GATA2 rs2335052 Polymorphism Predicts the Survival of Patients with Colorectal Cancer

<div><p>Background</p><p>GATA binding protein 2 (GATA2) is a transcription factor that has essential roles in hematologic malignancies and progression of various solid tumors. Our previous studies suggested that high GATA2 expression is associated with recurrence of colorectal cancer (CRC). However, the influence of GATA2 single nucleotide polymorphisms (SNPs) on the survival of CRC remains unknown.</p><p>Methods</p><p>We genotyped GATA2 SNP rs2335052 using Sanger sequencing after PCR amplification, and determined GATA2 expression by immunohistochemistry in a cohort of 180 CRC patients. Kaplan-Meier survival analysis and Cox proportional hazard regression were used to analyze the association between the GATA2 rs2335052 genotypes and the clinical outcome of CRC.</p><p>Results</p><p>We found that there was no significant correlation between the rs2335052 genotypes and the expression of GATA2. However, the Kaplan-Meier survival analysis suggested that the carriers of the A-allele of SNP rs2335052 were significantly associated with increased risk of recurrence and reduced disease-free survival (DFS), compared with those carrying the variant genotype of GG in rs2335052 (<i>P</i> = 0.021). Moreover, univariate and multivariate Cox regression analyses revealed that GATA2 SNP rs2335052 was an independent risk factor for the DFS of CRC patients.</p><p>Conclusion</p><p>Our results demonstrated that GATA2 SNP rs2335052 is an independent predictor for prognosis of CRC patients. This raised the possibility that SNP rs2335052 may serve as a potential indicator for predicting recurrence of CRC after curative colectomy.</p></div

Clinicopathological features with respect to determined genotypes of included GATA2 SNP rs2335052.

<p>Differences in categorical study variables between genotypes were tested for statistical significance with the Chi-squared test. Tumors were classified according to the guidelines of the American Joint Committee on Cancer (AJCC) staging system.</p><p>Clinicopathological features with respect to determined genotypes of included GATA2 SNP rs2335052.</p

Univariate and multivariate analyses of GATA2 rs2335052 genotypes in CRC patients with respect to DFS.

<p><i>HR</i> hazard ratio, <i>CI</i> confidence interval, <i>P</i> values in bold were statistically significant.</p><p>Univariate and multivariate analyses of GATA2 rs2335052 genotypes in CRC patients with respect to DFS.</p

Kaplan-Meier analysis of DFS according to GATA2 expression.

<p><b>(A)</b> Kaplan-Meier survival curve showed DFS for patients with GATA2-high tumors versus patients with GATA2-low tumors. Kaplan-Meier survival curves showed DFS for patients stratified by GATA2 rs2335052 GG <b>(B)</b>, and GG+AA <b>(C)</b> genotypes. The log-rank test was used to calculate <i>P</i> values.</p

Sequencing results of GATA2 rs2335052 genotypes.

<p>Sequencing results of GATA2 rs2335052 genotypes.</p

Kaplan-Meier analysis for DFS according to rs2335052 genotypes in CRC patients stratified by clinical stage.

<p>Kaplan-Meier curves indicated DFS for the subgroup of patients with stage I/II <b>(A)</b>, and stage III/IV <b>(B)</b> CRC. The log-rank test was used to calculate <i>P</i> values.</p

Immunohistochemical analysis of GATA2 expression in colorectal tissues.

<p>Representative image indicated strong GATA2 staining in CRC tissue, and negative GATA2 staining in matched noncancerous tissue. Magnification is 100×.</p

Tanshinone IIA Increases the Bystander Effect of Herpes Simplex Virus Thymidine Kinase/Ganciclovir Gene Therapy via Enhanced Gap Junctional Intercellular Communication

<div><p>The bystander effect is an intriguing phenomenon by which adjacent cells become sensitized to drug treatment during gene therapy with herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV). This effect is reported to be mediated by gap junctional intercellular communication (GJIC), and therefore, we postulated that upregulation of genes that facilitate GJIC may enhance the HSV-tk/GCV bystander effect. Previous findings have shown Tanshinone IIA (Tan IIA), a chemical substance derived from a Chinese medicine herb, promotes the upregulation of the connexins Cx26 and Cx43 in B16 cells. Because gap junctions are formed by connexins, we hypothesized that Tan IIA might increase GJIC. Our results show that Tan IIA increased GJIC in B16 melanoma cells, leading to more efficient GCV-induced bystander killing in cells stably expressing HSV-tk. Additionally, in vivo experiments demonstrated that tumors in mice with 10% HSV-tk positive B16 cells and 90% wild-type B16 cells became smaller following treatment with the combination of GCV and Tan IIA as compared to GCV or Tan IIA alone. These data demonstrate that Tan IIA can augment the bystander effect of HSV-tk/GCV system through increased gap junction coupling, which adds strength to the promising strategy that develops connexins inducer to potentiate the effects of suicide gene therapy.</p></div

Tan IIA in combination with GCV causes greater growth inhibition on a mixture of HSV-tk and WT B16 cells.

<p>HSV-tk B16 cells were mixed with WT B16 cell line at a ratio of 1∶9. The mixture of cells was left untreated or treated with Tan IIA (10 or 20 µM) for 24 h, and then was cultured with or without GCV (15.7 µM) for 48 h followed by MTT assay. *p<0.05, **p<0.01 compared with control; ^##p<0.01 compared with Tan IIA or GCV treatment alone.</p

Tan IIA enhances the bystander effect.

<p>Stable HSV-tk-EGFP and RFP B16 cell lines were mixed at a ratio of 1∶1. The mixture of cells was left untreated or treated with Tan IIA (10 or 20 µM) for 24 h, and then was cultured with or without GCV (15.7 µM) for 48 h. (A) Representative images as shown by fluorescence microscopy. The red fluorescence in living RFP cells mainly localizes in cytoplasm; the aggregation of red fluorescence indicates cell shrinkage which is the hallmark of apoptosis (white arrows); the clustered light spots indicate the formation of apoptosis bodies (yellow arrows). (B) The apoptosis of RFP cells was analyzed by flow cytometry with annexin V stain. (C) Quantification of three independent experiments. *p<0.05, **p<0.01 compared with control; ^#p<0.05, ^#p<0.01 compared with Tan IIA or GCV treatment alone.</p