabine were selected based on the following criteria: (I) interference with IDO; (II) direct cytotoxic activity against tumor cells (via induction of immunogenic cell death) in (pre-)clinical studies, (III) safe application with minimal toxic side effects (at least in low-doses) in patients, and (IV) enhanced antitumoral effects when given in conjunction with other drugs. These experiments identified Curcumin as very promising candidate for IDO-blocking cancer therapy. Biomass of tumor cells significantly reduced and oncolytic effects even increased when Curcumin was combined with Gemcitabine, a quite surprising finding. In functional analyses, senescence and apoptosis could be identified as underlying molecular mechanisms. Effects were cell line and treatment specific, but again largely independent from IDO-expression status. Cytotoxicity of Curcumin is mainly based on apoptosis induction, without significant impairment of the cell cycle. This was somewhat unexpected, since short-time treatment with Curcumin has been reported to decrease cyclin D1 expression and to induce G1-/S-phase arrest [The missing direct cytotoxic potential of Indoximod monotherapye arrest , 35. In vivo, IDO blockade by Indoximod breaks immune tolerance by increasing the level of inflammatory molecules, such as C-reactive protein and IL6 [Finally, a coculture system mimicking tumor-immune cell interactions was applied to test the impact of IDO-inhibition for reconstitution of immune function. and IL6 . We coul and IL6 , 36. Gem and IL6 . However colon ascendens. HROC60 cells show high abundance of immunosuppressive molecules, i.e., IDO, PD1/PD-L1. It is therefore of particular interest whether such Curcumin-based combinations provide a real opportunity for selected CRC patients in the future.Interestingly, there was a trend towards highest susceptibility of HROC60 cells, established from a 71-year-old male patient with a low-grade CIMP tumor in theHowever, it is worth mentioning that coculture experiments were performed in an allogeneic setting using naïve and thus “unprimed” immune cells from healthy volunteers, without tumor-induced T-cell dysfunction. Prospective studies will have to test if comparable strong oncolytic effects can be achieved in an autologous setting with partially exhausted lymphocytes from cancer patients.Although we were unsuccessful to directly link IDO-level with treatment response, we would like to bring forward the argument that this molecule plays a central role in tumor-induced immunosuppression. To this end, not only pharmacological agents, but also other means of IDO1 targeting may provide clinical benefits to patients. A recently performed phase I study even described disease stabilization in patients after application of an IDO1-peptide vaccine. In most patients with metastatic non-small cell lung carcinoma, detectable IDO-1-specific T cell responses were accompanied by significantly improved overall survival , 39. ComWe report effects of IDO-inhibition on a panel of patient-derived colorectal cancer (CRC) cell lines. IDO blockade was induced by different substances, either directly (Indoximod) or indirectly  interacting with IDO. We have shown that Curcumin-based treatment is promising by inducing apoptosis in CRC cell lines, with even higher cytotoxic activity when Gemcitabine is added in low and thus nontoxic doses. Coculture of tumor and immune cells revealed a boosted antitumoral effect of Curcumin-based therapies by massive activation of unspecific immune responses, counteracting IDO-induced immune tolerance.


We found that the expression of EGFR/ERBB2 and activation of ERBB pathway was significantly related to Lapatinib sensitivity. GO (Gene Oncology) analysis of top 10 pathways showed that the sensitivity of Lapatinib was positively correlated with cell keratin, epithelial differentiation, and cell-cell junction, while negatively correlated with signatures of extracellular matrix. Forty-four differentially expressed lncRNAs were found between the Lapatinib sensitive and resistant groups . Gene set variation analysis (GSVA) was performed based on 44 lncRNAs and genes in the top 10 pathways. Five lncRNAs were identified as hub molecules. Co-expression network was constructed by more than five lncRNAs and 199 genes in the top 10 pathways, and three lncRNAs  and 47 genes were identified as close-related molecules. The three lncRNAs in epithelium-derived cancers were differentially expressed between sensitive and resistant groups, but no significance was found in non-epithelium-derived cancer cells. Correlation analysis showed that SPINT1-AS1  and GIHCG  were correlated with the IC50 of epithelium-derived cancer cells. In further experiments, GIHCG knockdown enhanced cancer cell susceptibility to Lapatinib, while high level of SPINT1-AS1 was a sensitive biomarker of NCI-N87 and MCF7 cancer cells to Lapatinib. In conclusions, lncRNAs GIHCG and SPINT1-AS1 were involved in regulating Lapatinib sensitivity. Up-regulation of GIHCG was a drug-resistant biomarker, while up-regulation of SPINT1-AS1 was a sensitive indicator.Lapatinib is a small molecule inhibitor of EGFR (HER1) and ERBB2 (HER2) receptors, which is used for treatment of advanced or metastatic breast cancer. To find the drug resistance mechanisms of treatment for EGFR/ERBB2 positive tumors, we analyzed the possible effects of lncRNAs. In this study, using CCLE (Cancer Cell Line Encyclopedia) database, we explored the relationship between the lncRNAs and Lapatinib sensitivity/resistance, and then validated those findings through  Lapatinib is a small molecular drug that has been shown to be a dual tyrosine kinase inhibitor, which is involved in the EGFR/HER1 and ERBB2/HER2 pathways and suppresses the autophosphorylation of these receptors. Clinically, it has been used in combination therapy with capecitabine in patients with advanced or metastatic breast cancer that overexpressed ERBB2/HER2 in the cases of previous treatment with anthracyclines, taxanes, or trastuzumab (Herceptin)  are a large class of transcribed RNA molecules that are longer than 200 nucleotides but do not encode proteins. In addition to the regulation of diverse cellular processes, such as epigenetics, cell cycle, and cell differentiation, they have been found to play important roles in carcinogenesis, tumor development, and treatment resistance  is an open access resource with the most completely integrated datasets of cancer cells genomes and drug effectiveness. It includes the experimental datasets of drug treatment of 24 kinds of chemical compounds in almost 1,000 cancer cell lines of various human cancers  database , three were melanoma cell lines , three were hepatocarcinoma cell lines , three were thyroid cancer cell lines , two were breast cancer cell lines (MCF7 and SK-BR-3), two were pancreatic cancer cell lines (TCC-PAN2 and BxPC3), and two were colorectal cancer lines (DLD-1 and NCIH-747). Cell lines NCI-N87, MuM-2C, LM3, MV3, Huh7, SGC-7901, CAL-62, AGS, MCF7, C643, 97L, SK-BR-3, KHM-5M, A-375, TCC-PAN2, MKN-45, and BxPC3 were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences . Cell lines DLD-1 and NCIH-747 were purchased from The Global Bioresource Center ATCC . The cell lines were cultured in RPMI-1640 supplemented with 10% fetal bovine serum in a humidified incubator at 37°C with 95% air and 5% CO2.Nineteen cancer cell lines were used for validating experiments SPINT1-AS1 and GIHCG siRNAs were transfected into cancer cells by Lipofectamine 2000  according to the manufacturer's instructions. The siRNA sequences are shown in Table S1.Total RNA was isolated using the TRIzol solution . The cDNA was synthesized using Reverse Transcription kit . Real-time PCR was performed in 10 μl reaction mixtures with the HT 7900  using SYBR™ Select Master Mix . The sequences of primers were designed and synthesized by Sunny Biotech : The primer sequences are shown in Table S1.Five thousand cells of different cancers were placed in each well of 96-well plates (100 μl/well). Different concentrations of Lapatinib  were incubated for 48 h. After adding 10 μl CCK-8 for 2 h, OD value was measured at 450 nm by spectrophotometry .Gene Set Variation Analysis,) in R language, which utilizes non-parametric unsupervised method for evaluating gene set enrichment (GSE) in transcriptomic data . Student's t-tests were performed by GraphPad Prism 6.0. P < 0.05 was considered statistically significant.The “corrplot” and “pheatmap” package in R software were utilized for visualizing pearson correlation analysis and cluster analysis by “euclidean” method. The Benjamini and Hochberg method was used to calculate P. adjust value. By means of “clusterProfiler” package in R, GSEA  and GO (Gene Ontology) analyses were carried out to explore involved gene clusters. GSEA is a computational method based on previous publication by Subramanian et al. . GO analn = 302) and low_IC50 (n = 118). Since EGFR and ERBB2 are the targets of the Lapatinib drug, the expression levels of EGFR, and ERBB2 in high_IC50 and low_IC50 groups were analyzed. The expression levels of EGFR and ERBB2 were significantly higher in low-IC50 group than in high_IC50 . The distribution tendency of 22 types of solid cancer cell lines in high-IC50 (up to 8 μM) and low_IC50 (lower than 8 μM) groups is presented in P < 0.002, p. adjust = 0.064; regulation of ERBB signaling pathway NES = −1.69, P < 0.002, p. adjust = 0.064).The CCLE data of Lapatinib IC50 of the selected 420 cell lines was shown in P < 0.001, P. adjust < 0.001).To illustrate the mechanism of Lapatinib resistance, we selected genes with fold-change >1.5 times to perform GO analysis (Table S2). In the top 10 involved pathways, Lapatinib sensitivity was positively associated with cell keratin, epithelial differentiation, and cell-cell junction, while negatively related to signatures of extracellular matrix (P < 0.01). Then, we selected genes in the top 10 pathways and 44 differential lncRNAs for the construction of the co-expression network. The enrichment scores of the top 10 pathway genes in every cancer cell lines were calculated and determined by GSVA analysis. Five lncRNAs were highlighted as the hub factors in the top 10 regulating pathways  and non-epithelium derived group . The differential expression levels of the three lncRNAs between the two groups are presented in P < 0.05). In the non-epithelium groups, there was no significant difference of the three lncRNAs between Lapatinib high_IC50 and low_IC50 groups. Higher expressing level of SPINT1-AS1 was found in epithelium-derived cancer cells, and higher expressing levels of MAGI2-AS3 and GIHCG were observed in the non-epithelium group.We divided the 420 cancer cell lines into epithelium derived group .Differentially expressed genes (1.5-fold change) between the Lapatinib high_IC50 and low_IC50 groups in epithelial group (Table S3) were utilized to perform GO analysis. Enhanced signatures of cell keratin, epithelial differentiation, and cell-cell junction were observed in Lapatinib low_IC50 group, and decreased signature of extracellular matrix were observed in Lapatinib low_IC50 group , while higher expression levels of GIHCG were significantly related to higher Lapatinib IC50 .In validating experiments, we examined expression levels of GIHCG and SPINT1-AS1 in seven types of cancer cell lines  and Lapatinib IC50 of the same cancer cell lines. Correlation analysis showed that higher expression levels of SPINT1-AS1 were significantly associated with lower Lapatinib IC50 (The sensitive cancer cell lines of NCI-N87 (gastric cancer) and MCF7 (breast cancer), as well as the resistant cancer cell lines of NCIH-747(colon cancer) and BxPC3 (pancreatic cancer) were selected for a subsequent validating study. After knocking-down expression levels of GIHCG and SPINT1-AS1 by small interfering RNAs, Lapatinib IC50, and inhibitory rate of cancer cells were detected. Among three small interference sequences of GIHCG and SPINT1-AS1 mRNAs, siRNA sequence 3 of GIHCG Si3, , and siRG, suppression of GIHCG in Lapatinib resistant cancer cell lines NCIH-747 and BxPC3 could induce up-regulation of SPINT1-AS1 (P < 0.05), while knockdown of SPINT1-AS1 did not change the expression level of GIHCG (P > 0.05).Knocking-down of GIHCG could significantly enhance the sensitivity to Lapatinib in MCF7 and BxPC3 cancer cell lines , while dLncRNA is an important regulatory molecule in drug resistance during chemotherapy or gene targeted therapy (Li et al., PI3K/Akt, Ras/Raf/MEK/ERK1/2, and PLCγ pathways are downstream pathways of EGFR and ERBB2 and play important roles in cell proliferation and survival of multiple cancers (Roskoski, Although the role of lncRNAs in cancer progression and Lapatinib resistance have been reported in other studies (Russell et al., In conclusion, the current study proposed a group of lncRNAs related to Lapatinib sensitivity based on pan-cancer cell lines analysis. By subsequent experimental study, lncRNAs GIHCG and SPINT1-AS1 were firstly identified as crucial lncRNAs in regulating Lapatinib resistance or sensitivity in epithelium-derived cancer cell lines. SPINT1-AS1 is a Lapatinib sensitivity predictor, while GIHCG is a predictive molecule for Lapatinib resistance.The protocols used in this study were approved by Rui Jin Hospital Ethics Review Boards. Written informed consents were obtained from all human material donors in accordance with the Declaration of Helsinki. Animals were used according to the protocols approved by Rui Jin Hospital Animal Care and Use Committee.KL and YY conceived and designed the experiments. ZX, ShS, ZZ, JG, and QL performed the experiments. ZX, ZZ, SaS, WS, YY, and KL analyzed the data. ZX, ShS, ZZ, SaS, WS, YY, and KL contributed reagents, materials, and analysis tools. ZX, YY, and KL wrote the paper.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 current review focuses on the role of PTPs in controlling CD4+ T cell activity in the intestinal mucosa and how disruption of PTP activity in CD4+ T cells can contribute to intestinal inflammation.Protein tyrosine phosphatases (PTPs) play a critical role in co-ordinating the signaling networks that maintain lymphocyte homeostasis and direct lymphocyte activation. By dephosphorylating tyrosine residues, PTPs have been shown to modulate enzyme activity and both mediate and disrupt protein-protein interactions. Through these molecular mechanisms, PTPs ultimately impact lymphocyte responses to environmental cues such as inflammatory cytokines and chemokines, as well as antigenic stimulation. Mouse models of acute and chronic intestinal inflammation have been shown to be exacerbated in the absence of PTPs such as PTPN2 and PTPN22. This increase in disease severity is due in part to hyper-activation of lymphocytes in the absence of PTP activity. In accordance, human PTPs have been linked to intestinal inflammation. Genome wide association studies (GWAS) identified several PTPs within risk loci for inflammatory bowel disease (IBD). Therapeutically targeting PTP substrates and their associated signaling pathways, such as those implicated in CD4 The gastrointestinal track is a large mucosal surface at which the host's immune system is juxtaposed with a dense microbial population and a diverse array of dietary antigens. Immune recognition of enteric antigens however, is minimized by physical compartmentalization. Bacteria and dietary products are retained within the gut lumen, while the host immune system is localized in the mucosal tissue. This physical separation is preserved by the intestinal mucosal barrier, which includes a mucin layer, a single epithelial cell lining sealed by tight and adherens junctions and a continuous secretion of anti-inflammatory soluble mediators Figure  1–5).–5.1–5).The mucosal barrier is not absolute however, and a small number of bacteria do translocate from the lumen to the underlying lamina propria. In such instances, a complex network of innate and adaptive immune cells impedes the spread of intestinal bacteria in a manner that limits tissue damage , 7. ThisPhysical compartmentalization and mucosal immunity therefore establish and maintain microbiome-host mutualism . The los+ T cell development and function. Examples include IL23R, JAK2, and STAT3  have been shown to induce remission in both CD and UC patients , Ustekinumab  and Infliximab   –33. It s+ T cells are classified into distinct subsets based on their inducing cytokines, transcription factor expression, and effector cytokine secretion. The initial classification of CD4+ T cells as TH1 IFNγ producers vs. TH2 IL-4 producers, has been broadened to include multiple additional subsets (H9 (IL-9), TH17 , TH22 (IL-22), T follicular helper TFH (IL-21) cells, as well as thymic-derived and peripherally-induced T regulatory cells   –40 , co-stimulatory molecules, and the T cell receptor (TCR), converge to induce gene expression profiles that drive CD4+ T cell activation and differentiation into distinct subsets  expressed on antigen presenting cells. Upon TCR engagement, Src-family kinases  are activated and phosphorylate tyrosine residues within the immune-receptor tyrosine-based activation motifs (ITAMs) in the TCR-associated CD3 and zeta chains –46. Phos+ T cell differentiation  cells are generated primarily from CD4+ Foxp3− T cells exposed to antigen under tolerogenic conditions or during homeostatic proliferation .–62.+ T c+ T cell activation and differentiation. The current review will examine the role of protein tyrosine phosphatases (PTP) in safeguarding this network, and how PTP deletion can perturb CD4+ T cell function and consequently contribute to intestinal inflammation.Thus a network of signaling pathways, heavily dependent on tyrosine phosphorylation, directs CD4The PTP family comprises a heterogeneous set of enzymes that were first defined by Tonks and colleagues by their capacity to dephosphorylate phospho-tyrosine residues and by their structurally related phosphatase catalytic domain , 64. PTPThe human PTP family members are divided into distinct classes based on their structural and biochemical properties Figure . The majMore recent studies characterized enzymes using alternate nucleophilic catalytic residues which were then added to the list of PTP family members. These two additional classes are the aspartic-based and histidine-based acid phosphatase enzymes. Thus, within this classification frame, Alonso and Pulido expanded the PTP gene superfamily to 125 members .Tyrosine phosphorylation is a reversible mechanism that promotes protein–protein interactions and propagates signaling cascades in all cell types. It therefore stands to reason that the PTPs targeting these phosphorylated amino acids play important roles in modulating the strength and duration with which a signaling pathway is activated. Moreover, given that PTPs are a heterogeneous gene family  it is not surprising that PTPs have been associated with many distinct pathologies in both animal models and in human disease . Of impoThe immune system relies heavily on the use of protein phosphorylation to recognize and transmit intra- and extracellular signals. Signaling cascades initiated at the cell surface by receptor kinases, or from the recruitment of cytoplasmic kinases such as the JAK and Src-like kinase families, translate into multiple immune cell responses like cellular adhesion, division, migration and others. Hence, from the list of over 100 classical Cys-based PTPs, Arimura and Yagi demonstrated that between 58 and 76 of them are expressed in various cell lineages of the immune system  and thatMultiple genome wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) linked with IBD, in or near PTP coding units , 71. ExaPTPN22 is a non-receptor protein tyrosine phosphatase that is expressed primarily in hematopoietic cells . It has PTPN22 deficient mice exhibit a lymphoproliferative disease and accumulate memory-phenotype T cells with age, but do not display signs of spontaneous autoimmunity  tyrosine following TCR ligation and not due to changes to IL-2 or IL-15 receptor signaling  induced model, these studies attributed the increase in disease severity to changes in TLR4 signaling, type I interferon production and macrophage polarization , 95.PTPN2−/− CD4 and CD8 was identified as causing increased T cell activation due to hyper-phosphorylation of the activating tyrosine residue of Lck  presented higher expression of TH1 and TH17-related transcription factors and cytokines  in comparison to PTPN2 expressing CD patients. Such human studies solidify the important role of PTPN2 CD4+ T cell responses in the context of human IBD  is a cytoplasmic phosphatase expressed in all hematopoietic cell lineages throughout development and activation (me) and motheaten viable (mev) mice. These mice harbor mutations that result in undetectable and reduced PTPN6 expression respectively. Similar to PTPN2 deficient mice, me/me mice succumb to disease 2–3 weeks following birth . Similar to PTPN6, PTPN11 regulates signaling downstream of multiple surface receptors including growth factor receptors, cytokine receptors and integrins. Its broad tissue expression is in line with the embryonic lethality of PTPN11 knockout mouse at mid-gestation in –133.PTPN11 mutations in patients with multiple cancer types including leukemias, breast cancer and gastric cancer  differentiation is suppressed  mice were reconstituted with either WT or DUSP2−/− CD4+CD45RBhi T cells. In this model, an increase in body weight loss, severity of histopathology scoring, and colon thickening were observed following the transfer of DUSP2−/− T cells compared to controls. Such clinical signs were corroborated by a rise in the number of colon infiltrating CD4+ T cells expressing a TH17 gene signature. Moreover, such TH17 T cells in the inflamed tissue expressed elevated levels of pathogenic TH17-associated genes but not non-pathogenic genes  and is only increased minimally following PMA stimulation. The low levels of DUPS2 expression were found to be attributed to CpG methylation. Unfortunately, the levels of DUSP2 in colonic biopsies, or colonic T cells, were not quantified. As such, further evidence is required to definitely demonstrate that a reduction in T cell DUSP2 occurs in IBD patients and that this reduction impacts human T cell polarization in the disease state  signaling regulates multiple cellular responses to activation including T cell proliferation, cytokine production, survival and adhesion. CD4balance. –153.in vitro assays suggested a role for DUSP6 in the regulation of CD4+ T cell activation. Specifically, DUSP6 was shown to be upregulated following TLR4 stimulation and then restrained ERK activation and suppressed IFNγ production by TCR stimulation  that recognize and dephosphorylate the insulin receptor (IR). The regulation of IR activation by a PTP was hypothesized to decrease IR mediated signaling events, and subsequent entry of glucose into insulin receptive tissues. It was believed that a rise in the expression or activity of such a regulatory PTP would result in high levels of blood glucose in the circulation, which would then instigate type II diabetes. This hypothesis was confirmed by two seminal publications which described the PTP1B knockout mouse that presented with an increase in IR phosphorylation. These studies validated PTP1B as an outstanding candidate to be targeted for the treatment of type II diabetes , 159 andIn spite of all these reports, there are no small molecule inhibitors against PTP1B (or any PTP) that have been successfully developed beyond initial clinical trials. Multiple reviews have already examined the difficulties associated with developing PTP competitive inhibitors and this subject is beyond the scope of this review –163. In It is worthy to note that novel therapeutic strategies are also being developed to target PTPs. These include the forced dimerization and inhibition of receptors PTPs , 167, inex-vivo cell cultures. This allows for the modulation of PTP activity in a cell-type specific and temporary fashion. For example, we reported a protocol for dendritic cell (DC) vaccination that employs inhibitors of PTPN1-PTPN2 (Significant effort is also being made in the development of cell based therapies. Potential adverse effects that could be associated with systemically inhibiting PTPs are mitigated by using PTP inhibitors in N1-PTPN2 . This woFrom an IBD perspective, it is clear that inhibiting the PTPs presented above would most likely exacerbate disease by potentiating the effects of pro-inflammatory cytokines and promoting the differentiation of pathogenic T cells. Indeed this contention is supported by the fact that, biologics and small molecules that suppress cytokine receptor signaling have been clinically successful in IBD disease management. One example would be tofacitinib, a JAK inhibitor found to be effective in phase 2 and 3 trials in moderate to severe ulcerative colitis .Our understanding of PTP function in immune cells is expanding beyond cytokine receptor signaling, and our capacity to modulate cellular responses by titrating PTP expression is also evolving at a rapid pace. The cell-dependent context of positive or/and negative modulation bestowed by the nearly 80 PTPs expressed in immune cells, remains an exciting ground for study and clinical improvement. It remains to be seen if this renewed interest in PTP inhibitors would be applicable to inflammatory diseases such as UC and CD.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.