Mutation or not, what directly establishes a neoplastic state, namely cellular immortality and autonomy, still remains unknown and should be prioritized in our research.

Although the concept that cancer is caused by mutations has been widely accepted, there still are ample data deprecating it. For example, embryonic cells displaced in non-embryonic environments may develop to cancer, whereas cancer cells placed in embryonic environments may be reverted to phenotypic normal. Although many intracellular or extracellular aberrations are known to be able to initiate a lengthy tumorigenesis, the molecular or cellular alterations that directly establish a neoplastic state, namely cellular immortality and autonomy, still remain unknown. Hereditary traits are encoded not only by gene sequences but also by karyotype and DNA or chromosomal structures that may be altered via non-mutational mechanisms, such as post-translational modifications of nuclear proteins, to initiate tumorigenesis. However, the immortal and autonomous nature of neoplasms makes them "new" organisms, meaning that neoplasms should have mutations to distinguish themselves from their host patients in the genome. Neoplasms are malignant if they bear epigenetic or genetic alterations in mutator genes, i.e. the genes whose alterations accelerate other genes to mutate, whereas neoplasms are benign if their epigenetic or genetic aberrations occur only in non-mutator genes. Future mechanistic research should be focused on identifying the alterations that directly establish cellular immortality and autonomy. Benign tumors may have many fewer alterations and thus be much better models than cancers for such research. Future translational research should be aimed at identifying the cellular factors that control cancer cells' phenotypes and at establishing approaches of directing cancer cells towards differentiation, which should be a promising therapeutic tactic.

Interface Study on the Effect of Carbon and Boron Carbide Diffusion Barriers in Sc/Si Multilayer System.

Sc/Si multilayers are one of the promising material combinations commonly used in the spectral range of 35-50 nm. However, diffusion and silicidation at the interfaces of Sc/Si multilayers limit widespread applications of this material combination. To improve the properties of Sc/Si multilayers, the scheme of barrier layers is utilized. In this work, a series of Sc/Si multilayers with boron carbide and carbon barrier layers were designed and fabricated to compare the properties including interface quality and thermal stability. The effect on the multilayer structure and quality before and after annealing were investigated by using grazing-incidence X-ray reflection, X-ray diffraction, rocking-curve X-ray diffuse scattering, transmission electron microscopy, and selected area electron diffraction. The results indicate that severe interdiffusion and crystallization occur in the multilayer with a carbon barrier after annealing. However, a boron carbide barrier layer improves thermal stability up to 550 °C since the interfaces remain abrupt and clear after annealing. The multilayer quality is confirmed to be improved significantly.

Effect of membrane-bound complement regulatory proteins on tumor cell sensitivity to complement-dependent cytolysis triggered by heterologous expression of the α-gal xenoantigen.

Engineering malignant cells to express a heterologous α-gal antigen can induce heterograft hyperacute rejection, resulting in complement-dependent cytolysis (CDC) of tumor cells, which has been considered as a novel strategy for antitumor therapy. A549 cells engineered to express Galα1-3Galß1-4GlcNAc-R (α-gal) epitope exhibited strong resistance to CDC treated by normal human serum (NHS) in a previous study. We hypothesized that the expression of membrane-bound complement regulatory proteins (mCRPs) decay accelerating factor (CD55) and protectin (CD59) influenced the efficacy of the α-gal/NHS-mediated antitumor effect to tumor cells in vitro. The present study confirmed that A549 cells expressed high levels of CD55 and CD59, whereas Lovo cells expressed relatively low levels of these proteins. A549 and Lovo cells transfected with plasmids containing or lacking the α-gal epitope were evaluated for their susceptibility to CDC by NHS and detected using a trypan blue exclusion assay. α-gal-expressing Lovo (Lovo-GT) cells were almost completely killed by α-gal-mediated CDC following incubation with 50% NHS, whereas no cytolysis was observed in α-gal expressing A549 (A549-GT) cells. Abrogating CD55 and CD59 function from A549-GT cells by various concentrations of phosphatidylinositol-specific phospholipase C (PI-PLC) or blocking antibodies increased the susceptibility of cells to CDC, and the survival rate decreased significantly comparing to the controls (P<0.05). The findings of the present study indicated that using the α-gal/NHS system to eliminate tumor cells via inducing the complement cascade reaction might represent a feasible approach for the treatment of cancer. However, high levels of mCRP expression may limit the efficacy of this approach. Therefore, an improved efficacy of cancer cell killing may be achieved by combining strategies of heterologous α-gal expression and mCRP downregulation.

Baicalein inhibits hepatocellular carcinoma cells through suppressing the expression of CD24.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer death and is the most common type of liver cancer. Current therapies for hepatocellular carcinoma are still rather limited and novel therapeutic strategies are required. Baicalein, extracted from Scutellaria baicalensis, has anticancer effects on HCC in vitro and vivo. However, the detailed mechanisms are not well studied yet. In the present study, we evaluated anticancer effects of purified botanical extracts on HCC cells using high-throughput screening and investigated the effects of baicalein on HCC cells using proliferation and apoptosis assays, RT-PCR, and Western blot. Transfection was used to explore the underlying mechanisms of these effects. Our results showed that baicalein is the most efficient botanical extract in a HCC cell line as compared with the other 13 extracts. Baicalein significantly decreased the expression of c-Myc, a crucial regulator of cell proliferation, apoptosis and cellular transformation, in dose- and time-dependent manners in HCC cells. Moreover, baicalein inhibited HCC cell proliferation and induced apoptosis. The mRNA and protein expressions of CD24 were downregulated by baicalein in HCC cells and ectopic overexpression of CD24 reversed baicalein-induced inhibition of cell proliferation and survival. Taken together, our results demonstrate efficient anticancer effects of baicalein on HCC cells and indicate that baicalein suppresses cell growth and cell survival through downregulation of CD24.

Culture at a Higher Temperature Mildly Inhibits Cancer Cell Growth but Enhances Chemotherapeutic Effects by Inhibiting Cell-Cell Collaboration.

Acute febrile infections have historically been used to treat cancer. To explore the underlying mechanism, we studied chronic effects of fever on cancer cell growth and chemotherapeutic efficacy in cell culture. We found that culturing cancer cells at 39°C mildly inhibited cell growth by arresting the cells at the G1 phase of the cell cycle. When cells were seeded in culture dishes at a lower density, e.g. about 1000-2000 cells per 35-mm dish, the growth inhibition was much greater, manifested as many fewer cell colonies in the 39°C dishes, compared with the results at a higher density seeding, e.g. 20,000 cells per dish, suggesting that cell-cell collaboration as the Allee effect in cell culture is inhibited at 39°C. Withdrawal of cells from serum enhanced the G1 arrest at 39°C and, for some cell lines such as A549 lung cancer cells, serum replenishment failed to quickly drive the cells from the G1 into the S and G2-M phases. Therapeutic effects of several chemotherapeutic agents, including clove bud extracts, on several cancer cell lines were more potent at 39°C than at 37°C, especially when the cells were seeded at a low density. For some cell lines and some agents, this enhancement is long-lasting, i.e. continuing after the cessation of the treatment. Collectively these results suggest that hyperthermia may inhibit cancer cell growth by G1 arrest and by inhibition of cell-cell collaboration, and may enhance the efficacy of several chemotherapeutic agents, an effect which may persist beyond the termination of chemotherapy.

A study on the inhibitory effect of matrine on gastric cancer SGC-7901 cells.

The objective of this paper was to investigate the antitumour mechanism of action of matrine by studying its inhibitory effect on gastric cancer SGC-7901 cells. SGC-7901 cells were chosen, and cell-killing capacity of matrine on gastric cancer SGC-7901 cells was determined using MTT assay and single PI staining assay. The results showed that matrine had an inhibitory effect on gastric cancer SGC-7901 cells, which was somewhat dose-dependent. The study concluded that matrine has a significant in-vitro inhibitory effect on SGC-7901 tumour cells, influences cell cycle of SGC-7901 cells, and induces their apoptosis.

[Experimental study on A549 cell death mediated by xenoantigen α-gal 
in human serum].

BACKGROUND AND OBJECTIVE: The absence of α-gal in humans is caused by the inactivity of α-1,3GT gene. However, humans have pre-existing and abundant anti-gal antibodies. Xenotransplantation procedures have indicated the high potential of introducing α-1,3GT gene to synthesize α-gal for cancer gene therapy by mimicking hyper-acute rejection. The aim of this study is to construct a lung cancer A549 cell line that expressed α-gal, and to observe the antitumor mechanisms mediated by human serum. METHODS: A549 cells were transfected with pEGFP-N1-GT plasmids constructed in a previous study. A stable transgenic cell line, A549-GT, was then selected and cultivated. The biological characteristics of A549-GT cells, including morphology and proliferation, were examined. α-1,3GT mRNA expression was detected by RT-PCR. Direct immunofluorescence staining and flow cytometry (FCM) were used to analyze the synthesis of α-gal in A549-GT. The binding of human serum IgM and C3 with A549-GT were also detected. RESULTS: α-1,3GT mRNA was expressed in A549-GT. Direct immunofluorescence staining and FCM indicated a high and stable α-gal expression rate in A549-GT. Compared with parental A549 cells, the biological characteristics of A549-GT were unaltered. α-Gal expression was not detected in the human fetal lung fibroblast cell line MRC-5 even though A549-GT and its culture medium were cultivated with the enzyme. Immunofluorescence staining and FCM also indicated abundant binding between A549-GT treated with human serum and IgM/C3. CONCLUSIONS: α-Gal expression in tumor cells by gene transduction can induce complement-dependent cytototic antitumor effects.

Identification of the PAG1 gene as a novel target of inherent radioresistance in human laryngeal carcinoma cells.

Laryngeal carcinoma, as a malignant tumor that occurs in the head and neck region, is widely treated by radiation, but in some cases, the cancer is radioresistant to the radiotherapy. The reason for the radioresistant response needs to be clinically understood. We designed our present study to identify the molecules that may be involved in this radioresistant response. In this study, we initially established the inherent radioresistant (Hep-2max) and radiosensitive (Hep-2min) cell lines from the parental laryngeal cancer cell line Hep-2. Furthermore, using microarray analysis, we identified a novel inherent radioresistance-related gene, phosphoprotein associated with glycosphingolipid-enriched microdomains1 (PAG1). We showed that siRNA directed against PAG1 in a radioresistant (Hep-2max) cell line dramatically enhanced the radiosensitivity and IR-induced cell death. On the contrary, ectopic expression of PAG1 in radiosensitive (Hep-2min) cell lines led to radioresistance and suppressed the IR-induced cell death. Taken together, our results indicate that the PAG1 gene may be a novel, promising radiosensitization target for laryngeal carcinoma.

[Cloning of splicing variants of alpha1,3-galactosyltransferase cDNA of Chinese Banna Minipig inbred line and its expression in human cells].

OBJECTIVE: To study the transfection and expression of the splicing variants of alpha1, 3-galactosyltransferase cDNA of Chinese Banna Minipig Inbred Line (BMI) in human A549 cells. METHODS: Full length of alpha1,3-GT gene cDNA was amplified by RT-PCR from total RNA of BMI liver tissue and cloned into T-A cloning vector. Two different splicing variants of BMI alpha1,3-GT cDNA were confirmed by sequencing 15 positive clones and inserted respectively into pEGFP-N1 to construct eukaryotic expression vectors pN-GT1 and pN-GT2. The vectors were transfected into human lung adenocacinoma A549 cells and the expression of alpha1,3-GT gene was detected by RT-PCR. The expression of the a-Gal epitopes on transfected cells was confirmed under fluorescent microscope and by flow cytometry using FITC-BS-IB4 lectin. The binding of IgM and complement C3 in human serum to a-Gal on transfected cells were measured by flow cytometry using FITC-anti-IgM and FITC-anti-C3. RESULTS: There was no other splicing variants of alpha1,3-GT cDNA found in BMI except GT1 and GT2, which were 1116 bp and 1080 bp in length respectively, the latter lacks exon 5. The expression of BMI alpha1,3-GT mRNA and the synthesis of a-Gal on A549 cells transfected with either pN-GT1 or pN-GT2 were detected, and the binding of IgM nature antibodies and complements C3 in human serum on transfected A549 cells were observed. The expression level of alpha-Gal and the deposits of IgM and C3 on transffected cells showed no significant difference between pN-GT1 and pN-GT2. CONCLUSION: The splicing variants of alpha1,3-GT cDNA of BMI could express in human cells, which provide the basis for genetic manipulation of the alpha1,3-GT of BMI for future xenotransplantation studies.

[Establishment and identification of human lung adenocarcinoma cell line stably expressing the alpha1, 3-galaetosyltransferase gene from pig].

OBJECTIVES: To establish a human lung adenocarcinoma cell subline A549 that can stably express the Chinese Banna minipig inbred-line (BMI) alpha1 ,3-galactosyltransferase (alpha1 ,3GT) gene and alpha-galactosyl (Gala1-3Galb1-4GlcNAc-R, alpha-gal) epitopic, providing a cell model which expressed xenotransplantation antigens for the further research on the effect of complement dependent cytotoxic lysis of the tumor cells triggered by human natural serum. METHODS: The pEGFP-CMV-GT plasmid containing Banna minipig alpha1 ,3-GT gene was ransfected into A549 cells with lipofectin in vitro. After screened with G418,the single clones were got out and then amplified, the stable transfected cells was named A549-GT. The transcription of alpha1, 3-GT gene in A549-GT cells was detected by RT-PCR. Direct immunofluonrescence methods and flow cytometer were performed to observe the expression of alpha-gal and the binding conditions of IgM and complement C3 in human serum on A549-GT cells. The biological characters of A549-GT cells including morphology, proliferation, and tumorigenesis in nude mice were also examined. RESULTS: After G418 screening, A549-GT that stablely transfected with alpha1, 3-GT gene was obtained and has been passaged for 2 years. The expression of alpha1,3-GT mRNA and alpha-gal was detected continuously and stably in A549-GT. The expression rate of alpha-gal positive cells reached 80.1% +/- 3.2%. The binding of human serum IgM and C3 in human serum on A549-GT cells were founded. Compared with parental A549 cells, its biological characteristics did not change. CONCLUSION: A549-GT cell line stably and continuously expressing alpha1, 3-GT and alpha-gal was established successfully. It provided a useful cell model for the further study of pig alpha1,3-GT gene in tumor immunotherapy.

[Cloning of Chinese Banna minipig inbred-line alpha1,3-galactosyltransferase gene and construction of its recombinant eukaryotic expression vector].

This study sought to clone Chinese Banna minipig inbred-line (BMI) alpha1,3-galactosyltransferase (alpha1,3-GT) gene and construct its recombinant eukaryotic expression vector. Total RNA was isolated from BMI liver. Full length cDNA of alpha1,3-GT gene was amplified by RT-PCR and cloned into pMD18-T vector to sequence. Subsequently, alpha1,3-GT gene was inserted into pEGFP-N1 to construct eukaryotic expression vector pEGFP-N1-GT. Then the reconstructed plasmid pEGFP-N1-GT was transiently transfected into human lung cancer cell line A549. The expression of alpha1,3-GT mRNA in transfected cells was detected by RT-PCR. FITC-BS-IB4 lectin was used in the direct immunofluorescence method, which was performed to observe the alpha-Gal synthesis function of BMI alpha1,3-GT in transfected cells. The results showed that full length of BMI alpha1,3-GT cDNA was 1116 bp. BMI alpha1,3-GT cDNA sequence was highly homogenous with those of mouse and bovine, and was exactly the same as the complete sequence of those of swine, pEGFP-N1-GT was confirmed by enzyme digestion and PCR. The expression of alpha1,3-GT mRNA was detected in A549 cells transfected by pEGFP-N1-GT. The expression of alpha-Gal was observed on the membrane of A549 cells transfected by pEGFP-N1-GT. Successful cloning of BMI alpha1,3-GT cDNA and construction of its eukaryotic expression vector have established a foundation for further research and application of BMI alpha1,3-GT in the fields of xenotransplantation and immunological therapy of cancer.