IL7R Is Correlated With Immune Cell Infiltration in the Tumor Microenvironment of Lung Adenocarcinoma.

Tumor microenvironment plays an important role in the development, progression, and prognosis of lung adenocarcinoma. Exploring new biomarkers based on the immune microenvironment of lung adenocarcinoma can effectively predict the prognosis and provide effective clinical treatment. In this study, we used the ESTIMATE algorithm to score the immune and stromal components in lung adenocarcinoma data downloaded from the TCGA database. The result showed that the immune/stromal score was associated with clinical features and prognosis of lung adenocarcinoma patients. Interleukin-7 receptor (IL7R) is an important prognostic biomarker identified by intersection analysis of protein-protein interaction networks and Cox regression survival analysis. According to TCGA and Oncomine database analysis, IL7R expression in adenocarcinoma tissues was significantly lower than that in normal lung tissues and was further verified in clinical tissue samples. Survival analysis showed IL7R was an independent prognostic factor of lung adenocarcinoma. IL7R expression was positively correlated with the overall survival and progression-free survival of lung adenocarcinoma patients and negatively correlated with tumor size. Our results suggest that IL7R inhibits tumor growth by regulating the proportion of immune infiltrating cells in the tumor immune microenvironment. IL7R could be a beneficial prognostic marker in patients with lung adenocarcinoma and has great potential in immune therapy.

Changes in Methylation across Structural and MicroRNA Genes Relevant for Progression and Metastasis in Colorectal Cancer.

MiRs are important players in cancer and primarily genetic/transcriptional means of regulating their gene expression are known. However, epigenetic changes modify gene expression significantly. Here, we evaluated genome-wide methylation changes focusing on miR genes from primary CRC and corresponding normal tissues. Differentially methylated CpGs spanning CpG islands, open seas, and north and south shore regions were evaluated, with the largest number of changes observed within open seas and islands. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed several of these miRs to act in important cancer-related pathways, including phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt) and mitogen-activated protein kinase (MAPK) pathways. We found 18 miR genes to be significantly differentially methylated, with MIR124-2, MIR124-3, MIR129-2, MIR137, MIR34B, MIR34C, MIR548G, MIR762, and MIR9-3 hypermethylated and MIR1204, MIR17, MIR17HG, MIR18A, MIR19A, MIR19B1, MIR20A, MIR548F5, and MIR548I4 hypomethylated in CRC tumor compared with normal tissue, most of these miRs having been shown to regulate steps of metastasis. Generally, methylation changes were distributed evenly across all chromosomes with predominance for chromosomes 1/2 and protein-coding genes. Interestingly, chromosomes abundantly affected by methylation changes globally were rarely affected by methylation changes within miR genes. Our findings support additional mechanisms of methylation changes affecting (miR) genes that orchestrate CRC progression and metastasis.

Lower serum CA125 level, negative vascular invasion, and wild BRAF were strongly associated with better 2-year disease-free survival in patients with stage III colorectal cancer who received adjuvant chemotherapy.

BACKGROUND: Adjuvant chemotherapy plays important role in the comprehensive treatment of patients with stage III colorectal cancer. However, there is few molecular markers for predicting the therapeutic effect. OBJECTIVE: To identify factors that could predict adjuvant chemotherapy benefits in patients with stage III colorectal cancer. METHODS: The medical records of 294 patients were reviewed and analyzed using the Kaplan-Meier method and Cox analysis. RESULTS: Lower CA125 (⩽ 35 u/ml, P= 0.0015) serum levels, stage IIIa (P= 0.0027), 1-3 positive lymph nodes (P= 0.0256), negative vascular invasion (P= 0.0215), lower CA199 (⩽ 27 u/ml, P= 0.0038) serum levels, and wild-type BRAF status (P= 0.0125) were significantly associated with a higher 2-year DFS rate in patients with stage III colorectal cancer. However, in multivariate COX analysis, the association remained significant only for CA125 levels (vs. ⩽ 35 u/ml group, HR 3.341; 95% CI, 1.198-9.316; P= 0.0212), vascular invasion (vs. negative vascular invasion, HR, 2.349; 95% CI, 1.227-4.499; P= 0.01), and BRAF (V600E) (vs. wild Braf, HR, 7.794; 95% CI, 1.867-32.531; P= 0.0049). CONCLUSION: Lower CA125 serum levels, negative vascular invasion, and wild-type BRAF status were significantly associated with improved 2-year DFS rates among patient with stage III disease who received adjuvant chemotherapy.

TNM staging system may be superior to Lugano and Ann Arbor systems in predicting the overall survival of patients with primary gastrointestinal lymphoma.

PURPOSE: To assess the survival predicting value of TNM, Lugano, and Ann Arbor staging systems in patients with primary gastrointestinal lymphoma (PGL). METHODS: 101 patients with PGL were reviewed. All of them were staged according to TNM, Lugano, or Ann Arbor staging system. Five-year survival overall survival/OS rate was used as major clinical outcome. The prognostic value of different variables like depth of tumor infiltration (T), lymph node status (N), metastasis (M), sex, age, LDH, ECOG performance status (PS), subtypes, and tumor sites were assessed in relation to clinical outcome. RESULTS: The median follow-up time was 46.6 months (range 1.3-158.6). The estimated 5-year OS rate was 74.22%. In gastric lymphoma ,the 5-year OS rate was well correlated with stage in the TNM system (stage I 100.00%, stage II 87.18%, stage III 75.17%, and stage IV 16.67%. p<0.0001), but there were inverse 5-year OS or overlapped survival in the Lugano (81.48% in stage II, 85.71% in stage IIE) and Ann Arbor systems (69.47% in stage IIE, 66.67% in stage IIIE). In aggressive lymphomas, the 5-year OS of TNM stage I, stage II, stage III , and stage IV was 100.00%, 81.34%, 63.52%, and 16.00%, respectively (p=0.0002), but there were overlapped survival curves in Lugano and Ann Arbor systems. The 5-year OS of patients with T1 or T2 was significantly superior compared to patients with T3 or T4 (96.15 vs 67.92%, p=0.0087), and multivariate Cox analysis showed that T (p=0.0181) and M (p=0.0031) were the covariates prognostically significant for OS. CONCLUSION: TNM staging system may be superior to Lugano and Ann Arbor system in predicting OS of patients with PGL.

Zoledronic acid inhibits growth of hepatocellular carcinoma cells in vitro and in vivo.

BACKGROUND: Growing preclinical evidence shows that zoledronic acid (ZOL) exhibits direct antitumor activity in various cancer cell lines. However, the cytotoxic effects of ZOL on human hepatocellular carcinoma (HCC) cells have not been established. In the present study, we investigated the effect of ZOL on HCC both in vitro and in vivo. METHODS: Cytotoxicity and cell cycles were assessed with Sulforhodamine B colorimetric assay and flow cytometry. Expression levels of cell cycle phase-linked proteins were examined. The effect of ZOL on HCC in vivo was explored based on H22-subcutaneous injection (s.c.) and H22-intraperitoneal injection (i.p.) mice model. RESULTS: ZOL inhibited the growth of SK-HEP-1 and H22 cells and induced S-phase arrest through downregulating cdc2 protein and upregulating cyclin A. It inhibited the growth of s.c tumors, and increased the survival of both H22-s.c. and H22-i.p. mice in vivo. CONCLUSION: ZOL inhibits growth of HCC cells in vitro and in vivo.

Twist expression associated with the epithelial-mesenchymal transition in gastric cancer.

This study aimed to investigate the expression of Twist in gastric cancer tissues and its correlation between Twist and the epithelial-mesenchymal transition (EMT). By means of RT-PCR and Western blot, the mRNA and protein expressions of Twist, E-cadherin, and Vimentin in 61 gastric cancer tissues and adjacent normal tissues were detected. The positive rates of Twist, E-cadherin, and Vimentin mRNA expression in gastric cancer tissues were 73.9. 40.6, and 60.9 %, respectively; compared to the expression of these genes in adjacent normal tissues (2.9, 75.4, and 27.5 %), the differences were significant (p < 0.05). The E-cadherin protein expression level in gastric cancer tissues was significantly lower than that in the adjacent normal tissues (p < 0.05). After the transfection of Twist siRNA into the MKN45 cells, the protein expression of Twist was significantly reduced (p < 0.05), the protein expression of E-cadherin was significantly increased, and the number of cells that passed through the Transwell chamber was significantly lower than that in the non-transfected control group as well as the transfected control group (p < 0.05). Twist may be associated with the epithelial-mesenchymal transition in gastric cancer and the tumorigenesis, invasion, and metastasis of gastric cancer.

Antitumor activity of an adenovirus harboring human IL-24 in colon cancer.

Data have increasingly shown that melanoma differentiation associated gene-7 (Mda-7/IL-24) has growth suppression activity and can induce apoptosis in many tumor cells, but to our knowledge there have been few studies about its role in colon cancer. We examined its anti-cancer effect on colon cancer. We constructed a recombinant replication-deficient adenovirus carrying human melanoma differentiation associated gene-7 (Ad-IL-24) and examined its apoptosis-inducing efficacy on the colon cancer HT-29 cell line and on an oxaliplatin-resistant cell line HT-29/oxa, using a combination of flow cytometry, growth suppressive activity by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and xenografts. Furthermore, we tested the suppression activity of Mda-7/IL-24 on vascular endothelial growth factor (VEGF) and microvessel density (MVD), as well as the inductive effect on expression of the growth arrest and DNA damage gene (GADD) in xenograft tumors by immunohistochemistry. Melanoma differentiation associated gene-7 can inhibit the growth of colon cancer cell lines and induced apoptosis in about (5.6±0.3)% of HT-29 cells (P<0.05). Xenograft growth was retarded in vivo in mice treated with melanoma differentiation associated gene-7, but the tumor proliferation rate for this group was not significantly different in comparison to controls (P>0.05). Furthermore, melanoma differentiation associated gene-7 induced expression of a growth arrest and DNA damage (GADD) gene and reduced the expression of both VEGF and MVD in xenograft tumors. This study supports a potential therapeutic effect for melanoma differentiation associated gene-7 on colon cancer.

[Construction of adenovirus expressing anti-vascular endothelial growth factor hairpin ribozyme and its inhibition of growth of colorectal cancer HT-29 cells].

BACKGROUND AND OBJECTIVE: Oversexpression of vascular endothelial growth factor (VEGF) is correlated to poor prognosis of colorectal cancer. This study was to construct replication deficient adenovirus carrying anti-VEGF hairpin ribozyme and investigate its inhibitory effects on VEGF expression and growth of colorectal cancer HT-29 cells. METHODS: Anti-VEGF hairpin ribozyme was cloned into the transfer vector pAdTrack-CMV, and recombined with adenoviral backbone vector pAdEasy-1 in BJ5183 bacteria. The recombinant plasmids were packaged and amplified in 293 cells (named Ad-Rz). The expression of anti-VEGF ribozyme in HT-29 cells was examined by RT-PCR. Inhibition of Ad-RZ on VEGF mRNA and protein expressions were detected by real-time PCR and ELISA, respectively. Inhibition of cell growth was measured by MTT assay. HT-29 cell xenografts were established in nude mice and the microvessel density (MVD) was determined by CD34 staining. RESULTS: The anti-VEGF ribozyme was successfully inserted into the adenoviral vector. Anti-VEGF ribozyme was effectively expressed in HT-29 cells. After infection by Ad-RZ, the relative expression of VEGF mRNA was decreased to about(45+/-0.01)% of that of PBS control group in HT-29 cells (P<0.05); the amount of VEGF protein in the supernatant of Ad-Rz treated cells [(OD=(0.46+/-0.35)/million cells] was lower than that of PBS treated cells [(OD=(0.80+/-0.35)/million cells] (P<0.05). Although Ad-Rz did not significantly inhibit cell growth of HT-29 cells (P>0.05), it significantly inhibited tumor angiogenesis of HT-29 cell xenografts in nude mice, compared with PBS group (P<0.05). The proliferation rate of xenografts treated by Ad-Rz was lower than that treated by PBS, but the difference was not significant (P>0.05). CONCLUSION: Anti-VEGF hairpin ribozyme mediated by adenovirus could inhibit the expression of VEGF and the tumor angiogenesis in colorectal cancer HT-29 cells.

Another formula for calculating the gene change rate in real-time RT-PCR.

Relative quantification real-time PCR has become a routine method in molecular biology research to study the small amount gene expression. There are a few mathematical models for relative quantification data analysis in real-time PCR, and the formula 2(-DDCT ) is one of the most frequently used method. In this paper, we are to present another equation which directly calculates the change rate (R (c)) of gene expression, and give a example to compare the application of this model with 2(-DDCT).

Antitumor activity of an adenovirus harboring two therapeutic genes, anti-VEGF ribozyme and human IL-24, in colon cancer.

Increased vascular endothelial growth factor (VEGF) expression and tumor vascularization are correlated with both tumor progression and a poor clinical prognosis in colon cancer. There is increasing evidence that mda-7/IL-24 has potent growth suppression activity and induces apoptosis in many different tumor cells. We constructed a recombinant replication-deficient adenovirus carrying both an anti-VEGF hairpin ribozyme and human IL-24 (Ad-Rz/IL-24) with an internal ribosome entry site and tested its VEGF inhibition effect and antitumor activity. The results showed that Ad-Rz/IL-24 significantly inhibited VEGF expression and angiogenesis in vitro and in vivo. The proliferation rate of xenograft tumors treated with Ad-Rz/IL-24 was approximately 50% lower than the rate of the xenograft tumors treated with adenovirus carrying a single gene anti-VEGF hairpin ribozyme (Ad-Rz) or IL-24 (Ad-IL-24) alone. These findings demonstrated the potential therapeutic effect of this dual gene-expressing adenovirus for cancer gene therapy.