Mechanisms underlying the efficacy and limitation of dopa and tetrahydrobiopterin therapies for the deficiency of GTP cyclohydrolase 1 revealed in a novel mouse model.

Dopa and tetrahydrobiopterin (BH4) supplementation are recommended therapies for the dopa-responsive dystonia caused by GTP cyclohydrolase 1 (GCH1, also known as GTPCH) deficits. However, the efficacy and mechanisms of these therapies have not been intensively studied yet. In this study, we tested the efficacy of dopa and BH4 therapies by using a novel GTPCH deficiency mouse model, Gch1KI/KI, which manifested infancy-onset motor deficits and growth retardation similar to the patients. First, dopa supplementation supported Gch1KI/KI mouse survival to adulthood, but residual motor deficits and dwarfism remained. Interestingly, RNAseq analysis indicated that while the genes participating in BH4 biosynthesis and regeneration were significantly increased in the liver, no significant changes were observed in the brain. Second, BH4 supplementation alone restored the growth of Gch1KI/KI pups only in early postnatal developmental stage. High doses of BH4 supplementation indeed restored the total brain BH4 levels, but brain dopamine deficiency remained. While total brain TH levels were relatively increased in the BH4 treated Gch1KI/KI mice, the TH in the striatum were still almost undetectable, suggesting differential BH4 requirements among brain regions. Last, the growth of Gch1KI/KI mice under combined therapy outperformed dopa or BH4 therapy alone. Notably, dopamine was abnormally high in more than half, but not all, of the treated Gch1KI/KI mice, suggesting the existence of variable synergetic effects of dopa and BH4 supplementation. Our results provide not only experimental evidence but also novel mechanistic insights into the efficacy and limitations of dopa and BH4 therapies for GTPCH deficiency.

[Screening for EGFR mutations in lung cancer by a novel real-time PCR with double-loop probe and Sanger DNA sequencing].

OBJECTIVE: To map the frequency and types of EGFR gene mutations present in lung cancer tissues. To evaluate the clinical applicability of a novel real-time double-loop probe PCR of which the ADx-EGFR kit is based, and to compare its performance with traditional Sanger DNA sequencing in the detection of somatic mutations of tumor genes. METHODS: A total of 208 formalin-fixed paraffin-embedded (FFPE) tumor samples were tested. Genomic DNA of the tissue samples was extracted and purified, and subjected to both traditional PCR amplification, Sanger sequencing of EGFR gene in exon 18, 19, 20, 21, and ADx's EGFR mutation detection kit. The mutation rates for EGFR gene in exon 18, 19, 20, 21, as well as the frequency of each mutation detected by the two methods, were analyzed. RESULTS: The traditional Sanger DNA sequencing technique was successfully performed in 196 out of 208 (94.2%) lung cancer samples, and 22 samples (11.2%) showed EGFR gene mutations. ADx-EGFR kit was successfully used in the lung cancers of all of the 208 cases (100.0%), and 40 samples (19.2%) showed mutations. In the lung cancer samples analyzed, mutations were mainly detected in the exon 19 and exon 21 L858R point mutation, i.e. 4.8% (10/208) and 11.6% (23/208) of total mutations, respectively, and the remaining mutations were rare. CONCLUSIONS: The success rate of ADx-EGFR real-time PCR for formalin-fixed and paraffin-embedded tissues samples is significantly higher than that of Sanger sequencing (P < 0.01). There are significant differences between the two methods. ADx-EGFR real-time PCR shows a much higher successful detection rate and mutation rate of lung cancer tissues compared with that of Sanger sequencing. As a result, the real-time PCR with ADx-EGFR kit is proved to have a good clinical applicability and a strong advantage over the traditional Sanger DNA sequencing. It is an effective and reliable tool for clinical screening of somatic gene mutations in tumors.

Promoting effect of Tmsb4x on the differentiation of peripheral blood mononuclear cells to dendritic cells during septicemia.

BACKGROUND: Thymosin beta 4 × (Tmsb4x) has been highlighted as an important regulator in immune and inflammation responses. Promoted differentiation of mononuclear cells into dendritic cells (DCs) exert a beneficial effect on septicemia. Herein, we investigated the effects of Tmsb4x on the mononuclear cells to affect immune responses during septicemia. METHODS: Initially, we isolated peripheral blood samples from healthy individuals and patients with septicemia for extraction of mononuclear cells, followed by Tmsb4x expression quantification. A cell model was constructed with mononuclear cells through lipopolysaccharide stimulation. The viability and apoptosis were evaluated in response to Tmsb4x silencing or re-expression. Additionally, the proportion of DCs was assessed by determining levels of inflammatory factors as well as by flow cytometric analysis. A mouse septicemia model was developed for in vivo validation. RESULTS: Cell and animal models demonstrated decreased Tmsb4x expression in the setting of septicemia, which led to increased inflammatory response and reduced proportion of DCs, along with inhibited mononuclear cell viability and promoted apoptosis. However, restoration of Tmsb4x facilitated the differentiation of mononuclear cells into DCs. CONCLUSION: To conclude, upregulated Tmsb4x promoted the generation of DCs from mononuclear cells, which contributed to deep understanding of underpinning mechanisms in the development of septicemia.

Quantitative analyses of CD133 expression facilitate researches on tumor stem cells.

CD133 is regarded as a marker of tumor initiating cells in many tumors, including colorectal cancer. O'Brien and Ricci et al. have proved that in primary colorectal tumors there are colorectal tumor stem cells (initiating cells) which are marked by CD133 antigen. Using a genetic knockin lacZ reporter mouse model, Shmelkov et al. challenged this increasingly influential viewpoint and drew two important conclusions that challenge former opinions. First, CD133 is widely distributed throughout the full range of tumor epithelial cells in the colon as opposed to being limited to a few cells. Second, CD133 negative cells of colon tumors are also tumorigenic, and are more inclined to metastasize. Based on these two opinions, we hypothesize that the expression of CD133 is different among tumor cells, and that quantitative but not qualitative analyses of CD133 abundance are necessary to determine the relationship between CD133 expression and tumor stem cell characteristics. To verify this hypothesis, colorectal cancer cell line SW620 was cultured and sorted into CD133(Hi), CD133(Mid) and CD133(Low) subgroups using magnetic microbeads to compare their xenograft biological characteristics. The results showed that the CD133(Hi) subgroup of SW620 is more close to the tumor initiating cells in terms of biological characteristics than CD133(Mid) and CD133(low) subgroups, but the CD133(low) subgroup still maintains the ability of tumorigenicity. It supported that tumor initiating cells are more correlated to the abundance of CD133.

The proteasome subunit PSMA7 located on the 20q13 amplicon is overexpressed and associated with liver metastasis in colorectal cancer.

The proteasome subunit PSMA7 located on the 20q13 amplicon was found to be differentially expressed in colorectal cancer by semiquantitative RT-PCR. PSMA7 mRNA was overexpressed in 37.5% (12/32) colorectal cancer tissues while it was either of a low level or not expressed in matched normal mucosa. The aim of this study was to examine the PSMA7 protein expression in 62 colorectal cancer primary sites, 34 lymph node metastatic sites and 13 liver metastatic sites by immunohistochemistry and clarify the correlations of this expression with the clinicopathological parameters. PSMA7 high expression was detected in 38.8% (24/62) colorectal cancer primary sites, 52.9% (18/34) lymph node metastatic sites and 100% (13/13) liver metastatic sites but not in the normal colorectal tissues. The PSMA7 high expression was significantly correlated with liver metastasis (P=0.028). Survival was significantly lower in patients with a PSMA7 high expression than in those with a PSMA7 low expression (P=0.0012). Moreover, in multivariate analysis, the PSMA7 expression demonstrated an independent prognostic factor (P=0.004, relative risk 5.057; 95% confidence interval, 1.682-15.201). These results indicated that PSMA7 may play an important role in colorectal cancer progression and provide a unique target site for the development of therapeutic drugs. The evaluation of PSMA7 expression in primary colorectal cancer at the time of surgery may be a valuable tool for defining patients with a high risk of developing liver metastasis.

[High expression of proteasome subunit PSMA7 in colorectal cancer is significantly correlated with liver metastasis].

OBJECTIVE: To investigate the correlation between the proteasome subunit PSMA7 expression in colorectal cancer and its role in liver metastasis. METHODS: To identify the PSMA7 protein expression in 62 primary site colorectal cancers, 34 lymph node metastatic sites and 13 liver metastatic sites by immunohistochemistry and clarify the correlation of its expression with the clinicopathological parameters. RESULTS: High expression of PSMA7 was detected in 38.7% (24/62) of primary site colorectal cancer, 52.9% (18/34) of lymph node metastatic sites and 100% (13/13) liver metastatic sites but not in the normal colorectal tissue. High expression of PSMA7 was significantly correlated with liver metastasis (P = 0.028). The survival rate was significantly lower in patients with high expression of PSMA7 than in those with low expression of PSMA7 (P = 0.0008). As well, in multivariate analysis, PSMA7 expression demonstrated to be an independent prognostic factor (P = 0.004, relative risk 5.057; 95% confidence interval, 1.682-15.201). CONCLUSION: PSMA7 may play an important role in the colorectal cancer progression. Evaluation of PSMA7 expression in primary colorectal cancer at the time of surgery might be a valuable test in defining patients with a high risk of developing liver metastasis.

Relationship between expression of CD105 and growth factors in malignant tumors of gastrointestinal tract and its significance.

AIM: Angiogenesis is an important step in the growth of solid malignant tumors. A number of angiogenic factors have been found such as transforming growth factor beta1 (TGF-beta1) and vascular endothelial growth factor (VEGF). However, the roles of TGF-beta1 and VEGF in gastrointestinal carcinogenesis are still unclear. This study was to investigate the expressions of TGF-beta1 and VEGF in gastrointestinal tract malignant tumors, as well as their association with microvessel density (MVD). At the same time, we also observed the localization of TGF-beta1 and its receptor CD105 in gastric malignant tumors. METHODS: The expressions of TGF-beta1 and CD105 were detected in 55 fresh specimens of gastric carcinoma and VEGF and CD105 in 44 fresh specimens of colorectal carcinoma by immunohistochemical staining (S-ABC). TGF-beta1 and CD105 in 55 gastric carcinoma tissues on the same slide were detected by using double-stain Immunohistochemistry (DS-ABC). RESULTS: Among the 55 cases of gastric carcinoma tissues, 30 were positive for TGF-beta1 (54.55%). The MVD of TGF-beta1 strong positive group (++ approximately +++ 23.22 +/- 5.8) was significantly higher than that of weak positive group (+17.56 +/- 7.2) and negative group (- 17.46 +/- 3.9) (q=4.5, q=5.3207, respectively, P<0.01). In the areas of high expression of TGF-beta1, MVD and the expression of CD105 were also high. Among the 44 cases of colonic carcinoma tissues, 26 were positive for VEGF (59.1%). The expressions of both VEGF and CD105 (MVD) were related with the depth of invasion (F=5.438, P<0.05; F=4.168, P=0.05), lymph node metastasis (F=10.311, P<0.01; F=20.282, P<0.01) and Dukes stage (F=6.196, P<0.01; F=10.274, P<0.01), but not with histological grade (F=0.487, P>0.05). There was a significant correlation between the expression of VEGF and CD105 (MVD) (r=0.720, P<0.01). CONCLUSION: Over-expression of TGF-beta1 and VEGF acts as stimulating factors of angiogenesis in gastrointestinal tumors. CD105, as a receptor of TGF-beta1, can regulate the biological effect of TGF-beta1 in tumor angiogenesis. MVD marked by CD105 is more suitable for detecting newborn blood vessels.

Genotype CC of rs1800947 in the C-reactive protein gene may increase susceptibility to colorectal cancer: a meta-analysis.

BACKGROUND: Single nucleotide polymorphisms of C-reactive protein (CRP) have been shown to be related to circulating CRP level, risk and prognosis in cancer patients. However, accumulating evidence of rs1800947 involvement in risk of cancer is inconsistent. Thus, a meta-analysis was performed to obtain a more precise relationship. MATERIALS AND METHODS: The pooled odds ratio (OR) and its 95% confidence interval were assessed in 10 eligible articles with 12 studies containing 5,601 cancer cases and 8,669 cancer-free controls. RESULTS: No significant association was observed overall and in subgroups in comparison of genotype GC vs GG (PH=0.847, OR=0.939, 95%CI=0.810-1.087), GC/CC vs GG (PH=0.941, OR=1.021, 95%CI=0.901-1.157) and allele C vs G (PH=0.933, OR=1.026, 95%CI=0.909-1.159). However, statistically significance was evident in comparison of genotype CC vs GG in cancer risk (PH=0.586, OR=2.854, 95%CI= 1.413-5.763), especially in colorectal cancer (PH=0.481, OR=4.527, 95%CI= 1.664- 12.315). CONCLUSIONS: Genotype CC of rs1800947 in the CRP gene is strongly associated with increased cancer risk, particularly in colorectal cancer.

Comparative screening of K-ras mutations in colorectal cancer and lung cancer patients using a novel real-time PCR with ADx-K-ras kit and Sanger DNA sequencing.

We determined frequency/types of K-ras mutations in colorectal/lung cancer. ADx-K-ras kit (real-time/double-loop probe PCR) was used to detect somatic tumor gene mutations compared with Sanger DNA sequencing using 583 colorectal and 244 lung cancer paraffin-embedded clinical samples. Genomic DNA was used in both methods; mutation rates at codons 12/13 and frequency of each mutation were detected and compared. The data show that 91.4% colorectal and 59.0% lung carcinoma samples were detected conclusively by DNA sequencing, whereas 100% colorectal and lung samples were detected by ADx-K-ras kit. K-ras gene mutations were detected in 32.9-27.4% colorectal samples using kit and sequencing methods, respectively. Whereas 10.6-8.3% lung cancer samples were positively detected by kit and sequencing methods, respectively. Notably, 172/677 showed mutations and 467/677 showed wild type by both methods; 38 samples showed mutations with kit but wild type with sequencing. Mutations in colorectal samples were as follows: GGT → GAT/codon-12 (35.1%); GGC → GAC/codon-13 (26.6%); GGT → GTT/codon-12 (18.2%); and GGT → GCT/codon-12 (1.6%). Mutations in lung samples were as follows: GGT > GTT/codon-12 (40.9%) and GGT > GCT/codon-12 (4.5%). In conclusion, K-ras mutations involved 32.2% colorectal and 10.6% lung samples among this cohort. ADx-K-ras real-time PCR showed higher detection rates (P < 0.05). The kit method has good clinical applicability as it is simple, fast, less prone to contamination and hence can be used effectively and reliably for clinical screening of somatic tumor gene mutations.

Adenovirus-mediated small hairpin RNA targeting Bcl-XL as therapy for colon cancer.

Bcl-XL, an anti-apoptotic protein of Bcl-2 family, is overexpressed in colon cancers. To determine Bcl-XL's potential feasibility as a therapeutic target, we constructed a recombinant adenovirus that expressed a U6 promoter-driven small hairpin RNA (shRNA) targeting Bcl-XL (Ad/Bcl-XL shRNA) and evaluated the vector's ability to induce RNA interference in vivo and alter apoptosis induction in colon cancer cells and tumours. Ad/Bcl-XL shRNA effectively knocked down Bcl-XL expression in colon cancer cells and decreased their viability. Treatment with Ad/Bcl-XL shRNA but not control vectors led to dramatically increased cleavage of cellular apoptosis-related enzymes caspase-9, caspase-3 and poly(ADP-ribose) polymerase. Ad/Bcl-XL shRNA also significantly suppressed the growth of subcutaneous tumours derived from DLD1 cells in a nude mouse model and did so without causing any obvious damage to normal tissues or normal human fibroblasts. Together, our results support the feasibility of using adenovirus-mediated RNA interference therapy targeting Bcl-XL against colon cancers and warrant further studies of its safety and efficacy.