[Morphological differentiation of Garra orientalis(Cyprinidae)among different geographical populations].

Morphological differences were found among specimens of Garra orientalis from Minjiang River, Pearl River, Red River and Hainan Island. To confirm whether the morphological differences among these geographical populations had reached the level of subspecies or species, seventy-two specimens of Garra orientalis were divided into four groups according to the four drainage areas stated above. Ten external morphological characteristics were observed. Thirteen frame characteristics and twenty-three general characteristics were measured and analyzed for multivariate morphometrics. In scatter plots of principal components, the specimens from Hainan Island were relatively concentrated on the scatter plots, but were not completely separated from the other three drainages. The samples from the other three drainages were completely mixed together. These results indicated that the specimens from the different drainages could not be distinguished by morphological comparison and morphometric analysis. Species or subspecies level differentiation of Garra orientalis among geographical populations could instead be studied by molecular biological methods in the future.

Activation of AKT is associated with metastasis of nasopharyngeal carcinoma.

Although radiotherapy results of nasopharyngeal carcinoma (NPC) at an early stage are better than other tumors, there is still a portion of patients with NPC who die before 5 years after the treatment; the underlying mechanism remains to be further understood. This study aims to investigate the mechanism by which NPC cells escape from irradiation. Patients with NPC at stage I was included in this study. All the patients were treated with irradiation. NPC biopsies were obtained from each patient before and 1 week after the start of radiotherapy. Expression of AKT in NPC tissue was assessed by Western blotting. NPC cell line, SUNE-1 cells, was treated with irradiation. The levels of AKT in SUNE-1 cells were also assessed. The frequency of apoptotic SUNE-1 cells was evaluated by flow cytometry. The levels of AKT were markedly increased in NPC tissue after treatment with irradiation, which was significantly correlated with NPC metastasis and mortality. After irradiation, NPC cell line, SUNE-1 cells, expressed higher levels of AKT than control cells. The knockdown of AKT in SUNE-1 cells markedly increased apoptotic cell rate. Radiotherapy can increase the levels of AKT in NPC cells that are associated with NPC metastasis and increase in mortality.

[Association between single nucleotide polymorphism locus rs189037 in the promoter of ATM gene and nasopharyngeal carcinoma susceptibility in Cantonese].

OBJECTIVE: To evaluate the correlation between single nucleotide polymorphisms (SNP) in the promoter of ataxia-telangiectasia mutated (ATM) gene and the susceptibility of nasopharyngeal carcinoma in Cantonese. METHODS: A total of 176 NPC Cantonese NPC patients and 202 age-matched healthy controls were enrolled in this study. G/A genotyping of the SNP locus rs189037 in the promoter of ATM gene was performed by PCR and direct sequencing of the PCR products. RESULTS: The genotype frequency of heterozygote G/A was 50% (88/176) and 47.5% (96/202), and that of the homozygote variant A/A was 15.3% (27/176) and 18.3% (37/202) in NPC patients and healthy individuals, respectively. Statistics analysis revealed no evident correlations of the G/A and A/A genotypes to the clinical phenotypes of NPC in either NPC group or healthy control group (OR7equals;1.022, 95%CIequals;0.668-1.564 for G/A+A/A). Analysis after stratification by age and gender found no such correlations either. CONCLUSION: The genotype frequencies of SNP locus rs189037 in the promoter of ATM gene are similar between NPC patients and healthy subjects, suggesting that rs189037 is not related to the genetic susceptibility of NPC in Cantonese.

Silibinin activated ROS-p38-NF-κB positive feedback and induced autophagic death in human fibrosarcoma HT1080 cells.

Our previous results demonstrated that silibinin induced autophagic and apoptotic cell death dependent on reactive oxygen species (ROS especially H(2)O(2) and [image omitted] ) in HT1080 cells. In this study, we further show that p38-NF-κB pathway is involved in silibinin-induced ROS-mediated autophagy. Cells were pretreated with serum-free media for 24 h before being treated with silibinin. Generation of ROS and autophagy was detected in 15 min and 1 h, respectively. Development of autophagy was supported by an upregulated expression of Beclin-1 and conversion of light chain (LC3-I-LC3-II). Expression of p38/p-p38 and transposition of NF-κB from cytoplasm to nuclei were also increased. Inhibitors of p38 and NF-κB and scavengers of H(2)O(2) and O(2)(*-) reduced both generation of ROS and simultaneous occurrence of silibinin-induced autophagy. Besides, expression of p38/p-p38 and transposition of NF-κB from cytoplasm to nuclei were decreased by these two ROS scavengers. ROS and p38-NF-κB pathway were possibly cooperated in a positive feedback mechanism. Inhibition of p38, NF-κB, H(2)O(2), or O(2)(*-) rescued cells from silibinin-induced death in a long-term (12 h) manner. According to the previous study that silibinin-induced autophagy was a positive regulator of apoptotic cell death, it was possible that ROS and p38-NF-κB mediated silibinin-induced autophagy and eventually led to cell death.

Silibinin activated p53 and induced autophagic death in human fibrosarcoma HT1080 cells via reactive oxygen species-p38 and c-Jun N-terminal kinase pathways.

Our previous research demonstrated that hepatic-protectant silibinin induced autophagy in human fibro-sarcoma HT1080 cells through reactive oxygen species (ROS) pathway. Pifithrin-α (PFT-α), a specific inhibitor of p53, reduced autophagy and reversed silibinin's growth-inhibitory effect; besides, PFT-α decreased the activation of caspase-3, a crucial executor of apoptosis. Silibinin upregulated expression of p53/phosphorylated-p53 (p-p53) in a time-dependent manner. Catalase (scavenger of H(2)O(2)), superoxide dismutase (SOD) (scavenger of O(2)(•-)), and SB203580 (inhibitor of p38) attenuated upregulation of p53 expression, suggesting that p53 might be partially regulated by ROS-p38 pathway. On the other hand, c-Jun N-terminal kinase (JNK) increased autophagic death in silibinin-treated cells, and JNK/p-JNK expression was upregulated by silibinin time-dependently. Inhibition of JNK by SP600125 did not influence generation of ROS. Scavengers of H(2)O(2) or O(2)(•-) showed no effect on expression of JNK/p-JNK, indicating that JNK might not correlate with ROS in this process. However, activation of p53 was suppressed by SP600125; therefore the function of p53 was possibly controlled by JNK as well. Western blotting analysis showed that PFT-α reduced activation of extracellular regulated kinase1/2 (ERK1/2) and expression of protein kinase B (PKB, or Akt)/p-Akt. PD98059 (inhibitor of mitogen-activated protein kinase kinase (MEK)/ERK) and wortmannin (inhibitor of phosphoinositide 3-kinase (PI3K)/Akt) enhanced silibinin's cytotoxicity. Wortmannin augmented silibinin-induced autophagy, while PD98059 did not affect autophagic ratio. These results suggest that silibinin might induce p53-mediated autophagic cell death by activating ROS-p38 and JNK pathways, as well as inhibiting MEK/ERK and PI3K/Akt pathways.

Impact of prolonged fraction delivery times simulating IMRT on cultured nasopharyngeal carcinoma cell killing.

PURPOSE: To determine the impact of prolonged fraction delivery times (FDTs) simulating intensity-modulated radiotherapy (IMRT) on cultured nasopharyngeal carcinoma (NPC) cell killing. METHODS AND MATERIAL: Cultured NPC cell lines CNE1 and CNE2 were used in this study. The biological effectiveness of fractionated irradiation protocols simulating conventional external beam radiotherapy and IMRT (FDT of 15, 36, and 50 minutes) was estimated with standard colony assay, and the differences in cell surviving fractions after irradiation with different protocols were tested by use of the paired t test. The impact degree of prolonged FDTs (from 8 to 50 minutes) on cell killing was also assessed by the dose-modifying factors, which were estimated by comparing the effectiveness of intermittently delivered 2 Gy with that of continuously delivered 1.5 to 2 Gy. RESULTS: The cell surviving fractions of both CNE1 and CNE2 after fractionated irradiation simulating IMRT were higher than those simulating conventional external beam radiotherapy (p < 0.05). The dose-modifying factors for a fraction dose of 2 Gy increased from 1.05 to 1.18 for CNE1 and from 1.05 to 1.11 for CNE2 with the FDT being prolonged from 15 to 50 minutes. CONCLUSIONS: This study showed that the prolonged FDTs simulating IMRT significantly decreased the cell killing in both CNE1 and CNE2 cell lines, and these negative effects increased with the FDT being prolonged from 15 to 50 minutes. These effects, if confirmed by in vivo and clinical studies, need to be considered in designing IMRT treatments for NPC.

[Role of lung fibroblast activation in radiation lung injury].

OBJECTIVE: To investigate the role of lung fibroblast activation in radiation-induced lung injury. METHODS: Thirty-five male Wistar rats were exposed to a single-dose 30 Gy irradiation of the right hemithorax or sham right lung irradiation. At 1, 7, 14, 28, 56 or 84 days after the irradiation, the rats were sacrificed for examination of alpha-smooth muscle actin (alpha-SMA) expression in the bilateral lung tissues using immunohistochemistry. RESULTS: alpha-SMA expression in fibroblast increased significantly in the out-field and in-field lung tissues within 24 h after irradiation after the irradiation (P<0.001). CONCLUSION: Activation of the lung fibroblasts occurred within 24 h after irradiation and found in ont-field and in-field lung tissues, suggesting that radiation-induced lung injury may not have an obvious latency.

[Three-dimensional conformal radiotherapy combined with transcatheter arterial chemoembolization for massive primary liver cancer].

OBJECTIVE: To evaluate the outcomes of patients with unresectable massive primary liver cancer (PLC) receiving three-dimensional conformal radiotherapy (3-DCRT) combined with transcatheter arterial chemoembolization (TACE). METHODS: From January 2001 to December 2004, 84 patients with unresectable massive PLC (tumor size> or =10 cm) received 3-DCRT combined with TACE, including 49 cases in UICC/AJCC T(3) stage and 35 cases in T(4) stages. Lymph node metastasis was found in none of the patients, and portal vein tumor thrombosis (PVTT) was detected in 30 cases. Child-Pugh grade A of liver cirrhosis was present in 64 cases and grade B in 20 cases. The mean value of GTV was 705-/+430 cm(3) (170-2099 cm(3)). Following injections of fluorouracil and hydroxycamptothecine into the target artery of the tumor, the mixture of carboplatin, mitomycin (or pirarubicin) and super-liquefactive iodized oil was injected into the target artery. Gelatin sponge was used to embolize the artery. The procedure was repeated every 1.5-2 months according to the condition of the patients, and each patient received 1-3 such procedures. 3-DCRT was performed in all the patients, who received a total dose of 53.6-/+6.6 Gy (4-6 Gy per fraction at the interval of 48 h), and 3 fractions were given every week. RESULTS: Eight patients died in 3 months after 3-DCRT and were not evaluated. The total response rate (CR+PR) in these patients was 68.9% (51/74). The overall survival rates at 1, 2 and 3 years were 55.4%, 24.7% and 15.4%, respectively. T stage, GTV, PVTT and fraction size had no significant impact on the overall survival. Child-Pugh grade was found to have significant impact on the patients' survival (P=0.035, RR=2.440). CONCLUSION: 3-DCRT combined with TACE has definite therapeutic effect on advanced massive PLC, and Child-Pugh grade is an independent prognostic factor in such cases.

Influence of FDG-PET on computed tomography-based radiotherapy planning for locally recurrent nasopharyngeal carcinoma.

PURPOSE: Assuming F-18-fluoro-2-deoxy-d-glucose positron emission tomography (FDG-PET)/computed tomography (CT) to be more accurate in representing the true disease extent than CT alone, we prospectively designed this study to evaluate how the addition of FDG-PET influences CT-based radiotherapy planning for locally recurrent nasopharyngeal carcinoma. PATIENTS AND METHODS: All patients underwent FDG-PET/CT simulation scans. For each patient, the gross tumor volume (GTV) was separately delineated with or without the addition of PET information and defined as GTV PET/CT and GTV CT, respectively. Corresponding planning target volumes (PTV) were generated for the GTV CT (PTV(CT)) and GTV PET/CT (PTV PET/CT). Three-dimensional conformal radiotherapy plans were separately created for PTV CT and PTV PET/CT. To assess the potential geographic miss of the PET/CT-based disease in CT-based treatment planning, the size and location of the GTV PET/CT, PTV(PET/CT), and PTV(CT) were analyzed, and the three-dimensional conformal radiotherapy plans created using the PTV CT were evaluated with the GTV PET/CT and PTV PET/CT information. RESULTS: A total of 43 patients were enrolled in this study. Distant metastasis was found in 4 patients with the addition of the PET information. The 39 patients without distant metastasis proceeded to three-dimensional conformal radiotherapy planning. Inadequate coverage of the GTV PET/CT and PTV PET/CT by the PTV CT occurred in 7 (18%) and 20 (51%) patients, respectively. This resulted in <95% of the GTV(PET/CT) and PTV PET/CT receiving >or=95% of the prescribed dose in 4 (10%) and 13 (33%) patients, respectively. CONCLUSIONS: The addition of FDG-PET information might influence CT-based radiotherapy planning for locally recurrent nasopharyngeal carcinoma by altering the definition of the target volume, with the potential to avoid a geographic miss of true disease.

[Relationship between radiosensitivity of nasopharyngeal carcinoma and MDR1 gene polymorphism].

OBJECTIVE: To investigate the relationship between human multidrug resistancel gene (MDR1) polymorphisms and the radiosensitivity of nasopharyngeal carcinoma (NPC). METHODS: Blood samples were collected from 59 NPC patients, who were devided into radiosensitive or radioresistant groups according to their responses to radiation therapy. The genotypes for MDR1 polymorphisms (G2677T in exon 21 and C3435T in exon 26) and their haplotypes were determined by PCR and restriction fragment length polymorphism analysis. The results were further confirmed by sequencing. RESULTS: The 3435CC genotype was associated with a significantly better response to radiotherapy than combined 3435 CT and TT genotype (P=0.026). The 2677GG genotype was also associated with a better response in comparison with combined 2677 GT and TT genotype, but this relation was not statistically significant. Patients with 2677G-3435C haplotype had a significant better response to radiotherapy than those with the other haplotypes (P=0.017). CONCLUSION: The MDR1 G2677T and C3435T polymorphisms may help predict the response to radiotherapy in NPC patients.

[Radiosensitization of nasopharyngeal carcinoma cell line CNE1 by inhibiting the expression of ATM/PI3K functional region].

BACKGROUND & OBJECTIVE: It is reported that ATM gene is closely correlated to cellular radiosensitivity in several malignant tumors. Suppression of ATM protein expression leads to cellular radiosensitization. This study was to determine whether this effect also exists in nasopharyngeal carcinoma (NPC) cell line CNE1 by inhibiting the expression of ATM protein through antisense RNA of ATM/PI3K region, which is the most important functional fragment of ATM gene. METHODS: The recombinant pDOR-atm expressing antisense RNA of ATM/PI3K segment was constructed with retroviral vector pDOR. CNE1 cells were stably transfected with pDOR-atm by cationic liposome and named CNE1/pDOR-atm. Semi-quantitive RT-PCR was used to detect the level of ATM mRNA. Flow cytometry (FCM) was employed to analyze the percentage of positive cells and mean fluorescence density of protein expressing ATM. Cellular radiosensitivity was evaluated by colony survival assay (CSA) and linear-quadratic model in both CNE1/ pDOR-atm and control cells. RESULTS: The level of ATM mRNA index (RI) was 0.23+/-0.02 in CNE1/pDOR-atm group, and 0.51+/-0.03 in control group (P<0.05). The percentage of positive cells and mean fluorescence density of proteins expressing ATM were 70.8% and 1.81+/-0.12 in CNE1/pDOR-atm group, while 99.3% and 4.51+/-0.18 in control group(P<0.01). The expression of ATM mRNA and protein was inhibited by antisense RNA. The alpha value (one function from linear-quadratic model) of CNE1/pDOR-atm group was 0.40 Gy(-1), while it was 0.36 Gy(-1) in control group. The radiosensitizing ratio of surviving fraction at 2 Gy (SF(2)) was 3.0, indicating that antisense RNA group was more radiosensitive to X-ray than the controls. CONCLUSION: NPC cell line CNE1 could be radiosensitized by the down-regulation of ATM/PI3K expression.