Computed Tomography-Based Evaluation of Volume and Position Changes of the Target Region and Organs at Risk During Radiotherapy for Esophageal Cancer: A Pilot Study.

OBJECTIVE: To analyze changes in volume and position of target regions and organs at risk (OARs) during radiotherapy for esophageal cancer patients. METHODS: Overall, 16 esophageal cancer patients who underwent radiotherapy, including 10 cases of intensity-modulated radiation therapy (IMRT) and six of three-dimensional conformal radiotherapy (3D-CRT), were enrolled. The prescription doses for the planning target volumes (PTVs) were as follows: PTV1, 64 Gy/32 fractions; and PTV2, 46 Gy/23 fractions. Repeat computed tomography (CT) was performed for patients after the 5th, 10th, 15th, 20th, and 25th fractions. Delineation of the gross tumor volume (GTV) and OAR volume was determined using five repeat CTs performed by the same physician. The target and OAR volumes and centroid positions were recorded and used to analyze volume change ratio (VCR), center displacement (ΔD), and changes in the distance from the OAR centroid positions to the planned radiotherapy isocenter (distance to isocenter, DTI) during treatment. RESULTS: No patient showed significant changes in target volume (TV) after the first week of radiotherapy (five fractions). However, TV gradually decreased over the following weeks, with the rate slowing after the fourth week (40 Gy). The comparison of TV from baseline to 40 Gy (20 fractions) showed that average GTVs decreased from 130.7 ± 63.1 cc to 92.1 ± 47.2 cc, with a VCR of -29.21 ± 13.96% (p<0.01), while the clinical target volume (CTV1) decreased from 276.7 ± 98.2 cc to 246.7 ± 87.2 cc, with a VCR of -10.34 ± 7.58% (p<0.01). As TVs decreased, ΔD increased and DTI decreased. After the fourth week of radiotherapy (40 Gy), centroids of GTV, CTV1, and prophylactic CTV (CTV2) showed average deviations in ΔD of 7.6 ± 4.0, 6.9 ± 3.4, and 6.0 ± 3.0 mm, respectively. The average DTI of the heart decreased by 4.53 mm (from 15.61 ± 2.96 cm to 15.16 ± 2.27 cm). CONCLUSION: During radiotherapy for esophageal cancer, Targets and OARs change significantly in volume and position during the 2nd-4th weeks. Image-guidance and evaluation of dosimetric changes are recommended for these fractions of treatment to appropriate adjust treatment plans.

A comparative study of anti-aging properties and mechanism: resveratrol and caloric restriction.

Resveratrol and caloric restriction (CR) are the powerful therapeutic options for anti-aging. Here, their comparative effect on longevity-associated gene silencing information regulator (SIRT1) were evaluated in vitro and in vivo. IMR-90 cells treated with 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) were applied to establish a cellular senescence model, and rats treated with D-galactose (D-gal) were used as an aging animal model. Resveratrol and CR exhibited similar anti-aging activities, evidenced by inhibiting senescence and apoptosis, and restoring cognitive impairment and oxidative damage. Moreover, they could up-regulate telomerase (TE) activity, increase expressions of SIRT1, forkhead box 3a (Foxo3a), active regulator of SIRT1 (AROS) and Hu antigen R (HuR ), but decrease p53 and deleted in breast cancer 1 (DBC1) levels. However, 10 µM resveratrol in vitro and the high dose group in vivo showed relatively stronger activities of anti-aging and stimulating SIRT1 level than CR. In conclusion, resveratrol and CR showed similar anti-aging activities on SIRT1 signaling, implicating the potential of resveratrol as a CR mimetic.

Kruppel-like factor 2 inhibit the angiogenesis of cultured human liver sinusoidal endothelial cells through the ERK1/2 signaling pathway.

Kruppel-like factor 2 (KLF2) is a crucial anti-angiogenic factor. However, its precise role in hepatic angiogenesis induced by liver sinusoidal endothelial cells (LSECs) remain unclear. This study was aimed to evaluate the effect of KLF2 on angiogenesis of LSECs and to explore the corresponding mechanism. Cultured human LSECs were infected with different lentiviruses to overexpress or suppress KLF2 expression. The CCK-8 assay, transwell migration assay and tube formation test, were used to investigate the roles of KLF2 in the proliferation, migration and vessel tube formation of LSECs, respectively. The expression and phosphorylation of ERK1/2 were detected by western blot. We discovered that the up-regulation of KLF2 expression dramatically inhibited proliferation, migration and tube formation in treated LSECs. Correspondingly, down-regulation of KLF2 expression significantly promoted proliferation, migration and tube formation in treated LSECs. Additionally, KLF2 inhibited the phosphorylation of ERK1/2 pathway, followed by the function of KLF2 in the angiogenesis of LSECs disrupted. In conclusion, KLF2 suppressed the angiogenesis of LSECs through inhibition of cell proliferation, migration, and vessel tube formation. These functions of KLF2 may be mediated through the ERK1/2 signaling pathway.

A new lignan glycoside from Chamaecyparis obtusa var. breviramea f. crippsii.

A new lignan glycoside, (-)-(8S, 8'R)-thujastandin-4-O-beta-D-glucopyranoside (1), together with fourteen known lignanoids (2-15) and one coumarin (16) were isolated from the branches and leaves of Chamaecyparis obtusa var. breviramea f. crippsii. Their structures were elucidated by extensive spectroscopic and spectrometric analysis. Compound 16 exhibited cytotoxicity against A549, BGC-823 and Hela cell lines with IC50 values of 25.9, 20.9 and 18.5 microM, respectively.

HER2 mRNA status contributes to the discrepancy between gene amplification and protein overexpression in gastric cancer.

BACKGROUND: Human epidermal growth factor receptor 2 (HER2) is an important proto-oncogene of prognostic use in gastric cancer (GC). Fluorescence in-situ hybridization (FISH) and immunohistochemistry (IHC) are the main clinical methods of detection of HER2, but consistency between the methods is poor and the cause of the discrepancy is unclear. AIM: To investigate the involvement of HER2 mRNA status in the disparity between gene amplification and protein overexpression. METHODS: We investigated HER2 gene, mRNA, and protein profiles in gastric precancer and cancer tissues by use of the molecular approaches FISH, real-time polymerase chain reaction, and IHC. The relationships between HER2 and matrix metalloproteinase 9 (MMP9) and Smad7 expression were analyzed and the involvement of HER2 in the interaction between tumor cells and lymphocytes was investigated by coculturing GC cell lines with peripheral blood mononuclear cells (PBMCs). RESULTS: HER2 protein expression was significantly increased in cancer compared with precancer (P = 0.003), and the corresponding mRNA levels were significantly lower in precancer and cancer tissues than in normal tissues (κ = 0.290, P = 0.025). HER2 mRNA levels were significantly higher in tumor than in peritumor tissue (P = 0.028), and were positively correlated with MMP9 and Smad7 mRNA levels in tumor tissues. HER2 mRNA expression in GC cell lines was increased by coculture with PBMCs. CONCLUSIONS: Different HER2 mRNA profiles, possibly in relation to contact between tumor cells and lymphocytes, might help to explain the discrepancy between gene amplification and protein overexpression results.

Transforming growth factor-ß1 and -ß2 in gastric precancer and cancer and roles in tumor-cell interactions with peripheral blood mononuclear cells in vitro.

Transforming growth factor-ß1 (TGF-ß1) and -ß2 are correlated with poorer prognosis in gastric cancer (GC), which act in both tumor and immune cells. However, their expressions in precancer and tumor-cell interactions with peripheral blood mononuclear cells (PBMCs) remain unclear. Protein levels of TGF-ß1 and -ß2 were analyzed by immunohistochemistry and corresponding mRNA levels were determined by quantitative real-time polymerase chain reaction in 93 surgical and biopsy specimens. Serum TGF-ß concentration was detected by enzyme-linked immunosorbent assays. AGS and MKN45 cell lines were directly or indirectly cocultured with PBMCs in vitro. TGF-ß and Smad molecules were detected after cocultures and the growths of GC cells and PBMCs were assessed by cell proliferation assay. The results showed positive staining for TGF-ß1 was detected in 20% of control samples, 52.3% of precancer, 59.1% of early GC and 66.7% of advanced GC samples, correlated with lesion progression (χ²â€Š= 9.487, P = 0.002). All tissues were positive for TGF-ß2. TGF-ß1 mRNA levels were increased in advanced cancers, while TGF-ß2 increased earlier. TGF-ß1 mRNA levels were higher in tumor than in peritumor, which positively correlated with Smad2 and Smad7. Serum TGF-ß levels were significantly higher in patients with early and advanced cancers compared to controls (TGF-ß1∶50.08±4.38 and 45.76±5.00 vs. 27.78±6.11 ng/mL; TGF-ß2∶133.61±21.90 and 111.34±15.76 vs. 59.41±15.42 ng/mL, both P<0.05). The levels of TGF-ß1 mRNA and cytokine secretion were higher in GC cells after direct coculture compared to indirect culture. TGF-ß1 was decreased and TGF-ß2 was increased in PBMCs after cocultures. Moreover, TGF-ß1 inhibited the viability of PBMCs but not cancer cells. Collectively, neoplastic transformation may be an early event involving the increase of TGF-ß1 in the general and local environment. TGF-ß1 production is promoted by the direct interaction between GC cells and PBMCs, which might facilitate cancer development.

FoxP3 inhibits proliferation and induces apoptosis of gastric cancer cells by activating the apoptotic signaling pathway.

Forkhead Box Protein 3 (FoxP3) was identified as a key transcription factor to the occurring and function of the regulatory T cells (Tregs). However, limited evidence indicated its function in tumor cells. To elucidate the precise roles and underlying molecular mechanism of FoxP3 in gastric cancer (GC), we examined the expression of FoxP3 and the consequences of interfering with FoxP3 gene in human GC cell lines, AGS and MKN45, by multiple cellular and molecular approaches, such as immunofluorescence, gene transfection, CCK-8 assay, clone formation assay, TUNEL assay, Flow cytometry, immunoassay and quantities polymerase chain reaction (PCR). As a result, FoxP3 was expressed both in nucleus and cytoplasm of GC cells. Up-regulation of FoxP3 inhibited cell proliferation and promoted cell apoptosis. Overexpression of FoxP3 increased the protein and mRNA levels of proapoptotic molecules, such as poly ADP-ribose polymerase1 (PARP), caspase-3 and caspase-9, and repressed the expression of antiapoptotic molecules, such as cellular inhibitor of apoptosis-1 (c-IAP1) and the long isoform of B cell leukemia/lymphoma-2 (Bcl-2). Furthermore, silencing of FoxP3 by siRNA in GC cells reduced the expression of proapoptotic genes, such as PARP, caspase-3 and caspase-9. Collectively, our findings identify the novel roles of FoxP3 in inhibiting proliferation and inducing apoptosis in GC cells by regulating apoptotic signaling, which could be a promising therapeutic approach for gastric cancer.

[A new monoterpenoid glucoside from the twigs of Chamaecyparis obtusa var. breviramea f. crippsii].

To investigate the chemical constituents of Chamaecyparis obtusa var. breviramea f. crippsii, various column chromatography and spectroscopic methods were used for the isolation and elucidation of compounds. One new monoterpenoid glucoside, (4S)-4-isopropylcyclohex-l-enecarboxylic acid 4-O-beta-D-glucopyranoside (1), together with five known compounds, (4R)-p-menth-l-ene-7, 8-diol 7-O-beta-D-glucopyranoside (2), skimmin (3), 7-[[6-O-(6-deoxy-alpha-L-mannopyranosyl)-beta-D-glucopyranosyl]oxy]-2H-1-benzopyran-2-one (4), stigmast-4-en-3-one (5) and 1, 4-benzenedicarboxylic acid 1-butyl-4-(2-methylpropyl) ester (6) were isolated and identified from the twigs of this plant. All compounds were isolated from this plant for the first time. The methanol extract of this plant showed cytotoxicity on cancer cell lines A549, BGC-823, Du145 and MDA-MB-231 with IC50 values of 0.94, 1.07, 0.95 and 0.96 microg x mL(-1), respectively. Yet, compounds 1, 2 and 3 showed no cytotoxicity on cancer cell lines HeLa, BGC-823 and A549.

Decreased expression of retinoblastoma protein-interacting zinc-finger gene 1 in human esophageal squamous cell cancer by DNA methylation.

BACKGROUND: To study the expression of the RIZ1 (Retinoblastoma protein-interacting zinc-finger gene 1) gene and investigate the promoter region methylation status of RIZ1 gene in the human esophageal squamous cell carcinoma (ESCC) cell lines of KYSE150, KYSE510, TE13, EC9706, CaEsl7, and EC109. To investigate the influence of DNMT (DNA methyltransferase) 5-aza-CdR(5-aza-2'-deoxycytidine) on the transcription of the RIZ1 gene in one cell line whose RIZ1 gene promoter region methylation was detected, and to investigate its influence on the cell proliferation. METHODS: Real-time PCR (Real-time quantitative PCR) and an immunohistochemistry technique was used to get the expression of RIZ1 in specimens from 6 human ESCC cell lines and 28 ESCC patients (tumor tissues and adjacent non-cancerous tissues). MSP (Methylation-specific PCR) was used to investigate the promoter region methylation status of the RIZ1 gene in the 6 ESCC cell lines. One cell line, whose RIZ1 gene promoter region methylation was detected, was chosen for the next studies in which it was treated it by with 5-aza-CdR. Real-time PCR was used to investigate its influence on the transcription of RIZ1 gene and MTT (methyl thiazolyl tetrazolium) was used to detect if 5-aza-CdR inhibits the proliferation of the cell line. RESULTS: In the 28 ESCC patient samples, RIZ1 expression was significantly lower in the tumor tissues than that in their adjacent non-cancerous tissues (p < 0.05). Consistently, immunohistochemistry analyses of RIZ1 protein expression showed that in the ESCC tissues RIZ1 protein expression was also significantly lower than in the adjacent tissues. In the human ESCC tissues the rate of expression accounts for 0% (0/12), and in the adjacent noncancerous tissues the rate of expression was 66.7% (8/12), the correlation was highly significant (chi2 = 12.000, p < 0.05). Promoter methylation of the RIZ1 gene was detected in TE13, CaEsl7, EC109. The cell line TE13 was chosen for the next studies. The expression of RIZ1 mRNA in TE-13 was up-regulated after having been treated with 5-aza-CdR. 5-aza-CdR inhibited cell proliferation of TE-13 in a time and concentration-dependent manner. CONCLUSIONS: Promoter methylation may play an important role in the epigenetic silencing of RIZ1 gene expression. Methylation of the RIZ1 promoter and loss of RIZ1 expression in human ESCC are independent biomarkers. Their determination may offer guidance for selecting appropriate diagnoses and treatments. RIZ1 may be a potential tumor suppressor in human ESCC.

Study on RIZ1 gene promoter methylation status in human esophageal squamous cell carcinoma.

AIM: To investigate the promoter region methylation status of retinoblastoma protein-interacting zinc finger gene 1 (RIZ1) in the human esophageal squamous cell carcinoma (ESCC) cell lines and tissues and verify the relationship between methylation of RIZ1 and oncogenesis, tumor progression and metastasis etc of ESCC. METHODS: Methylation-specific polymerase chain reaction (MSP) was used to investigate the promoter region methylation status of RIZ1 in 6 ESCC cell lines. One cell line where RIZ1 promoter region methylation was detected was selected for the next study, where the cell line was treated with 5-aza-CdR. Real-time polymerase chain reaction was used to investigate its influence on the transcription of RIZ1. Experiments using frozen pathological specimens from 47 ESCC patients were performed using the same MSP methodology. RESULTS: Promoter methylation of RIZ1 gene was detected in TE13, CaEs17 and EC109 cell lines and the cell line TE13 was chosen for further study. The expression of RIZ1 mRNA in TE-13 was up-regulated after treatment with 5-aza-CdR. The rate of methylation in carcinomas tissues was significantly higher than those in matched neighboring normal and distal ending normal tissue, and the deviation of data was statistically significant (χ(2) = 24.136, P < 0.01). Analysis of the gender, age familial history, tumour deviation, tumour saturation, lymph gland displacement and clinical staging of 47 samples from ESCC patients showed that the fluctuation of data was not statistically significant. CONCLUSION: Promoter methylation may play an important role in the epigenetic silencing of RIZ1 gene expression in human ESCC. RIZ1 is considered to be a potential tumor suppressor gene and may be a biological parameter for testing early stage human ESCC.