MTA2 promotes the metastasis of esophageal squamous cell carcinoma via EIF4E-Twist feedback loop.

Metastasis-associated protein 2 (MTA2) is frequently amplified in many types of cancers; however, the role and underlying molecular mechanism of MTA2 in esophageal squamous cell carcinoma (ESCC) remain unknown. Here, we reported that MTA2 is highly expressed in ESCC tissue and cells, and is closely related to the malignant characteristics and poor prognosis of patients with ESCC. Through in vitro and in vivo experiments, we demonstrated that MTA2 significantly promoted ESCC growth, metastasis, and epithelial-mesenchymal transition (EMT) progression. This integrative analysis combined with expression microarray showed that MTA2 could interact with eukaryotic initiation factor 4E (EIF4E), which positively regulates the expression of Twist, known as a master regulator of EMT. Moreover, the results of chromatin immunoprecipitation revealed that MTA2 was recruited to the E-cadherin promoter by Twist, which reduced the acetylation level of the promoter region and thus inhibited expression of E-cadherin, and subsequently promoted the aggressive progression of ESCC. Collectively, our study provided novel evidence that MTA2 plays an aggressive role in ESCC metastasis by a novel EIF4E-Twist positive feedback loop, which may provide a potential therapeutic target for the management of ESCC.

High expression of TCN1 is a negative prognostic biomarker and can predict neoadjuvant chemosensitivity of colon cancer.

Transcobalamin (TCN1) is a vitamin B12 (cobalamin)-binding protein that regulates cobalamin homeostasis. Recent studies and bioinformatic analyses have found that TCN1 is highly expressed in cancer tissues and is associated with tumour aggressiveness and poor prognosis. The present study aimed to detect TCN1 as a novel biomarker for prognosis and chemosensitivity of colon cancer. Next-generation sequencing showed that TCN1 was one of several upregulated mRNAs in colon cancer, which was verified by further bioinformatics analyses. Western blotting (n = 9) and quantitative real time polymerase chain reaction (qRT-PCR, n = 30) revealed that TCN1 was highly expressed in colon cancer tissues at both the protein and mRNA level. A total of 194 cases of colon cancer were examined by immunohistochemistry and revealed that TCN1 expression level was related to advanced stages (P < 0.005). Kaplan-Meier analysis verified that patients with lower TCN1 expression usually had longer overall survival (P = 0.008). In addition, TCN1 was highly expressed in pulmonary metastatic tumour tissues (n = 37, P = 0.025) and exhibited higher levels in right-sided colon cancer than in left-sided colon cancer (P = 0.029). TCN1 expression in specimens that had received neoadjuvant chemotherapy decreased compared with that in colonoscopy biopsy tissues (n = 42, P = 0.009). Further bioinformatics analyses verified that apoptosis pathways might have a role in high TCN1 expression. All the studies revealed that TCN1 expression in colon cancer was significantly associated with malignant biological behaviour. Therefore, TCN1 could be used as a novel biomarker for colon cancer aggressiveness and prognosis and might also be a potential biomarker for predicting neoadjuvant chemosensitivity.

Enzyme-injected method of enzymatic dispersion at low temperature is effective for isolation of smooth muscle cells from human esophagogastric junction.

The present study was conducted to examine the feasibility of in vitro isolation and primary culture of smooth muscle cells (SMCs) from the esophagogastric junction (EGJ). Smooth muscles of EGJ were harvested from 23 patients with esophageal cancer during esophagostomy from January 2015 to December 2017. Enzymatic dispersion (ED) was performed for isolation. Collagenase II and Trypsin/EDTA were applied by enzyme injection (EI) into tissue fragments or immersion of tissue fragments into enzyme solution. Growth characteristics and proliferation [Cell Counting Kit-8 (CCK-8)] of cells were recorded for both smooth muscle cell medium (SMCM) and DMEM/F12 containing 10% newborn bovine serum (10%-F12). All ED methods could isolate primary cells; EI was the most effective method with low collagenase II concentration (0.5 mg/ml) at 4˚C for 14-24 h. Primary cells demonstrated mainly spindle- and long-spindle-shaped with 'hills and valleys' morphology. The CCK-8 assay in SMCM showed better proliferation results than in 10%-F12. After passaging for 4-8 generations in SMCM or 2-4 generations in 10%-F12, cells enlarged gradually with passages and lost spindle structures. mRNA and proteins of α-smooth muscle actin (α-SMA), smooth muscle 22 α (SM22α), vimentin, desmin, CD90 and proliferating cell nuclear antigen were detected in tissues and cells with different levels of expression. SMCs of esophageal circular muscle, esophageal longitudinal muscle, gastric circular muscle near sling in gastric bottom and gastric circular muscle near clasp in lesser gastric curvature, all cultured in 10%-F12, exhibited superior smooth muscle phenotypes compared with SMCs cultured in SMCM in terms of α-SMA, SM22α and vimentin expression. The EI method of ED at low temperature appears effective for isolation and primary culture of SMCs from human EGJ in vitro.

The lncRNA FEZF1-AS1 Promotes the Progression of Colorectal Cancer Through Regulating OTX1 and Targeting miR-30a-5p.

Long noncoding RNAs (lncRNAs) participate in and regulate the biological process of colorectal cancer (CRC) progression. Our previous research identified differentially expressed lncRNAs in 10 CRC tissues and 10 matched nontumor tissues by next-generation sequencing (NGS). In this study, we identified an lncRNA, FEZF1 antisense RNA 1 (FEZF1-AS1), and further explored its function and mechanism in CRC. We verified that FEZF1-AS1 is highly expressed in CRC tissues and cell lines. Through functional experiments, we found that reduced levels of FEZF1-AS1 significantly suppressed CRC cell migration, invasion, and proliferation and inhibited tumor growth in vivo. Mechanistically, we discovered that reduced levels of the lncRNA FEZF1-AS1 inhibited the activation of epithelial-mesenchymal transition (EMT); the overexpression of orthodenticle homeobox 1 (OTX1) partially rescued the FEZF1-AS1-induced inhibition of protein expression. It indicated that FEZF1-AS1 may play a role in the occurrence and development of CRC by regulating the FEZF1-AS1/OTX1/EMT pathway. Furthermore, it was reported that FEZF1-AS1 is located in both the nucleus and cytoplasm of HCT116 cells. Dual-luciferase reporter assays verified that FEZF1-AS1 directly binds miR-30a-5p and negatively regulated each other. Further, we showed that 5'-nucleotidase ecto (NT5E) is a direct target of miR-30a-5p, and the inhibition of miR-30a-5p expression partially rescued the inhibitory effect of FEZF1-AS1 on NT5E. Our results indicated that the mechanism by which FEZF1-AS1 positively regulates the expression of NT5E is through sponging miR-30a-5p. Our study demonstrated that lncRNA FEZF1-AS1 is involved in the development of CRC and may serve as a diagnostic and therapeutic target for CRC patients.

p-Hydroxylcinnamaldehyde from cochinchinamomordica seed reverses resistance to TRAIL in human oesophageal squamous cell carcinoma via the activation of the p38 mitogen-activated protein kinase signalling pathway.

BACKGROUND: Our previous studies have showed that p-Hydroxylcinnamaldehyde (CMSP) could induce the differentiation of ESCC cells via the cAMP-RhoA-MAPK signalling pathway, which suggests a new potential strategy for ESCC treatment. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in several tumour cells by binding to the death receptors DR4 and DR5. However, TRAIL has little effect on oesophageal squamous cell carcinoma (ESCC) cells due to the loss of the receptors. The present study determined the effect of CMSP, the firstly found chemical constituent of Cochinchinamomordica seed (CMS), on TRAIL-induced apoptosis and its mechanism in ESCC cells. METHODS: MTS assays were performed to examine the CMSP- and TRAIL-mediated inhibition of ESCC cell growth. Flow cytometry and Hoechst 33258 staining assays were used to detect apoptosis in ESCC cells treated with CMSP combined with TRAIL. Western blotting was used to determine the effect of CMSP on the expression of p38, p-p38, DR4, DR5, Bid and caspase-3/8 in ESCC cells treated with CMSP combined with TRAIL. Additionally, immunodeficient Balb-c/null mouse model was used to determine the chemotherapeutic efficacy of CMSP and TRAIL against ESCC tumour xenograft growth in vivo. RESULTS: We found that the combination of CMSP and TRAIL had a greater inhibitory effect on ESCC cell viability in vitro than CMSP or TRAIL alone. CMSP enhanced the TRAIL-induced apoptosis in ESCC cells by upregulating the expression of DR4 and DR5 via the p38 MAPK signalling pathway. Furthermore, the increased expression of DR4 and DR5 upon TRAIL-induced apoptosis in ESCC cells was mediated at least in part by subsequent caspase-3 and caspase-8 activation. Moreover, the in vivo model showed that tumour growth was significantly slower in CMSP and TRAIL combination-treated mice than in mice treated with CMSP or TRAIL alone. CONCLUSION: Taken together, our findings indicate that CMSP as an extract from TCM, might be as a potential sensitizer of TRAIL and thus provide a novel strategy for the clinical treatment of ESCC.

Antitumor Effect of Periplocin in TRAIL-Resistant gastric cancer cells via upregulation of death receptor through activating ERK1/2-EGR1 pathway.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a member of the tumor necrosis factor family, induces apoptosis in a variety of cancer cells. However, gastric cancer (GC) cells are insensitive to TRAIL usually. In the previous study, we showed that Periplocin could induce apoptosis in GC cells via the activation of ERK1/2-EGR1 pathway. In the present study, we have shown that the combination of Periplocin and TRAIL had a greater inhibitory effect on gastric cancer cell viability in vitro and in vivo than Periplocin or TRAIL alone. Through upregulating the expression of DR4 and DR5 at transcriptional and protein levels, Periplocin enhanced the sensitivity of gastric cancer cells to TRAIL. Furthermore, enhanced activity of ERK1/2-EGR1 pathway was responsible for upregulating of DR4 and DR5 uponPeriplocin treatment, subsequently reducing the expression of Mcl-1 and Bcl2 and activating Bid and caspase-3/8. Collectively, these data implied that Periplocin might act as a sensitizer of TRAIL and could be a potential strategy for the treatment of GC.

The tumor-suppressive function of miR-1296-5p by targeting EGFR and CDK6 in gastric cancer.

We aimed to confirm the role of miR-1296-5p in gastric cancer and to identify its target genes. The expression of miR-1296-5p was measured in gastric cancer tissues and cell lines. The function of miR-1296-5p was examined by the overexpression and inhibition of its expression in typical gastric cell lines as well as SGC-7901 and MGC-803 cells. The targets of miR-1296-5p were identified by a luciferase activity assay. We found that miR-1296-5p was down-regulated in gastric cancer tissue and cell lines, and low expression levels of miR-1296-5p were associated with advanced clinical stage. Moreover, miR-1296-5p inhibited cell proliferation, migration, and invasion in SGC-7901 and MGC-803 cells. Then, we identified CDK6 and EGFR as novel targets of miR-1296-5p by a luciferase activity assay. Furthermore, the overexpression of miR-1296-5p suppressed the expression of CDK6 and EGFR. Our results indicated a tumor-suppressive role of miR-1296-5p through the translational repression of oncogenic CDK6 and EGFR in gastric cancer.

The histone deacetylase inhibitor panobinostat exerts anticancer effects on esophageal squamous cell carcinoma cells by inducing cell cycle arrest.

Esophageal squamous cell carcinoma (ESCC) is a common malignancy without effective therapy. Histone deacetylase inhibitors (HDACIs) have been demonstrated as an emerging class of anticancer drugs for a range of haematological and solid tumours. However, the effect of HDACIs has not yet been investigated on ESCC cells. In this study, HDACIs were initially considered to have anticancer activity for ESCC, due to the high expression of HDAC genes in ESCC cell lines by analysing expression data of 27 ESCC cell lines from the Broad-Novartis Cancer Cell Line Encyclopedia. Next, we used five ESCC cell lines and one normal immortalized esophageal epithelial cell line to screen three HDACIs, panobinostat (LBH589), vorinostat (SAHA), and trichostatin A (TSA), for the ability to inhibit growth. Here, we report that LBH589 more effectively suppressed cell proliferation of ESCC cell lines, in a dose-dependent manner, than TSA and SAHA, as well as had lower toxicity against the SHEE normal immortalized esophageal epithelial cell line. Further experiments indicated that LBH589 treatment significantly inhibited TP53 (mutated TP53) expression, both at the mRNA and protein level, and simultaneously increased p21 and decreased cyclin D1 expression. Taken together, we propose that LBH589 inhibits ESCC cell proliferation mainly through inducing cell cycle arrest by increasing p21 and decreasing cyclin D1 in a p53-independent manner. SIGNIFICANCE OF THE STUDY: In this study, the antitumor activity of HDACIs LBH589, SAHA, and TSA on ESCC was characterized, with LBH589 displaying the most potent anti-proliferative activity while not harming normal immortalized esophageal epithelial cells. Furthermore, we propose that LBH589 exerts its anti-proliferative effect by inducing cell cycle arrest. The ability to specifically target cancer cells indicates therapeutic potential for use of LBH589 in the treatment of ESCC.

Differentially expressed lncRNAs and mRNAs identified by NGS analysis in colorectal cancer patients.

Long noncoding RNAs (lncRNAs) play an important role in gene regulation, but their impact on the pathogenesis of colorectal cancer and the biological function of cancer cells is unclear. In this study, we used next-generation sequencing to study the differences in the expression profiles of lncRNAs and mRNAs in colorectal cancer tissues. We analyzed the differentially expressed genes by Gene Ontology/Kyoto Encyclopedia of Genes and Genomes (GO/KEGG) enrichment and predicted new lncRNA functions. Our results revealed that compared with lncRNAs and mRNAs in nontumor colorectal tissues, 1019 lncRNAs (512 upregulated, 507 downregulated) and 3221 mRNAs (1606 upregulated, 1615 downregulated) were differentially expressed in tumor colorectal tissues (fold change >2 and P < 0.05). We validated some of these genes by qPCR. Furthermore, we identified some new lncRNAs differently expressed in colorectal cancer samples from patients in northern China. We confirmed the function of lncRNA-FIRRE-201 and SLCO4A1-AS1-202 in colorectal cancer cells to provide an experimental basis for studies on their roles in the occurrence and development of colorectal cancer and in the regulation of networks.

C/D-Box Snord105b Promotes Tumorigenesis in Gastric Cancer via ALDOA/C-Myc Pathway.

BACKGROUND/AIMS: Small nucleolar RNAs (snoRNAs) play an important role in carcinogenesis. In this study, we identified a C/D box snoRNA, snord105b, and further investigated the function and mechanism of the snord105b in gastric cancer (GC). METHODS: The expression level of snord105b in GC tissures, sera and cell lines were detected by qRT-PCR. Cell viability was assessed using MTS assay. Transwell and wound healing assay were performed to evaluate migration and invasion, and protein expression was examined by western blotting. ChIRP and MS analysis was used to seek for the special binding protein of snord105b. RESULTS: The snord105b was upregulated and associated with tumor size, differentiation, and pathological stage in GC. Snord105b affected proliferation, migration and invasion in multiple GC cell lines. The oncoqenic activity of snord105b was also confirmed with in vivo data. Mechanistically, snord105b specifically bound to ALDOA and affected C-myc, which plays a key role in carcinogenesis and tumor development. CONCLUSION: Snord105b appears to be a novel oncogene and is clinically and functionally involved in the development of GC. Targeting snord105b and its pathway may provide new biomarkers or potential treatments for patients with GC.

miR-200c regulates crizotinib-resistant ALK-positive lung cancer cells by reversing epithelial-mesenchymal transition via targeting ZEB1.

Crizotinib is an orally administered drug for the treatment of patients with anaplastic lymphoma kinase (ALK)-positive locally advanced or metastatic non­small cell lung cancer (NSCLC). Despite the impressive efficacy of crizotinib in the treatment of ALK­positive lung cancer, acquired resistance eventually develops in the majority of patients. The microRNA (miR)­200c reverses the resistance of lung cancer cells to various chemotherapeutic drugs and molecular targeted drugs, however, whether it can reverse the resistance of crizotinib remains unknown. The present study established a crizotinib resistant cell line (NCI­2228/CRI), which was derived from the parental NCI­2228 cell line by long­term exposure to increasing concentrations of crizotinib. Through overexpression and suppression of miR­200c expression, the characteristics associated with epithelial­mesenchymal transition (EMT), including morphology, EMT marker proteins and cellular mobility, were investigated. Cell viability and invasion assays demonstrated that high expression of miR­200c significantly inhibited the proliferation, migration and invasion of NCI­2228 cells compared with the negative control. A luciferase reporter assay indicated that miR­200c directly targeted the 3'­untranslated region of zinc finger E­box binding homeobox 1. Additionally, reverse transcription­quantitative polymerase chain reaction analysis demonstrated that the mRNA levels of N­cadherin and Vimentin were decreased in NCI­2228 cells transfected with miR­200c mimic compared with negative control cells, whereas the mRNA level of E­cadherin was increased. In addition, EMT was reversed by miR­200c, which suggests that miR­200c may serve a role in mediating the sensitivity of NCI­2228/CRI cells to crizotinib. The present study may therefore contribute to improving the sensitivity of ALK positive lung cancer cells to crizotinib.

p-Hydroxylcinnamaldehyde induces the differentiation of oesophageal carcinoma cells via the cAMP-RhoA-MAPK signalling pathway.

p-Hydroxylcinnamaldehyde (CMSP) has been identified as an inhibitor of the growth of various cancer cells. However, its function in oesophageal squamous cell carcinoma (ESCC) and the underlying mechanism remain unclear. The aim of the present study was to characterize the differentiation effects of CMSP, as well as its mechanism in the differentiation of ESCC Kyse30 and TE-13 cells. The function of CMSP in the viability, colony formation, migration and invasion of Kyse30 and TE-13 cells was determined by MTS, colony-formation, wound healing and transwell assays. Western blotting and pull-down assays were used to investigate the effect of CMSP on the expression level of malignant markers of ESCC, as well as the activity of MAPKs, RhoA and GTP-RhoA in Kyse30 and TE-13 cells. We found that CMSP could inhibit proliferation and migration and induce Kyse30 and TE-13 cell differentiation, characterized by dendrite-like outgrowth, decreased expression of tumour-associated antigens, as well as the decreased expression of malignant markers. Furthermore, increased cAMP, p-P38 and decreased activities of ERK, JNK and GTP-RhoA, were detected after treatment with CMSP. These results indicated that CMSP induced the differentiation of Kyse30 and TE-13 cells through mediating the cAMP-RhoA-MAPK axis, which might provide new potential strategies for ESCC treatment.

P-Hydroxycinnamaldehyde Induces B16-F1 Melanoma Cell Differentiation via the RhoA-MAPK Signaling Pathway.

BACKGROUND/AIMS: Due to its antitumor and gastroprotective properties, cochinchina momordica seed (CMS), has been widely used to treat cancer patients in Asia. Our previous reports have shown that CMS is able to induce the differentiation of B16-F1 melanoma cells. However, its functional component and mechanism remain unclear and are addressed in this study. METHODS AND RESULTS: CMSP (p-hydroxycinnamaldehyde isolated from CMS) inhibited the proliferation, migration and invasiveness of B16-F1 cells both in vivo and in vitro. CMSP also induced the differentiation of B16-F1 cells, as characterized by dendrite-like outgrowth, increased melanogenesis and enhanced tyrosinase activity. Furthermore, CMSP treatment reduced the level of malignant markers of melanoma, specifically S-100B and melanoma-derived growth regulatory protein precursor (MIA), in a concentration-dependent manner. According to a western blot analysis, B16-F1 cells treated with CMSP exhibited a sustained increase in p-P38 and decreased activities of ERK and JNK. Our data further indicated that the downregulation of GTP-RhoA, which was mediated by increased cAMP release, was involved in CMSP-induced changes in MAPK, while LPA (Lysophosphatidic acid) partially reversed CMSP-induced B16 cell differentiation. CONCLUSION: These results demonstrated that CMSP-induced differentiation of B16F1 cells may occur through the RhoA-MAPK axis, which suggests a new potential strategy for melanoma treatment.

Periplocin Extracted from Cortex Periplocae Induced Apoptosis of Gastric Cancer Cells via the ERK1/2-EGR1 Pathway.

BACKGROUND/AIMS: Periplocin is extracted from the traditional herbal medicine cortex periplocae, which has been reported to suppress the growth of cancer cells. However, little is known about its effect on gastric cancer cells. METHODS: Gastric cancer cells were treated with periplocin, and cell viability was assessed using MTS assay. Flow cytometry and TUNEL staining were performed to evaluate apoptosis, and protein expression was examined by western blotting. Microarray analysis was used to screen for changes in related genes. RESULTS: We found that periplocin had an inhibitory effect on gastric cancer cell viability in a dose-dependent manner. Periplocin inhibited cell viability via the ERK1/2-EGR1 pathway to induce apoptosis. Periplocin also inhibited the growth of tumor xenografts and induced apoptosis in vivo. CONCLUSION: Our results show that periplocin inhibits the proliferation of gastric cancer cells and induces apoptosis in vitro and in vivo, indicating its potential to be used as an antitumor drug.

The tumor-suppressive function of miR-1 by targeting LASP1 and TAGLN2 in esophageal squamous cell carcinoma.

OBJECTIVE: This study determined the expression of microRNA-1 in esophageal squamous cell carcinoma (ESCC) tissue and cell lines to evaluate its effects on clinicopathological parameters and its target genes LASP1 and TAGLN2. METHODS: The expression of miR-1, lasp1, and tagln2 was detected in 55 ESCC tissues and adjacent normal tissues by reverse transcription-polymerase chain reaction (RT-PCR). The association between miR-1, lasp1, and tagln2 expression and clinicopathological characteristics was observed. MicroRNA-1 (mimics-miR-1) and its inhibitor (Inhibitor-miR-1) were transfected into esophageal cancer cells KYSE 510 and Eca 109; cell proliferation, migration, and invasion assays were carried out. Plasmid construction and dual-luciferase reporter assay were also carried out to indicate whether LASP1 and TAGLN2 were miR-1 target genes. The expression of LASP1 and TAGLN2 was detected with Western blot methods in cell lines, by immunohistochemistry in ESCC tissue. RESULTS: The gene expression level of microRNA-1 in cancer tissues was significantly lower than that in adjacent normal tissues (P < 0.01). The expression of miR-1 in ESCC was correlated with involvement of lymph nodes (P = 0.002), histologic classification (P = 0.000), and vessel invasion (P = 0.022). The expression of lasp1 and tagln2 increased in cancer tissues compared with in adjacent normal tissues (P < 0.05). MiR-1 suppresses the cell growth, migration, and invasion in vitro. The expression of LASP1 and TAGLN2 decreased in mimics-miR-1 transfected cells, and increased in inhibitor-miR-1 transfected cells. Luciferase reporter assay confirmed that LASP1 and TAGLN2 mRNA actually had the target sites of miR-1. CONCLUSIONS: miR-1 suppresses cell proliferation, invasiveness, metastasis, and progression of ESCC by binding its targeted genes LASP1 and TAGLN2.

Inhibitory effect of Embelin on human acute T cell lymphoma Jurkat cells through activation of the apoptotic pathway.

It has previously been shown that Embelin inhibits proliferation, promotes apoptosis, and increases sensitivity and reduces resistance to chemotherapy drugs, in various types of tumor cells. The present study examined the effects of Embelin on the proliferation of human acute T cell lymphoma Jurkat cells. Jurkat cells were treated with various concentrations of Embelin and the effects of Embelin on the inhibition of growth of Jurkat cells were evaluated. Expression of X-linked inhibitor of apoptosis protein (XIAP); poly ADP ribose polymerase; caspase-3; caspase-8; caspase-9; the proapoptotic protein, Bax; and the antiapoptotic proteins, Bcl-xl and Bcl-2, were assessed. The results showed that Embelin significantly inhibited the growth of human acute T cell lymphoma Jurkat cells. Following treatment with 5, 10 or 20 mM Embelin for 48 h, cell viability was 82.31, 58.65 and 37.62%, respectively, which was significantly reduced compared with that of the control group and the 0.1% DMSO control group (P<0.01). Furthermore, the caspase-3 inhibitor, z-DEVD-fmk, and the caspase-9 inhibitor, Ac-LEHD-CHO, reversed this inhibitory effect. It was also shown that the apoptotic rate of cells treated with Embelin was significantly elevated. Subsequently, it was demonstrated that Embelin downregulated the expression of XIAP and the proapoptotic Bcl2 family members, Bcl-2 and Bcl-xl, while it concomitantly upregulated that of the antiapoptotic protein, Bax. These results showed that Embelin inhibited growth and induced apoptosis of Jurkat cells in vitro, by activating the endogenous caspase-dependent apoptotic pathway through inhibition of XIAP and proapoptotic Bcl-2 family members.

[Effect of Tanreqing injection on immune activity of peripheral blood lymphocytes of patients with lung cancer].

To investigate the effect of Tanreqing injection on immune activity of peripheral blood lymphocytes of patients with lung cancer. The peripheral blood lymphocytes of patients with lung cancer and healthy persons were separated by the density gradient centrifugation method for subsequent experiments, with those from healthy persons as the positive control. The effect of Tanreqing injection on stimulating the proliferation of lymphocytes with phytohemagglutinin (PHA) was determined by MTT method. The effect of Tanreqing injection on the lymphocyte secretions of IFN-γ and TNF-α and the subset ratio of lymphocytes cultured separately or with Tanreqing injection of different concentrations were examined by ELISA and flow cytometry (FCM) respectively. In addition, the LDH release assay was used to detect the cytotoxicity of cytotoxic T cells (CTL) and natural killer cells (NK). According to the findings, all of immunological indexes of lymphocytes from patients with lung cancer were weaker than that of healthy persons, but with the obvious increases in proliferation activity and IFN-γ and TNF-α secretions of lymphocytes co-cultured with Tanreqing Injection (P < 0.05). Among lymphocyte subsets co-cultured with Tanreqing Injection, CD3+, CD3+ CD4+ and CD3- CD16 + 56+ cell ratios notably increased, whereas CD4+ CD25+ Treg cell ratio obviously decreased (P < 0.05). In the meantime, Tanreqing injection can markedly promote the cytotoxicities of CTL and NK (P < 0.05). In conclusion, Tanreqing injection shows a significant effect in promoting the immune activity of lymphocytes from patients with lung cancer and their anti-tumor immunity.

Prevention effects of Schisandra polysaccharide on radiation-induced immune system dysfunction.

In this study, we investigate the efficacy of SP (Schisandra polysaccharide) in prevention of radiation-induced immune dysfunction and discussed the underlying mechanisms with a Bal/bc mouse model. The data demonstrated that SP could reverse the decreases in the number of white blood cells and lymphocytes in peripheral blood. In addition, the immunoglobulin G (IgG) and complement C3 in blood serum were all decreased after radiation and SP could restore this radiation disorder. Furthermore, SP could reverse the deregulation of CD3(+)CD4(+) and CD3(+)CD8(+) T cell subsets in peripheral blood and thymus of mice after radiotherapy. We also performed terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) and Immunohistochemistry (IHC) to investigate the apoptosis and underlying mechanisms of SP in thymus. Data showed that radiation-induced apoptosis of thymocytes could be reversed by SP through inducing upregulation of Bcl-2 expression and downregulation of Fas and Bax levels. Furthermore, SP has no any side-effects on immunity of normal mice. In conclusion, our results indicated that SP could effectively prevent immune injury during radiotherapy by protecting the immune system. This valuable information should be of assistance in choosing a rational design for therapeutic interventions of prevention immune system damage in the radiation treatment.

Optimization of reference genes for normalization of the quantitative polymerase chain reaction in tissue samples of gastric cancer.

For an exact comparison of mRNA transcription in different samples or tissues with real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR), it is crucial to select a suitable internal reference gene. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and beta-actin (ACTB) have been frequently considered as house-keeping genes to normalize for changes in specific gene expression. However, it has been reported that these genes are unsuitable references in some cases, because their transcription is significantly variable under particular experimental conditions and among tissues. The present study was aimed to investigate which reference genes are most suitable for the study of gastric cancer tissues using qRT-PCR. 50 pairs of gastric cancer and corresponding peritumoral tissues were obtained from patients with gastric cancer. Absolute qRT-PCR was employed to detect the expression of GAPDH, ACTB, RPII and 18sRNA in the gastric cancer samples. Comparing gastric cancer with corresponding peritumoral tissues, GAPDH, ACTB and RPII were obviously up-regulated 6.49, 5.0 and 3.68 fold, respectively. Yet 18sRNA had no obvious expression change in gastric cancer tissues and the corresponding peritumoral tissues. The expression of GAPDH, ß-actin, RPII and 18sRNA showed no obvious changes in normal gastric epithelial cells compared with gastric cancer cell lines. The carcinoembryonic antigen (CEA), a widely used clinical tumor marker, was used as a validation gene. Only when 18sRNA was used as the normalizing gene was CEA obviously elevated in gastric cancer tissues compared with peritumoral tissues. Our data show that 18sRNA is stably expressed in gastric cancer samples and corresponding peritumoral tissues. These observations confirm that there is no universal reference gene and underline the importance of specific optimization of potential reference genes for any experimental condition.