Targeting GPC3high cancer-associated fibroblasts sensitizing the PD-1 blockage therapy in gastric cancer.

Cancer-associated fibroblasts (CAFs) are an important part of tumour microenvironment, but its role in immunotherapy of gastric cancer (GC) is still needed to further study. In this study, we firstly distinguish the GC related CAFs via single cell sequencing dataset. CAFs in deep layers of GC tissues gain more developmental potential. Moreover, we found Glypican-3 (GPC3) is up-regulated in the CAFs subgroups of the advanced GC and correlated with poor prognosis in GC patients. In addition, higher GPC3 expression GC patients have higher TIDE (Tumour Immune Dysfunction and Exclusion) score, dysfunction and exclusion score. independent GC cohort also show GC patients with GPC3high CAFs have lower response rate to PD-1 therapy. GPC3 secreted from CAFs up-regulated PD-L1, TIM3, CD24, CYCLIN D1, cMYC and PDK mRNA expression level in HGC-27 cells. At last, in vivo model demonstrate that targeting GPC3high CAFs sensitizing the PD-1 blockage therapy in GC. In conclusion, GPC3 expression in CAFs is a critical prognostic biomarker, and targeting GPC3high cancer-associated fibroblasts sensitizing the PD-1 blockage therapy in GC.Key messagesGlypican-3 (GPC3) is up-regulated in the CAFs subgroups of the advanced gastric cancer.Gastric cancer patients with GPC3high CAFs have lower response rate to PD-1 therapy.Targeting GPC3high CAFs sensitizing the PD-1 blockage therapy in gastric cancer.

MicroRNA-200b-3p restrains gastric cancer cell proliferation, migration, and invasion via C-X-C motif chemokine ligand 12/CXC chemokine receptor 7 axis.

This study was conducted to investigate the impact of microRNA (miR)-200b-3p on viability, migration, and invasion of gastric cancer (GC) cells and its mechanism. Quantitative real-time PCR (qRT-PCR) was conducted to measure miR-200b-3p expression in GC tissues and cells; besides, the relationship between miR-200b-3p expression and overall survival time (OS) was analyzed with OncomiR database; cell counting kit-8 (CCK-8), colony formation assay, flow cytometry, scratch healing assay, and Transwell assay were performed to detect the proliferation, cell cycle progression, migration, and invasion of GC cells; a lung metastasis model in nude mice was used to examine the effect of miR-200b-3p on the metastasis of GC cells in vivo; the interplay between miR-200b-3p and C-X-C motif chemokine ligand 12 (CXCL12) mRNA 3' UTR was predicted by bioinformatics and verified with a dual-luciferase reporter gene assay; besides, the expression of CXCL12 and CXC chemokine receptor 7 (CXCR7) was probed by Western blot. It was found that miR-200b-3p expression was down-regulated in GC tissues, which was remarkably associated with the lymph node metastasis and decrease of differentiation of GC; transfection with miR-200b-3p mimics restrained the growth, migration, and invasion of GC cells in vitro, induced cell cycle arrest, and inhibited CXCL12 and CXCR7 expression levels; transfection of miR-200b-3p inhibitors worked oppositely in vitro and promoted lung metastasis in vivo. CXCL12 was confirmed as the downstream target of miR-200b-3p and was negatively modulated by miR-200b-3p. In conclusion, miR-200b-3p inhibited GC progression via regulating CXCL12/CXCR7 axis.

Chromodomain-helicase-DNA-binding protein 1-like (CHD1L) silencing inhibits gastric cancer cell proliferation, invasion, and migration.

BACKGROUND: We aimed to investigate the effect of chromodomain-helicase-DNA-binding protein 1-like (CHD1L) silencing on the biological behavior of gastric cancer cells. METHODS: Small hairpin RNA (shRNAs) targeting CHD1L were designed and transduced into BGC-823 human gastric cancer cells. Expression of p53, p21, nerve growth factor IB (Nur77), and ARHGEF9 was assessed by western blotting, and the effect of CHD1L silencing on gastric cancer cell proliferation, apoptosis, and migration was examined by MTT, flow cytometry, and wound healing assays, respectively. RESULTS: In CHD1L-shRNA-1-treated cells, the expression of p53, p21, Nur77, and ARHGEF9 was significantly upregulated, and the number of apoptotic cells was significantly increased compared to the shRNA-negative control (NC; P<0.05). Additionally, the number of cells in the G1 phase was significantly increased among CHD1L-shRNA-1-treated cells. In contrast, the number of cells in the S phase was significantly decreased among CHD1L-shRNA-1-treated cells compared to shRNA-NC-treated cells (P<0.05). Following CHD1L silencing, there were 58.63±10.97 invading cells compared to 144.95±12.68 and 148.49±17.86 in the shRNA-NC and untreated groups, respectively (P<0.05). After 24 h, CHD1L-silenced BGC-823 cells migrated 0.54±0.34 µm compared to 1.34±0.26 and 1.31±0.31 µm in the shRNA-NC and untreated groups, respectively (P<0.05). CONCLUSIONS: CHD1L silencing significantly inhibited the proliferation, invasion, and migration of BGC-823 gastric cancer cells and induced apoptosis. Knockdown of CHD1L may present a novel approach for treating gastric cancer.

The diagnostic performance of serum LIM homeobox transcription factor 1 alpha in patients with gastric cancer.

The study was conducted to investigate the diagnostic performance of serum LIM homeobox transcription factor 1 alpha (LMX1A) in patients with gastric cancer (GC).The serum level of LMX1A in GC, benign, and healthy groups was measured using quantitative real time PCR (qRT-PCR) and compared with the student t test. The associations of serum LMX1A levels with clinical parameters were analyzed with chi-square test. The diagnostic value of serum LMX1A in GC was evaluated by receiver operating characteristic (ROC) curve.The level of serum LMX1A in GC group (1.309 ±â€Š0.553) was significantly lower than that in the benign group (2.174 ±â€Š0.676) and healthy group (2.598 ±â€Š0.826) (P < .01 for both). The decreased level of LMX1A was associated with large tumor size (P = .009), positive lymph node metastasis (P = .027), and advanced TNM stages (P = .002). Receiver operating characteristic (ROC) analysis demonstrated that serum LMX1A could discriminate GC patients from the healthy individuals, with the area under the curve (AUC) of 0.889 (95% confidence interval [CI] = 0.838-0.938) combining with the sensitivity and specificity of 82.68% and 82.61%. Additionally, serum LMX1A also exhibited high accuracy in discriminating between GC patients and benign gastric disease cases (AUC = 0.842, 95% CI = 0.782-0.901), with the sensitivity of 81.89% and specificity of 72.41%.Serum LMX1A may be an effective biomarker for early detection of GC.

GSDME mediates caspase-3-dependent pyroptosis in gastric cancer.

Gastric cancer is a malignancy that starts from the cells in the stomach with relatively low overall survival rate. Chemotherapy following resection surgery has been recommended as a curative strategy for gastric cancer. However, the mechanism of the chemotherapy drugs on gastric cancer is not completely understood. Pyroptosis is a form of programmed cell death and plays critical role in immunity. The role of pyroptosis on cancer cells is less known. In this study, we treated SGC-7901 and MKN-45 with 5-FU and found that the cell viability was significantly decreased. The release of LDH and the percentage of PI and APC Annexin-V double positive cells after 5-FU treatment were elevated compared to control group. Moreover, there were large bubbles blowing from the membrane of 5-FU-treated cells and the cleavage of GSDME but not GSDMD, which were blocked by the silence or specific inhibitor of caspase-3. Additionally, GSDME knockout by CRISPR-Cas9 switched 5-FU induced pyroptosis into apoptosis in SGC-7901. In conclusion, our findings firstly revealed that GSDME switches chemotherapy drug-induced caspase-3 dependent apoptosis into pyroptosis in gastric cancer cells.

MED23 in endocrinotherapy for breast cancer.

We investigated the role of the transcriptional mediator subunit 23 (MED23) in everolimus drug resistance, invasion and metastasis during breast cancer treatment and its molecular mechanism. We also evaluated the endocrinotherapy and prevention method for breast cancer. Breast cancer cell strains were established that can continuously express MED23, as well as inducible MED23-shRNA expression plasmids. The inductive agent, doxycycline (Dox), was added to the water for long-term silencing of MED23 in intratumoral cells. We conducted experiments on the role of MED23 in the regulation of invasion and metastasis of breast cancer using cell culture, western blotting, MTT proliferation experiment, fluorescent quantitative PCR and chromatin immunoprecipitation (ChIP). The silencing of MED23 significantly inhibited cellular growth and proliferation as well as soft agar cloning. Silencing of MED23 strengthened the sensitivity of the everolimus-resistant breast cancer cell strains BT474 and MCF-7/ADM cells to everolimus medication. The silencing of MED23, in combination with everolimus, inhibits the cell cycle progress of breast cancer cells. ChIP indicated that the mutual regulation of HER2 and MED23 also participates in the formation of the everolimus drug resistance mechanism. Therefore, MED23 plays an important role in everolimus drug resistance, invasion, and metastasis of breast cancer. As a potential molecular therapeutic target of breast cancer, MED23 overcomes drug resistance in clinical endocrinotherapy and controls the distal relapse and metastasis in breast cancer by the targeted silencing of MED23.

Expression of Inhibitor of Differentiation-1 and Its Effects on Angiogenesis in Gastric Cancer.

OBJECTIVE: To explore the effects of recombinant human endostatin (endostar, ES) and cisplatin on the growth of gastric cancer-transplanted tumor in nude mice and the expression of microvessel density (MVD). METHODS: Human gastric cancer SGC-7901 cells were subcutaneously injected into the armpit of nude mice to prepare cancer-bearing nude mice. A total of 32 cancer-bearing nude mice were divided into four groups (each group with 8 mice). The four groups included control group and other three groups in which mice were treated with 5 mg/kg of ES (group E), 5 mg/kg of cisplatin (group Ci), and 5 mg/kg of ES combined with 5 mg/kg of cisplatin (group C), respectively. MVD was determined by immunohistochemistry, and the expressions of mRNA and protein of inhibitor of differentiation-1 (ID1) and vascular endothelial growth factor (VEGF) were detected with reverse transcription polymerase chain reaction (RT-PCR) and western blot, respectively. Apoptosis was observed with transmission electron microscope. RESULTS: Compared with control group, the sizes and weights of tumors were significantly decreased in other three groups (all p < 0.05). MVD was significantly lower in groups E, Ci, and C than in control group, and in groups E and C than in group Ci (all p < 0.05). Compared with control group, the expressions of mRNA and protein of ID1 and VEGF significantly decreased in groups E and C (all p < 0.05). There were no significant differences in the expressions of mRNA and protein of ID1 and VEGF between group Ci and control group. There was apoptosis in groups E and C, but no apoptosis was found in group Ci and control group. CONCLUSION: ES can inhibit the growth of gastric cancer cells through suppressing angiogenesis and promoting apoptosis of tumor cell. This study provides a new idea for the treatment of gastric cancer.

Oxymatrine mediates Bax and Bcl-2 expression in human breast cancer MCF-7 cells.

UNLABELLED: The aim of this study was to explore the effects of oxymatrine in treating breast cancer patients using biomolecular methodology. Human breast cancer MCF-7 cells were treated with oxymatrine at concentrations of 0 (control), 25, 50 and 100 µg/mL. Apoptosis assay by Annexin/PI staining was performed to examine the effects of oxymatrine on apoptotic rates of MCF-7 cells at time points of 24 h, 48 h, and 72 h after treatment. Real-time PCR was performed for the mRNA abundance of Bax and Bcl-2 after the cells were treated with oxymatrine at concentration of 0, 25, 50, and 100 µg/mL at the time points of 24, 48, and 72 h. Western blotting was performed when the cells were treated with oxymatrine at various concentrations for 72h. High concentration of oxymatrine at 100 µg/mL enhanced apoptosis by 6.4-fold at 72 h compared with control (33.16% vs. 4.47%; t= 9.82, p< 0.001). Oxymatrine at 100 µg/mL up regulated Bax mRNA abundance by 169 % at 72 h (t = 18.32, p = 0.001), and reduced Bcl-2 mRNA abundance by 24 % at 72 h (t = 6.30, p = 0.001) compared with control. Oxymatrine enhanced the expression of Bax protein while reduced the expression of Bcl-2 protein. Oxymatrine treatment showed pro-apoptotic effects in breast cancer MCF-7 cells, and these effects correlated with the up regulation of Bax transcription and protein expression and the down regulation of Bcl-2 transcription and protein expression in a time- and dose-dependent manner. CONCLUSION: Oxymatrine had effects in promoting apoptosis in human breast cancer MCF-7 cells by mediating the mRNA and protein expression levels of Bax and Bcl-2.

Expression of tissue factor pathway inhibitor-2 in gastric stromal tumor and its clinical significance.

The aim of this study was to explore the expression of tissue factor pathway inhibitor-2 (TFPI-2) in gastric stromal tissue and its clinical significance. TFPI-2 expression was detected by immunohistochemical analysis, RT-PCR and western blotting in tumor, peritumoral and gastric normal tissues from 72 patients with gastric stromal tumors. The level of TFPI-2 expression was observed to be significantly higher in gastric normal tissue than in peritumoral tissue, and was significantly higher in peritumoral tissue than in tumor tissue (P<0.01). As the NIH grade increased, the level of TFPI-2 expression decreased (P<0.01). A low expression level of TFPI-2 was closely associated with invasion and metastasis of gastric stromal tumors. In conclusion, the level of TFPI-2 expression was higher in gastric normal tissue than in gastric stromal tumors. Low expression levels of TFPI-2 may be associated with invasion and metastasis of gastric stromal tumors.

The evaluation of anti-angiogenic effects of Endostar on rabbit VX2 portal vein tumor thrombus using perfusion MSCT.

BACKGROUND: There were many treatments for hepatocellular carcinoma with portal vein tumor thrombus (PVTT), in which targeted anti-angiogenic drug therapy is becoming a popular research topic. However, an objective and non-invasive method that can evaluate the treatment effects is still lacking. METHODS: Eighteen New Zealand white rabbits implanted with VX2 tumor thrombus in portal vein were randomly assigned into 3 groups: Endostar, saline, or control, six in each group. Multi-slice CT (MSCT) perfusion scanning was performed to measure the differences in blood flow (TBF), tissue blood volume (TBV), and capillary permeability time the surface (PS) before and after Endostar treatment, between Endostar and saline treatment. Two weeks after treatment, both Endostar and saline groups underwent CT perfusion scan. The rabbits then were sacrificed by air embolism, and specimens of tumor thrombosis were collected. Immunohistochemistry assay was also performed to compare the expression of vascular endothelial growth factor (VEGF) in PVTT after Endostar, saline and placebo treatment. RESULTS: In Endostar group, PVTT CT perfusion parameters (TBF, TBV, PS) significantly decreased after the treatment (p <0.05). Post-treatment PVTT CT perfusion parameters (TBF, TBV, PS) were significantly lower in Endostar group than in Saline group (p <0.05). VEGF is mainly expressed in cytoplasma. After Endostar treatment, the expression of VEGF in PVTT was markedly reduced. There was also significant difference on post-treatment VEGF protein expression measured by Immunohistochemistry assay between Endostar group and control group (p <0.05). Post-treatment PVTT CT perfusion parameters (TBF, TBV, PS) were positively correlated with VEGF protein expression in all 3 groups (rs > 0, p <0.05). CONCLUSIONS: Multi-slice CT perfusion imaging can evaluate the anti-angiogenic effects of Endostar for the VX2 tumor thrombus in portal vein, and provide quantitative functional information.

Expression of protease-activated receptor-2 in human gastric stromal tumor and its clinicopathological significance.

BACKGROUND/AIMS: To explore the expression of protein-activated receptor-2 (PAR-2) in human gastric stromal tumor and its clinicopathological significance. METHODOLOGY: The expression of PAR-2 was detected with immunohistochemisty, RT-PCR and Western blot in tumor tissue, peritumoral tissue and gastric normal tissue from 72 patients with gastric stromal tumor. RESULTS: PAR-2 expression was significantly higher in peritumoral tissue (p < 0.05) and tumor tissue (p < 0.01) than in gastric normal tissue, and significantly higher in tumor tissue than in peritumoral tissue (p < 0.01). With the increase in NIH grade, PAR-2 expression was elevated in tumor tissues. PAR-2 expression was strongly associated with mucosal invasion. CONCLUSIONS: PAR-2 expression is significantly higher in gastric stromal tumor tissue than in peritumoral tissue and gastric normal tissue. The high expression of PAR-2 may be associated with the invasion and metastasis of gastric stromal tumor.

Clinical study of tumor angiogenesis and perfusion imaging using multi-slice spiral computed tomography for breast cancer.

OBJECTIVES: To explore the correlation between multi-slice spiral CT (MSCT) perfusion parameters and the expression of vascular endothelial growth factor (VEGF) as well as matrix metalloproteinase-2 (MMP- 2) in breast cancer. METHODS: Forty five breast cancer patients and 16 patients with benign breast tumor, both confirmed by pathology examination, were enrolled. All underwent MSCT perfusion imaging to obtain perfusion maps and data for parameters including blood flow (BF), blood volume (BV) and permeability surface (PS). Cancer patients did not receive treatment prior to surgery. The expression of VEGF and MMP-2 were examined with both immunohistochemistry and Western blotting. RESULTS: The levels of VEGF and MMP-2 by immunohistochemistry were significantly higher in the breast cancer group (P < 0.01) than the benign tumor group. Relative OD values from Western blotting were also higher in cancer cases (P < 0.05). Similarly, the mean MSCT perfusion parameters (BF, BV, PS) were significantly higher in the breast cancer group (P < 0.01), BF and BV positively correlating with VEGF expression (r = 0.878 and 0.809 respectively, P < 0.01); PS and VEGF and MMP-2 expression were also positively correlated (r= 0.860, 0.786 respectively, P < 0.01). CONCLUSION: There is a correlation between breast cancer MSCT perfusion parameters and VEGF andMMP-2 expression, which might be useful for detection of breast lesions, qualitative diagnosis of breast cancer, and evaluation of breast cancer treatment.