Establishment and validation of a prediction model for the probability of malignancy in solid solitary pulmonary nodules in northwest China.

BACKGROUND AND OBJECTIVES: To construct a prediction model of solitary pulmonary nodules (SPNs), to predict the possibility of malignant SPNs in patients aged 15-85 years in northwest China for clinical diagnostic and therapeutic decision-making. METHODS: The features of SPNs were assessed by multivariate logistic regression, followed by visualization using a nomogram. Hosmer lemeshow was applied to evaluate the fitting degree of the model. The area under the receiver operating characteristic (ROC) curve was identified to determine the discriminative ability of the model. RESULTS: Lobulation, spiculation, pleural-tag, carcinoembryonic antigen, neuron-specific enolase, and total serum protein were independent predictors of malignant pulmonary nodules (p < .05). Lobulation (100 points) scored the highest in the nomogram, and the Hosmer-Lemeshow goodness-of-fit statistic was 0.805 (p > .05). The area under curve (AUC) of the modeling and validation groups using logistic regression were 0.859 (95% CI, 0.805-0.903) and 0.823 (95% CI, 0.738-0.890), respectively. Moreover, the AUC of our model was higher than that of the Mayo model, VA model, and Peking University (AUC 0.823 vs. 0.655 vs. 0.603 vs. 0.521). CONCLUSION: Our prediction model is more suitable for predicting the possibility of malignant SPNs in northwest China, and can be calculated using a nomogram to determine further treatments.

[Construction and Identification of the cDNA Expression Library for Human Esophageal Cancer Cells].

OBJECTIVES: To construct a cDNA phage expression library for human esophageal cancer cells. METHODS: After the total RNA were obtained from esophageal cancer cells, the mRNA were separated with magnetic beads adsorption method, and the single-strand and double-strand cDNA were synthesized through reverse transcription. With the undesirable cDNA fragments removed, the remaining cDNA (linked withEcoR1 aptamer and phosphorylated its 5'end) combined with the carrier of T7 Select10-3b. The recombinant phage were packaged in vitro for preliminary cDNA library. PCR was used to identify the size of inserted cDNA. RESULTS: The constructed original cDNA phage expression library for human esophageal cancer cells was consisted of 2.01×106 pfu/mL bacteriophages with a recombination rate of 100%. The length of the inserted cDNA fragments were range from 300 bp to 1 500 bp. CONCLUSIONS: The cDNA phage expression library of human esophageal cell is successfully constructed to meet the currently recognized standards, and can be well used to screen cDNA-cloned genes of human esophageal cancer antigens by serological analysis of recombinantly expressed cDNA clone (SEREX).

[Combination of phage display and SEREX for screening early lung cancer associated antigens].

OBJECTIVE: To screen out effective lung cancer associated antigens for early diagnosis in order to improve the level of early diagnosis. METHODS: A T7 phage display cDNA library of human early lung cancer was developed. And then differential phage clones were picked out to be sequenced and bioinformatically analyzed. With the 8 screened differential phage clones a lung cancer associated antigen microarray was established to evaluate the single or combined roles of all the selected antigens in the diagnosis of lung cancer by the reaction of the antigens plus serum from normal subjects and patients with lung cancer, respectively. RESULTS: The titer of the constructed cDNA library was 3.71×10 (6); pfu/ml and the number of phage was 1.11×10 (6); pfu, with a recombination rate of cDNA library over 90%. Nine differential phage clones were initially screened out, but the genes of two antigens (A42 and A83) were found the same. Bioinformatics analyses showed that the genes of the 8 antigens were known before and they were all proven to be related with tumor except A64. The positive reaction rates of the 8 antigens with serum from lung cancer patients were significantly higher than that with serum from normal subjects (Ps<0.05). When keeping specificity no less than 60%, the sensitivity of each antigen in predicting lung cancer alone was under 70% and the areas under curve (AUC) of the antigens were all under 0.8. However, when all the antigens were combined to detect lung cancer, the sensitivity and specificity was 90.8% and 94.1%, respectively, and AUC reached up to 0.969. CONCLUSION: A T7 phage display cDNA library with a good quality of capacity, recombination rate and representativeness of human early lung cancer was successfully developed, and 8 lung cancer associated antigens were screened out. A combination of the 8 antigens can greatly improve their value to diagnose lung cancer with a higher sensitivity and specificity (both above 90%)．

Anti-angiogenesis effect of generation 4 polyamidoamine/vascular endothelial growth factor antisense oligodeoxynucleotide on breast cancer in vitro.

OBJECTIVE: To study the effects of the generation 4 polyamidoamine/vascular endothelial growth factor antisense oligodeoxynucleotide (G4PAMAM/VEGFASODN) compound on the expressions of vascular endothelial growth factor (VEGF) and its mRNA of breast cancer cells and on the inhibition of vascular endothelial cells. METHODS: We examined the morphology of G4PAMAM/VEGFASODN compound and its pH stability, in vitro transfection efficiency and toxicity, and the expressions of VEGF and its mRNA. Methyl thiazolyl tetrazolium assay was used to detect the inhibitory function of the compound on vascular endothelial cells. RESULTS: The compound was about 10 nm in diameter and was homogeneously netlike. From pH 5 to 10, it showed quite a buffered ability. The 48-h transfection rate in the charge ratio of 1:40 was 98.76%, significantly higher than that of the liposome group (P<0.05). None of the transfection products showed obvious toxicity on the cells. The expressions of both VEGF protein and its mRNA after G4PAMAM/VEGFASODN transfection decreased markedly. CONCLUSION: With a low toxicity, high safety, and high transfection rate, G4PAMAM/VEGFASODN could be a promising gene vector. Specifically, it inhibits VEGF gene expression efficiently, laying a basis for further in vivo animal studies.

Correction to: RIP1 protects melanoma cells from apoptosis induced by BRAF/MEK inhibitors.

Since publication of this paper, the authors have noticed that there were errors in Fig. 2A (the GAPDH of Mel-CV, Mel-CV.S, Mel-RMu and Mel-RMu.S), Fig. 2C (the GAPDH of Mel-CV.S and Mel-RMu.S), Fig. 3F (the GAPDH of Mel-CV.S and Mel-RMu.S), Fig. 3J(the GAPDH of Mel-RMu.S), Fig. 5C (the ERK1/2 of patient#3(post)), and Fig. 5F (the RIP1 of Mel-CV.S and Mel-RMu.S, the GAPDH of Mel-CV and Mel-RMu). As a result of the misfiling of the data during preparation of figures, incorrect images were inadvertently inserted in these figures. The correct figures are given below. The corrections do not alter the conclusions of the paper.

Efficacy and safety of gemcitabine-oxaliplatin combined with huachansu in patients with advanced gallbladder carcinoma.

AIM: To evaluate the efficacy and safety of gemcitabine-oxaliplatin (GEMOX) combined with huachansu (cinobufagin) injection treatment in patients with locally advanced or metastatic gallbladder carcinoma (GBC), and to assess the quality of life (QOL) of such patients. METHODS: Twenty-five patients with locally advanced or metastatic GBC were treated with intravenous gemcitabine (1000 mg/m2) over 30 min on days 1 and 8, 2 h infusion of oxaliplatin (120 mg/ m2) on day 1, and 2-3 h infusion of huachansu (20 mL/ m2) on days -3-11, every 3-4 wk. Treatment was continued until occurrence of unacceptable toxicity or disease progression. QOL of patients was assessed by the EORTC QLQ-C30 at baseline, at the end of the first, third and sixth chemotherapy cycles, and 1 mo after the treatment. RESULTS: Among the 25 patients with a median age of 64 years (range 42-78 years), 23 were evaluable in the study. A total of 137 cycles of therapy were performed and the median cycle was 5 (range 1-8) per patient. Out of the 23 patients whose response could be evaluated, 8 partial responses (PR) were observed (34.8%), while 7 patients (30.4%) demonstrated a stable disease (SD). The disease control rate was 65.2%. Progression of cancer was observed in 8 (34.8%) patients. The median progression-free and overall survival time was 5.8 mo (95% CI: 4.5-7.1 mo) and 10.5 mo, respectively. The therapy was well tolerated, with moderate myelosuppression as the main toxicity. Anemia grade 2 was seen in 16.0%, neutropenia grade 3 in 8.0% and thrombocytopenia grade 3 in 24.0% of patients, respectively. Non-hematologic toxicity ranged from mild to moderate. No death occurred due to toxicity. The QOL of patients was improved after chemotherapy, and the scores of QOL were increased by 10 to 20 points. CONCLUSION: GEMOX combined with huachansu (cinobufagin) injection is well tolerated, effective, thus improving the QOL of patients with advanced GBC.

[Expression of ER alpha and SMRT in apoptosis of breast cancer cells induced by tamoxifen].

OBJECTIVE: To observe the expression of ER alpha and SMRT in ER alpha-positive and -negative cell lines before and after treatment with tamoxifen (TAM). METHODS: Breast cancer T47D cells (ER alpha-positive) and MDA-MB-231 cells (ER alpha-negative) were treated with TAM, cell viability was measured by MMT assay before and after TAM treatment. Flow cytometry (FCM) was applied to analyze apoptosis rate and cell cycle. Immunohistochemistry and Western blot were used to test ER alpha and SMRT expression in T-47D and MDA-MB-231 cells with and without TAM treatment. RESULT: Proliferation rate of T-47D and MDA-MB-231 decreased after 0.10 mmol/L TAM treatment for 48 h compared with control group (P <0.05), especially that of T47D cells. The result of FCM showed that sub-diploid apoptosis peak was found in both cell lines after TAM treatment. Immunohistochemistry and Western blot indicated that T-47D cells presented ER alpha++ and SMRT++, and ER alpha expression decreased after TAM treatment, meanwhile, that of SMRT increased. MDA-MB-231 cells presented ER alpha-, SMRT-, and both expression levels increased slightly after TAM treatment. CONCLUSION: TAM can inhibit the proliferation of breast cancer cells by inducing cell apoptosis,which is associated with alteration of ER alpha and SMRT expression.

[Anti-angiogenesis effect of G4PAMAM/VEGFASODN on breast cancer in vitro].

OBJECTIVE: To investigate the effect of G4PAMAM/VEGFASODN compound on expression of VEGF and VEGF mRNA in MDA-MB-231 breast cancer cells and the growth inhibition of vascular endothelial cells. METHODS: The diameter of G4PAMAM/VEGFASODN granule was measured by transmission electron microscopy, and its stability under different pH was also observed. The efficiency of transfection in vitro was detected by flow cytometer and the positively transfected cells were detected by laser scanning confocal microscope. The survival rate and the inhibitory rate of vascular endothelial cells were determined by MTT assay. The expression of protein VEGF was detected by immunohistochemical method and the expression of VEGF mRNA was detected by RT-PCR. RESULT: The diameter of G4PAMAM/VEGFASODN granules was about 10 nm and it arranged as homogeneous netlike. Under pH 5-10 G4PAMAM/VEGFASODN presented highly stable and no dissociation was observed with different charge ratios. The 48-hour transfection rate of G4PAMAM/VEGFASODN in charge ratio of 1:40 was significantly higher than that of the lipofectamine group. None of the transfection products in each group showed cell toxicity. The staining of VEGF protein in the cytoplasm of MDA-MB-231 cells after G4PAMAM/ASODN transfection weakened markedly, and the positive expression rate decreased. Meanwhile, the VEGF mRNA expression was also decreased. CONCLUSION: With good stability and transfection rate, compound G4PAMAM/VEGFASODN can be a promising new nanometer vector of gene transfer system. The G4PAMAM/VEGFASODN can inhibit the expression of VEGF gene specifically and efficiently, which may be used for in vivo animal experiment.

RIP1 protects melanoma cells from apoptosis induced by BRAF/MEK inhibitors.

Many recent studies have uncovered the necessary role for the receptor-interacting protein kinase 1 (RIP1) in regulating apoptosis and necrosis that cells undergo in response to various cellular stresses. However, the functional significance of RIP1 in promoting cancer cells survival remains poorly understood. Here, we report that RIP1 was upregulated and contributed to both intrinsic and acquired resistance of melanoma cells to BRAF/MEK inhibitors through activation of NF-κB. Strikingly, Snail1-mediated suppression of CYLD played a crucial role in promoting RIP1 expression upon ERK activation, particularly, in melanoma cells with acquired resistance to BRAF inhibitors. In addition, RIP1 kinase activity was not required for melanoma cells to survive BRAF/MEK inhibition as RIP1 mediated NF-κB activation through its intermediate domain. Collectively, our findings reveal that targeting RIP1 in combination with BRAF/MEK inhibitors is a potential approach in the treatment of the disease.

Alterations of serum cholinesterase in patients with gastric cancer.

AIM: To understand the correlation of serum cholinesterase (CHE) activity with gastric cancer and to assess their clinical significance. METHODS: The velocity method was adopted to detect the activity of serum CHE in patients with gastric cancer and in patients with non-malignant tumor as controls. RESULTS: The serum CHE activity in the treatment group was significantly lower than that in the control group with a very significant difference between the two groups (83.3:113.1,P = 0.0003). Age was significantly associated with the incidence of gastric cancer. CONCLUSION: Serum CHE activity has a close relation with the incidence of gastric cancer.

Clinical significance of expression of apoptotic signal proteins in gastric carcinoma tissue.

AIM: To evaluate the expressions of apoptotic signal proteins FADD, TRADD, FasL, Fas, and NFkappaB in gastric carcinoma tissues and their clinical significance. METHODS: Western blot immune trace method was adopted to detect the expressions of apoptotic signal proteins FADD, TRADD, FasL, Fas, and NFkappaB in 55 tissue specimens of gastric carcinoma. RESULTS: Five apoptotic signal proteins had different expressions in the gastric carcinoma samples and their expressions were not correlated to age (P = 0.085). Expressions of the FADD, FasL, Fas, and NFkappaB proteins reduced with increase of the volume of tumor with the exception of increased expression the TRADD protein (64.7-71.1%, P = 0.031). With gradual increase of the malignancy of gastric carcinoma tissues, expressions of the FADD, FasL, and Fas proteins decreased (78.6-28.0%, P = 0.008; 78.6-65.9%, P = 0.071; 100.0-46.3%, P = 0.014), while expressions of the TRADD and NFkappaB proteins increased (42.9-78.1%, P = 0.063; 78.6-79.1%, P = 0.134). With gradual increase of serum CEA, expression of the FADD protein decreased (62.5-34.0%, P = 0.073), but expressions of the TRADD, FasL, Fas, and NFkappaB proteins increased (0.0-80.8%, P = 0.005; 62.5-70.2%, P = 0.093; 0.0-70.2%, P = 0.003; 62.5-80.9%, P = 0.075). When compared to the tissues of gastric carcinoma without metastasis, the positive rate of expressions of the FADD and FasL proteins increased, whereas expressions of the TRADD, FADD, and NFkappaB proteins decreased. There was no significant difference between them (P = 0.095). CONCLUSION: Gastric carcinoma is endurable to Fas-related apoptosis and apoptotic signal proteins are differently expressed in gastric carcinoma.

Expression of estrogen receptor and estrogen receptor messenger RNA in gastric carcinoma tissues.

AIM: To study estrogen receptor (ER) and estrogen receptor messenger RNA (ERmRNA) expression in gastric carcinoma tissues and to investigate their association with the pathologic types of gastric carcinoma. METHODS: The expression of ER and ERmRNA in gastric carcinoma tissues (15 males and 15 females, 42-70 years old) was detected by immunohistochemistry and in situ hybridization, respectively. RESULTS: The positive rate of ER (immunohistochemistry) was 33.3 % in males and 46.7 % in females. In Borrmann IV gastric carcinoma ER positive rate was greater than that in other pathologic types, and in poorly differentiated adenocarcinoma and signet ring cell carcinoma the positive rates were greater than those in other histological types of both males and females (P<0.05). The ER was more highly expressed in diffused gastric carcinoma than in non-diffused gastric carcinoma (P<0.05). The ER positive rate was also related to regional lymph nodes metastases (P<0.05), and was significantly higher in females above 55 years old, and higher in males under 55 years old (P<0.05). The ERmRNA (in situ hybridization) positive rate was 73.3 % in males and 86.7 % in females. The ERmRNA positive rates were almost the same in Borrmann I, II, III and IV gastric carcinoma (P>0.05). ERmRNA was expressed in all tubular adenocarcinoma, poorly differentiated adenocarcinoma and signet ring cell carcinoma (P<0.05). The ERmRNA positive rate was related to both regional lymph nodes metastases and gastric carcinoma growth patterns, and was higher in both sexes above 55 years old but without statistical significance (P>0.05). The positive rate of ERmRNA expression by in situ hybridization was higher than that of ER expression by immunohistochemistry (P<0.05). CONCLUSION: ERmRNA expression is related to the pathological behaviors of gastric carcinoma, which might help to predict the prognosis and predict the effectiveness of endocrine therapy for gastric carcinoma.

Tumor stem cells: A new approach for tumor therapy (Review).

Recent studies have demonstrated the existence of a minority of tumor cells possessing the stem cell properties of self-renewal and differentiation in leukemia and several solid tumors. However, these cells do not possess the normal regulatory mechanisms of stem cells. Following transplantation, they are capable of initiating tumorigenesis and are therefore known as 'tumor stem cells'. Cellular origin analysis of tumor stem cells has resulted in three hypotheses: Embryonal rest hypothesis, anaplasia and maturation arrest. Several signaling pathways which are involved in carcinogenesis, including Wnt/ß-catenin, Notch and Oct-4 signaling pathways are crucial in normal stem cell self-renewal decisions, suggesting that breakdown in the regulation of self-renewal may be a key event in the development of tumors. Thus, tumors can be regarded as an abnormal organ in which stem cells have escaped from the normal constraints on self-renewal, thus, leading to abnormally differentiated tumor cells that lose the ability to form tumors. This new model for maligancies has significance for clinical research and treatment.

Effect of VEGF-targeted antisense gene therapy on retinoblastoma cell line SO-RB50 in vitro and in vivo.

AIM: To evaluate the possibility of generation 4 polyamidoamine (G4PAMAM) dendrimers acting as the delivery system of vascular endothelial growth factor (VEGF) antisense oligodeoxynucleotides (VEGFASODN), and to investigate the anti-tumor effect of G4PAMAM/VEGFASODN complex on the cultured cells and the mouse tumor xenograft model. METHODS: The transfection efficiency was assessed by Flow cytometry (FCM). Thiazolyl tetrazolium (MTT) assay was performed to determine the relative growth rate (RGR) of the cells after transfection. Then a mouse tumor xenograft model of human retinoblastoma was established. Different interventions were given to the mice by intratumoral injection and the tumor growth was monitored. The expression of VEGF mRNA was detected by reverse transcription PCR (RT-PCR), the expression of VEGF protein was determined by western blot analysis, and the microvessel density (MVD) was measured by immunohistochemistry (IHC) staining. RESULTS: G4PAMAM/VEGFASODN exhibited a high transfection rate in vitro, and the transfection rates of different doses of G4PAMAM/VEGFASODN groups increased with higher doses. This effect was accompanied by a dose-depended reduction in cell viability. The tumor growth in the tumor-bearing athymic mice was significantly inhibited in the G4PAMAM/VEGFASODN group. The expressions of VEGF mRNA and protein were obviously inhibited in the G4PAMAM/VEGFASODN group (P<0.05), and the MVD of the G4PAMAM/VEGFASODN group was lower than that of the other groups (P<0.05). CONCLUSION: VEGFASODN can be delivered into the cultured and transplanted retinoblastoma cells efficiently by G4PAMAM, suppress the expressions of VEGF mRNA and protein, and reduce the MVD of tumor tissues. The G4PAMAM/VEGFASODN complex has antitumor properties in vitro and in vivo.

TNF related apoptosis-inducing ligand and its receptors in ocular tumors.

Most of the ocular tumors have poor prognosis, and they remain a difficult problem in the area of ophthalmology. With the rapid development of molecular biology and immunologic techniques and the deep research on ocular tumor related genes, it becomes possible to diagnose and treat malignant tumors from the molecular level. The tumor necrosis factor related apoptosis-inducing ligand (TRAIL), a member of the tumor necrosis factor (TNF) super family, is a promising candidate, either alone or in combination with established cancer therapies, since it can initiate apoptosis through the activation of their death receptors. The ability of TRAIL to selectively induce apoptosis of transformed, virus-infected or tumor cells but not normal cells promotes the development of TRAIL-based cancer therapy. Here, we will review TRAIL and its receptors' structure, function, mechanism of action and application in ocular tumors therapy.

Secretory expression of Par-4 SAC-HA2TAT following adeno-associated virus-mediated gene transfer induces apoptosis in HepG2 cells.

Prostate apoptosis response-4 (Par-4) is a tumor-suppressor protein that induces apoptosis in cancer cells, but not in normal cells. The cancer-specific pro-apoptotic action of Par-4 is encoded in its centrally located SAC domain. In this study, to further enhance the anti-cancer effect of Par-4 in order to overcome the limitations of peptide therapy, a recombinant adeno-associated virus was constructed using the following strategies: the secretory expression of therapeutic peptide, a HA2TAT-mediated cytosolic delivery technique, and an adeno-associated virus gene transfer system. To test the hypothesis that Par-4 has an additive bystander effect as an anti-cancer therapy, we designed a secretory protein by adding a secretory signal peptide NT4(Si) to the Par-4 SAC-HA2TAT peptide gene sequence [NT4(Si)-Par-4 SAC-HA2TAT]. The results indicated that, compared to the normal NIH3T3 cell line, AAV-NT4(Si)-Par-4 SAC-HA2TAT significantly suppressed cell growth and induced rapid cell death in HepG2 cells in a time-dependent manner through successful gene transfer and secretory expression of therapeutic peptide at 48 h post-transfection. In addition, the secretory properties of Par-4 may greatly increase its effectiveness in cancer therapy when delivered in vivo.

Generation 4 polyamidoamine dendrimers is a novel candidate of nano-carrier for gene delivery agents in breast cancer treatment.

Polyamidoamine dendrimer (PAMAM-D) is a new gene vector developed in recent years. In this study, we successfully prepared G4PAMAM and detected its unique structure by NMR, FITR and TEM. We revealed that G4PAMAM could bind to human erythrocytes and BSA through electrostatic interaction respectively, and caused haemolysis and reduced bioavailability. However, G4PAMAM-VEGF-ASODN (antisense oligodeoxynucleotides) complex could prevent G4PAMAM from binding to the erythrocytes and BSA and remained stable as a conjugate, therefore the toxicity of the complex was reduced. Meanwhile, we showed that G4PAMAM could be used as a gene vector to deliver AODNs into breast cancer MDA-MB-231 cells without significant cell toxicity, and it enhanced cellular uptake of ODNs. In vivo experiment of human breast tumor xenograft mice model, G4PAMAM also showed more efficiency of accumulating VEGF-ASODN to inhibit the tumor vascularization of breast tumor tissue than naked AODN. Furthermore, G4PAMAM could protect DNA in cytoplasm from digestion of restriction enzymes, which was important to become an effective tool in gene research and therapy.

Combined treatment of oxaliplatin and capecitabine in patients with metastatic esophageal squamous cell cancer.

AIM: To investigate the efficacy and side effects of the combined therapy of oxaliplatin and capecitabine in patients with metastatic esophageal squamous cell cancer (ESCC) and the survival of the patients. METHODS: Sixty-four patients (median age of 63 years) with histological or cytological confirmation of ESCC received oxaliplatin 120 mg/m(2) intravenously on day 1 and capecitabine 1000 mg/m(2) orally twice daily on days 1 to 14 in a 21-d treatment cycle as palliative chemotherapy. Each patient received at least two cycles of treatment. The efficacy, side effects and patient survival were evaluated. RESULTS: The partial response (PR) rate was 43.8% (28/64). Stable disease (SD) rate was 47.9% (26/64), and disease progression rate was 15.6% (10/64). The clinical benefit rate (PR + SD) was 84.4%. The main toxicities were leukopenia (50.0%), nausea and vomiting (51.6%), diarrhea (50.0%), stomatitis (39.1%), polyneuropathy (37.5%) and hand-foot syndrome (37.5%). No grade 4 event in the entire cohort was found. The median progression-free survival was 4 mo, median overall survival was 10 mo (95% CI: 8.3-11.7 mo), and the 1- and 2-year survival rates were 38.1% and 8.2%, respectively. High Karnofsky index, single metastatic lesion and response to the regimen indicated respectively good prognosis. CONCLUSION: Oxaliplatin plus capecitabine regimen is effective and tolerable in metastatic ESCC patients. The regimen has improved the survival moderately and merits further studies.