Outward-oriented sites within clustered CTCF boundaries are key for intra-TAD chromatin interactions and gene regulation.

CTCF plays an important role in 3D genome organization by adjusting the strength of chromatin insulation at TAD boundaries, where clustered CBS (CTCF-binding site) elements are often arranged in a tandem array with a complex divergent or convergent orientation. Here, using Pcdh and HOXD loci as a paradigm, we look into the clustered CTCF TAD boundaries and find that, counterintuitively, outward-oriented CBS elements are crucial for inward enhancer-promoter interactions as well as for gene regulation. Specifically, by combinatorial deletions of a series of putative enhancer elements in mice in vivo or CBS elements in cultured cells in vitro, in conjunction with chromosome conformation capture and RNA-seq analyses, we show that deletions of outward-oriented CBS elements weaken the strength of long-distance intra-TAD promoter-enhancer interactions and enhancer activation of target genes. Our data highlight the crucial role of outward-oriented CBS elements within the clustered CTCF TAD boundaries in developmental gene regulation and have interesting implications on the organization principles of clustered CTCF sites within TAD boundaries.

Molecular Characteristics of T Cell-Mediated Tumor Killing in Hepatocellular Carcinoma.

Background: Although checkpoint blockade is a promising approach for the treatment of hepatocellular carcinoma (HCC), subsets of patients expected to show a response have not been established. As T cell-mediated tumor killing (TTK) is the fundamental principle of immune checkpoint inhibitor therapy, we established subtypes based on genes related to the sensitivity to TKK and evaluated their prognostic value for HCC immunotherapies. Methods: Genes regulating the sensitivity of tumor cells to T cell-mediated killing (referred to as GSTTKs) showing differential expression in HCC and correlations with prognosis were identified by high-throughput screening assays. Unsupervised clustering was applied to classify patients with HCC into subtypes based on the GSTTKs. The tumor microenvironment, metabolic properties, and genetic variation were compared among the subgroups. A scoring algorithm based on the prognostic GSTTKs, referred to as the TCscore, was developed, and its clinical and predictive value for the response to immunotherapy were evaluated. Results: In total, 18 out of 641 GSTTKs simultaneously showed differential expression in HCC and were correlated with prognosis. Based on the 18 GSTTKs, patients were clustered into two subgroups, which reflected distinct TTK patterns in HCC. Tumor-infiltrating immune cells, immune-related gene expression, glycolipid metabolism, somatic mutations, and signaling pathways differed between the two subgroups. The TCscore effectively distinguished between populations with different responses to chemotherapeutics or immunotherapy and overall survival. Conclusions: TTK patterns played a nonnegligible role in formation of TME diversity and metabolic complexity. Evaluating the TTK patterns of individual tumor will contribute to enhancing our cognition of TME characterization, reflects differences in the functionality of T cells in HCC and guiding more effective therapy strategies.

Chemokine CXCL1 may serve as a potential molecular target for hepatocellular carcinoma.

The purpose of this study was to screen for changes in chemokine and chemokine-related genes that are expressed in hepatocellular carcinoma (HCC) as potential markers of HCC progression. Total RNA was extracted from tumor and peritumor tissues from mice with HCC and analyzed using a PCR microarray comprising 98 genes. Changes in gene expression of threefold or more were screened and subsequently confirmed by immunohistochemical analyses and western blotting. Furthermore, whether chemokine knockdown by RNA interference (RNAi) could significantly suppress tumor growth in vivo was also evaluated. Finally, total serum samples were collected from HCC patients with HBV/cirrhosis (n = 16) or liver cirrhosis (n = 16) and from healthy controls (n = 16). The serum mRNA and protein expression levels of CXCL1 in primary liver cancer patients were detected by qRT-PCR and western blot analysis, respectively. Several genes were up-regulated in tumor tissues during the progression period, including CXCL1, CXCL2, CXCL3, and IL-1ß, while CXCR1 expression was down-regulated. CBRH-7919 cells carrying CXCL1 siRNA resulted in decreased tumor growth in nude mice. The differences in serum CXCL1 mRNA and protein levels among the HCC, hepatic sclerosis (HS), and control groups were significant (P < 0.001). The mRNA and protein levels of CXCL1 in the HCC group were up-regulated compared with the HS group or the control group (P < 0.001). Several chemokine genes were identified that might play important roles in the tumor microenvironment of HCC. These results provide new insights into human HCC and may ultimately facilitate early HCC diagnosis and lead to the discovery of innovative therapeutic approaches for HCC.

Targeted silencing of CXCL1 by siRNA inhibits tumor growth and apoptosis in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is an aggressive malignancy and a major cause of cancer-related mortality worldwide. Our previous study shows that chemokine (C-X-C motif) ligand 1 (CXCL1) was upregulated and CXCR1 was downregulated in tumor tissues as compared to peritumor tissues by chemotaxis assay. As the status of CXCL subgroups and their receptors affect progression of HCC, we evaluated potential mechanisms of CXCL1 associated with anticancer effects in HCC based on our previous study. The effects of targeting CXCL1 by RNA interference (RNAi) on the proliferation and apoptosis of CBRH-7919 cells were observed in vitro and in vivo. Additionally, whether CXCL1 knockdown significantly reduce the activity of STAT3, NF-κB and HIF-1 or not were also estimated. RNAi of CXCL1 in the CBRH-7919 cells decreased the growth of tumors in nude mice by inhibited cells proliferation and induced apoptosis. In conclusion, these findings suggest that CXCL1 plays critical roles in the growth and apoptosis of HCC. RNAi of CXCL1 inhibits the growth and apoptosis of tumor cells, which indicates that CXCL1 may be a potential molecular target for use in HCC therapy.

Inflammatory microenvironment and expression of chemokines in hepatocellular carcinoma.

AIM: To study the inflammatory microenvironment and expression of chemokines in hepatocellular carcinoma (HCC) in nude mice. METHODS: CBRH-7919 HCC cells were injected into the subcutaneous region of nude mice. Beginning two weeks after the challenge, tumor growth was measured every week for six weeks. The stromal microenvironment and inflammatory cell infiltration was assessed by immunohistochemistry in paired tumor and adjacent peritumoral samples, and macrophage phenotype was assessed using double-stain immunohistochemistry incorporating expression of an intracellular enzyme. A chemokine PCR array, comprised of 98 genes, was used to screen differential gene expressions, which were validated by Western blotting. Additionally, expression of identified chemokines was knocked-down by RNA interference, and the effect on tumor growth was assessed. RESULTS: Inflammatory cell infiltrates are a key feature of adjacent peritumoral tissues with increased macrophage, neutrophil, and T cell (specifically helper and activated subsets) infiltration. Macrophages within adjacent peritumoral tissues express inducible nitric oxide synthase, suggestive of a proinflammatory phenotype. Fifty-one genes were identified in tumor tissues during the progression period, including 50 that were overexpressed (including CXCL1, CXCL2 and CXCL3) and three that were underexpressed (CXCR1, Ifg and Actb). RNA interference of CXCL1 in the CBRH-7919 cells decreased the growth of tumors in nude mice and inhibited expression of CXCL2, CXCL3 and interleukin-1ß protein. CONCLUSION: These findings suggest that CXCL1 plays a critical role in tumor growth and may serve as a potential molecular target for use in HCC therapy.

First line chemotherapy with weekly docetaxel and cisplatin in elderly patients with advanced non-small cell lung cancer: a multicenter phase II study.

INTRODUCTION: We report outcomes for a phase II study of the combination of weekly docetaxel and cisplatin in elderly patients with advanced non-small cell lung cancer. METHODS: Patients with chemotherapy-naive, stage IIIB/IV, an Eastern Cooperative Oncology Group performance status of 0 or 1, ages 70 years or older, were eligible. Chemotherapy consisted of cisplatin (25 mg/m2) on days 1, 8, and 15 and docetaxel (20 mg/m2) on days 1, 8, and 15 every 4 weeks. RESULTS: Forty-six (95.8%) of the 48 patients were assessable for response, 1 case of complete response and 18 cases of partial response were confirmed, giving an overall response rate of 39.6% (95% confidence interval [CI], 25.7-53.5%). The median time to progression and overall survival for all patients was 5.0 months (95% CI, 4.1-5.7 months) and 10.9 months (95% CI, 9.6-12.2 months), respectively. The most severe hematologic adverse event was anemia, which occurred with grade 3 intensity in 6 (13.0%) patients and grade 4 in 2 (4.3%) patients. Neutropenia occurred with grade 3 intensity in 4 (8.7%) patients. Grade 3 asthenia, diarrhea, neuropathy, stomatitis, and nausea/vomiting were observed in 2 (4.3%), 5 (10.9%), 5 (10.9%), 5 (10.9%), and 3 (6.5%) patients, respectively. Yet, no grade 4 nonhematologic toxicity was observed. CONCLUSIONS: The combination of weekly docetaxel and cisplatin is a well-tolerated treatment modality with encouraging activity and survival outcome in previously untreated elderly patients with advanced non-small cell lung cancer.

Identification of lung cancer patients by serum protein profiling using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry.

OBJECTIVE: Diagnosis of lung cancer at an early disease stage is important for successful treatment and improving the outcome of patients. To improve its prognosis, we attempted to explore novel tools for screening serum biomarkers to distinguish lung cancer from healthy individuals by serum protein profiles and a classification tree algorithm. METHODS: Serum samples were applied to metal affinity protein chips to generate mass spectra by surface-enhanced laser desorption/ionization (SELDI) time-of-flight mass spectrometry. Protein peak identification and clustering were performed using the Biomarker Wizard software. Proteomic spectra of serum samples from 89 lung cancer patients and age- and sex-matched 68 healthy individuals were used as a training set and a classification tree with 3 distinct protein masses was generated by using Biomarker Pattern software. The validity of the classification tree was then challenged with a blind test set including another 62 lung cancer patients and 34 healthy individuals. We additionally determined Cyfra21-1 and carcinoembryonic antigen in all the serum samples included in this study using an electrochemiluminescent immunoassay. RESULTS: The software identified an average of 48 mass peaks/spectrum and 3 of the identified peaks at 5808, 5971, and 7779 d were used to construct the classification tree. The classification tree separated effectively lung cancer from healthy individuals, achieving a sensitivity of 91% (81 of 89) and a specificity of 97% (66 of 68). The blind test challenged the model with a sensitivity of 89% (55 of 62) and a specificity of 91% (31 of 34), and a positive predictive value of 90% (86 of 96), respectively. The specificity of Cyfra21-1 and the sensitivity provided by Cyfra21-1 and carcinoembryonic antigen used individually or in combination were significantly lower than that of the SELDI marker pattern (P < 0.05 or P < 0.005, respectively). CONCLUSION: The results suggest that SELDI time-of-flight mass spectrometry technique can correctly distinguish lung cancer patients from healthy individuals and shows great potential for the development of a screening test for the detection of lung cancer.

Anti-tumor activities and apoptosis-regulated mechanisms of bufalin on the orthotopic transplantation tumor model of human hepatocellular carcinoma in nude mice.

AIM: To investigate anti-tumor activities and apoptosis-regulated mechanisms of bufalin in the orthotopic transplantation tumor model of human hepatocellular carcinoma in nude mice. METHODS: BEL-7402 cells of human hepatocellular carcinoma were inoculated to form subcutaneous tumors, and were implanted into the liver to establish orthotopic transplantation tumor models of human hepatocellular carcinoma in nude mice. Seventy-five animals were randomized divided into five groups (n = 15). Bufalin was injected intraperitoneally into three groups at doses of 1.5 mg/kg (BF1), 1 mg/kg (BF2) and 0.5 mg/kg (BF3) for d 15-24, respectively. The NS group was injected an equal volume of saline as above and adriamycin was injected intraperitoneally into the ADM group at a dose of 8.0 mg/kg for d 15. Ten mice in each group were killed at d 25 and the survival time in each group was calculated. We also observed the morphologic alterations in the myocardium, brain, liver, kidney and tumor tissues by pathology and electron microscopy, measured the apoptotic rate by TUNEL staining method, and detected the expression of apoptosis-regulated genes bcl-2 and bax by immunohistochemical staining and RT-PCR in tumor tissues. RESULTS: The tumor volumes in each group of bufalin were reduced significantly (35.21 +/- 12.51 vs 170.39 +/- 25.29; 49.83 +/- 11.46 vs 170.39 +/- 25.29; 83.99 +/- 24.63 vs 170.39 +/- 25.29, P < 0.01, respectively), and the survival times were prolonged in group BF1-2 (31.8 +/- 4.2 vs 23.4 +/- 2.1 and 29.4 +/- 3.4 vs 23.4 +/- 2.1, P < 0.05, respectively), and necrosis was mainly in severe or moderate degree in group BF1-2. No morphological changes were detected in the myocardium, brain, liver and kidney tissues. Apoptotic characteristics could be seen in group BF1-2. The positive rates of bcl-2 and bax protein expression of each group by immunohistochemical staining were 10.0%, 10.0%, 20.0%, 10.0% and 20.0%; 90.0%, 80.0%, 80.0%, 40.0% and 30.0%, respectively. Loss of expression of bcl-2 mRNA in each group was to be found and the density of bax mRNA was increased progressively with increase of dose of bufalin by RT-PCR. CONCLUSION: Bufalin has significant anti-tumor activities in the orthotopic transplantation tumor model of human hepatocellular carcinoma in nude mice with no marked toxicity and was able to induce apoptosis of transplanted tumor cells. This apoptosis may be mediated mainly via up-regulating the expression of apoptosis-regulated gene bax, which may be involved in its anti-tumor mechanism of bufalin.

[Inhibition action of bufalin on human transplanted hepatocellular tumor and its effects on expressions of Bcl-2 and Bax proteins in nude mice].

OBJECTIVE: To investigate the anti-tumor effect of bufalin and its regulation on Bcl-2 and Bax proteins in orthotopically transplanted tumor of human hepatocellular carcinoma in nude mice. METHODS: Orthotopically transplanted tumor of human hepatocellular carcinoma was established in nude mice. The mice were randomly divided into five groups: high-dose bufalin-treated group (1.5 mg/kg), medium-dose bufalin-treated group (1 mg/kg), low-dose bufalin-treated group (0.5 mg/kg), adriamycin-treated group (8.0 mg/kg), and normal saline-treated group. After 25 days, mice were sacrificed. The tumor volume was measured, and the pathological changes of tumor tissues were detected by HE staining to observe the tumor necrosis degree. Cell morphological changes were also observed by an electron microscopy. Label index of tumor cell apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and the expressions of Bcl-2 and Bax proteins were determined by immunohistochemical method. RESULTS: The tumor volume in the bufalin-treated groups was shrunk significantly compared with that in the normal saline-treated group (P<0.01). The survival time of the bufalin-treated groups was prolonged compared with that of the adriamycin-treated group and the normal saline-treated group P<0.05. Apoptotic characteristics could be seen in tumor tissues of the bufalin-treated groups. The label index of tumor cell apoptosis in the bufalin-treated groups (5.87+/-2.13, 8.86+/-2.96 and 10.60+/-3.42 in low-, medium- and high-dose groups respectively) was higher than that in the adriamycin-treated group (3.28+/-0.98) (P<0.05, P<0.01). The expression of Bax was up-regulated, while no changes were detected as to Bcl-2 protein in tumors of the bufalin-treated groups. CONCLUSION: Bufalin has significant anti-tumor effect on the orthotopically transplanted tumor of human hepatocellular carcinoma in nude mice. Its effect might be related to up-regulation of Bax protein and inducement of the tumor cell apoptosis.

Growth arrest and apoptosis of the human hepatocellular carcinoma cell line BEL-7402 induced by melittin.

OBJECTIVE: The aim of this study was to investigate the cellular effects of melittin on the growth and apoptosis of human hepatocellular carcinoma (HCC) cells and to provide the molecular mechanism for potential application of a recombinant adenovirus carrying the melittin gene (Ad-rAFP-Mel) in the treatment of liver cancer. METHODS: Human HCC cells (BEL-7402) were infected with Ad-rAFPMel at different times. In vitro cell growth was determined by MTT assay. Cellular apoptosis was evaluated quantitatively and qualitatively by phase-contrast microscopy, transmission electron microscopy, DNA ladder electrophoresis, TUNEL staining and flow cytometry. RESULTS: Ad-rAFP-Mel infection had an inhibitory effect on the proliferation of BEL-7402 cells. The morphological changes of apoptosis were confirmed by microscopy and DNA electrophoresis. The ultrastructural characteristics of apoptotic cells, such as chromatin condensation and nuclear fragmentation, were also observed by electron microscopy in the Ad-rAFP-Mel-infected cells. Ad-rAFPMel infection markedly induced cellular apoptosis, and Fas expression on Bel-7402 cells infected by Ad-rAFPMel was up-regulated. CONCLUSION: The fact that melittin can induce apoptosis of the HCC cell line BEL-7402 leads us to consider adenovirus-mediated delivery of melittin as a promising approach for the treatment of HCC. However, the underlying mechanism needs to be further investigated.

[Establishment and biological characteristics of orthotopic transplantation tumor model of hepatocellular carcinoma in mice].

OBJECTIVE: To study the feasibility of the establishment of the orthotopic transplantation tumor model of hepatocellular carcinoma in mice and its tumor biological characteristics. METHODS: H22 cells of hepatocellular carcinoma were inoculated to form ectopic transplanted model in mice by subcutaneous injection. Then the subcutaneous tumors were implanted into the liver of mice, and the orthotopic transplantation tumor model of hepatocellular carcinoma was established. RESULTS: The successful rate of the orthotopic transplantation tumor model was 95.6% and the spontaneous metastatic rate was 81.8%, the rate of mass ascites was 40.9% and the natural extinctive rate was 0%. The natural survival time in the orthotopic transplantation tumor model was 28 days and the proliferation of tumor in transplanted model was accelerated after 2 weeks or so. CONCLUSION: The orthotopic transplantation tumor model in mice is an ideal model for studying the metastatic mechanism and screening anti-tumor drugs for liver cancer, just because of its high successful rate and high spontaneous metastatic rate with no natural extinction.