Expression of IDO1 in Tumor Microenvironment Significantly Predicts the Risk of Recurrence/Distant Metastasis for Patients With Esophageal Squamous Cell Carcinoma.

In this study, we aimed to explore immune markers predicting locoregional recurrence/distant metastasis (R/M) for patients with esophageal squamous cell carcinoma (ESCC) post-surgical intervention by using a novel high-throughput spatial tool to quantify multiple immune proteins expressed in ESCC and lymphocytes in tumor microenvironment (TME-L). First, formalin-fixed paraffin-embedded tissues from surgical patients with ESCC (n = 94) were constructed on a microarray, which was then divided into discovery (n = 36) and validation cohorts (n = 58). Using a newly developed GeoMx digital spatial profiling tool, 31 immune proteins in paired ESCC and TME-L, morphologically segmented by PANCK and CD45, respectively, from the discovery cohort were quantified, releasing 2,232 variables. Next, the correlation matrix was analyzed using the Corrplot package in R Studio, resulting in 6 closely correlated clusters. The Least Absolute Shrinkage and Selection Operator regression scoring model predictive of R/M risk with superior specificity was successfully established based on the 3 following hierarchically clustered immune proteins: ARG1 in ESCC/PANCK+, STING, and IDO1 in TME-L/CD45+. Moreover, the expression of IDO1 in TME-L, rather than in ESCC, significantly predicted the R/M risk score with an area under curve of 0.9598. In addition, its correlation with R/M status was further validated by dual immunohistochemistry staining of IDO1 and CD45 in discovery and validation cohorts. Above all, our findings not only provide a more accurate scoring approach based on quantitative immune proteins for the prediction of R/M risk, but also propose that IDO1 in TME-L potentially plays a driving role in mediating R/M in ESCC.

MYC-targeted genes predict distant recurrence in patients with ocular adnexal extranodal marginal zone lymphoma.

Ocular adnexal extranodal marginal zone lymphoma (OA-EMZL) is the most frequent subtype of ocular adnexal lymphoma, with a high propensity for recurrence. Distant recurrence (DR) as an essential prognostic event has unique clinical risk factors, but whether distinct molecular features exist remains poorly understood. Here, we identified potential biomarkers using proteomic analysis of 27 OA-EMZL samples. The MYC-targeted genes PCNA, MCM6, and MCM4 were identified as candidates. MYC-targeted genes were further identified as the most significantly activated gene set in patients with DR. The candidate genes were verified in samples from 11 patients with DR and 33 matched controls using immunohistochemistry. The 3-year and 5-year AUC values of MCM6 (0.699 and 0.757) were higher than those of Ki-67 (0.532 and 0.592). High expressions of MCM6 and MCM4 were significantly associated with shorter distant recurrence-free survival (Log-rank p = 0.017, Log-rank p = 0.0053). Multivariate Cox regression identified MCM6 expression as an independent risk factor for DR (HR, 6.86; 95% CI, 1.32-35.79; P = 0.02). Knockdown of c-Myc in B cells resulted in decreased MCM6 and MCM4 expression and reduced proliferative capacity. Our results suggest that activation of the MYC-targeted gene is a distinct molecular feature of DR in OA-EMZL. MYC-targeted gene, MCM6, is a promising pathological biomarker for DR.

Gold Nanotriangle-Assembled Nanoporous Structures for Electric Field-Assisted Surface-Enhanced Raman Scattering Detection of Adenosine Triphosphate.

A reliable, rapid, cost-effective, and simple method for the detection of biomolecules would greatly promote the research of analytical detection of single molecules. A nanopore-based analytical technique is promising for detecting biomolecules. Conventional electrochemical nanopores cannot distinguish biomolecules precisely because of their fast translocation speed and limited electrochemical information. Therefore, it is highly desirable to develop electrochemical surface-enhanced Raman scattering (SERS) nanopores to obtain multidimensional information. Herein, we designed and fabricated gold nanotriangle (AuNT)-assembled porous structures at the tip of a glass capillary using dithiol adenosine triphosphate (ATP) aptamers as cross-linking molecules. The AuNTs exhibited an edge length of 57.3 ± 6.2 nm and thickness of about 15 nm. The gold nanoporous structure (GPS) showed a strong ion rectification even at a high concentration of electrolyte (2 M) and a high SERS activity. Based on these designed structures, SERS and electrochemistry techniques were combined to control the rapid movement of ATP to the vicinity of the GPS by an applied potential of +1 V, where ATP was concentrated by ATP aptamers and the molecular signals were amplified by SERS. As a result, the GPS successfully detected ATP at a concentration as low as 10-7 M.

Red meat consumption and risk for dyslipidaemia and inflammation: A systematic review and meta-analysis.

Aim: The study (PROSPERO: CRD42021240905) aims to reveal the relationships among red meat, serum lipids and inflammatory biomarkers. Methods and results: PubMed, EMBASE and the Cochrane databases were explored through December 2021 to identify 574 studies about red meat and serum lipids markers including total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), C-reactive protein (CRP) or hypersensitive-CRP (hs-CRP). Finally, 20 randomized controlled trials (RCTs) involving 1001 people were included, red meat and serum lipid markers and their relevant information was extracted. The pooled standard mean difference (SMD) was obtained by applying a random-effects model, and subgroup analyses and meta-regression were employed to explain the heterogeneity. Compared with white meat or grain diets, the gross results showed that the consumption of red meat increased serum lipid concentrations like TG (0.29 mmol/L, 95% CI 0.14, 0.44,P<0.001), but did not significantly influence the TC (0.13 mmol/L, 95% CI -0.07, 0.33, P = 0.21), LDL-C (0.11 mmol/L, 95% CI -0.23, 0.45, P = 0.53), HDL-C (-0.07 mmol/L, 95% CI -0.31, 0.17, P = 0.57),CRP or hs-CRP (0.13 mmol/L, 95% CI -0.10, 0.37,P = 0.273). Conclusion: Our study provided evidence to the fact that red meat consumption affected serum lipids levels like TG, but almost had no effect on TC, LDL-C, HDL-C and CRP or hs-CRP. Such diets with red meat should be taken seriously to avoid the problem of high lipid profiles. Systematic review registration: [https://www.crd.york.ac.uk/PROSPERO], identifier [CRD42021240905].

Occurrence, transport and environmental risk assessment of pharmaceuticals and personal care products (PPCPs) at the mouth of Jiaozhou Bay, China based on stir bar sorptive extraction.

In recent years, research on pharmaceuticals and personal care products (PPCPs) in the marine environment has attracted increasing attention worldwide. However, more work is needed to improve PPCPs detection methods, specifically for seawater environments. An analytical method based on stir bar sorptive extraction (SBSE) had been developed and fully optimized for the pretreatment and detection of ten widely used PPCPs that are commonly found in seawater samples. By optimizing several variables including the material of the stir bars, extraction temperature, extraction time, ionic strength, desorption solvent, and desorption time, the optimized method has achieved excellent results in the detection and quantification of target PPCPs with detection limits ranging from 0.03 to 1 ng/L. The distribution of target PPCPs at the mouth of Jiaozhou Bay was successfully determined by this method, and the concentrations and detection frequencies of PPCPs varied greatly from N.D. to 449.36 ng/L and from 9.1 % to 100 %, respectively. Moreover, the distributions of PPCPs were explained by the Lagrangian particle-tracking model, and the results showed that the Tuandao sewage treatment plant had the most significant impact on the study area. The environmental risk assessment results showed that several target PPCPs might pose risks to aquatic organisms. In particular, triclocarban should receive more attention and the risk quotients of the mixtures (MRQ) should not be ignored.

Loss of LEF-1 expression as a diagnostic indicator for extranodal NK/T-cell lymphoma: An immunohistochemical study of 88 cases.

PURPOSE: Extranodal natural killer (NK)/T-cell lymphoma (ENKTL) is not a uniform entity but consists of various disease subtypes associated with Epstein-Barr virus (EBV) infection. Lymphoid enhancer binding factor-1 (LEF-1), a member of the T-cell factor/LEF family of transcription factors, plays a significant role in NK-cell and T-cell development. We aimed to explore the expression of LEF-1 in ENKTL and evaluate the applicability of LEF-1 in the diagnosis of ENKTL. METHODS: The expression of LEF-1 was investigated in tissue samples harvested from patients with ENKTL by immunohistochemistry. RESULTS: LEF-1 staining was not observed in 85 of 88 ENKTL cases (97%). Eighty-six of the 88 cases (98%) were positive for CD56, whereas all the tested cases were negative for both CD16 and CD27. Of the cytotoxic T-cell-like features studied, 81 cases (92%) were negative for CD8, 85 of 88 cases (97%) were immunoreactive for the recombinant linker for activation of T cells (LAT), all (100%) were immunoreactive for TIA-1, 85 (97%) were immunoreactive for granzyme B and 65 (74%) were immunoreactive for perforin. CONCLUSION: Loss of LEF-1 expression is a highly specific diagnostic indicator of ENKTL.

A new monoclonal antibody that blocks dimerisation and inhibits c-kit mutation-driven tumour growth.

PURPOSE: Imatinib, a small-molecule tyrosine kinase inhibitor, has shown good clinical activity by inhibiting adenosine triphosphate (ATP) binding to the receptor. Unfortunately, majority of patients eventually develop drug resistance, which limits the long-term benefits of the tyrosine kinase inhibitors and poses a significant challenge in the clinical management of GIST. The aim of our study was to explore the feasibility of blocking KIT dimerisation upstream of the phosphorylation in imatinib-resistant GIST. METHOD: KITMAb was prepared using hybridoma technique. The biological function of KITMAb was examined in KIT-dimer-expressing cells constructed by transfecting with liposomes using enzyme linked immunosorbent assay (ELISA), immunohistochemistry, western blot, MTT, Annexin V/FITC, and flow cytometry assay, respectively. RESULTS: KIT-dimer was expressed in 293 cells transfected with c-kit mutated-type pcDNA3.1. Treatment of KIT-dimer-expressing cells with the KITMAb significantly decreased the expression of both KIT-dimer and other phosphorylated proteins of KIT downstream signalling pathway. Furthermore, KITMAb slowed down cell growth and reduced the proportion of cells in the proliferative phase (S + G2-M). Finally, we also found that KITMAb treatment accelerated cell apoptosis. These results indicate that KITMAb strongly inhibits KIT receptor dimerisation-mediated signalling pathway and cell growth responses in vitro. CONCLUSIONS: We demonstrate c-kit mutation-driven KIT auto-dimerisation prior to tyrosine kinase phosphorylation as same as the procedure in ligand-dependent signalling pathway and describe a monoclonal antibody, KITMAb, with strong affinity to the dimerisation domain of KIT that blocks the important step in both the KIT signalling pathways. Further, the results suggest that treatment with KITMAb may be potentially therapeutic in imatinib-resistant GIST.

17-allylamino-17-demethoxygeldanamycin impeded chemotherapy through antioxidant activation via reducing reactive oxygen species-induced cell death.

Hyperthermia enhances the anticancer effects of thymidylate synthase (TYMS) inhibitors (raltitrexed, RTX) and improves the precise biochemical mechanisms partially through enhancement of intracellular drug absorption. Recent research focuses on the potential anticancer drug target Heat Shock Protein 90 (HSP90), which could increase the sensitivity of cancer cells to TYMS inhibitors; however, with different HSP90 inhibitors, several research studies finally showed a poor efficacy in preclinical or clinical research. Here, we showed that 17-allylamino-17-demethoxygeldanamycin (17-AAG, HSP90 inhibitor) affects the efficacy of chemotherapy through antioxidant activation-induced resistance. In this study, we found that RTX, alone or in combination with hyperthermia, triggers reactive oxygen species (ROS) exposure and thus induces cell death. Also, the addition of hyperthermia showed more ROS exposure and function. The pharmacologic inhibition of HSP90 reversed the effects of chemotherapeutical treatments, while the overexpression of HSP90 showed no relation with these effects, which demonstrated that dysregulation of HSP90 might have a significant impact on chemotherapeutic treatments. The addition of 17-AAG increased the activation of antioxidant with increased antioxidant enzymes, thus affecting the RTX efficacy.

FoxM1 is regulated by both HIF-1α and HIF-2α and contributes to gastrointestinal stromal tumor progression.

BACKGROUND: FoxM1 plays important regulatory roles in a variety of diseases. However, the functional role of FoxM1 and mechanisms responsible for its expression in gastrointestinal stromal tumor (GIST) is not thoroughly understood. METHODS: FoxM1 protein expression and biological function were examined in human GIST tissues and cells using immunohistochemistry, quantitative real-time PCR, western blot, CCK-8, wound-healing- and Matrigel invasion assays, respectively. The role of hypoxia-inducible factor (HIF) signaling in FoxM1 expression was investigated using chromatin immunoprecipitation and luciferase reporter and in vivo tumor growth assays. RESULTS: FoxM1 was highly expressed in highly proliferative and migratory/invasive GIST specimens. Upregulation of FoxM1 was positively correlated with the expression of HIF-1α and HIF-2α in GIST specimens, and hypoxia-induced FoxM1 expression in GIST cells. Functionally, ectopic expression of FoxM1 significantly promoted GIST cell proliferation, cell cycle progression, migration and invasion, whereas the knockdown of endogenous FoxM1 of hypoxic GIST cells had the opposite effects. Molecularly, FoxM1 was transcriptionally regulated by HIF-2α under normoxia, whereas it was upregulated by both HIF-1α and HIF-2α under hypoxia. The xenograft tumor data further confirmed the regulated effect of HIF-1α and HIF-2α on FoxM1, and demonstrated that the simultaneous downregulation of both HIF-1α and HIF-2α inhibited GIST tumor growth. CONCLUSIONS: Our data demonstrated the critical role of FoxM1 in promoting GIST progression and uncovered a novel HIF-1α/HIF-2α-FoxM1 axis. These findings identify FoxM1 as a possible new molecular target for designing novel therapeutic treatments to control GIST progression.

Expression profiles of stemness genes in gastrointestinal stromal tumor.

Gastrointestinal stromal tumor (GIST) is believed to originate from intestinal cells of Cajal or their stem cell precursors, and expresses stemness-related markers, such as CD117, CD34, DOG1 and nestin. To further characterize phenotypic features of GISTs, we examined expression profiles of a panel of stemness genes in GISTs, by analyzing existing gene expression profiling datasets. Our results showed that mRNA levels of B-lymphoma moloney murine leukaemia virus insertion region-1 (BMI1), kruppel-like factor 4 (KLF4), sal-like protein 4 (SALL4) and telomerase reverse transcriptase (TERT) were significantly unregulated in GISTs. Subsequently, protein expression of BMI1 and TERT was identified in GIST specimens by immunohistochemistry. Especially, we found that high expression of nuclear BMI1 was associated with large tumor size (P = .0239), high mitotic count (P < .01), high Ki-67 index (P = .0357), advanced National Institute of Health (NIH) criteria (P = .0025) and advanced World Health Organization (WHO) classification (P < .01) in GISTs. Functional and pathway enrichment analysis showed that most of BMI1's coexpressed genes were involved in tumor growth-related process, such as regulation of cell cycle and proliferation. Furthermore, we confirmed RAS oncogene family (RAB18) and limb development membrane protein 1 (LMBR1) genes as novel targets for BMI1 in GIST cells. These results provide valuable information for the expression profiles of stemness genes in GISTs, and identified nuclear BMI1 as an important marker of GIST cell proliferation and progression.

Fibroblast growth factor receptor-1 is essential for regulation of mitochondrial biogenesis via control of microRNA let-7b / 中华内分泌代谢杂志

Objective To explore the role of fibroblast growth factor receptor ( FGFR ) 1 in endothelial homeostasis via an induction of microRNA let-7s, with effects on AcSDKP(N-acetyl-seryl-aspartyl-lysyl-proline) and associated mitochondrial biogenesis. Methods Blocking FGFR1 signaling pathway, Western blot and immunofluorescence staining were used to measure mitochondrial fusion ( mitofusin-2, MFN2;optic atrophy protein 1, OPA1 ) and fission ( dynamin-related protein-1, DRP1 ) proteins and mitochondrial biogenesis by MitoTraker Green. Also real-time quantitative PCR(qPCR) was performed to test microRNA let-7' expression. Results FGFR1 signaling pathway was critical for AcSDKP maintaining mitochondrial biogenesis through induction of microRNA let-7b. In endothelial cells, the AcSDKP restored the triple[TGF-β2, interleukin (IL)-1β, tumor necrosis factor (TNF)-α]-suppressed microRNA let-7b-5p expression and associated with mitochondrial biogenesis. Such effect of AcSDKP was lost in either fibroblast growth factor receptor substrate 2 (FRS2) siRNA or neutralizing FGFR1 treated-cells. Similarly, AcSDKP lost its effect on mitochondrial biogenesis in microRNA let-7b-5p inhibitor-treated-cells. In addition, microRNA let-7b-5p mimic reversed the FRS2 siRNA-suppressed mitochondrial biogenesis in endothelial cells. Conclusion These findings demonstrated that FGFR1 is critical for maintaining mitochondrial biogenesis through control of microRNA let-7b-5p in endothelial cells.

A study of peritoneal metastatic xenograft model of colorectal cancer in the treatment of hyperthermic intraperitoneal chemotherapy with Raltitrexed.

Peritoneal metastasis of colorectal cancer is one of the most incident and fateful diseases among relapse cases. It shows a certain resistance to systemic chemotherapy. The perfusion system in clinic is complex and hard to be used in fundamental researches. This study aims at evaluating the effect of an improved hyperthermic intraperitoneal chemotherapy with Raltitrexed used in tumor-bearing mice with peritoneal metastatic colorectal carcinoma. The results showed that no severe adverse effect was observed. All control animals developed extensive peritoneal and mesenteric metastatic nodes. Tumor sites in the treatment groups were reduced significantly. The administration dose of Raltitrexed influenced concentration in systemic blood and peritoneal tissues. Temperature promoted the intracellular absorption of Raltitrexed significantly. Our findings reveal that hyperthermic intraperitoneal chemotherapy is an efficient therapy in treating peritoneal metastatic carcinoma in nude mice. It can effectively reduce the extension of carcinoma cells from macro and micro examination. The combination of hyperthermia and Raltitrexed resulted in an improved therapeutic effect on animal models.

Endoplasmic reticulum-targeting photosensitizer Hypericin confers chemo-sensitization towards oxaliplatin through inducing pro-death autophagy.

Hypericin is an endoplasmic reticulum (ER)-located photosensitizer, which causes oxidative damage to ER during photodynamic therapy (PDT). Hypericin-mediated PDT (HY-PDT) has been confirmed to enhance chemo-sensitivity of oxaliplatin (L-OHP) in colon cancer cells. The present study reveals that autophagy plays a key role in chemosensitization during HY-PDT. We proved pro-death autophagy was required for sensitization and HY-PDT/L-OHP antitumor synergism. High dosage of HY-PDT induced autophagic cell death; while low dose of HY-PDT predominantly triggered protective autophagy and promoted cell proliferation. Low dose of HY-PDT reduced the cytotoxicity of L-OHP in oxaliplatin-resistant colon cancer cells. Different level of autophagy therefore contributed to the opposite effect of HY-PDT on cell fate and chemo-sensitivity. Furthermore, we revealed the role of CHOP as a regulator connecting pro-survival and pro-death autophagy under ER damage. High dose of HY-PDT induced massive ROS generation and severe ER stress, which then led to induction of CHOP. CHOP thereby activated CHOP/TRIB3/Akt/mTOR cascade and triggered autophagic cell death. Additionally, when apoptotic pathway was blocked, cells treated with high dose of HY-PDT preferentially underwent death through autophagic pathway. On the other hand, suppression of autophagy made cells more vulnerable to apoptosis under low dose of HY-PDT. These results provided new evidences for the clinical application of ER-targeting PDT in modifying chemosensitivity of colorectal cancer therapy.

Inhibition of polypyrimidine tract-binding protein 3 induces apoptosis and cell cycle arrest, and enhances the cytotoxicity of 5- fluorouracil in gastric cancer cells.

BACKGROUND: Human polypyrimidine tract binding protein 3 (PTBP3) was first discovered in 1999 and has been well characterised as a differentiation regulator. However, its role in human cancer has rarely been reported. Our previous study revealed increased PTBP3 protein level in gastric cancer tissues. Downregulation of PTBP3 suppressed the proliferation and differentiation of gastric cancer cells in vivo. METHODS: PTBP3 mRNA levels in human gastric cancer and adjuvant non-tumour tissues were detected. Apoptosis and 5-FU effect were determined in PTBP3-silenced gastric cancer cells. Underlying molecular mechanisms were investigated. RESULTS: MRNA expression of PTBP3 was upregulated in gastric cancer tissues, especially in those at an advanced stage. PTBP3 silencing led to apoptosis, under which modulation of PTB and thereby switch of Bcl-x pre-mRNA splicing pattern might be an important mechanism. Further research found that inhibition of PTBP3 expression enhanced the chemosensitivity of gastric cancer cells towards 5-FU treatment. This was mediated by reduced expression of histone deacetylase 6 (HDAC6), which further inhibited the phosphorylation of Akt and the expression of thymidylate synthase (TYMS), the critical determinant of 5-FU cytotoxicity. CONCLUSIONS: PTBP3 might serve as a biomarker of gastric cancer or potential target for anti-cancer therapy.

[Comparisons of risk factors for low birth weight between Han and Uygur full-term infants].

OBJECTRIVE: To compare the differences in risk factors for low birth weight (LBW) between Han and Uygur full-term infants and to provide a basis for the prevention of LBW in newborn infants. METHODS: Eighty-seven full-term LBW infants (38 Hans and 49 Uygurs) between March 2013 and June 2014 were selected as the case group, and 186 full-term normal birth weight infants (92 Hans and 94 Uygurs) were selected as the control group. A questionnaire survey was performed to investigate the related factors for LBW. Multivariate logistic regression analysis was carried out to determine the risk factors for LBW. RESULTS: The birth weights in Uyghur LBW infants were lower than in Han ones (P<0.05). Multivariate logistic regression analysis showed that drinking (OR=2.472, P=0.015) and smoking (OR=2.323, P=0.007) by the father, pregnancy complications (OR=14.377, P<0.001), and times of pregnancy (OR=2.995, P=0.001) were the risk factors for LBW in Han infants, while drinking by the father (OR=1.968, P=0.007), times of pregnancy (OR=1.953, P=0.005), pregnancy complications (OR=10.283, P=0.002), and poor indoor environment (OR=1.367, P=0.027) were the risk factors for LBW in Uyghur infants. CONCLUSIONS: There are differences in physical growth between Han and Uygur LBW infants. Han and Uygur infants share the same traditional risk factors for LBW, such as father's harmful behaviors like drinking, times of pregnancy, and pregnancy complications, however, the indoor environment also plays a role in the occurrence of LBW in Uygur infants.

Comparisons of risk factors for low birth weight between Han and Uygur full-term infants / 中国当代儿科杂志

<p><b>OBJECTRIVE</b>To compare the differences in risk factors for low birth weight (LBW) between Han and Uygur full-term infants and to provide a basis for the prevention of LBW in newborn infants.</p><p><b>METHODS</b>Eighty-seven full-term LBW infants (38 Hans and 49 Uygurs) between March 2013 and June 2014 were selected as the case group, and 186 full-term normal birth weight infants (92 Hans and 94 Uygurs) were selected as the control group. A questionnaire survey was performed to investigate the related factors for LBW. Multivariate logistic regression analysis was carried out to determine the risk factors for LBW.</p><p><b>RESULTS</b>The birth weights in Uyghur LBW infants were lower than in Han ones (P<0.05). Multivariate logistic regression analysis showed that drinking (OR=2.472, P=0.015) and smoking (OR=2.323, P=0.007) by the father, pregnancy complications (OR=14.377, P<0.001), and times of pregnancy (OR=2.995, P=0.001) were the risk factors for LBW in Han infants, while drinking by the father (OR=1.968, P=0.007), times of pregnancy (OR=1.953, P=0.005), pregnancy complications (OR=10.283, P=0.002), and poor indoor environment (OR=1.367, P=0.027) were the risk factors for LBW in Uyghur infants.</p><p><b>CONCLUSIONS</b>There are differences in physical growth between Han and Uygur LBW infants. Han and Uygur infants share the same traditional risk factors for LBW, such as father's harmful behaviors like drinking, times of pregnancy, and pregnancy complications, however, the indoor environment also plays a role in the occurrence of LBW in Uygur infants.</p>

Expression of stem cell factor in gastrointestinal stromal tumors: Implications for proliferation and imatinib resistance.

KIT autophosphorylation caused by mutation of KIT is considered to be a critical mechanism for the oncogenesis of gastrointestinal stromal tumors (GISTs). However, little is known regarding whether stem cell factor (SCF), the KIT ligand, is able to induce the proliferation of GIST cells by activating the wild-type KIT receptor in GISTs. Imatinib, a tyrosine kinase inhibitor, has been demonstrated to be effective as treatment for the majority of GISTs. However, primary resistance to imatinib in GISTs with wild-type KIT and acquired resistance in GISTs with mutant KIT are becoming increasingly significant problems. The aims of this study were to detect the expression and function of SCF in 68 GIST samples, and to explore the relationship between SCF activity and imatinib resistance using immunohistochemical staining and western blot analysis. Results showed abundant expression of SCF in GISTs and demonstrated that SCF is capable of enhancing GIST cell proliferation. Similar to its ineffectiveness in wild-type GISTs, imatinib also failed to inhibit SCF-induced KIT activation in GISTs with mutant KIT. We also found increased SCF expression in GIST cells treated with imatinib. Overall, our results indicated that SCF-induced KIT activation is a novel essential pathway for the proliferation of GISTs. Imatinib was not able to inhibit the activity of SCF, while it promoted the expression of SCF, which may have contributed to acquired imatinib resistance.

The expression of KIT receptor dimers in gastrointestinal stromal tumors independent of c-kit mutation and SCF expression is associated with high-risk stratification.

Although the dimerization of KIT, a receptor tyrosine kinase, plays a major role in a number of tumors, correlations between the clinicopathological parameters and KIT receptor dimers have not been identified. In the current study, a method for the detection of KIT receptor dimer expression was described and correlations between the clinicopathological parameters and KIT receptor dimers were analyzed. A single center cohort study of 49 patients with gastrointestinal stromal tumors (GISTs) was conducted to analyze the expression of KIT receptor dimers by SDS-PAGE, Native-PAGE and modified Native-PAGE. Immunohistochemistry was used to examine the expression of ki-67, c-kit and stem cell factor (SCF). Mutations of the c-kit gene were examined in 48 GISTs according to the polymerase chain reaction (PCR) and direct sequencing methods. Based on the data, a signal for the KIT receptor monomer was obtained by SDS-PAGE. Faint bands were observed on the nitrocellulose membrane by Native-PAGE, while clear bands were identified for KIT receptor dimers and monomers using modified Native-PAGE (15 out of 49 cases). The tumor size was larger in KIT receptor dimer-positive cases compared with that in KIT receptor dimer-negative cases. Analysis of KIT receptor dimer expression levels and risk stratification demonstrated that KIT receptor dimer-positive cases belonged to the higher risk classification. In addition, there was no significant correlation between the existence of KIT receptor dimers and c-kit gene mutations, including SCF expression. In conclusion, this study established a method for the detection of the existence of KIT receptor dimers in tissues and confirmed that KIT receptor dimers were correlated with risk stratification. Data also indicated that ligand-dependent SCF/KIT dimerization is an independent crucial mechanism in GIST cell proliferation and increases the risk of GIST. Therefore, blocking KIT dimerization may prove to be an effective approach for the treatment of GISTs.

A novel PDGFRA mutation in gastrointestinal stromal tumours, L839P, is sensitive to imatinib in vitro.

Evidence suggests that different types of mutation in gastrointestinal stromal tumours (GISTs) correlate with different response rates to imatinib (Glivec, STI571). The purpose of this study was to explore the sensitivity of the PDGFRA(L839P) mutant, a novel gain-of-function mutation isoform related to GISTs, to imatinib in vitro. The eukaryotic expression vectors pcDNA3.1-PDGFRA(Wild), pcDNA3.1-PDGFRA(D842V) and pcDNA3.1-PDGFRA(L839P) were constructed and transfected into Chinese hamster ovary (CHO) cells by liposome methods. The responses of cells with PDGFRA(Wild), PDGFRA(L839P) and PDGFRA(D842V) mutants to imatinib were determined by methyl thiazolyl tetrazolium (MTT) assay, western blotting and apoptosis assays. Reults of the MTT assay revealed that the growth rate of CHO(PDGFRA(L839P)) cells decreased to approximately 60% when exposed to 1 µM imatinib and to approximately 50% with 5 µM imatinib. However, the growth rate of CHO(PDGFRA(D842V)) cells did not significantly change with 5 µM imatinib. Western blot analysis indicated that 1 µM imatinib completely blocked the phosphorylation of PDGFRA(L839P), but did not affect PDGFRA(D842V) phosphorylation. Apoptosis analysis suggested that the percentage of apoptotic CHO(PDGFRA(L839P)) cells increased approximately 4-fold (from 5.90 to 25.2%) with 1 µM imatinib. Although the treatment of CHO(PDGFRA(D842V)) and CHO(PDGFRA(Wild)) cells with 5 µM imatinib resulted in a slight increase in the number of apoptotic cells, the percentage of apoptotic cells remained approximately 10% of the total population. Our findings showed that the PDGFRA gene mutation isoform L839P is sensitive to inhibition by imatinib. Screening for PDGFRA mutations in GISTs is essential to identify the response to treatment with imatinib.

Stem cell factor-mediated wild-type KIT receptor activation is critical for gastrointestinal stromal tumor cell growth.

AIM: To clarify the biological role of stem cell factor (SCF)-mediated wild-type KIT receptor activation in gastrointestinal stromal tumor (GIST) growth. METHODS: The co-expression of wild-type KIT receptor and SCF was evaluated in 51 GIST samples using mutation analysis and immunohistochemistry, and the results were correlated with clinicopathological parameters, including the mitotic count, proliferative index (Ki-67 immunohistochemical staining), mitotic index (phospho-histone H3 immunohistochemical staining) and apoptotic index (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling). Using primary cultured GIST cells, the effect of SCF-mediated wild-type KIT receptor activation was determined by western blotting, methyl thiazolyl tetrazolium (MTT), and apoptosis assays. RESULTS: We found that wild-type KIT receptor and SCF protein were expressed in 100% and 76.5% of the 51 GIST samples, respectively, and the co-expression of wild-type KIT receptor and SCF was associated with known indicators of poor prognosis, including larger tumor size (P = 0.0118), higher mitotic count (P = 0.0058), higher proliferative index (P = 0.0012), higher mitotic index (P = 0.0282), lower apoptosis index (P = 0.0484), and increased National Institutes of Health risk level (P = 0.0012). We also found that the introduction of exogenous SCF potently increased KIT kinase activity, stimulated cell proliferation (P < 0.01) and inhibited apoptosis (P < 0.01) induced by serum starvation, while a KIT immunoblocking antibody suppressed proliferation (P = 0.01) and promoted apoptosis (P < 0.01) in cultured GIST cells. CONCLUSION: SCF-mediated wild-type KIT receptor activation plays an important role in GIST cell growth. The inhibition of SCF-mediated wild-type KIT receptor activation may prove to be particularly important for GIST therapy.