Palliative whole-brain radiotherapy and health- related quality of life for patients with brain metastasis in cancer.

OBJECTIVE: To assess the use of palliative whole-brain radiotherapy (WBRT) in the treatment of brain metastases (BMs) and to evaluate the health-related quality of life (HRQOL) of these patients. MATERIALS AND METHODS: We conducted a retrospective study of 46 patients with BMs who were treated with WBRT at the First Affiliated Hospital of Xi'an Jiaotong University between January 2013 and January 2015. External beam radiotherapy techniques were used to deliver 40 Gy in 20 fractions or 30 Gy in ten fractions with a 10 MV photon beam from a linear accelerator to the whole brain. Data were stored and analyzed using SPSS version 17.0. RESULTS: Of the 46 patients, the survival time of patients in our study was 10.8±0.55 months: 11.8±0.46 months in patients with WBRT, 11.75±1.00 in patients with WBRT + chemotherapy, and 3±0.79 months in patients with supportive care, respectively (P<0.01). The HRQOL scores of all the patients were 70±1.16 (before therapy) and 76.83±1.04 (after therapy) (P<0.01). The HRQOL scores of the patients with WBRT were 72.23±0.88 (before therapy) and 78.49±0.87 (after therapy) (P<0.01). There was no central nervous system toxicity; only two (4.3%) patients were found to have BM hemorrhage. Radiation necrosis happened in one patient (2.2%). CONCLUSION: Effective treatment options for patients with BMs are important. WBRT was evaluated to ensure survival outcomes and QOL were enhanced after therapy for patients with BMs.

Analysis of the characteristics and prognosis of advanced non-small-cell lung cancer in older patients.

OBJECTIVE: Lung cancer is still the leading cause of cancer-related deaths worldwide. However, most elderly patients with advanced non-small-cell lung cancer (NSCLC) have been undertreated and the outcome related to age is controversial. A retrospective analysis was conducted for advanced NSCLC in order to investigate the characteristics and prognosis of older patients. METHODS: Medical records were collected from 165 patients with NSCLC (stages IIIA-IIIB) who had been treated with concurrent chemoradiotherapy (CRT) or radiotherapy from January 2009 to January 2011. The cases were divided into two age groups 1) patients ≥70 years old; 2) patients <70 years old. There were 73 patients in group I, 92 in group II. Patient characteristics, treatment toxicities, and prognosis were evaluated. RESULTS: Of the 165 patients analyzed, 34 patients (34/73) in group I received concurrent CRT while 47 (47/92) in group II completed that treatment. No significant difference was observed in the reason for patients who discontinued CRT in two groups (P>0.05). In the patients with adenocarcinoma, more cases were found in group II than that in group I; the more squamous cell carcinoma and the more smokers with squamous cell carcinoma were seen in older group (P<0.05). With a median follow-up of 20.5 months, the 1-year survival for group I and II were 49.3% and 40.2% respectively (P=0.243). Two-year survival for the two groups was 20.5% and 16.3% (P=0.483); 3-year survival was 9.6% and 9.8% (P=0.967). There was no significant difference between two groups statistically in survival by univariate analysis (P>0.05). The therapy-related toxicities in group I seem to be similar to the group II (P>0.05). CONCLUSION: More adenocarcinoma patients were found in youthful lung cancer and the more smokers with squamous cell carcinoma were seen in older group. Age is not the important factor for the selection and allocation of treatment in advanced NSCLC. The same prognosis and toxicities had been shown in older and young. Age may not be an independent increased risk of death in advanced NSCLC.

Serum dickkopf-1 is a novel serological biomarker for the diagnosis and prognosis of pancreatic cancer.

PURPOSE: To identify whether Dickkopf-1 (DKK1) could be a potential biomarker for early detection and prognosis in patients with pancreatic cancer (PC). METHODS: Serum was collected from 140 patients with pancreatic adenocarcinoma and 92 control patients without pancreatic adenocarcinoma. Serological levels of DKK1 were examined by enzyme-linked immunosorbent assay (ELISA). The sensitivity and specificity was compared with carbohydrate antigen 19-9 (CA19-9). A 2-year follow-up was monitored to evaluate the correlation between DKK1 serum levels and overall survival. The expression of DKK1 in PC tumor tissues was also evaluated using immunohistochemistry staining. RESULTS: Serum levels of DKK1 and CA19-9 were elevated in PC patients in the early-stage cases. These levels increased with the advancement of clinical stage. There was significant difference in DKK1 serum levels between early and advanced PC stages. Receiver operating characteristic curve (ROCC) analysis showed that DKK1 was significantly better than CA19-9 in differentiating patients with PC from the controls (area under the curve (AUC) 0.919 versus 0.853, respectively), especially in distinguishing early-stage cancer from chronic pancreatitis (CP). The expression of DKK1 in PC tissues correlated with its expression in serum samples. The overall survival rate was 24.4% in the group with higher DKK1 levels and was found to be significantly different from the group with lower DKK1 levels (33.3%). CONCLUSION: DKK1 may be a novel diagnostic/prognostic biomarker for PC.

MiR-21 promotes intrahepatic cholangiocarcinoma proliferation and growth in vitro and in vivo by targeting PTPN14 and PTEN.

Intrahepatic cholangiocarcinoma (ICC) constitutes the second-most common primary hepatic malignancy. MicroRNAs (miRNAs) play important roles in the pathogenesis of ICC. However, the clinical significance of miR-21 levels in ICC remains unclear. Here, we investigated the role of miR-21 in ICC and found that its expression was significantly upregulated in serum of ICC patients. Serum miR-21 levels robustly distinguished ICC patients from control subjects. Further experiments showed that inhibition of miR-21 suppressed ICC cell proliferation in vitro and tumor growth in vivo. Specifically, inhibition of miR-21 induced cell cycle arrest and apoptosis. Moreover, PTPN14 and PTEN were identified as direct and functional targets of miR-21. Finally, we showed high expression levels of miR-21 were closely related to adverse clinical features, diminished survival, and poor prognosis in ICC patients. This study revealed functional and mechanistic links between miR-21 and tumor suppressor genes, PTPN14 and PTEN, in the pathogenesis of ICC. MiR-21 not only plays important roles in the regulation of cell proliferation and tumor growth in ICC, but is also a diagnostic and prognostic marker, and a potential therapeutic target for ICC.

The intensity of radiotherapy-elicited immune response is associated with esophageal cancer clearance.

Radiation therapy is one of the standard therapeutic modalities for esophageal cancer, achieving its main antitumor efficacy through DNA damage. However, accumulating evidence shows that radiotherapy can substantially alter the tumor microenvironment, particularly with respect to its effects on immune cells. We hypothesized that the immune response elicited by radiotherapy may be as important as the radiation itself for successful treatment. More specifically, immunomodulatory cytokines may enhance the effectiveness of radiotherapy. To investigate this hypothesis, we measured changes in the serum interferon-gamma (IFN- γ ) and interleukin-2 (IL-2) concentrations during radiotherapy and compared these modifications with outcomes. We found that serum concentrations of IL-2 and IFN- γ were positively associated with local response to radiotherapy in esophageal cancer. More generally, the intensity of the radiotherapy-elicited immune response was positively associated with local response to radiotherapy in esophageal cancer. Changes in serum IL-2 and IFN- γ concentrations were further associated with increased risks of acute hematologic toxicity and acute organ toxicity of the esophagus, lung, and skin. These results suggest that deciphering the mechanisms of radiotherapy-elicited immune response may help in the development of therapeutic interventions that would enhance the efficacy of radiotherapy and convert some ineffective responses to effective responses.

The potential clinical applications and prospects of microRNAs in lung cancer.

Lung cancer is the major cause of cancer deaths worldwide due to its late diagnosis and poor outcome. Understanding genomic medicine may widen our vision into the oncogenesis of lung cancer and may open the door to improvements in the clinical management of lung cancer. It is well known that almost half of all genes are regulated by microRNAs (miRNAs). This review focuses on the role of miRNAs in lung cancer and also touches on the value of miRNA-based novel therapies for lung cancers.

Cerium oxide nanoparticles in cancer.

With the development of many nanomedicines designed for tumor therapy, the diverse abilities of cerium oxide nanoparticles (CONPs) have encouraged researchers to pursue CONPs as a therapeutic agent to treat cancer. Research data have shown CONPs to be toxic to cancer cells, to inhibit invasion, and to sensitize cancer cells to radiation therapy and chemotherapy. CONPs also display minimal toxicity to normal tissues and provide protection from various forms of reactive oxygen species generation. Differential cytotoxicity is important for anticancer drugs to distinguish effectively between tumor cells and normal cells. The antioxidant capabilities of CONPs, which enable cancer therapy protection, have also resulted in the exploration of these particles as a potential anticancer treatment. Taken together, CONPs might be a potential nanomedicine for cancer therapy and this review highlights the current research into CONPs as a novel therapeutic for the treatment of cancer.

Serum SALL4 is a novel prognosis biomarker with tumor recurrence and poor survival of patients in hepatocellular carcinoma.

AIM: Sal-like protein 4 (SALL4), is reexpressed in tissues of a subgroup of HCC associated with poor prognosis. Reports of SALL4 serological levels linked to HCC patients are meager and unclear in the prognosis of this malignancy. METHODS: Immunohistochemistry and optical microscopy protocols were used to examine the presence of SALL4 in liver tissues from the following patients: 38 HCC, 11 chronic hepatitis B virus (HBV), 13 liver cirrhosis, and 12 healthy controls. Additionally, enzyme-linked immunosorbent assay (ELISA) was used to measure the SALL4 levels in serum samples isolated from patients as follows: 127 with HCC, 27 with HBV, 24 with liver cirrhosis, and 23 normal controls. RESULTS: Analysis of liver tissues sections from HCC patients (18 out 38; 47.4%) showed positive staining for SALL4 and its expression did no correlate with any of the clinicopathologic characteristics. HCC patients displayed higher levels (50.4%) of SALL4 protein in serum, compared with the three control groups. Moreover, SALL4 concentration reached the maximum level after one week after treatment and dropped quickly after one month. These HCC patients showing high SALL4 serum levels had poor prognosis, evidenced by both tumor recurrence and overall survival rate. CONCLUSIONS: High SALL4 serum levels are a novel biomarker in the prognosis of HCC patients.

The evaluation of older patients with cervical cancer.

OBJECTIVE: The number of elderly patients being diagnosed with cervical cancer is increasing, and the outcome of cervical cancer related to age is controversial. We conducted a retrospective analysis in patients treated for advanced cervical cancer in order to investigate patient characteristics and prognosis of older patients. METHODS: Medical records were collected of 159 patients with cervical cancer who had been treated with radiotherapy or combined radiotherapy and chemotherapy from January 2007 to January 2009. The patients were divided into two age groups: (1) patients ≥65 years old, and (2) patients <65 years old. There were 52 women in group 1, 107 in group 2. Prognosis, patient characteristics, treatment, and toxicities were evaluated. RESULTS: With a median follow-up of 36.5 months, local control for groups 1 and 2 was 88.5% and 79.4%, respectively. Disease-free survival for the two groups was 71.2% and 67.3%; overall survival was 73.1% and 72.9%. As shown by univariate analyses, there was no statistically significant difference between the two groups (P > 0.05). Seventy-six patients had human papillomavirus (HPV) at diagnosis (twelve women ≥65 years, 64 women ≤65 years; P = 0.000). Forty-two women tested positive for HPV 16, while 32 women tested positive for HPV 18 respectively. Pelvic and/or paraaortic lymph-node metastasis was found in 25 patients (eight in group 1, 17 in group 2; P = 0.960) on computed tomography scan. Of the 159 patients analyzed, sixteen patients (16/52) in group 1 received concurrent chemotherapy, while 96 (96/107) in group 2 completed that treatment. CONCLUSIONS: Cervical cancer has the same prognosis in old and young women. Age may not be an independent increased risk of death in women with cervical cancer, and the age-group is at lower risk for virulent HPV strands (HPV 16/18) compared to younger patients. Treatment recommendations were implemented less often for older patients. Radiotherapy remained the most common treatment chosen for elderly patients. This confirms that there is a stronger need to pay attention to the elderly patient.

The role of CDK1 in apoptin-induced apoptosis in hepatocellular carcinoma cells.

Apoptin, a small protein derived from the chicken anemia virus, specifically induces apoptosis in transformed cells or tumor cells but not in normal cells. Thus, apoptin is involved in a general, tumor-specific pathway. Apoptin-induced apoptosis presumably requires additional interaction partners that activate specific signaling pathways in cancer cells. A number of molecules interact with apoptin and play an important role in the nuclear localization of apoptin or its tumor-selective cytotoxicity. Our data indicated that apoptin selectively kills HepG2 hepatocellular carcinoma (HCC) cells but has no effect on the normal liver cell line HL-7702. Analyses of human HCC tissue samples confirmed that CDK1 (cyclin-dependent kinase 1) activity was detected in primary malignancies but not in healthy paraneoplastic tissues. shRNA knockdown of CDK1 significantly reduced the tumor-specific killing effects of apoptin, suggesting that CDK1 plays an important role in the regulation of apoptin-induced apoptosis. Furthermore, the majority of apoptin translocated to the cytoplasm from the nucleus after knockdown of CDK1. Collectively, our results revealed for the first time that apoptin interacts with CDK1 in the complex process of tumorigenesis. The link between CDK1 and apoptin may be a novel cellular signaling pathway to modulate apoptosis in cancer; therefore, apoptin may have pharmacological potential to be directly employed for cancer therapy.

Molecular beacons: a novel optical diagnostic tool.

As a result of the efforts of the Human Genome Project and the rise in demand for molecular diagnostic assays, the development and optimization of novel hybridization probes have focused on speed, reliability, and accuracy in the identification of nucleic acids. Molecular beacons (MBs) are single-stranded, fluorophore-labeled nucleic acid probes that are capable of generating a fluorescent signal in the presence of target, but are dark in the absence of target. Because of the high specificity and sensitivity characteristics, MBs have been used in variety of fields. In this review, MBs are introduced and discussed as diagnostic tools in four sections: several technologies of MBs will be illustrated primarily; the limitation of MBs next; the third part is new fashions of MBs; and the last one is to present the application of MBs in disease diagnosis.

Systemic delivery of lentivirus-mediated secretable TAT-apoptin eradicates hepatocellular carcinoma xenografts in nude mice.

Apoptin, a chicken anemia virus-derived protein, has been shown to induce apoptosis in various human cancer cell lines, but not in normal cells, thus making it a candidate for the development of novel therapeutic strategies. To enable the efficient transduction of tumor cells with apoptin, we have developed a novel mammalian expression system for the secretion of apoptin in vitro. We have previously shown the efficient and tumor-specific killing of cells by adding a secretory signal peptide (SP) to the N terminus of transacting activator of transcription (TAT)-apoptin (SP-TAT-apoptin). In addition, our report showed the successful secretion of high levels of TAT-apoptin/GFP into the culture medium from HUVEC cells infected by lentivirus LV-SP-TAT-apoptin/GFP. To obtain sustained apoptin-induced tumor cell death in vivo, we injected the LV-SP-TAT-apoptin viruses via the tail vein for systemic delivery of the viruses; viruses expressing LV-SP-TAT-GFP were used as a negative control. Markedly, almost all the hepatocellular carcinoma xenograft tumors disappeared following the treatment while the xenografts that received the control LV-SP-TAT-GFP viruses continued to grow. Moreover, the animal studies presented in this paper demonstrate a low toxicity of SP-TAT-apoptin in vivo, confirming and extending the results of the in vitro studies. Taken together, our data strongly suggest that systemic delivery of lentivirus-mediated secretable TAT-apoptin is feasible to eradicate liver cancer in vivo.

[Construction and identification of adenoviral vectors expressing shRNA targeting CDK1 gene].

AIM: To construct three adenoviral vectors harboring shRNA targeting CDK1 gene, and identify its inhibitory effect on the gene expression of CDK1 in hepatoma carcinoma HepG2 cells. METHODS: Three shRNA sequences targeting CDK1 mRNA were designed. The shRNA sequences were annealed and linked with linearized pSIREN-RetroQ-ZsGreen. The recombinants were identified by PCR and DNA sequencing. CDK1-shRNA plasmid was then transfected into the cultured HepG2 cell line with lipofectamine 2000. RT-PCR and Western blot were used to detect the mRNA and protein expression of CDK1, respectively. RESULTS: The small hair-pin RNA sequences were successfully inserted into pSIREN-RetroQ-ZsGreen vector, and the sequences were identified by DNA sequencing. Further, Realtime PCR and Western blot results validated that the three small hair-pin RNAs effectively knockdowned the expression of endogenous CDK1 in HepG2 cells. CONCLUSION: CDK1-shRNA can be effectively transfected into HepG2 cells, and induce post-transcriptional gene silencing of CDK1, which enables further functional study on CDK1.

Role of Twist in vasculogenic mimicry formation in hypoxic hepatocellular carcinoma cells in vitro.

Vasculogenic mimicry (VM) refers to the unique ability of highly aggressive human tumor cells to form matrix-rich networks de novo when cultured on a three-dimensional matrix, thus mimicking embryonic vasculogenesis. Some studies have shown that tumor hypoxia can promote tumor cells to form vessel-like tubes in vitro and express genes associated with VM. Although, the mechanisms involved in hypoxia-induced VM remain elusive, we hypothesized that the epithelial-mesenchymal transition (EMT) regulator Twist may play a major role in hypoxia-induced VM. We investigated this hypothesis in vitro by pretreating hepatocellular carcinoma cells under hypoxic conditions. Following the hypoxia treatment, the cells formed typical pipe-like VM networks. Moreover, the expression of VM markers was increased. Hypoxia-induced VM was accompanied by the increased expression of Twist. Twist siRNA reversed the effects of hypoxia on VM. These results suggest that the overexpression of Twist correlates to hypoxia-induced VM in hepatocellular carcinoma cells.

Apoptin sensitizes radiation-induced cell death via classic mitochondrial, caspase and p53-dependent signaling in HepG2 cells.

Resistance or insensitivity to radiation therapy is one of the hallmarks of hepatocellular carcinoma. Sensitizing radioresistant cancer by combining radiation with other therapeutics to induce apoptosis has been widely investigated. Our previous study showed that chicken anaemia virus-derived apoptin protein induced the apoptosis of hepatic carcinoma HepG2 cells. In the present study, we demonstrated that apoptin sensitizes cells to radiation-induced apoptosis using a lentivirus-apoptin expression system in hepatic carcinoma HepG2 cells. Combination therapy with radiation and apoptin dramatically induced mitochondrial cytochrome c release and the cleavage of caspases -9, -3 and -7. Our findings are also the first to show that the combination of radiation and apoptin up-regulates p53 expression. Thus, apoptin treatment represents a potential method for enhancing the effectiveness of radiotherapy in poorly responding hepatocellular carcinoma.

Lowered HGK expression inhibits cell invasion and adhesion in hepatocellular carcinoma cell line HepG2.

AIM: To investigate the effects of RNA interference targeting hepatocyte progenitor kinase-like kinase (HGK) in the invasion and adhesion of hepatocellular carcinoma (HCC) cell line HepG2. METHODS: Three paired insert DNA fragments specific to HGK gene and one negative control DNA fragment were synthesized and inserted into RNAi-Ready pSIREN-RetroQ-ZsGreen vector. Western blotting assay and real-time reverse transcriptase polymerase chain reaction (RT-PCR) were used to screen the vector with a highest inhibitory rate. The vector was used to generate recombinant retrovirus specific to HGK. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2h-tetrazolium bromide (MTT) assay was used to examine cell growth; wound closure assay and cell adhesion assay were employed to investigate cell migration and adhesion respectively; and transwell assay and three-dimensional culture invasion assay were used to detect cell invasion. The expressions of matrix metalloproteinase (MMP)-2, MMP-9 and nuclear factor (NF)-κB were detected by Western blotting assay. RESULTS: The real time RT-PCR and Western blotting assay showed that cells transfected with retrovirus mediating RNAi targeting of HGK (RV-shHGK)-1 vector had the strongest inhibition of HGK protein, with an inhibition rate of 76%, and this vector was used to generate recombinant retrovirus RV-shHGK-1. Cell adhesion assay and MTT assay found that cell adhesion and growth of the cells infected with RV-shHGK-1 were significantly lower than those of the control cells (P < 0.05). Wound closure assay, transwell assay and three-dimensional culture invasion assay showed that the cell invasiveness was significantly less in HGK knockdown cells than in the control cells (P < 0.05). The expressions of MMP-2, MMP-9 and NF-κB were inhibited in HepG2 cells infected with RV-shHGK-1. CONCLUSION: Down-regulation of HGK can obviously inhibit the migration and invasion of HepG2 cells in vitro. HGK may be a new therapeutic target for treatment of HCC.

[The effect of the fused gene of SP-TAT-Apoptin transfected by lentivirus on HepG2 cells].

AIM: To Package the recombinant lentivirus containing the fused gene of SP-TAT-Apoptin to infect HepG2 cell and measure the efficiency of apoptosis. METHODS: The eukaryotic expression vector of SP-TAT-Apoptin fused gene and other packaging plasmids were transfected into 293FT cells by Lipofectamine(TM);2000 reagent. The supernatant of the cultured 293FT cells was harvested and virus titration was determined by real time PCR. The expression of the fused gene of SP-TAT-Apoptin in 293FT cells infected by the recombinant lentivirus was examined by immunofluorescence histochemistry method. At the same time, the apoptosis rates of the HepG2 cells infected by the recombinant lentivirus were determined by using flow cytometer. RESULTS: The 293FT cells infected by the recombinant lentivirus could express the fused protein SP-TAT-Apoptin. Anexin-V PI assay showed that SP-TAT-Apoptin carried by the recombinant lentivirus could cause the HepG2 cell apoptosis, and its apoptosis rate was significantly more than paired control group and SP-TAT-Apoptin carried by liposomes only. CONCLUSION: The recombinant lentivirus of SP-TAT-Apoptin is successfully packaged and it can induce HepG2 cells to apoptosis.

[SP-TAT-Apoptin induces G1 arrest in HepG2 cells].

AIM: To investigate the effect of SP-TAT-Apoptin in inducing HepG2 cells apoptosis and the possible application on hepatocellular carcinoma gene therapy. METHODS: Recombinant gene SP-TAT-Apoptin was amplified by PCR and cloned into the eukaryotic vector plenti6-V5-D-TOPO. After the recombinant plasmid was identified by restriction enzyme digestion analysis and DNA sequencing, CHO cells were stably transfected with SP-TAT-Apoptin gene and the culture supernatant was collected. Then the expression of the fusion protein was detected by RT-PCR and Western blot. HepG2 cells were co-cultured with the supernatant. At various times post co-culture, HepG2 cells were detected by FCM. RESULTS: The secretory Tat-Apoptin has an additive bystander effect as an anti-cancer therapy in vitro. The recombinant Apoptin was able to be secreted from transfected cells and re-enter adjacent un-transfected HepG2 cells, it can induce HepG2 cells apoptosis and induce G0/G1 arrest. CONCLUSION: SP-TAT-Apoptin can induce HepG2 cell apoptosis and cell cycle G1 arrest.

Secretory Transactivating Transcription-apoptin fusion protein induces apoptosis in hepatocellular carcinoma HepG2 cells.

AIM: To determine whether SP-TAT-apoptin induces apoptosis and also maintains its tumor cell specificity. METHODS: In this study, we designed a secretory protein by adding a secretory signal peptide (SP) to the N terminus of Transactivating Transcription (TAT)-apoptin (SP-TAT-apoptin), to test the hypothesis that it gains an additive bystander effect as an anti-cancer therapy. We used an artificial human secretory SP whose amino acid sequence and corresponding cDNA sequence were generated by the SP hidden Markov model. RESULTS: In human liver carcinoma HepG2 cells, SP-TAT-apoptin expression showed a diffuse pattern in the early phase after transfection. After 48 h, however, it translocated into the nuclear compartment and caused massive apoptotic cell death, as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and annexin-V binding assay. SP-TAT-apoptin did not, however, cause any cell death in non-malignant human umbilical vein endothelial cells (HUVECs). Most importantly, the conditioned medium from Chinese hamster ovary (CHO) cells transfected with SP-TAT-apoptin also induced significant cell death in HepG2 cells, but not in HUVECs. CONCLUSION: The data demonstrated that SP-TAT-apoptin induces apoptosis only in malignant cells, and its secretory property might greatly increase its potency once it is delivered in vivo for cancer therapy.